HK40103960A - Carbon-source regulated protein production in a recombinant host cell - Google Patents

Description

Production of carbon source-regulated proteins in recombinant host cells

Divisional application

This application is a divisional application of application number 202080008585.5, filed on January 10, 2020, entitled “Production of carbon source-regulated proteins in recombinant host cells”.

Technical Field

This invention relates to the production of a target protein (POI) in a recombinant host cell comprising a heterologous expression cassette for expressing a target gene (GOI) encoding the POI, wherein the host cell is engineered to reduce the expression of the FLO8 protein.

Background Technology

Proteins produced in recombinant host cell cultures have become increasingly important as diagnostic and therapeutic agents. To this end, cells are engineered and/or selected to produce exceptionally high levels of the desired recombinant or heterologous protein. Optimization of cell culture conditions is crucial for the successful commercial production of recombinant or heterologous proteins.

In cell culture, both prokaryotic and eukaryotic host cells have been successfully used to produce target proteins (POIs). Eukaryotic host cells, particularly mammalian host cells, yeast, or filamentous fungi or bacteria, are commonly used as production hosts for biopharmaceutical proteins and for bulk chemicals. The most prominent examples are yeasts such as *Saccharomyces cerevisiae*, *Pichia pastoris*, or *Hansenula polymorpha*, filamentous fungi such as *Aspergillus awamori* or *Trichoderma reesei*, or mammalian cells such as CHO cells. Methylotrophic yeasts (such as *Pichia pastoris*) are known for their efficient secretion of heterologous proteins. Pichia pastoris has been reclassified into a new genus, Komagataella, and divided into three species: Komagataella pastoris, Komagataella phaffii, and Komagataella pseudopastoris. Strains commonly used for biotechnology applications belong to the two proposed species, Komagataella pastoris and Komagataella phaffii. Strains GS115, X-33, CBS2612, and CBS7435 are Komagataella phaffii, while the SMD series of protease-deficient strains (e.g., SMD1168) are classified as Komagataella pastoris, serving as the reference strain for all available Pichia pastoris strains (Kurtzman 2009, *Journal of Industrial Microbiology and Biotechnology* 36(11):1435-8). Mattanovich et al. (Microbial Cell Factories, 2009, 8:29 doi:10.1186/1475-2859-8-29) described the genome sequencing of Pasteurella multocida strain DSMZ 70382 and analyzed its secreted proteins and sugar transporters.

WO2015/158808A2 discloses a recombinant host cell that has been engineered to overexpress accessory proteins.

WO2015/158800A1 discloses the ability to enhance the expression and/or secretion of POIs in host cells by engineering downexpression of certain proteins (referred to as KO proteins), said proteins being endogenous to the host cells and having been shown to reduce protein production yield upon overexpression. Therefore, these KO proteins have been selected as knockout targets for enhancing yield. In turn, downexpression of KO proteins in Pichia pastoris host cell lines has been found to increase the yield of model proteins by 1.2 to 2.4 times. Inducible (pAOX1) or constitutive (pGAP) promoters have been used. KO proteins have been identified as Pichia pastoris homologs of Saccharomyces cerevisiae FLO8 protein, Saccharomyces cerevisiae HCH1 protein, and KO3a Pichia pastoris homolog of Saccharomyces cerevisiae SCJ1 protein.

Rebnegger et al. (Applied and Environmental Microbiology, 2016, 82(15):4570-4583) described glucose-restricted chemostat cultures of Pichia pastoris flo8 deletion mutant to prevent filter clogging.

Promoters used for protein production in recombinant host cells are either regulated (e.g., induced by the addition of methanol to the culture medium, methanol-controlled) or remain active (constitutive). Methanol-controlled promoters lead to technological limitations, such as waste heat or oxygen supply in the reactor.

WO20137050551A1 discloses a series of carbon-source-regulated promoters (named pG1-pG8) of Pichia pastoris, and the induction of protein production when carbon sources are restricted in cell cultures.

WO2017021541A1 discloses a variant of the carbon-source-regulated promoter (designated pG1) of Pichia pastoris, which is regulated by a carbon source other than methanol, i.e. is not controlled by methanol, for example, is inhibited in the presence of a carbon source during the growth phase, and is induced by restricting the carbon source during the production phase.

Prielhofer et al. (Microbial Cell Factory, 2013; 12(5): 1-10) described a modifiable and induced Pichia pastoris promoter in the absence of methanol.

Prielhofer et al. (Biotechnology and Bioengineering 2018; 115:2479–2488) described a glucose-regulated _PGTH1 promoter and engineered variants with significantly enhanced inducibility compared to the wild-type promoter.

EP2669375A1 discloses yeast with improved protein expression through high-level expression of the MPP1 homolog.

Hye Young Kim et al. (Biochemical and Biophysical Research Communications, 2014, 449: 202-207) described the roles of two domains of the Flo8 activator in the transcriptional activation of a set of target genes, and the mode of action of Flo8 through interaction with the Mss11 activator.

EP2952584A1 discloses improved protein production through overexpression of certain polynucleotides.

EP2258855A1 discloses certain leader and secretory signal sequences of Pichia pastoris.

WO2010099195A1 discloses a genetically modified Pichia pastoris strain and the co-expression of heterologous and chaperone proteins.

Summary of the Invention

The object of this invention is to improve protein production in recombinant host cells and increase protein production yield. Another object of this invention is to provide a method for producing recombinant or heterologous proteins in host cells, wherein the risk of morphological changes in host cells during the production process is reduced.

This objective is achieved through the subject matter as claimed.

According to the present invention, a recombinant host cell comprising an endogenous gene encoding an FLO8 protein, the FLO8 protein comprising an amino acid sequence identified as SEQ ID NO:1 or a homolog thereof, the host cell being engineered by one or more gene modifications to reduce (or eliminate) the expression of the polynucleotide compared to a host cell prior to the one or more gene modifications, and the host cell comprising a heterologous expression cassette comprising a target gene (GOI) for expressing the GOI under the control of an expression cassette promoter (ECP).

Specifically, ECP can be modulated using non-methanol carbon sources.

Specifically, ECP can be suppressed by non-methanol carbon sources.

Specifically, ECP can be inhibited by inhibiting a carbon source, such as a non-methanol-based inhibiting carbon source, such as glucose or glycerol, and can be induced (de-inhibited) by reducing the amount of the inhibiting carbon source.

Specifically, ECP is not induced by methanol.

Specifically, a non-methanol carbon source is any carbon source other than methanol that is suitable for host cell culture. Specifically, a non-methanol carbon source is not methanol.

Specifically, non-methanol carbon sources are carbon sources other than methanol. In particular, ECP is not methanol-controlled. Although cell cultures or cell media may or may not contain methanol, ECP, as used herein, is not regulated by any amount of methanol, and in particular is not methanol-induced, and therefore is not methanol-controlled. Specifically, ECP can be fully induced in methanol-free cell cultures or cell media.

For the purposes described herein, the term "FLO8 protein" should refer to both, including the protein identified as the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that has some homology with SEQ ID NO:1. However, homologous sequences are also referred to as FLO8 homologs.

Specifically, the FLO8 homolog has any one of 25%, 30%, or 35% sequence identity with SEQ ID NO:1, for example, any one of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity, or 100% identity with SEQ ID NO:1. Specifically, as further disclosed herein, sequence identity is determined, for example, when comparing full-length sequences.

Specifically, the FLO8 protein or its corresponding homolog includes a LisH domain that includes at least one of 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:50, or has 100% identity with SEQ ID NO:50.

According to one specific aspect, the LisH domain of the FLO8 protein includes at least one having 80%, 85%, 90%, or 95% sequence identity with any naturally occurring LisH domain of the FLO8 protein in eukaryotic organisms or with a corresponding FLO8 ortholog, or having 100% identity with such naturally occurring LisH domains, specifically LisH domains of yeast or fungi, particularly filamentous fungi, such as LisH domains comprising any one or a combination of the following:

a) SEQ ID NO:50, which is the LisH domain of the FLO8 protein of *Phomys fargesii* (SEQ ID NO:50);

b) SEQ ID NO:51, LisH domain of FLO8 protein from *Saccharomyces cerevisiae* (BLASTp: 96% identity with SEQ ID NO:50 (23/24aa));

c) SEQ ID NO:52, which is the LisH domain of the FLO8 ortholog of the Saccharomyces cerevisiae strain (BLASTp: 54% identity with SEQ ID NO:50 (13/24aa));

d) SEQ ID NO:53, which is the LisH domain of another Saccharomyces cerevisiae strain FLO8 ortholog (BLASTp: 54% identity with SEQ ID NO:50 (13/24aa));

e) SEQ ID NO:54, which is the LisH domain of the FLO8 ortholog of Yarrowia lipolytica strain (BLASTp: 71% identity with SEQ ID NO:50 (12/17aa));

f) SEQ ID NO:55, which is the LisH domain of an FLO8 ortholog of Ogataea polymorpha (BLASTp: 62% identity with SEQ ID NO:50 (8/13aa)); or

g) SEQ ID NO:56, which is the LisH domain of the FLO8 ortholog of Aspergillus niger strain (BLASTp: 73% identity with SEQ ID NO:50 (8/11aa)).

Typically, the LisH domain of the FLO8 protein is 25-27 amino acids long and shares some sequence identity with the human protein LIS1, as described in the database Pfam, ID number PF08513 and the corresponding sequence. In molecular biology, the LisH domain is a protein domain found in a large number of eukaryotic proteins with broad functions. The structure of the LisH domain in the N-terminal region of LIS1 describes it as a dimerization motif (The dimerization mechanism of LIS1 and its implication for proteins containing the LisH motif). Mateja A, Cierpicki T, Paduch M, Derewenda ZS, Otlewski J. 2006. Journal of Molecular Biology (J Mol Biol.) 357(2):621-31.

It is particularly understood that the FLO8 homolog is endogenous to the host cells used to produce recombinant host cells as described further herein. In particular, the FLO8 protein is a species-endogenous ortholog of the host cell species.

Specifically, if the host cell is *Pichia pastoris*, particularly *Poma pastoris* and *Poma freundii*, then the FLO8 protein is derived from *Pichia pastoris*, particularly *Poma pastoris* or *Poma freundii*. Alternatively, the FLO8 protein comprises a homologous (or orthologous) sequence of such a FLO8 protein derived from *Pichia pastoris*, particularly *Poma pastoris* or *Poma freundii*, which is endogenous to the wild-type host cell, if it is from another source or species. For example, if the host cell is *Poma freundii*, then the endogenous FLO8 protein comprises or consists of the amino acid sequence identified as SEQ ID NO:1. According to another example, if the host cell is *Pasteurella pastoris*, the endogenous FLO8 protein comprises or consists of the amino acid sequence identified as SEQ ID NO:3, which has 91% identity with SEQ ID NO:1. However, if the host cell is a different species (besides *Pasteurella pastoris* and/or *Pasteurella frenulum*), the endogenous FLO8 protein sequence in the host cell is a homolog of SEQ ID NO:1, and for the purposes described herein, the expression of such homologs (the homologous sequence of SEQ ID NO:1) in the host cell is reduced.

Specifically, any or every homologous sequence is characterized in that the FLO8 protein in the corresponding wild-type host cell has the same qualitative function as the FLO8 protein in Pichia pastoris, particularly Kommas pastoris or Kommas freundii, such as as a transcription factor, particularly a DNA-binding transcription activator involved in the regulation of cell adhesion, flocculation, invasive growth or starch catabolism, although its quantitative activity may differ from that of the FLO8 protein in wild-type Kommas pastoris or Kommas freundii.

Specifically, the corresponding homologous sequences are from species different from *Pichia pastoris*, particularly *Kumaria pastoris* or *Kumaria fremilum*, for example, another yeast or filamentous fungal cell, preferably a yeast of the genus *Kumaria* or *Pichia*, or *Saccharomyces genus* or any methyltrophic yeast. However, the host cell can be an animal cell, vertebrate cell, mammalian cell, human cell, plant cell, bacterial cell, nematode cell, invertebrate cell such as insect cell or mollusc cell or stem cell, and the corresponding FLO8 protein and its homologs described herein are endogenous to the corresponding host cell, but their expression is reduced or eliminated as described herein.

Specifically, the FLO8 protein homolog is endogenous or derived from the Pichia pastoris species, or endogenous or derived from any other yeast, fungus, or bacterium, and has 25% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, and in certain cases at least any one of 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%, or has 100% identity with SEQ ID NO:1 or SEQ ID NO:2. Specifically, when an exogenous FLO8 protein (or the gene encoding the exogenous FLO8 protein) is added to a culture of a flo8 knockout strain of Pichia pastoris or Saccharomyces cerevisiae, if the exogenous FLO8 protein is functional, then the exogenous FLO8 protein is identified as a homolog of the FLO8 protein, and the knockout strain is from a different source than the exogenous FLO8 protein, thus demonstrating that the missing endogenous FLO8 protein has a functional replacement.

Specifically, the FLO8 protein is derived from Pichia pastoris, and is encoded by genes endogenous in the host cell, Pichia pastoris.

Specifically, the FLO8 protein is derived from *Morchella frenulum* and comprises or consists of SEQ ID NO:1. Specifically, this type of FLO8 protein is encoded by a gene endogenous in a host cell, wherein the host cell is *Morchella frenulum*. Specifically, the FLO8 protein is encoded by a nucleotide sequence identified as SEQ ID NO:2.

Specifically, the FLO8 protein is derived from *Kombucha sinensis* and has at least 90% or 91% sequence identity with SEQ ID NO:1. Specifically, the FLO8 protein is derived from *Kombucha sinensis* and comprises or consists of SEQ ID NO:3. Specifically, such FLO8 proteins are encoded by genes endogenous to a host cell, wherein the host cell is *Kombucha sinensis*.

Specifically, the FLO8 protein is derived from *Saccharomyces cerevisiae* and includes at least 35% sequence identity with SEQ ID NO:1. Specifically, the FLO8 protein is derived from *Saccharomyces cerevisiae* and includes or consists of SEQ ID NO:5 or SEQ ID NO:6. Specifically, such FLO8 proteins are encoded by genes endogenous to a host cell, wherein the host cell is *Saccharomyces cerevisiae*.

Specifically, the FLO8 protein is derived from *Yarrowia lipolytica* and includes at least 40% or 44% sequence identity with SEQ ID NO:1. Specifically, the FLO8 protein is derived from *Yarrowia lipolytica* and includes or consists of SEQ ID NO:7. Specifically, such FLO8 proteins are encoded by genes endogenous to a host cell, wherein the host cell is *Yarrowia lipolytica*.

Specifically, the FLO8 protein is derived from *Henrynheideum helicobacter* and includes at least 30% or 34% sequence identity with SEQ ID NO:1. Specifically, the FLO8 protein is derived from *Henrynheideum polymorphum* and includes or consists of SEQ ID NO:8. Specifically, such FLO8 proteins are encoded by genes endogenous to a host cell, wherein the host cell is *Henrynheideum polymorphum*.

Specifically, the FLO8 protein is of Aspergillus genus and includes at least 25% or 26% sequence identity with SEQ ID NO:1. Specifically, the FLO8 protein is of Aspergillus niger and includes or consists of SEQ ID NO:9. Specifically, such FLO8 proteins are encoded by genes endogenous to a host cell, wherein the host cell is Aspergillus niger.

Specifically, the host cell undergoes gene modification through one or more gene modifications, including (one or more) chromosomal mutations, which respectively reduce the transcription and/or translation of the polynucleotide encoding the FLO8 protein, and/or otherwise reduce the expression of the polynucleotide and the production of the FLO8 protein.

Specifically, the one or more gene modifications include (i) one or more endogenous polynucleotides or a portion thereof; or (ii) disruption, substitution, deletion or knockout of expression control sequences.

According to one specific aspect, the one or more gene modifications are gene modifications of one or more endogenous polynucleotides of the host cell described herein, the endogenous polynucleotides being such as those encoding polynucleotides, including, for example, those encoding the FLO8 protein (or genes), particularly wild-type (unmodified or native) proteins that are naturally present in the host cell species, type or strain.

According to one particular aspect, the one or more gene modifications are gene modifications that express control sequences (including, for example, promoters, ribosome binding sites, transcription or translation initiation and termination sequences), or enhancer or activator sequences.

Various methods can be used to engineer host cells to reduce the expression of endogenous polynucleotides, such as genes encoding the FLO8 protein. These methods include, for example, disrupting the polynucleotide encoding the FLO8 protein, disrupting promoters operatively linked to such polynucleotides, replacing such promoters with another promoter having lower promoter activity, and modifying or regulating (e.g., activating, upregulating, inactivating, repressing, or downregulating) regulatory sequences that regulate the expression of such polynucleotides. Examples of such methods include using RNA-guided ribonuclease targeting relevant sequences as used in CRISPR-based methods of modifying host cells. These regulatory sequences are selected from the group consisting of promoters, ribosome binding sites, transcription initiation or termination sequences, translation initiation or termination sequences, enhancer or activator sequences, repressor or inhibitor sequences, signal or leader sequences, particularly those that control protein expression and/or secretion.

Specifically, compared to a corresponding host without such genetic modification, the one or more genetic modifications include one or more genomic mutations, the genomic mutations including the deletion or inactivation of a gene or genomic sequence that reduces the expression of a gene or a portion of a gene by at least 50%, 60%, 70%, 80%, 90%, or 95%, or even completely eliminates its expression by, for example, gene knockout.

Specifically, the one or more gene modifications include genomic mutations that constitutively weaken or otherwise reduce the expression of one or more endogenous polynucleotides.

Specifically, the one or more gene modifications include genomic mutations that introduce one or more inducible or repressible regulatory sequences that conditionally weaken or otherwise reduce the expression of one or more endogenous polynucleotides. Such conditionally active modifications are particularly targeted at regulatory elements and genes that are active and/or expressed depending on cell culture conditions.

Specifically, when a POI is generated, the expression of one or more endogenous polynucleotides is reduced, thereby reducing the expression of the polynucleotide encoding the FLO8 protein. Specifically, during gene modification, the expression of the FLO8 protein is reduced under the conditions of the host cell culture that generates the POI.

Specifically, compared to host cells without the modification, host cells are genetically modified to reduce the amount (e.g., level or concentration) of the FLO8 protein by at least any one of 50%, 60%, 70%, 80%, 90%, or 95% (mol/mol), or even by 100%, for example, to an undetectable amount, thereby completely eliminating the production of the FLO8 protein, for example, by knocking out the gene. According to one specific embodiment, the host cell is genetically modified to include one or more deletions of genomic sequences, particularly those encoding the FLO8 protein or its corresponding homologs. Such host cells are typically provided as deletion or knockout strains.

According to a specific implementation, once the host cells described herein are cultured in a cell culture, the amount of total FLO8 protein in the host cells or host cell cultures is reduced by at least any one of 50%, 60%, 70%, 80%, 90%, or 95% (mol/mol), or even reduced by 100%, for example, to an undetectable amount, compared to a reference amount expressed or produced by host cells before or without such gene modification, or to a reference amount produced in the corresponding host cell culture, or to host cells before or without said modification.

When comparing the effects of the genetic modifications described herein on reducing the production of the FLO8 protein in host cells, comparisons are typically made with comparable host cells before or without such genetic modifications. Comparisons are usually made with the same host cell species or type that has not undergone such genetic modifications and has been engineered to produce recombinant or heterologous POIs, particularly when cultured under conditions that produce the POIs. However, comparisons can also be made with the same host cell species or type that has not been further engineered to produce recombinant or heterologous POIs.

According to one specific aspect, the reduction of the FLO8 protein or its corresponding homologs is determined by a reduction in the amount (e.g., level or concentration) of the FLO8 protein in the cell. Specifically, the amount of the FLO8 protein or its corresponding homologs is determined by suitable methods, such as Western blotting, immunofluorescence imaging, flow cytometry, or mass spectrometry, particularly wherein the mass spectrometry is liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS), as described, for example, by Doneanu et al. (MAbs. 2012; 4(1):24–44).

According to one specific aspect, the recombinant host cell comprises only one or more heterologous expression cassettes, such as multiple copies of the expression cassette, including at least 2, 3, 4, or 5 copies (gene copy number, GCN). For example, the recombinant host cell comprises at most 2, 3, 4, or 5 copies. Each copy may include the same or different sequences or consist of the same or different sequences, but includes an ECP operatively linked to the GOI.

According to one specific aspect, the heterologous expression cassette comprises or consists of an artificial fusion of polynucleotides, the polynucleotides including an ECP operatively linked to a GOI, and optional other sequences, such as signal sequences, leader sequences, or termination sequences. Specifically, expression cassettes that are heterologous or artificial to the host cell are used, particularly those comprising a promoter (ECP) and a GOI, wherein the promoter and GOI are heterologous to each other and do not exist in nature in this combination, for example, wherein either (or only one) of the promoter and GOI is artificial or heterologous to the other described herein and/or to the host cell; the promoter is an endogenous promoter and the GOI is a heterologous GOI; or the promoter is an artificial or heterologous promoter and the GOI is an endogenous GOI; wherein both the promoter and GOI are artificial, heterologous, or from different sources, such as cells from a different species or type (strain) compared to the host cell described herein. Specifically, in cells used as host cells as described herein, ECPs are not naturally associated with and/or operatively linked to the GOI.

According to one specific aspect, ECP is inducible in the presence of a growth-limiting amount of non-methanol carbon source, preferably in the absence of methanol; and is inhibitable in the presence of an excess of non-methanol carbon source above the growth-limiting amount. Specifically, GOI expression via a heterologous expression cassette can be induced by inducible ECP.

Preferably, the ECP is carbon source-adjustable, such as being inhibited in the presence of any one of the following amounts of carbon source (referred to herein as promoter-inhibition amounts) above 1, 1.5, 2, 2.5, or 3 g/L in the cell culture medium or supernatant, and being induced or de-inhibited in the absence of a detectable carbon source, or being induced or de-inhibited in the presence of any one of the following amounts of carbon source (referred to herein as promoter-inducible amounts) at a level of up to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 g/L in the cell culture medium or supernatant. Such amounts in the cell culture medium or supernatant are specifically understood to be detectable during feeding and consumption by the host cells. Typically, when POIs are generated under growth-limited conditions, the cell culture is fed with a supplemental carbon source, but the amount is immediately consumed by the cells during POI generation. Therefore, no or only a small amount remains in the cell culture medium or supernatant, for example, at most 1.0 g/L.

Specifically, the carbon source for adjusting ECP is any carbon source other than methanol, and is referred to as the non-methanol carbon source in this paper.

Specifically, the non-methanol carbon source is carbohydrates.

Specifically, the non-methanol carbon source is selected from sugars, polyols, alcohols, or any mixture of one or more of the foregoing.

Specifically, the sugar can be any one or more monosaccharides, such as hexoses like glucose, fructose, galactose, or mannose, or disaccharides like sucrose; or alcohols or polyols such as ethanol, or any diol, or triols such as glycerol, or any mixture of the foregoing. Specifically, any such non-methanol carbon source can be used in cell cultures in an amount that produces the POI under the control of ECP.

According to one specific aspect, the ECP includes at least one first core regulation region and at least one second core regulation region. Specifically, the ECP includes at least two of the first core regulation regions and/or the second core regulation regions. Specifically, the ECP includes a finite number of first core regulation regions and second core regulation regions, wherein the number of first core regulation regions is only one, two, or three, and the number of second core regulation regions is only one, two, or three. Specifically, the ECP includes an equal number of first core regulation regions and second core regulation regions; for example, wherein the number of first core regulation regions is one, and the number of second core regulation regions is one; wherein the number of first core regulation regions is two, and the number of second core regulation regions is two; or wherein the number of first core regulation regions is three, and the number of second core regulation regions is three.

Specifically, the first core regulatory region has at least 75% sequence identity with SEQ ID NO:17, such as at least 80% or at least 90% sequence identity, and/or the second core regulatory region has at least 75% sequence identity with SEQ ID NO:18, such as at least 80% or at least 90% sequence identity.

Specifically, the first core regulation region and the second core regulation region each have a length of 8nt-16nt.

Specifically, the first core regulatory region has a length of 8 to 10 nt, particularly 9 nt. Specifically, the first core regulatory region includes, or consists of, a modification of, the nucleotide sequence identified as SEQ ID NO:17 or a nucleotide sequence identified as SEQ ID NO:17, wherein the modification is at most one or two point mutations, particularly one of which is a substitution, insertion, or deletion of a nucleotide.

Specifically, the second core regulatory region has a length of 14 to 16 nt, particularly 15 nt. Specifically, the second core regulatory region includes, or consists of, a modification of, the nucleotide sequence identified as SEQ ID NO:18 or the nucleotide sequence identified as SEQ ID NO:18, wherein the modification is at most one, two, or three point mutations, particularly one of which is any one of a substitution, insertion, or deletion of a nucleotide.

Specifically, the ECP includes at least one first core regulation region and at least one second core regulation region in any order, preferably very close to each other, for example, having any one of up to 20nt, 19nt, 18nt, 17nt, 16nt, 15nt, 14nt, 13nt, 12nt, 11nt or 10nt between the closest first core regulation region and the second core regulation region.

Specifically, the ECP includes a first core regulatory region and a second core regulatory region connected by spacers, and in particular separated by nucleotide sequences (referred to herein as the "spacer core region") of at least any one of 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, and 10 nt in length, and/or at most any one of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nt. Specifically, the spacer core region has at least 75% sequence identity with SEQ ID NO:36, such as at least 80% or at least 90% sequence identity, and/or includes, or is composed of, modifications of, SEQ ID NO:36 or a nucleotide sequence identified as SEQ ID NO:36, wherein the modifications are one, two, or three of up to four point mutations, particularly one of which is a substitution, insertion, or deletion of a nucleotide. Specifically, the spacer core region comprises or is composed of nucleotide sequences, wherein the majority of the nucleotides (at least 50% or at least 60%) are selected from G, C or T.

Specifically, the ECP includes at least one region consisting of a nucleotide sequence comprising three consecutive elements from the 5' end to the 3' end: (i) a first core regulatory region, (ii) a spacer core region, and (iii) a second core regulatory region, referred to herein as the "main regulatory region".

Specifically, the ECP includes at least one or two, or only one or two, main regulatory regions, each main regulatory region comprising or composed of a nucleotide sequence having at least any one of 85%, 90%, or 95% sequence identity with SEQ ID NO:35. Such main regulatory regions preferably consist of a first core regulatory region, a spacer core region, and a second core regulatory region.

Specifically, ECP includes at least one polynucleotide sequence that has at least one of 85%, 90%, or 95% sequence identity with SEQ ID NO:35.

Specifically, as described herein, the ECP comprises only one, two, or three main regulatory regions, particularly two or three, wherein the two or three regulatory regions may be identical or different from each other. Specifically, the ECP comprises only two main regulatory regions, which are separated by a nucleotide sequence of any length of 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt and/or at most 500 nt, 450 nt, 400 nt, 350 nt, or 300 nt, particularly a nucleotide sequence in the range of 100 nt to 300 nt (referred to herein as the “spacer main region”). Specifically, the spacer main region comprises a nucleotide sequence of at least any one of lengths 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, and 100 nt, the nucleotide sequence having at least 60% sequence identity with SEQ ID NO:37, such as at least any one of at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity, and/or includes, or consists of, modifications of, SEQ ID NO:37 or a nucleotide sequence identified as SEQ ID NO:37, or is composed of modifications of SEQ ID NO:37 or a nucleotide sequence identified as SEQ ID NO:37, wherein the modifications are multiple point mutations, which are any one of one or more, up to 30, 25, 20, 15, or 10 point mutations, particularly one of which is any one of a substitution, insertion, or deletion of a nucleotide.

According to one particular aspect, the ECP includes at least one T motif consisting of a nucleotide sequence, wherein the majority of the nucleotides are thymine (T), preferably at least any one of 50%, 60%, 70%, 80%, 90%, or 100% is T, for example, including or consisting of any one of SEQ ID NO:19-34, optionally without any extension of the T motif by one or more additional (or adjacent) thymines at either the 5' or 3' end of the T motif.

Specifically, ECP includes only one or two of the T motifs, or at least two of the T motifs, and at most four or three T motifs, wherein the T motifs are the same as or different from each other.

Specifically, the ECP includes at least one T motif upstream or downstream of the main control region, for example, one (or only one or two) T motifs upstream of the main control region and one (or only one or two) T motifs downstream of the main control region. However, according to one specific embodiment, the ECP includes at least one of the T motifs, particularly including only one or two T motifs within the spacer sub-main region.

Specifically, the ECP has at least any one of 350bp, 400bp, 450bp, 500bp, 550bp, 600bp, 650bp, 700bp, 850bp, 900bp, 950bp or 1000bp, for example, a length of up to 2000bp or up to 1500bp.

Specifically, the ECP includes a 3'-terminal nucleotide sequence, for example, at most 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 nt, or 5 nt in length, including a 3'-terminus comprising at least a portion of a translation initiation site, for example, a sequence having at least any one of 60%, 70%, 80%, 85%, or at least 90% identity with any one of SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40. The translation initiation site may be a Kozak concordant sequence in eukaryotes and a suitable promoter sequence supporting gene expression.

According to one specific aspect, ECP includes at least one of the following sequences having 60%, 65%, 70%, 75%, or 80% sequence identity with at least any one of 300 (continuous) nt, particularly 85%, 90%, or 95% sequence identity, or having 100% identity with at least any one of 300 nt, particularly 300 nt, 350 nt, 400 nt, 450 nt, 500 nt, 550 nt, 600 nt, 650 nt, 700 nt, 850 nt, 900 nt, 950 nt, or 1000 nt. Within the region containing the transcription factor binding site (TFBS), and/or within the 3' end sequence of any one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 or any one of SEQ ID NO:41-45, up to the full length of any of the aforementioned nucleotide sequences, especially if the full length is less than 1000 nt.

According to one specific aspect, ECP includes at least any one of the following sequence identity values of 60%, 65%, 70%, 75%, or 80%, particularly at least any one of the following sequence identity values of 85%, 90%, or 95%, or 100% with any one of the full-length sequences SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, or any one of SEQ ID NO:41-45.

A specific implementation refers to an ECP, which comprises or consists of SEQ ID NO:10 or SEQ ID NO:11, or comprises or consists of a nucleotide sequence, said nucleotide sequence having at least any one of the full-length sequences of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 or any one of SEQ ID NO:41-45 having 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity, or having 100% identity.

Specifically, the ECP promoter includes at least a portion or fragment of SEQ ID NO:10 or SEQ ID NO:11, or is composed of at least a portion or fragment of SEQ ID NO:10 or SEQ ID NO:11, with a length of at least any one of 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp or 1000bp, particularly including TFBS and/or the 3' end.

Specifically, either the first core regulatory region or the second core regulatory region of the ECP, or the main regulatory region of the ECP, contains one or more TFBSs. Specifically, each of the first core regulatory region and the second core regulatory region, or both of the first core regulatory region and the second core regulatory region together, or each of the main regulatory regions of the ECP, includes a TFBS or at least a portion thereof that is considered functional and recognized by the corresponding transcription factor.

Specifically, TFBS is recognized by any one or more transcription factors selected from the group consisting of Rgt1 (e.g., including or consisting of SEQ ID NO:47), Cat8-1 (e.g., including or consisting of SEQ ID NO:48), and Cat8-2 (e.g., including or consisting of SEQ ID NO:49).

TFBS are characterized by certain shared sequences, which can vary for the same factor. Specific transcription factor identification is as follows:

Rgt1 is a glucose-responsive transcriptional activator and repressor that regulates the expression of several glucose transporter (HXT) genes. The Rgt1 of *Pichia pastoris* includes the amino acid sequence SEQ ID NO:47.

Cat8-1 and Cat8-2 are zinc cluster transcription activators that bind to carbon source-responsive elements and are essential for the deexpression of multiple genes under non-fermentation growth conditions. The amino acid sequences of Cat8-1 and Cat8-2 in *Pichia pastoris* are SEQ ID NO:48 and SEQ ID NO:49, respectively.

According to one specific aspect, ECP includes at least two, three, four, five, six, seven, or eight TFBS, wherein each TFBS is individually identified by any one of Rgt1, Cat8-1, or Cat8-2.

Specifically, the ECP is characterized by an increased promoter strength compared to the reference promoter, wherein

- The promoter strength is the same as or greater than that of the reference promoter, particularly when in the induced state, by at least any one of the following: 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.3 times, 3.5 times, 3.8 times, 4 times, 4.5 times, 5 times, 5.5 times, or at least 6 times.

Specifically, native pGAP promoters of host cells (particularly pGAP promoters that are endogenous to and naturally present in host cells for recombinant POI production, such as native pGAP promoters of *Pichia pastoris* used to control GAPDH expression in *Pichia pastoris*, comprising or consisting of SEQ ID NO:46) can be used as a reference in *Pichia pastoris* host cells to determine the strength of improved ECP promoters. Such reference promoters can be used in parallel control experiments using the same host cells and expression system, or as internal controls within the same host cell culture. Such quantitative promoter function control experiments are preferably performed in *Pichia pastoris* host cell cultures, particularly in recombinant *Pichia pastoris* expressing model proteins such as GFP or eGFP, compared to reference promoters. Promoter strength compared to a reference promoter was determined using the following standard: *Pichia pastoris* strains expressing eGFP under the control of the tested promoter were screened in 24-well plates at 25°C with shaking at 280 rpm, using 2 mL of culture per well. Glucose feed beads (6 mm, Kuhner, CH) were used to generate glucose-limited growth conditions. eGFP expression in cells under induced conditions was analyzed (YP+1 feed bead, for 20–28 hours).

According to one specific aspect, the relative promoter or transcriptional strength or rate of the ECP described herein is compared with the natural pGAP promoter of cells of the same species or strain used as hosts for producing POIs.

Specifically, the reference promoter is the host cell's natural pGAP promoter. For example, the natural pGAP promoter of *Pichia pastoris* (which is an unmodified endogenous promoter sequence in *Pichia pastoris*, such as one used to control GAPDH expression in *Pichia pastoris* (GS115), for example including or consisting of the sequence identified as SEQ ID NO:46) can be used as a reference promoter in *Pichia pastoris*. If *Pichia pastoris* is used as a host cell for generating recombinant POIs as described herein, the transcriptional intensity or rate of the ECP described herein can be conveniently compared with such a natural pGAP promoter of *Pichia pastoris*.

Exemplary natural pGAP promoter sequence of Pichia pastoris (GS115) (SEQ ID NO:46)

# name PAS* PIPA* GS115 Description pGAP TDH3 PAS_chr2-1_0437 PIPA02510 Glyceraldehyde-3-phosphate dehydrogenase

*PAS: ORF name in Pichia pastoris GS115; PIPA: ORF name in Pichia pastoris strain DSMZ70382.

According to another embodiment, the natural pGAP promoter of *Saccharomyces cerevisiae* can be used as a reference promoter, which is an unmodified endogenous promoter sequence in *Saccharomyces cerevisiae*, such as for controlling GAPDH expression in *Saccharomyces cerevisiae*. If *Saccharomyces cerevisiae* is used as a recombinant host cell for generating POIs as described herein, the transcriptional intensity or rate of the ECP described herein can be conveniently compared with such a natural pGAP promoter of *Saccharomyces cerevisiae*.

Specifically, promoter strength, compared to a reference promoter, is determined by the expression level and/or transcriptional intensity of a point of interest (POI), such as a model protein (e.g., green fluorescent protein, GFP, including, for example, enhanced GFP, eGFP, gene bank registry number U57607)). Preferably, the transcriptional analysis is quantitative or semi-quantitative, preferably using qRT-PCR, DNA microarrays, RNA sequencing, and transcriptome analysis.

Specifically, ECP is further characterized by the promoter induction ratio, which is characterized by the high transcriptional intensity in the fully induced state compared to the low level in the repressed state.

Promoter induction ratio, specifically referring to the induction of transcription, includes further translation and optional expression of the POI. Transcription is typically defined as a measure of promoter strength, and specifically refers to the amount of transcript obtained upon complete induction of the promoter. Transcript abundance can be determined by transcriptional strength in a fully induced state, obtained, for example, under glucose-restricted chemostat culture conditions and expressed relative to the transcription rate of a reference promoter.

The induction ratio is a key parameter determining the carbon source regulation of ECP and sets the promoter activity or strength in the inducible state relative to the promoter activity or strength in the inhibited state. For example, the expression level of reporter proteins (e.g., GFP or eGFP) and/or the transcription level in the inhibited state are measured after inhibition by excess glycerol, and the expression level and/or transcription level of model proteins in the inducible state are measured after induction by glucose restriction.

If culture conditions provide approximately maximum induction, for example at glucose concentrations below 0.4 g/L, preferably below 0.04 g/L, and specifically below 0.02 g/L, the ECP promoter is considered derepressed and fully induced. A fully induced promoter preferably exhibits at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, and at least 100% of the transcriptional level/intensity, or even higher at least 150% or at least 200% of the transcriptional level/intensity, compared to the native pGAP promoter. The transcriptional level/intensity can be determined, for example, by the amount of transcript of a reporter gene (such as eGFP) when the clone is cultured in liquid culture. Alternatively, the transcription rate can be determined by the transcriptional intensity of a naturally controlled gene on a microarray, where the microarray data shows the difference in expression levels between a repressed and derepressed state compared to a control, and the high signal intensity in the fully induced state.

Specifically, the induction ratio can be determined by the ratio of the expression level in the induced state to that in the inhibited state (e.g., the expression level of a model protein, such as GFP or eGFP). The induction ratio compared to a reference promoter can be determined by the following standard assay: Pichia pastoris strains expressing eGFP under the control of the promoter to be tested are screened in 24-well plates with 2 mL of culture per well at 25°C and shaking at 280 rpm. Glucose feed beads (6 mm, Kuhner, CH) are used to generate glucose-limited growth conditions. eGFP expression in cells is analyzed during inhibition (YP + 1% glycerol, exponential phase) and induction (YP + 1 feed bead, lasting 20–28 hours).

Specifically, the ECP promoter in the de-repressed (inducible) state has promoter activity or strength (e.g., transcriptional activity or transcriptional strength) that is at least any one of 1.5, 2.0, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times higher than in the repressed state. Therefore, the corresponding induction rate can be at least any one of 1.5, 2.0, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

Surprisingly, the transcriptional activity (or transcriptional strength) of the ECP described herein (when fully induced, e.g., under glucose restriction induction) in the flo8 knockout strain was found to be significantly higher than that in the wild-type strain, which includes the flo8 locus and produces the FLO8 protein. These values were at least any one of 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher. This significant increase in transcriptional strength in the host cells described herein allowed for higher levels of GOI expression under ECP control in the flo8 deletion mutant. Conversely, transcription from pGAP or pAOX was not increased in the flo8 deletion mutant (as determined in the comparative examples).

According to one specific aspect, the heterologous expression cassette is contained in a self-replicating vector or plasmid, or integrated into the chromosome of the host cell.

Expression cassettes can be introduced into host cells and integrated into the host cell genome (or any chromosome thereof) as intrachromosomal elements, for example, at a specific integration site or randomly, thereby selecting high-producing host cell lines. Alternatively, expression cassettes can be integrated into extrachromosomal genetic elements (such as plasmids or artificial chromosomes, such as yeast artificial chromosomes (YAC)). According to one specific embodiment, expression cassettes are introduced into host cells via vectors, particularly expression vectors, which are introduced into the host cells through suitable transformation techniques. For this purpose, GOIs can be ligated into expression vectors.

Preferred yeast expression vectors (which are preferably used for expression in yeast) are selected from the group consisting of plasmids derived from pPICZ, pGAPZ, pPIC9, pPICZalfa, pGAPZalfa, pPIC9K, pGAPHis, pPUZZLE, or GoldenPiCS.

Techniques for transfecting or transforming host cells for the introduction of vectors or plasmids are well known in the art. These may include electroporation, protoplasts, lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA coprecipitation), viral infection, and in particular the use of modified viruses such as, for example, modified adenoviruses, microinjection, and electroporation.

Transformants as described herein can be obtained by introducing expression cassettes, vectors, or plasmid DNA into a host and selectively expressing relevant proteins or selection markers. Host cells can be treated with conventional methods for transforming host cells to introduce heterologous or exogenous DNA, such as electroporation, protoplastation, lithium acetate, and their modifications. Pichia pastoris transformation is preferably performed via electroporation. Preferred transformation methods for microbial uptake of recombinant DNA fragments include chemical transformation, electroporation, or protoplastation.

Specifically, the expression cassette includes an ECP operatively linked to a GOI encoding a POI, and optionally further includes a signal and leader sequence necessary for the expression and production of the POI as a secreted protein.

According to one particular aspect, the expression cassette further includes a nucleotide sequence encoding a signal peptide capable of secreting a POI, preferably wherein the nucleotide sequence encoding the signal peptide is fused adjacent to or directly fused to the 5' end of the GOI.

Specifically, the signal peptide was selected from the group consisting of: the signal sequence of the α-mating factor precursor peptide from Saccharomyces cerevisiae, the signal peptide from the acid phosphatase gene (PHO1) from Pichia pastoris, and extracellular protein X (EPX1) (Heiss, S., V. Puxbaum, C. Gruber, F. Altmann, D. Mattanovich & B. Gasser, Microbiology 2015; 161(7):1356-68).

Specifically, any signal and/or preamble sequence as described in WO2014067926 A1 may be used, particularly SEQ ID NO:59 or SEQ ID NO:60.

Specifically, signal sequences as described in WO2012152823 A1 can be used, particularly the signal sequence of the natural α-mating factor of Saccharomyces cerevisiae identified as SEQ ID NO:61 or a mutant thereof.

According to one specific aspect, the host cells described herein may undergo one or more further genetic modifications, for example, to improve protein production.

Specifically, the host cell is further engineered to modify one or more genes affecting proteolytic activity, which is used to produce protease-deficient strains, particularly those deficient in carboxypeptidase Y activity. Specific examples are described in WO1992017595A1. Further examples of protease-deficient Pichia pastoris strains with functional defects in vacuolar proteases such as protease A or protease B are described in US6153424A. Additional examples are Pichia pastoris strains with a deletion of ade2, and/or a deletion of one or both of the protease genes PEP4 and PRB1, provided by, for example, Thermo Fisher Scientific.

Specifically, as disclosed in WO2010099195A1, host cells are engineered to modify the nucleic acid sequence encoding at least one functional gene product, particularly a protease, said functional gene product being selected from the group consisting of PEP4, PRB1, YPS1, YPS2, YMP1, YMP2, YMP1, DAP2, GRHI, PRD1, YSP3 and PRB3.

Overexpression or underexpression of genes encoding cofactors have been specifically applied to enhance GOI expression, for example as described in WO2015158800A1.

Overexpression of the following genes has been shown to increase POI secretion in Pichia pastoris: PP7435_Chr3-0607, PP7435_Chr3-0933, PP7435_Chr2-0220, PP7435_Chr3-0639, PP7435_Chr4-0108, PP7435_Chr1-1232, PP7435_Chr1-1225, PP7435_Chr1-0667, and PP7435_Chr4-0448.

Low expression of the following genes has been shown to increase POI secretion in Pichia pastoris: PP7435_Chr1-0176, PP7435_Chr3-1062, and PP7435_Chr4-0252.

Specifically, the host cell can be engineered to overexpress any one or more cofactors and increase the production of the corresponding protein identified by any of SEQ ID NO:62-71, thereby further increasing POI production. POIs can be any eukaryotic, prokaryotic, or synthetic peptides, polypeptides, proteins, or metabolites of the host cell.

Specifically, POIs are heterologous to the host cell species.

Specifically, POI is a secreted peptide, polypeptide, or protein, that is, secreted from the host cell into the cell culture supernatant.

Specifically, POI is a eukaryotic protein, preferably a mammalian-derived or related protein, such as a human protein or a protein including a human protein sequence, or a bacterial protein or a bacterial-derived protein.

Preferably, the POI is a therapeutic protein that functions in mammals.

Under certain circumstances, POIs are multimeric proteins, especially dimers or tetramers.

According to a specific aspect, POI is a peptide or protein selected from the group consisting of antigen-binding proteins, therapeutic proteins, enzymes, peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccine antigens, growth factors, hormones, cytokines, process enzymes, and metabolic enzymes.

Specifically, antigen-binding proteins are selected from the following group:

a) Antibodies or antibody fragments, such as chimeric antibodies, humanized antibodies, bispecific antibodies, Fab, Fd, scFv, dimer, trimer, Fv tetramer, minibodies, single-domain antibodies such as VH, VHH, IgNAR or V-NAR.

b) Antibody mimics, such as Adnectins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, monomers, or NanoCLAMPs; or

c) Fusion proteins including one or more immunoglobulin folding domains, antibody domains, or antibody mimics.

A specific point of interest (POI) is an antigen-binding molecule, such as an antibody or a fragment thereof, particularly an antibody fragment that includes an antigen-binding domain. Antibodies in specific POIs include monoclonal antibodies (mAbs), immunoglobulins (Ig) or immunoglobulin G (IgG), heavy chain antibodies (HcAbs) or fragments thereof, such as fragment-antigen binding (Fab), Fd, single-chain variable fragments (scFv), or engineered variants thereof, such as, for example, Fv dimers (dimers), Fv trimers (trimers), Fv tetramers, or microantibodies, and single-domain antibodies such as VH, VHH, IgNAR, or V-NAR, or any protein that includes an immunoglobulin folding domain. Additional antigen-binding molecules may be selected from antibody mimics or (alternative) scaffold proteins, such as, for example, engineered Kunitz domains, Adnectins, affinity molecules, Affiline, Antiticalins, or DARPins.

According to one specific aspect, the POI is, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxin), alglucosidase alpha, daptomycin, YH-16, human chorionic gonadotropin alpha, filgrastim, cetrorelix, interleukin-2, aldesleukin, teceleulin, and denileukin. Diftitox, Interferon α-n3 (injection), Interferon α-nl, DL-8234, Interferon, Suntory (γ-1a), Interferon γ, Thymosin α1, Tasonermin, DigiFab, ViperaTAb, EchiTAb, CroFab, Nesiritide, Abatacept, Alefacept, Rebif, Eptoterminalfa, Teriparatide (osteoporosis), Calcitonin (injectable for bone diseases), Calcitonin (nasal injection, osteoporosis), Etanercept, Hemoglobin Glutamate 250 (bovine), Drotecogin alpha, Collagenase, Carperitide, Recombinant Human Epidermal Growth Factor (topical gel, wound healing), DWP401, Dabepoetin alpha alpha), erythropoietin omega, erythropoietin beta, erythropoietin alpha, desirudin, recombinant hirudin, bivalirudin, nonacog alpha, mononine, eptacog alpha (activated), recombinant factor VIII+VWF, recombinate, recombinant factor VIII, factor VIII (recombinant), alphnmate, octocog alpha), Factor VIII, palifermin, indikinase, tenecteplase, alteplase, pamiteplase, reteplase, nateplase, monteplase, follitropin alpha, rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, iniglucerase, galsulfase, leucotropin, molgramostirn, triptorelin acetate Acetate), Histrelin (subcutaneous implant, Hydrox), Deslorelin, Nafarelin, ATRIGEL Leuprorelin Extended-Release Depot, DUROS Leuprorelin Implant, Goserelin, Eutropin, KP-102 Program, Growth Hormone, Mecasermin (for growth failure), Enlfavirtide, Org-33408, Insulin Glargine, Insulin Glutamate, Insulin (inhalation), Insulin Lispro, Insulin Deternir, Insulin (oral, RapidMist), Mecasermin Linfiber rinfabate, anaproleukin, celmoleukin, 99mTc-aproxypeptide injection, myelopid, betaseron, glatiramer acetate, Gepon, sargramostim, oprelvekin, human leukocyte-derived alpha interferon, Bilive, recombinant insulin, recombinant human insulin, insulin aspart, mecasenin, Roferon-A, interferon-α2, Alfaferone, compound alpha interferon-1, interferon α, Avonex recombinant human luteinizing hormone, dornase Alpha), Trafermin, Ziconotide, Tatirelin, Diboterminalfa, Atosiban, Becaplermin, Eptifibatide, Zemaira, CTC-111, Shanvac-B, Quadrivalent HPV Vaccine, Octreotide, Lanreotide, Anestril, Agalsidase Beta, Agalsidase Alpha, Laronidase, Prezatide Copper Acetate (topical gel), rasburicase, ranibizumab, Actimmune, PEG-intron, Tricomin, recombinant house dust mite allergy desensitization injection, recombinant human parathyroid hormone (PTH) 1-84 (sc, osteoporosis), erythropoietin delta, transgenic antithrombin III, granditropin, hyaluronidase, recombinant insulin, interferon-α (oral lozenges), GEM-21S, vapreotide, idursulfase, omnapatrilat, recombinant serum albumin, certolizumab. pegol, glucarpidase, recombinant human C1 esterase inhibitor (angioneuropathy), lanoteplase, recombinant human growth hormone, enfuvirtide (needle-free injection, Biojector 2000), VGV-1, interferon (α), lucinactant, aviptadil (inhalation, lung disease), icatibant, ecallantide, omiganan, Aurograb, pexiganan acetate Acetate), ADI-PEG-20, LDI-200, degarelix, Cintredelinbesudotox, Favld, MDX-1379, ISAtx-247, liraglutide, teriparatide (for osteoporosis), tissue factor pathway inhibitor tifacogin, AA4500, T4N5 liposome emulsion, catumaxomab, DWP413, ART-123, Chrysalin, and more. Aminoplase, amediplase, corifollitropin alpha, TH-9507, teduglutide, Diamyd, DWP-412, growth hormone (continuously released injection), recombinant G-CSF, insulin (inhalation, air), insulin (inhalation, Technosphere), insulin (inhalation, AERx), RGN-303, DiaPep277, interferon beta (for hepatitis C virus infection (HCV)), interferon alpha-n3 (oral), belacept, transdermal insulin Insulin patches, AMG-531, MBP-8298, Xerecept, opebacan, AIDSVAX, GV-1001, LymphoScan, ranpirnase, Lipoxysan, lusupultide, MP52 (β-triphosphate carrier, bone regeneration), melanoma vaccine, sipuleucel-T, CTP-37, Insegia, vitespen, human thrombin (freezing, surgical bleeding), coagulation. Enzymes, TransMID, alfimeprase, Puricase, terlipressin (intravenous, for hepatorenal syndrome), EUR-1008M, recombinant FGF-I (injectable, for vascular diseases), BDM-E, rotigaptide, ETC-216, P-113, MBI-594AN, duramycin (inhalation, for cystic fibrosis), SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman Birk inhibitor concentrate, XMP-629, 99mTc-Hynic-annexin V, kahalalide F F), CTCE-9908, teverelix (delayed release), ozarelix, rornidepsin, BAY-504798, interleukin-4, PRX-321, Pepscan, iboctadekin, recombinant human lactoferrin, TRU-015, IL-21, ATN-161, cilengitide, and more. Albuferon, Biphasix, IRX-2, Omega interferon, PCK-3145, CAP-232, Parretide, huN901-DMI, Ovarian cancer immunotherapy vaccine, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16, Multi-epitope peptide melanoma vaccine (MART-1, gp100, tyrosinase), Nemifitide, rAAT (inhalation) rAAT (dermatology), CGRP (inhalation, asthma), pegsunercept, thymosin β4, plitidepsin, GTP-200, ramoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (oral, Eligen), calcitonin (oral, osteoporosis), examorelin, capromorelin, Car deva, velafermin, 131I-TM-601, KK-220, T-10, ularitide, depelestat, hematide, Chrysalin (topical), rNAPc2, recombinant factor V111 (pegylated liposomes), bFGF, pegylated recombinant staphylococcal kinase variant, V-10153, sonoLysis prolyse (ultrasound-degraded prourokinase), NeuroVax, CZEN-002, islet cell regeneration therapy, rGLP-1, BIM-51077, LY-548806, exenatide (controlled release, Medisorb), AVE-0010, GA-GCB, avorelin, ACM-9604, linaclotide. eacetate, CETi-1, Hemospan, VAL (injectable), rapid-acting insulin (injectable, Viadel), intranasal insulin, insulin (inhalation), insulin (oral, Alligator), recombinant methionine-based human leptin, Pitrakinra (subcutaneous injection, eczema), Pitrakinra (inhaled dry powder, asthma), multileukin, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10 (autoimmune diseases/inflammation), talactoferrin (topical), rEV-131 (ophthalmology), rEV-131 (respiratory diseases) (Diabetes), oral recombinant human insulin (diabetes), RPI-78M, oprelvekin (oral), CYT-99007CTLA4-Ig, DTY-001, valategrast, interferon α-n3 (topical), IRX-3, RDP-58, Tauferon, bile salt-stimulated lipase, Meripase, alanine phosphatase, EP-2104R, melanotan-II, bremelanotide, ATL-104, recombinant human microfibrinolytic enzyme, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, Malaria vaccine (virovirus, PeviPRO), ALTU-135, Parvovirus B19 vaccine, Influenza vaccine (recombinant neuraminidase), Malaria/HBV vaccine, Anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine (oral), HPV vaccine, Tat toxoid, YSPSL, CHS-13340, PTH (1-34) liposomal cream (Novasome), Ostabolin-C, PTH analogue (topical, psoriasis), MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85A vaccine (tuberculosis), FAR A04, BA-210, Recombinant FIV vaccine, AG-702, OxSODrol, rBetV1, Der-p1/Der-p2/Der-p7 allergen-targeted vaccine (dust mite allergy), PR1 peptide antigen (leukemia), mutant ras vaccine, HPV-16E7 lipopeptide vaccine, labyrinthin vaccine (adenocarcinoma), CML vaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptide, telbermin (dermal) Dermatology, diabetic foot ulcers), rupintrivir, reticulose, rGRF, HA, α-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensin-based therapeutic vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral, tuberculosis), DRF-7295, ABT-828, ErbB2 specific immunotoxin (anticancer), DT3SSIL-3, TST-10088, PRO-1762, Combotox, cholecystokinin-β/gastrin receptor-binding peptide, 111In-hEGF, AE-37. Trastuzumab-DM1, Antagonist G, IL-12 (Recombinant), PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (Topical), L-19-based Radioimmunotherapy (Cancer), Re-188-P-2045, AMG-386, DC/1540/KLH Vaccine (Cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 Vaccine (Peptide), NA17.A2 Peptide, Melanoma Vaccine (Pulse Antigen Therapy), Prostate Cancer Vaccine, CBP-501, Recombinant Human Lactoferrin (Dry Eye), FX-06, AP-214 WAP-8294A (injectable), ACP-HIP, SUN-11031, Peptide YY [3-36] (obesity, intranasal), FGLL, ataxic, BR3-Fc, BN-003, BA-058, Human parathyroid hormone 1-34 (nasal cavity, osteoporosis), F-18-CCR1, AT-1100 (celiac disease/diabetes), JPD-003, PTH (7-34) liposome cream (Novasome), Nilemycin (ophthalmology, dry eye), CAB-2, CTCE-0214, GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR- 013, Factor XIII, Aminocandin, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, Teverelix (immediate release), EP-51216, hGH (controlled release, Biosphere), OGP-I, Sifuvirtide, TV4710, ALG-889, ORG-41259, rhCC10, F-991, Thymopentin (lung disease), r(m)CRP, liver-selective insulin, subalin, L19-IL-2 fusion protein, elafin, NMK-150, ALTU-13 9. EN-122004, rhTPO, thrombopoietin receptor agonist (thrombocytopenia), AL-108, AL-208, nerve growth factor antagonist (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide (eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion vaccine (Therapore), EP-1043, pediatric pneumococcal vaccine, malaria vaccine, meningococcal group B vaccine, neonatal group B streptococcal vaccine, anthrax vaccine, HCV vaccine (gpE1+gpE2+MF-59), otitis media treatment, HCV vaccine (core). Antigen + ISCMATRIX), hPTH(1-34) (transdermal, ViaDerm), 768974, SYN-101, PGN-0052, Aviscumnine, BIM-23190, Tuberculosis vaccine, Multi-epitope tyrosinase peptide, Cancer vaccine, Enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, Vascular-targeted TNF (solid tumors), Desmopressin (oral controlled release), Onarcisic acid or TP-9201, Adalimumab (HUMIRA), Infliximab (REMICADE) ^Rituximab (RITUXAN ^™ / MAB THERA ^™ ), etanercept (ENBREL™), bevacizumab (AVASTIN ^™ ), trastuzumab (HERCEPTIN ^™ ), pegrigrastim (NEULASTA ^™ ), or any other suitable point of interest ( ^POI ), including biosimilars and biomodifiers.

According to one specific aspect, the host cell can be any animal cell, vertebrate cell, mammalian cell, human cell, plant cell, nematode cell, invertebrate cell such as insect cell or mollusc cell, derived from any of the aforementioned stem cells or fungal cells or yeast cells. Specifically, the host cell is a cell selected from the genera of the group consisting of Pichia pastoris, Hansenula polymorpha, Kumaria martensii, Saccharomyces, Kluyveromyces, Candida, Ogataea, Yarrowia, and Geotrichum, especially Saccharomyces cerevisiae, Pichia pastoris, Ogataea minuta, Kluyveromces lactis, Kluyveromes marxianus, Yarrowia lipolytica, or Hansenula polymorpha, or cells of filamentous fungi such as Aspergillus pumilus or Trichoderma reesei. Preferably, the host cell is a methyltrophic yeast, more preferably *Pichia pastoris*. In this document, *Pichia pastoris* is used synonymously with all *Pichia pastoris*, *Pichia freundii*, and *Pichia pastoris* species.

According to one specific aspect, the host cell is

a) Yeast cells selected from genera belonging to the group consisting of *Pichia pastoris*, *Hansenula polymorpha*, *Pichia komarginata*, *Saccharomyces cerevisiae*, *Saccharomyces kluyveri*, *Candida*, *Henrynchoda*, *Yersinia*, and *Dithia*, such as *Pichia* (e.g., *Pichia pastoris*, *Pichia methanolica*, *Pichia kluyveri*, and *Pichia angusta*), *Pichia komarginata* (e.g., *Pichia pastoris*, *Pichia pseudopasteurella*, or *Pichia freundii*), and *Saccharomyces cerevisiae* (e.g., *Saccharomyces cerevisiae*, *Saccharomyces kluyveri*, *Saccharomyces cerevisiae ... Kluyveromyces uvarum), Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), Candida (e.g., Candida utilis, Candida cacaoi, Candida booidinii), Gastromyces (e.g., Gastromyces fermentans), Hansenula polymorpha, Yersinia lipolytica, or Schizosaccharomyces pombe; or

b) Cells of filamentous fungi such as Aspergillus bubomori or Trichoderma reesei.

The preferred strain is Pichia pastoris. Specifically, the host cell is a Pichia pastoris strain selected from the group consisting of CBS 704, CBS 2612, CBS 7435, CBS 9173-9189, DSMZ 70877, X-33, GS115, KM71, KM71H and SMD1168.

Sources: CBS 704 (＝NRRL Y-1603＝DSMZ 70382), CBS 2612 (＝NRRL Y-7556), CBS7435 (＝NRRL Y-11430), CBS 9173-9189 (CBS strains: CBS-KNAW Fungal Biodiversity Centre, Centralealbureau voor Schimmelculturen, Utrecht, Netherlands) and DSMZ 70877 (German Collection of Microorganisms and Cell Cultures)); strains from Invitrogen, such as X-33, GS115, KM71, KM71H and SMD1168.

Examples of preferred Saccharomyces cerevisiae strains include W303, CEN.PK, and the BY-series (EUROSCARF set). All of the above strains have been successfully used to generate transformants and express heterologous genes.

According to one specific aspect, the eukaryotic host cell can be a fungal cell (e.g., Aspergillus (such as Aspergillus niger, Aspergillus fumigatus, Aspergillus orzyae, Aspergillus nidula)), Acremonium (such as Acremonium thermophilum)), Chaetomium (such as Chaetomium thermophilum)), gold Chrysosporium (e.g., *C. thermophile*), Cordyceps (e.g., *C. militaris*), Corynascus, Ctenomyces, Fusarium (e.g., *F. oxysporum*), Glomerella (e.g., *G. graminicola*), and Sarcoptera (… Hypocrea (e.g., *H. jecorina*), Magnaphorthe (e.g., *M. orzyae*), Myceliophthora (e.g., *M. thermophile*), Nectria (e.g., *N. heamatococca*), Neurospora (e.g., *N. crassa*), Penicillium (… icillium), sporotrichum (such as S. thermophile), Thievaria (such as T. terrestris, T. heterothallica), Trichoderma (such as T. reesei) or Verticillium (such as V. dahlia)

According to one specific aspect, the mammalian cell is a human, rodent, or bovine cell, cell line, or cell strain. Examples of specific mammalian cells suitable as host cells as described herein are mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, MDCK, NIH3T3, PC12, BHK (juvenile hamster kidney cells), VERO, SP2/0, YB2/0, Y0, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK-293, VERO, PER.C6, HeLA, EB1, EB2, EB3, oncolytic, or hybridoma cell lines. Preferably, the mammalian cell is a CHO cell line. In one embodiment, the cell is a CHO cell. In one embodiment, the cells are CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11CHO cells, DUKX CHO cells, CHO-S, CHO FUT8 knockout CHOGS knockout cells, CHO FUT8 GS knockout cells, CHOZN, or CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1SV GS knockout cells. CHO FUT8 knockout cells are, for example, CHOK1 SV (Lonza Biologics, Inc.). Eukaryotic cells also include avian cells, cell lines, or cell strains, such as, for example, EB14, EB24, EB26, EB66, or EBv13.

According to another specific aspect, eukaryotic cells are insect cells (e.g., Sf9, Mimic ^™ Sf9, Sf21, HighFive™ (BT1-TN-5B1-4) or BT1-Ea88 cells), algal cells (e.g., Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina, or Ochromonas)), or plant cells (e.g., from monocotyledons (e.g., corn, rice, wheat, or Setaria), or from dicotyledons (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella). (Patens) or Arabidopsis cells).

According to one specific aspect, the host cell is a prokaryotic cell, such as a bacterial cell. Specifically, the host cell is a Gram-positive cell, such as Bacillus, Streptomyces, Staphylococcus, or Lactobacillus. Suitable Bacillus species include, for example, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus natto, or Bacillus megaterium. In an embodiment, the cell is Bacillus subtilis, such as Bacillus subtilis 3NA and Bacillus subtilis 168. Bacillus can be obtained, for example, from the Bacillus Genetic Stock Center, Biological Sciences 556, 484 West 12th Avenue, Columbus, Ohio 43210-1214.

In one embodiment, the prokaryotic cells are Gram-negative cells, such as Salmonella spp. or Escherichia coli, such as, for example, TG1, TG2, W3110, DH1, DHB4, DH5a, HMS 174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue and Origami, as well as those derived from Escherichia coli strain B, such as, for example, BL-21 or BL21(DE3), all of which are commercially available.

According to a specific implementation plan, prokaryotic cells were selected from a group consisting of Escherichia coli, Bacillus subtilis, and Pseudomonas.

Suitable host cells are commercially available, for example, from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH), Braunschweig, Germany, or the American Type Culture Collection (ATCC).

According to one particular aspect, the present invention provides a method for increasing the yield of a target protein (POI) by reducing the expression of a gene encoding the FLO8 protein in a host cell, the target protein (POI) being produced by a host cell expressing a target gene (GOI) encoding the POI under the control of a promoter that can be regulated or inhibited by a non-methanol carbon source (particularly ECP as described herein), the FLO8 protein comprising an amino acid sequence identified as SEQ ID NO:1 or a homolog thereof, particularly the gene encoding the FLO8 protein, the FLO8 protein being endogenous to the host cell.

Specifically, compared with comparative host cells expressing the GOI, the yield increased by at least one of the following: 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 11.5-fold, or 12-fold.

a) It is not engineered to reduce the expression of the gene encoding the endogenous FLO8 protein, or the expression of the gene encoding the endogenous FLO8 protein is not modified; and optionally

b) Wherein the promoter controlling the expression of the GOI is a constitutive promoter, particularly the GAP promoter, or a methanol-inducible promoter, particularly the AOX1 promoter.

Specifically, the method for increasing POI production described herein uses recombinant host cells as further described herein.

According to another specific aspect, the present invention provides a method for producing said POI encoded by a target gene (GOI) by culturing recombinant host cells as further described herein under conditions for producing a target protein (POI).

According to another specific embodiment, the present invention provides the use of the host cells described herein for generating POIs.

Specifically, host cells are cell lines cultured in cell cultures, particularly production host cell lines.

According to one specific implementation scheme, the cell line is cultured under batch, fed-batch, or continuous culture conditions. The culture can be carried out in microtiter plates, shake flasks, or bioreactors, and optionally begins with a batch phase as the first step, followed by a fed-batch phase or a continuous culture phase as the second step.

Specifically, the method includes the following steps:

a) Culturing host cells under growth conditions; and further steps.

b) Under growth-restricted conditions, the host cells are cultured in the presence of a second non-methanol carbon source of up to 1 g/L to induce the expression of the GOI to produce the POI.

Specifically, step b) follows step a).

Specifically, the first carbon source is a non-methanol carbon source, which is referred to in this paper as the basic carbon source.

Specifically, in the first step, host cells are cultured in a cell culture medium containing a first carbon source under growth conditions, for example, in an amount sufficient to allow host cells to grow in the cell culture, optionally until the amount of carbon source is consumed, and further culture can be carried out under growth-limited conditions.

Specifically, the second carbon source is a non-methanol carbon source referred to in this paper as the supplementary carbon source.

Specifically, the first carbon source and/or the second carbon source are selected from sugars, polyols, alcohols, or any one or more of the foregoing, as further described herein.

According to a specific implementation, the basic carbon source differs from the supplementary carbon source, for example, in quantity and/or quality. Quantitative differences typically provide different conditions for inhibiting or de-inhibiting promoter activity.

According to another specific embodiment, the basic carbon source and the supplementary carbon source comprise the same type of molecules or carbohydrates, preferably at different concentrations. According to another specific embodiment, the carbon source is a mixture of two or more different carbon sources.

Any type of organic carbon source can be used, especially those commonly used for host cell culture, particularly for eukaryotic host cell culture. According to one specific embodiment, the carbon source is a hexose such as glucose, fructose, galactose, or mannose; a disaccharide such as sucrose; an alcohol such as glycerol or ethanol; or a mixture thereof.

According to a particularly preferred embodiment, the basic carbon source is selected from the group consisting of glucose, glycerol, ethanol, or mixtures thereof. According to a preferred embodiment, the basic carbon source is glycerol.

According to another specific embodiment, the supplementary carbon source is a hexose such as glucose, fructose, galactose, and mannose; a disaccharide such as sucrose; an alcohol such as glycerol or ethanol; or a mixture thereof. According to a preferred embodiment, the supplementary carbon source is glucose.

Specifically,

a) The basic carbon source is selected from the group consisting of glucose, glycerol, ethanol, and mixtures thereof; and

b) Supplemental carbon sources are hexoses such as glucose, fructose, galactose or mannose, disaccharides such as sucrose, alcohols such as glycerol or ethanol, or any mixture of the foregoing.

The two culture steps specifically involve culturing the cell line in the presence of the carbon source. For example, the culture of host cells under growth conditions (step a) is performed using a basal carbon source; and the culture of host cells under growth-limited conditions (step b) is performed using a supplemental carbon source, for example, in a limited amount such that the cell culture medium during culture (step b) contains at most 1 g/L or even no detectable amount of supplemental carbon source in the cell culture medium or supernatant.

De-inhibition (or induction) conditions can be appropriately achieved through specific means. Step two (b) optionally employs a feed medium that provides little or no supplemental carbon source in the cell culture medium or supernatant. Specifically, the feed medium is chemically defined and methanol-free.

Specifically, in the second step b), a feed medium is used to provide a supplementary carbon source at a growth-limiting amount to maintain the specific growth rate in the range of 0.0001 ^h⁻¹ to 0.2 ^h⁻¹ , preferably 0.005 ^h⁻¹ to 0.15 ^h⁻¹ .

The feed medium can be added to the culture medium in liquid form or in an alternative form (such as a solid, e.g., as tablets or other sustained-release agents, or a gas). However, according to a preferred embodiment, the limited amount of supplementary carbon source added to the cell culture medium can even be zero. Preferably, under limited carbon substrate conditions, the detectable concentration of supplementary carbon source in the culture medium is 0-1 g/L, preferably less than any one of 0.9 g/L, 0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1 g/L, preferably less than 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L, 50 mg/L, 40 mg/L, 30 mg/L, and 2 g/L. Any one of 0 mg/L or 10 mg/L, or even less than 9 mg/L, 8 mg/L, 7 mg/L, 6 mg/L, 5 mg/L, 4 mg/L, 3 mg/L, 2 mg/L or 1 mg/L, or specifically 1-50 mg/L or 1-10 mg/L, particularly preferably 1 mg/L or even lower, for example below the detection limit as measured by a suitable standard assay, for example determined as the residual concentration in the culture medium when consumed by the growing cell culture.

In a preferred method, a limited supplemental source provides the residual amount in the cell culture, which is below the detection limit, such as at the end of the production phase or in the output of the fermentation process, preferably at the time of harvesting the fermentation product, in the fermentation broth.

Specifically, step a) cultivation is carried out in a batch stage; and step b) cultivation is carried out in a fed batch or continuous cultivation stage.

Specifically, the host cells are grown in a carbon-rich medium that contains a basal carbon source during a high growth rate phase (under growth conditions) step a) (e.g., at least 50% or at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or up to the maximum growth rate) and generates POI during a low growth rate phase (under growth-limiting conditions) step b) (e.g., less than 90% of the maximum growth rate, preferably less than 80%, less than 70%, less than 60%, less than 50%, or less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, or less than 0.2%), while limiting the carbon source, particularly by supplying a defined basal medium containing only the amount of carbon source completely consumed during the production phase to maintain cell culture.

Specifically, POI is expressed under the growth-restricting conditions, for example by culturing cell lines at a growth rate below the maximum growth rate, typically below 90% of the maximum cell growth rate, preferably below 80%, below 70%, below 60%, below 50%, below 40%, below 30%, below 20%, below 10%, below 5%, below 3%, below 2%, below 1%, below 0.5%, below 0.4%, below 0.3%, or below 0.2%. Typically, the maximum growth rate is determined individually for each type of host cell.

Specifically, a batching phase is performed until the basal carbon source initially added to the cell culture is consumed by the cell line. The dissolved oxygen (DO) spike method can be used to determine the amount of basal carbon source consumed during the batching phase.

According to one specific implementation, the batching phase is characterized by a continuous decrease in the oxygen partial pressure (pO2) signal, and the end of the batching phase is characterized by an increase in pO2. Typically, when the basic carbon source is consumed during the batching phase and no additional carbon source is added as is usually done during batching, the oxygen partial pressure (pO2) signal will continuously decrease until, for example, below 65%, such as, for example, 30%. When the basic carbon source is consumed, pO2 can increase to, for example, above 30%, such as, for example, above 65%, or higher, indicating the appropriate time point to switch to a fed batching system using the feed medium to add an additional carbon source under carbon source-limited conditions.

Specifically, during the batching phase, the pO2 saturation is reduced to below 65% or lower, and then at the end of the batching, the saturation is increased to above 65% or higher. Specifically, the batching phase continues until the increase in the oxygen partial pressure (pO2) signal exceeds 65% saturation, specifically exceeding any one of 70%, 75%, 80%, or 85%.

Specifically, the process will be carried out in batches over approximately 10 to 36 hours.

The term "about" regarding culture time should refer to +/- 5% or +/- 10%.

For example, a specific batch performance time of approximately 10 to 36 hours could be 18 to 39.6 hours, specifically 19 to 37.8 hours.

According to one specific implementation, 40-50 g/L glycerol, particularly 45 g/L, is used as the basal carbon source in the batch culture medium for the batch phase, and incubation is carried out at 25°C for approximately 27-30 hours, or at 30°C for approximately 23-36 hours, or at any temperature between 25°C and 30°C for a period of 23-36 hours. Decreasing the glycerol concentration in the batch medium shortens the batch phase, while increasing the glycerol concentration in the batch medium can even prolong the batch phase. As a substitute for glycerol, approximately the same amount of glucose can be used, for example.

In a typical system for cell culture and POI expression, where a batch phase is followed by a fed-batch phase, specifically, the culture in the fed-batch phase lasts for any one of the following durations: approximately 15 to 80 hours, approximately 15 to 70 hours, approximately 15 to 60 hours, approximately 15 to 50 hours, approximately 15 to 45 hours, approximately 15 to 40 hours, approximately 15 to 35 hours, approximately 15 to 30 hours, approximately 15 to 35 hours, approximately 15 to 25 hours, or approximately 15 to 20 hours; preferably approximately 20 to 40 hours. Specifically, the culture in the fed-batch phase lasts for any one of the following durations: approximately 80 hours, approximately 70 hours, approximately 60 hours, approximately 55 hours, approximately 50 hours, approximately 45 hours, approximately 40 hours, approximately 35 hours, approximately 33 hours, approximately 30 hours, approximately 25 hours, approximately 20 hours, or approximately 15 hours.

Any fed-batch culture lasting less than 120 hours, less than 100 hours, or at most 80 hours (which leads to successful POI production and thus high yields) is referred to herein as “rapid fermentation.” Specifically, volumetric product formation rate (rP) is the amount of product (mg) formed per unit volume (L) and per unit time (hour) (mg(L h) ^⁻¹ ). Volumetric product formation rate is also known as space-time yield (STY) or volumetric productivity.

Specifically, fed-batch culture as described herein is performed such that the space-time yield is approximately 30 mg(L h) ^⁻¹ (meaning 30 mg(L h) ^⁻¹ +/- 5% or +/- 10%). Specifically, a space-time yield of approximately 30 mg(L h) ^⁻¹ is obtained within approximately 30 hours of fed-batch culture; specifically, at least any one of 27 mg(L h)⁻¹, 28 mg(L h)⁻¹, 29 mg(L h) ^⁻¹ , 30 mg(L h) ^⁻¹ , 31 mg(L h) ^⁻¹ , 32 mg(L h)⁻¹, or 33 mg(L h) ^⁻¹ can be obtained within any fed-batch time of less than 33 hours, ³² hours, 31 hours, 30 hours, 29 hours, ²⁸ hours, 27 hours, ²⁶ hours, or 25 hours.

Specifically, the batching stage is carried out as the first step a), and the replenishment batching stage is carried out as the second step b).

Specifically, in the second step b), the feed medium used in the feed batching stage provides a supplemental carbon source for growth-limiting amounts to maintain the specific growth rate in the range of 0.0001 ^h⁻¹ to 0.2 ^h⁻¹ , preferably less than any one of 0.2 ^h⁻¹ , 0.15 ^h⁻¹ , 0.1 ^h⁻¹ or 0.15 ^h⁻¹ .

Specifically, the culture method, which includes batch and fed-batch culture steps, can particularly use yeast host cells, such as any yeast of the genera *Saccharomyces*, *Pichia*, or *Kommasidra*, or yeast from genera other than *Pichia*, such as *Kluyveromyces lactis*, *Z. rouxii*, *Pichia stipitis*, *H. polymorpha*, or *Y. lipolytica*, preferably *Pichia pastoris* or *Kommasidra pastoris*.

According to another specific aspect, the present invention provides a method for generating a target protein (POI) in a host cell, comprising the following steps:

a) Genetically engineer host cells to reduce the expression of endogenous genes encoding the FLO8 protein, said FLO8 protein comprising the amino acid sequence identified as SEQ ID NO:1 or its homologs;

b) Introducing a heterologous expression cassette into a host cell, the heterologous expression cassette comprising encoding or expressing the target gene (GOI) of the POI under the control of an expression cassette promoter (ECP), the expression cassette promoter being operatively linked to the GOI, the ECP being regulated or inhibited by a non-methanol carbon source;

c) Culture the host cells under conditions that generate the POI;

d) Optionally isolate the POI from the cell culture; and

e) Optionally purify the POI.

Specifically, the steps a) of the method described herein are performed before, after, or accompanying step b).

According to one specific aspect, before being engineered for the production of POIs, host cells are first genetically modified to reduce the expression of the FLO8 protein or its corresponding homologs. According to one specific embodiment, wild-type host cells are genetically modified according to step a) of the methods described herein. Specifically, host cells are provided when the one or more genetic modifications are introduced into a wild-type host cell line to reduce the expression of the FLO8 protein or its corresponding homologs.

According to another aspect, host cells are first engineered to produce heterologous or recombinant POIs, and then further genetically modified to reduce the FLO8 protein or its corresponding homologs. According to one specific embodiment, wild-type host cells may first be engineered to contain an expression cassette for POI production. Such engineered host cells can then be further modified to reduce the FLO8 protein or its corresponding homologs as described herein.

According to another aspect, the host cell undergoes engineering for POI production and genetic modification for reducing the FLO8 protein or its corresponding homologs in one method step, for example, using appropriate expression cassettes, reagents, and tools in one or more reaction mixtures.

Specifically, the method employs method steps to generate recombinant host cells as further described herein.

Specifically, the heterologous expression cassette contains ECPs as further described herein.

Specifically, POIs can be produced by culturing host cells in a suitable culture medium, isolating the expressed POI from the cell culture, particularly from the cell culture supernatant or culture medium, during cell isolation, and purifying it using methods suitable for the expressed product, especially after isolating the POI from the cells and purifying it using suitable methods. Thus, a purified POI formulation can be prepared.

Attached Figure Description

Figure 1 : The sequence referred to in this article.

Detailed Implementation

The following are the meanings of certain terms used throughout this specification.

As used herein, the term "carbon source" (also known as "carbon substrate") refers to a fermentable carbon substrate, typically a source of carbohydrates suitable as an energy source for microorganisms (such as those capable of being metabolized by a host organism or a production cell line), particularly those selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and alcohols (including glycerol), provided in purified form, in minimal media, or in feedstocks, such as complex nutrient materials. Carbon sources can be used as a single carbon source or as a mixture of different carbon sources, as described herein.

In this paper, non-methanol carbon sources are understood to be any carbon source other than methanol, especially carbon sources that do not contain methanol.

As used herein, a “basic carbon source” is generally a carbon source suitable for cell growth, such as nutrients for host cells, particularly eukaryotic cells. The basic carbon source may be provided in a medium (such as a basal medium or a composite medium), but may also be provided in a chemically defined medium containing a purified carbon source. The basic carbon source is typically provided in amounts that support cell growth, particularly during the growth phase of the culture process, for example, to obtain a cell density of at least 5 g/L cell dry mass, preferably at least 10 g/L cell dry mass, or at least 15 g/L cell dry mass, exhibiting, for example, a viability of greater than 90%, preferably greater than 95%, during a standard subculture step.

The basal carbon source is typically used in excess or surplus, which is understood as providing an excess of energy to increase biomass, for example during the culture of cell lines with high specific growth rates, such as during the growth phase of cell lines in batch or fed-batch culture processes. This surplus specifically exceeds the limited amount of supplemental carbon source (as used under growth-limited conditions) to achieve a measurable residual concentration in the fermentation broth, which is typically at least 10 times higher than during periods when the supplemental carbon source is fed in limited quantities, preferably at least 50 times or at least 100 times higher.

As described herein, "supplementary carbon sources" generally refer to supplementary substrates that facilitate the production of fermentation products through production cell lines, particularly during the production phase of the culture process. The production phase specifically follows the growth phase, such as in batch, fed-batch, and continuous cultures. Supplementary carbon sources can specifically be included in the feed of fed-batch processes. Supplementary carbon sources are typically used in cell cultures under carbon-limited substrate conditions, i.e., when carbon sources are used in limited quantities.

The term "limited amount" or "finite carbon source" is understood in this text to specifically refer to the type and amount of carbon substrate that facilitates the production of fermentation products through production cell lines, particularly in cultures with controlled growth rates below the maximum growth rate. The production phase specifically follows the growth phase, for example, in batch, fed-batch, and continuous cultures. Cell culture processes can employ batch, continuous, and fed-batch methods. Batch culture is a process in which a small amount of seed culture is added to the culture medium, and cells are allowed to grow without the addition of additional medium or the removal of culture medium during the culture period. Continuous culture is a process in which culture medium is continuously added to and removed during the culture period. Continuous culture also includes perfusion culture. Fed-batch culture (which is an intermediate between batch and continuous culture and is also called semi-batch culture) is a process in which culture medium is added continuously or sequentially during the culture period, but unlike continuous culture, the culture medium is not continuously removed.

A particularly preferred approach is the fed-batch process, which involves introducing growth-limiting nutrient substrates into the culture. Fed-batch strategies (including single-batch or repeated-batch fermentation) are commonly used in bioindustry processes to achieve high cell densities in bioreactors. The controlled addition of carbon substrates directly impacts the culture's growth rate and helps prevent metabolic spillover or the formation of unwanted metabolic byproducts. Under carbon-limited conditions, the carbon source can specifically be included in the feed of a fed-batch process. Therefore, carbon substrates are provided in a limited quantity.

Similarly, in chemostats or continuous cultures as described in this article, the growth rate can be strictly controlled.

The limited amount of carbon source is specifically understood herein as the amount of carbon source necessary to maintain the production cell line under growth-limited conditions (e.g., during the production phase or production mode). Such a limited amount can be used in fed-batch processes where the carbon source is contained in the feed medium and supplied to the culture at a low feed rate for continuous energy delivery, such as to generate POI, while maintaining the biomass at a low specific growth rate. Typically, the feed medium is added to the fermentation broth during the production phase of the cell culture.

The limit of a carbon source can be determined, for example, by the amount of carbon source residue in the cell culture medium, which is below a predetermined threshold or even below the detection limit as measured in standard (carbohydrate) assays. Residual amounts are typically determined in the fermentation broth at the time of harvesting the fermentation product.

The limited amount of carbon source can also be determined by defining the average feed rate of the carbon source to the fermenter, for example, by determining the amount added throughout the entire culture process (e.g., during the fed-batch phase) at each culture time, to determine the average amount calculated each time. This average feed rate is kept low to ensure that the supplemented carbon source is fully utilized through cell culture, for example, between 0.6 g ^{L⁻¹ h⁻¹} (g of carbon source per L of initial fermentation volume ^and h of time), preferably between 1.6 ^{g L⁻¹ h⁻¹} and 20 g ^{L⁻¹ h⁻¹} .

The limited amount of carbon source can also be determined by measuring the specific growth rate, which is kept low during the production phase, for example below the maximum specific growth rate, for example within a predetermined range, such as between 0.001 ^h⁻¹ and 0.20 ^h⁻¹ or between 0.005 ^h⁻¹ and 0.20 ^h⁻¹ , preferably between 0.01 ^h⁻¹ and 0.15 ^h⁻¹ .

Specifically, a chemically defined feed medium that does not contain methanol is used.

The term "chemically defined" for cell culture media (such as basal or feed media in fed-batch processes) should refer to a medium suitable for in vitro cell culture to produce cell lines, wherein all chemical components and (poly)peptides are known. Typically, chemically defined media are completely free of animal-derived components and represent a pure and consistent cell culture environment.

As used in this article, the term "host cell" should refer to a single cell, a single-cell clone, or a cell line of a host cell.

As used herein, the term "cell line" refers to an established clone of a specific cell type that has acquired the ability to proliferate over an extended period of time. Cell lines are typically used to express endogenous or recombinant genes, or products of metabolic pathways, to produce peptides or cellular metabolites mediated by such peptides. "Production host cell line" or "production cell line" is generally understood to be a cell line prepared for cell culture in a bioreactor to obtain products of a production process, such as POIs.

As described in this article, the host cells that produce POIs are also called "production host cells", and the corresponding cell lines are called "production cell lines".

The specific implementation described herein refers to a production host cell line engineered to express, and/or have reduced expression of, an endogenous gene encoding the FLO8 protein, and is characterized by a high yield of POI production under the control of a carbon-source-regulated promoter (such as the ECP promoter described herein), particularly a promoter that can be induced without the addition of methanol to the cell culture. Such host cells have been shown to stably express POI without significant morphological alterations.

The term "host cell" should be applied specifically to any eukaryotic or prokaryotic cell or organism suitable for recombinant purposes to produce POIs or host cell metabolites. It is well known that the term "host cell" does not include humans. Specifically, host cells as described herein are derivatives of artificial organisms and natural (wild-type) host cells. It is well known that the host cells, methods, and uses described herein (e.g., particularly those containing one or more gene modifications, the heterologous expression cassette or construct, the transfected or transformed host cells, and the recombinant protein) are not naturally occurring, "artificial," or synthetic, and therefore not considered a result of "natural laws."

As used herein, the term “cell culture” or “culturing” refers to the maintenance of cells in an artificial, such as in vitro, environment, under conditions conducive to cell growth, differentiation, or continued survival, in the active or quiescent state of the cells, particularly in a controlled bioreactor, according to methods known in the art.

When culturing cell cultures using a suitable culture medium, the cells are brought into contact with the culture medium or substrate in the culture vessel under conditions suitable for supporting cell culture. As described herein, culture media suitable for the growth of host cells such as eukaryotic cells, particularly yeast or filamentous fungi, are provided. Standard cell culture techniques are well known in the art.

The cell cultures described herein specifically utilize techniques for producing secreted POIs, such as obtaining POIs in a cell culture medium, which can be separated from the cell biomass, referred to herein as "cell culture supernatant," and can be purified to obtain POIs of higher purity. When proteins (such as, for example, POIs) are produced and secreted by host cells in a cell culture, it should be understood herein that such proteins are secreted into the cell culture supernatant and can be obtained by separating the cell culture supernatant from the host cell biomass and optionally further purifying the protein to produce a purified protein formulation.

Cell culture media provide the nutrients required to maintain and grow cells in controlled, artificial, and in vitro environments. The properties and composition of cell culture media vary depending on specific cell requirements. Important parameters include osmolality, pH, and nutrient formulation. Nutrient feeding can be carried out continuously or discontinuously according to methods known in the art.

Batch culture is a cell culture mode in which all the nutrients required to culture the cells are contained in the initial culture medium, and no additional nutrients are supplied during fermentation. In fed-batch culture, following the batch stage, a feeding stage occurs where one or more nutrients are supplied to the culture via feed. Although the feeding mode is critical and important in most processes, the host cells and methods described herein are not limited to any one cell culture mode.

Using the host cells and corresponding cell lines described herein, recombinant POIs can be generated by culturing in a suitable culture medium, isolating the expressed products or metabolites from the culture, and optionally purifying them using a suitable method.

Several different methods for generating POIs, as described herein, are preferred. Cultures of transfected or transformed cells can be prepared by transfecting or transforming host cells with an expression vector containing recombinant DNA encoding the relevant protein; the cultures can be grown to induce transcription and POI production; and the POIs can be recovered for expression, processing, and optionally secretion.

In some implementations, the cell culture process is a fed-batch process. Specifically, host cells transformed with nucleic acid constructs encoding the desired recombinant POI are cultured in the growth phase and then transitioned to the production phase to produce the desired recombinant POI.

In another embodiment, the host cells described herein are cultured in a continuous mode (e.g., using a chemostat). The continuous fermentation process is characterized by a defined, constant, and continuous rate of feeding fresh culture medium into the bioreactor, whereby the culture medium is simultaneously removed from the bioreactor at the same defined, constant, and continuous removal rate. By maintaining the culture medium, feed rate, and removal rate at the same constant level, the cell culture parameters and conditions in the bioreactor are kept constant.

As described herein, stable cell cultures are specifically understood to be cell cultures that maintain genetic properties, particularly maintaining POI production at high levels, for example at least μg, even after about 20 generations of culture, preferably at least 30 generations, more preferably at least 40 generations, and most preferably at least 50 generations. Specifically, this provides stable recombinant host cell lines, which is considered a significant advantage when used for industrial-scale production.

The cell cultures described herein are particularly advantageous for industrial-scale production methods, for example, in terms of volume and technical systems, when combined with nutrient-based feeding culture models, especially fed-batch or batch processes, or continuous or semi-continuous processes (e.g., chemostats).

The host cells described in this article are typically tested for their ability to express GOIs for POI production by testing POI yield using any of the following tests: ELISA, activity assay, HPLC or other suitable assays such as SDS-PAGE and Western blotting, or mass spectrometry.

To determine the effect of gene modification on the low or reduced expression of genes encoding the FLO8 protein or its homologs in the corresponding cell cultures, such as its effect on POI production, host cell lines can be cultured in microtiter plates, shake flasks, or bioreactors using fed-batch or chemostat fermentation compared to strains without such gene modification in the corresponding cells.

The production method described herein particularly permits fermentation at a pilot or industrial scale. The industrial process scale will preferably be at least 10 L, especially at least 50 L, preferably at least 1 ^m³ , more preferably at least 10 ^m³ , and most preferably at least 100 ^m³ .

Industrial-scale production conditions are preferred, which refer, for example, fed-batch culture in reactor volumes of 100 L to 10 ^m³ or larger (with a typical process time of several days), or continuous processes in fermenter volumes of about 50-1000 L or larger, wherein the dilution rate is about 0.02-0.15 ^h⁻¹ .

The apparatus, facilities, and methods used for the purposes described herein are particularly suitable for culturing any desired cell lines, including prokaryotic and/or eukaryotic cell lines. Furthermore, in embodiments, the apparatus, facilities, and methods are suitable for culturing any cell type, including suspension cells or anchor-dependent (adherent) cells, and are suitable for being configured for manufacturing operations for producing pharmaceuticals and biopharmaceutical products such as peptide products (POIs), nucleic acid products (e.g., DNA or RNA), or cells and/or viruses, such as those for cell and/or viral therapies.

In some embodiments, cells express or produce products, such as recombinant therapeutic or diagnostic products. Examples of cell-generated products, as described in more detail herein, include, but are not limited to, points of interest (POIs) exemplified herein, including antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimics (peptide molecules that specifically bind to antigens but are not structurally associated with antibodies, such as, for example, DARPins, affinity molecules, adnectins, or IgNAR), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), or viral therapeutics (e.g., anticancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cell therapeutics (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines, or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (such as siRNA) or DNA (such as, for example, plasmid DNA), antibiotics, or amino acids. In embodiments, the apparatus, facilities, and methods can be used to produce biosimilars.

As described above, in some embodiments, the apparatus, facilities, and methods allow the production of eukaryotic cells, such as mammalian cells or lower eukaryotic cells, such as yeast cells or filamentous fungal cells, or prokaryotic cells, such as Gram-positive cells or Gram-negative cells, and/or products of eukaryotic or prokaryotic cells, such as POIs, including proteins, peptides, or antibiotics, amino acids, nucleic acids (such as DNA or RNA), which are synthesized by said cells in a large-scale manner. Unless otherwise stated herein, the apparatus, facilities, and methods may include any desired volume or production capacity, including but not limited to small-scale, pilot-scale, and full-scale production capabilities.

Furthermore, and unless otherwise stated herein, apparatus, facilities, and methods may include any suitable reactor, including but not limited to stirred tanks, airlift reactors, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and/or sputtered bed bioreactors. As used herein, “reactor” may include fermenter or fermentation unit, or any other reaction vessel, and the term “reactor” may be used interchangeably with “fermenter.” For example, in some aspects, exemplary bioreactor units may perform one or more of the following: feeding of nutrients and/or carbon sources, injection of suitable gases (e.g., oxygen), inlet and outlet flows of fermentation or cell culture media, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and _CO2 levels, maintenance of pH levels, agitation (e.g., stirring), and/or cleaning/sterilization. Exemplary reactor units (such as fermentation units) may comprise multiple reactors within a unit; for example, a unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each unit, and/or a facility may comprise multiple units within a facility having one or more reactors. In various embodiments, the bioreactor may be suitable for batch, semi-feed batch, fed batch, perfusion, and/or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactor may have a volume between about 100 mL and about 50,000 L. Non-limiting examples include 100mL, 250mL, 500mL, 750mL, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 15L, 20L, 25L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, 200L, 250L, 300L, 350L, 400L, 450L, 500L, and 550L. Volumes of 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and/or 50,000 liters. Furthermore, suitable reactors can be multi-purpose, single-purpose, disposable, or non-disposable, and can be formed from any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and chromium-nickel-iron alloys, plastics, and/or glass.

In implementations, and unless otherwise stated herein, the apparatus, facilities, and methods described herein may also include any suitable unit operations and/or equipment not otherwise mentioned, such as operations and/or equipment for separating, purifying, and isolating such products. Any suitable facilities and environments may be used, such as conventional fixed facilities, modular facilities, mobile facilities, and temporary facilities, or any other suitable structure, facility, and/or layout. For example, in some implementations, a modular cleanroom may be used. Furthermore, and unless otherwise stated, the apparatus, systems, and methods described herein may be housed and/or performed in a single location or facility, or alternatively, in one or more locations and/or facilities.

Suitable techniques may include cultivation in a bioreactor, beginning with a batch stage, followed by a short-exponential fed-batch stage at a high specific growth rate, and further followed by a fed-batch stage at a low specific growth rate. Another suitable cultivation technique may include a batch stage followed by a fed-batch stage at any suitable specific growth rate or combination of specific growth rates, such as a growth rate decreasing with POI production time, or a growth rate increasing with POI production time. Yet another suitable cultivation technique may include a batch stage followed by a continuous cultivation stage at a low dilution rate.

A preferred embodiment includes providing a batch culture of biomass, followed by a fed batch culture for high-yield POI production.

Preferably, the host cells as described herein are cultured in a bioreactor under growth conditions to obtain a cell density of at least 1 g/L cell dry weight, more preferably at least 10 g/L cell dry weight, preferably at least 20 g/L cell dry weight, and preferably 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, or 80 g/L cell dry weight. Providing such yields for biomass production at pilot or industrial scale is advantageous.

Growth media that allow for the accumulation of biomass (especially basal growth media) typically include carbon, nitrogen, sulfur, and phosphate sources. These media often also include trace elements and vitamins, and may further include amino acids, peptones, or yeast extracts.

Preferred nitrogen sources include _{NH₄H₂PO₄} , _{NH₃ ,} or _{( NH₄} ) _{₂SO₄ ;}

Preferred sulfur sources include _MgSO4 , ( _NH4 ) _{2SO4 ,} or _{K2SO4 ;}

Preferred _{phosphate sources include NH₄H₂PO₄ or H₃PO₄ or NaH₂PO₄} , _{KH₂PO₄ , Na₂HPO₄} or _{K₂HPO₄ ;}

Other typical culture medium components include KCl, _CaCl2 , and trace elements such as Fe, Co, Cu, Ni, Zn, Mo, Mn, I, and B.

Preferably, the culture medium is supplemented with vitamin _B7 ;

Typical _growth media for Pichia pastoris include glycerol, sorbitol or glucose, _NH4H2PO4 , _MgSO4 , _KCl , _CaCl2 , biotin, and trace elements.

During the production phase, the production medium is used exclusively in conjunction with a limited amount of supplemental carbon source.

Preferably, the host cell line is cultured in a mineral medium with a suitable carbon source, thereby further significantly simplifying the isolation process. Examples of preferred mineral media are those containing an available carbon source (e.g., glucose, glycerol, or sorbitol), salts containing macroelements (potassium, magnesium, calcium, ammonium, chloride, sulfate, phosphate) and microelements (copper, iodine, manganese, molybdate, cobalt, zinc, and iron salts, as well as boric acid), and optionally vitamins or amino acids, for example, to supplement auxotrophic cells.

Specifically, cells are cultured under conditions suitable for achieving the expression of the desired POI, which can be purified from the cells or culture medium, depending on the expression system and the nature of the expressed protein, such as whether the protein is fused to a signal peptide and whether the protein is soluble or membrane-bound. As those skilled in the art will understand, culture conditions will vary depending on factors including the type of host cell and the specific expression vector used.

Typical production media include a supplemental carbon _source , as well as additional _NH4H2PO4 , _MgSO4 , _KCl , _CaCl2 , biotin, and trace elements.

For example, the feed for supplemental carbon sources added to fermentation may include a carbon source having up to 50% by weight of available sugars.

Fermentation is preferably carried out at a pH in the range of 3 to 8.

The typical fermentation time is about 24 to 120 hours, with a temperature range of 20°C to 35°C, preferably 22°C to 30°C.

POI is preferably expressed using conditions that produce a yield of at least 1 mg/L, preferably at least 10 mg/L, preferably at least 100 mg/L, and most preferably at least 1 g/L.

As used herein, the term "expression" or "expression cassette" refers to a nucleic acid molecule containing a desired coding sequence and an operable control sequence, enabling a host transformed or transfected with these sequences to produce the encoded protein or host cell metabolite. For transformation, the expression system may be contained in a vector; however, the associated DNA may also be integrated into the host cell chromosome. Expression can refer to secreted or non-secretory expression products, including peptides or metabolites.

Expression cassettes are conveniently provided as expression constructs, for example, in the form of “vectors” or “plasmids,” which are typically recombinant nucleotide sequences (i.e., recombinant genes) transcribed and cloned in a suitable host organism and the DNA sequence required to translate their mRNA. Expression vectors or plasmids typically include an origin for autonomous replication or a site for genome integration in a host cell, selective markers (e.g., amino acid synthesis genes or genes conferring antibiotic resistance, such as ziomycin, kanamycin, G418, or hygromycin, norsin), multiple restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator, these components being operatively linked together. As used herein, the terms “plasmid” and “vector” encompass both autonomously replicating nucleotide sequences and genomes integrating nucleotide sequences, such as artificial chromosomes, for example, yeast artificial chromosomes (YAC).

Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, and specially designed plasmids. The preferred expression vectors described herein are those suitable for expressing recombinant genes in eukaryotic host cells, and are selected based on the host organism. Suitable expression vectors typically include regulatory sequences suitable for expressing DNA encoding a POI in eukaryotic host cells. Examples of regulatory sequences include promoters, operators, enhancers, ribosome binding sites, and sequences controlling the initiation and termination of transcription and translation. The regulatory sequence is typically operatively linked to the DNA sequence to be expressed.

To allow the expression of recombinant nucleotide sequences in host cells, the expression cassettes or vectors described herein include an ECP, typically a promoter nucleotide sequence adjacent to the 5' end of the coding sequence, for example, upstream and adjacent to the target gene (GOI), or, if a signal or leader sequence is used, upstream and adjacent to said signal and leader sequences, respectively, to promote the expression and secretion of the POI. The promoter sequence typically regulates and initiates the transcription of downstream nucleotide sequences (particularly including the GOI) operatively linked to it.

The specific expression construct described herein includes a promoter that is operatively linked to a nucleotide sequence encoding a POI under the transcriptional control of the promoter. Specifically, the promoter is not naturally associated with the coding sequence of the POI.

The specific expression constructs described herein comprise a polynucleotide encoding a POI linked to a leader sequence that causes the POI to be secreted from the host cell. When the POI used for recombinant expression and secretion is a non-naturally secreted protein and therefore lacks a natural secretion leader sequence, or when its nucleotide sequence has been cloned without its natural secretion leader sequence, the presence of such a secretion leader sequence in the expression vector is generally required. In general, any secretion leader sequence that effectively induces the secretion of a POI from the host cell can be used. The secretion leader sequence can be derived from yeast sources, such as yeast α-factors like MFa from *Saccharomyces cerevisiae*, or yeast phosphatases, from mammalian or plant sources, or others.

In specific implementations, polyclonal vectors, which are vectors with multiple cloning sites, can be used. Specifically, the desired heterologous gene can be integrated or incorporated at the multiple cloning site to prepare an expression vector. In the case of polyclonal vectors, the promoter is typically located upstream of the multiple cloning site.

The recombinant host cells described in this article are specially engineered to reduce the amount of endogenous FLO8 protein or its corresponding homologs or orthologs in the host cells, particularly by reducing the expression of the corresponding coding gene sequences, thereby resulting in low gene expression.

As used herein, the terms “gene expression” or “expressed polynucleotide” refer to steps that include at least one of the following groups: transcription of DNA into mRNA, mRNA processing, mRNA maturation, mRNA export, translation, protein folding, and/or protein transport.

The term “reduced expression” generally refers to “low expression” and typically means any amount below the expression level presented by a reference standard, which is the expression level of a polynucleotide in host cells before it is engineered to reduce the expression of that polynucleotide, or otherwise expressed in the same type or species of host cells without such engineering. Reduced expression as described herein specifically refers to the polynucleotide or gene encoding the defined FLO8 protein, particularly for a gene endogenous in the host cell before engineering. Specifically, the corresponding gene product is the defined FLO8 protein as described herein. When host cells are engineered by genetic modification to reduce the expression of said gene, the expression level of said gene product or peptide is lower than the expression level of the same gene product or peptide in the host cell before genetic modification or in a comparable host without genetic modification. “Lower than” includes, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher. The terms “reduced expression” or “low expression” also include no expression of the gene product or peptide.

According to the specific implementation described herein, the host cell is engineered to knock down or delete (for inactivation or deletion of the gene or part thereof) an endogenous host cell gene encoding the FLO8 protein (including, for example, corresponding homologs or orthologs as defined herein), or to confer on the host cell the ability to express or produce the FLO8 protein from other (coding or non-coding) nucleotide sequences.

Specifically, a missing strain was provided, in which the nucleotide sequence was disrupted.

As used herein, the term "disruption" refers to a significant reduction to complete elimination of the expression of one or more endogenous proteins in a host cell, such as by knockdown or knockout. This can be measured by the presence of the one or more endogenous proteins in the host cell culture or culture medium, such as by mass spectrometry, where the total amount of the endogenous protein may be less than a threshold or undetectable.

The term “disruption” specifically refers to the result of genetic engineering modifications by means of at least one step in the group consisting of gene silencing, gene knockdown, gene knockout, delivery of dominant-negative constructs, and conditional gene knockout, and/or by gene alterations concerning a specific gene.

As used herein in the context of gene expression, the terms “knockdown,” “reduction,” or “depletion” refer to experimental methods that result in a reduction in the expression of a given gene compared to its expression in control cells. Gene knockdown can be achieved through a variety of experimental techniques, such as introducing nucleic acid molecules into cells to hybridize with portions of the mRNA of the gene that causes its degradation (e.g., shRNA, RNAi, miRNA), or altering the gene sequence in a manner that results in reduced transcription, decreased mRNA stability, or reduced mRNA translation.

The complete suppression of expression of a given gene is called "knockout". Gene knockout means that no functional transcripts are synthesized from the gene, resulting in the loss of the function normally provided by the gene. Gene knockout is achieved by altering the DNA sequence that causes the gene or its regulatory sequence, or a portion thereof, to be disrupted or missing. Knockout techniques include using homologous recombination to replace, interrupt, or delete key parts or the entire gene sequence, or using DNA-modifying enzymes such as zinc finger enzymes or macronucleases to introduce double-strand breaks into the DNA of the target gene, as described, for example, by Gaj et al. (Trends Biotechnol. 2013; 31(7):397-405).

Specific implementation methods utilize one or more knockout plasmids or cassettes that have been transformed or transfected into host cells. The target gene in the host cell can be disrupted through homologous recombination. This procedure is typically repeated until all alleles of the target gene are stably removed.

One specific method for knocking out a specific gene, as described herein, is the CRISPR-Cas9 method, as described, for example, in Weninger et al. (J. Biotechnol. 2016, 235:139-49). Another method includes the cleavage marker method, as described, for example, in Heiss et al. 2013 (Appl Microbiol Biotechnol. 97(3):1241-9).

Another implementation involves target mRNA degradation by transfecting host cells with small interfering RNA (siRNA) and targeting mRNA encoding target proteins expressed endogenously by the host cells.

Gene expression can be suppressed or reduced by directly interfering with it, including but not limited to suppressing or reducing DNA transcription (e.g., by using specific promoter-associated repressors, by site-specific mutagenesis of a given promoter, by promoter exchange), or suppressing or reducing translation (e.g., by post-transcriptional gene silencing induced by RNAi or non-coding RNA). Expression of dysfunctional or inactive gene products with reduced activity can be achieved, for example, by site-specific or random mutagenesis, insertion, or deletion within the coding gene.

Inhibition or reduction of the activity of a gene product can be achieved, for example, by applying an inhibitor to or incubating with the corresponding enzyme before or concurrently with protein expression. Examples of such inhibitors include, but are not limited to, inhibitory peptides, antibodies, aptamers, fusion proteins or antibody mimics targeting the enzyme, or their ligands or receptors, or inhibitory peptides or nucleic acids, or small molecules with similar binding activity.

Gene silencing, gene knockdown, and gene elimination refer to techniques that reduce gene expression through gene modification or by treating the gene with oligonucleotides having sequences complementary to the mRNA transcript or gene. If DNA is modified, the result is a knockdown or elimination of the organism. If the change in gene expression is caused by an oligonucleotide that binds to mRNA or temporarily binds to the gene, this results in a temporary change in gene expression without altering the chromosomal DNA, and this is called transient knockdown.

In transient knockdown (also covered by the term above), the binding of the oligonucleotide to an active gene or its transcript leads to reduced expression by blocking transcription (in the case of gene binding), degradation of the mRNA transcript (e.g., by small interfering RNA (siRNA) or antisense RNA), or blocking mRNA translation.

Other methods for gene silencing, knockdown, or elimination are known to those skilled in the art from relevant literature, and their application in the context of this invention is considered conventional. Gene knockout refers to a technique in which gene expression is completely blocked, i.e., the corresponding gene is inactive, or even removed. Methodological approaches to achieve this objective are diverse and known to those skilled in the art. An example is the generation of dominant-negative mutants of a given gene. Such mutants can be generated by site-directed mutagenesis (e.g., deletion, partial deletion, insertion, or nucleic acid substitution), by using suitable transposons, or by other methods known to those skilled in the art from relevant literature, and therefore their application is considered conventional in the context of this invention. One example is knockout using targeted zinc finger nucleases. The corresponding kit is provided by Sigma Aldrich as “CompoZR Knockout ZFN.” Another approach involves the use of transcription activator-like effector nucleases (TALENs).

Delivery of the dominant-negative construct involves introducing a sequence encoding a dysfunctional gene expression product, for example, through transfection. This coding sequence is functionally coupled to a strong promoter, causing the expression of the dysfunctional enzyme to exceed the native expression of the gene expression product, which in turn results in an effective physiological defect in the corresponding activity of the gene expression product.

Conditional gene knockout allows for the blocking of gene expression in a tissue- or time-specific manner. For example, this can be achieved by introducing a short sequence called a loxP site around the target gene. Similarly, other methods are known to those skilled in the art from the relevant literature, and their application in the context of this invention is considered conventional.

Another approach is gene alteration, which may result in a dysfunctional gene product or a gene product with reduced activity. This approach involves introducing frameshift mutations, nonsense mutations (i.e., the introduction of premature stop codons), or mutations leading to amino acid substitutions that dysfunction the entire gene product or result in reduced activity. Such gene alterations can be produced, for example, through mutagenesis (e.g., deletion, partial deletion, insertion, or nucleic acid substitution), nonspecific (random) mutagenesis, or site-directed mutagenesis. Protocols describing the practical applications of gene silencing, gene knockdown, gene knockout, delivery of dominant-negative constructs, conditional gene knockout, and/or gene alteration are generally available to those skilled in the art and are within their conventional scope. Therefore, the technical teachings provided herein are entirely feasible with respect to all conceivable methods that result in the inhibition or reduction of gene expression, or the expression of a dysfunctional or inactive gene product, or with reduced activity.

The gene modifications described herein can be performed using tools, methods and techniques known in the art, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2001).

As used herein, the term "endogenous" refers to molecules and sequences, particularly endogenous genes or proteins, that exist in wild-type (natural) host cells prior to modification to reduce the expression of the corresponding endogenous gene and/or reduce the production of the endogenous protein. Specifically, endogenous nucleic acid molecules (e.g., genes) or proteins that are indeed present in (and obtainable from) a particular host cell, as found in nature, are understood to be "endogenous to the host cell" or "endogenous to the host cell." Furthermore, cells that "endogenously express" a nucleic acid or protein express that nucleic acid or protein, as in the same particular type of host found in nature. Additionally, host cells that "endogenously produce" nucleic acids, proteins, or other compounds produce said nucleic acids, proteins, or compounds, as in the same particular type of host cells found in nature.

Therefore, even if an endogenous protein is no longer produced by the host cell, such as in a knockout mutant of the host cell where the protein-coding gene is inactivated or missing, the protein is still referred to as "endogenous" in this paper.

As used herein with respect to nucleotide sequences, constructs such as expression cassettes, amino acid sequences, or proteins, the term "heterologous" refers to a compound that is foreign to a given host cell, i.e., "exogenous," such as a compound not found in nature within the host cell; or one that is naturally found in a given host cell, e.g., "endogenous," but is heterologous in the context of or integrated into such heterologous constructs, e.g., using heterologous nucleic acids fused to or bound to endogenous nucleic acids. Heterologous nucleotide sequences, such as those found endogenously, can also be produced in cells in non-natural amounts, e.g., greater than expected or greater than naturally found amounts. Heterologous nucleotide sequences, or nucleic acids containing heterologous nucleotide sequences, may differ in sequence from endogenous nucleotide sequences but encode the same protein found endogenously. Specifically, a heterologous nucleotide sequence is a nucleotide sequence not found in nature that has the same relationship to the host cell. Any recombinant or artificial nucleotide sequence is understood to be heterologous. Examples of heterologous polynucleotides are nucleotide sequences not naturally associated with a promoter, for example, to obtain a heterozygous promoter, or nucleotide sequences operatively linked to a coding sequence, as described herein. As a result, hybrid or chimeric polynucleotides can be obtained. Another example of a heterologous compound is a polynucleotide encoding a POI operatively linked to a transcriptional control element (e.g., a promoter), where the endogenous, naturally occurring POI coding sequence is typically not operatively linked to said transcriptional control element.

As used herein, the term "operably linked" refers to the association of nucleotide sequences on a single nucleic acid molecule (e.g., a vector or expression cassette) such that the function of one or more nucleotide sequences is influenced by at least one other nucleotide sequence present on said nucleic acid molecule. Through operably linked, a nucleic acid sequence is functionally related to another nucleic acid sequence on the same nucleic acid molecule. For example, when a promoter is capable of influencing the expression of a coding sequence, it is operably linked to the coding sequence of a recombinant gene. As another example, when a nucleic acid encoding a signal peptide is capable of expressing a secreted form of protein, such as a preform or mature protein of a mature protein, said nucleic acid is operably linked to a nucleic acid sequence encoding a POI. Specifically, such operably linked nucleic acids can be linked immediately, i.e., without any additional elements or nucleic acid sequences between the nucleic acid encoding the signal peptide and the nucleic acid sequence encoding the POI.

If a promoter controls the transcription of a coding sequence, then the “promoter” sequence is generally understood to be operatively linked to the coding sequence. If the promoter sequence is not naturally associated with the coding sequence, its transcription is not controlled by the promoter in natural (wild-type) cells, or the sequence recombines with different consecutive sequences.

Promoters that can be regulated (especially repressible) by non-methanol carbon sources and used for the purposes described herein are referred to herein as “ECP”. Therefore, this disclosure with respect to “ECP” should also refer to “promoters that can be regulated (or repressed) by non-methanol carbon sources”, and vice versa.

As described herein, the ECP specifically initiates, regulates, or otherwise mediates or controls the expression of POI-encoding DNA. The promoter DNA and encoding DNA can originate from the same gene or different genes, and can come from the same or different organisms.

As described herein, ECPs are specifically understood as adjustable promoters, particularly carbon-source adjustable promoters with different promoter strengths in repressed and induced states, especially non-methanol carbon-source adjustable promoters, such as ECPs that can be repressed by non-methanol carbon sources and, in particular, cannot be induced by methanol. Specifically, by using ECPs that are transcriptionally active in the absence of methanol, POIs can be generated under the transcriptional control of the ECP without the need to add methanol to the host cell culture.

The strength of an ECP specifically refers to its transcriptional strength, which is represented by the initiation efficiency of transcription occurring at that promoter at a high or low frequency. The higher the transcriptional strength, the more frequently transcription will occur at that promoter. Promoter strength is a defining characteristic of a promoter because it determines the frequency of transcription of a given mRNA sequence, effectively assigning some genes a higher transcriptional priority than others, resulting in higher transcript concentrations. For example, genes encoding large quantities of desired proteins typically have relatively strong promoters. RNA polymerases can only perform one transcription task at a time and therefore must prioritize their work for efficiency. Differences in promoter strength are chosen to achieve this prioritization.

The ECPs used in this article are relatively strong in the fully induced state, which is generally understood to be approximately the state of maximum activity. The relative strength is usually determined relative to a comparison promoter (referred to as the reference promoter in this article), which can be a standard promoter, such as the corresponding pGAP promoter of the cell used as the host cell.

The frequency of transcription is generally understood as the transcription rate, determined, for example, by the amount of transcript in a suitable assay (e.g., RT-PCR or Northern blotting). For example, the transcriptional strength of the promoter according to the invention was determined in host cells of Pichia pastoris and compared with the native pGAP promoter of Pichia pastoris.

The strength of promoter expression of the target gene is generally understood as expression intensity or the ability to support high expression levels/rates. For example, the expression and/or transcription intensity of the promoter of the present invention were measured in host cells of Pichia pastoris and compared with the native pGAP promoter of Pichia pastoris.

Comparative transcriptional intensity compared to a reference promoter can be determined using standard methods, such as by measuring the amount of transcript, for example using a microarray, or in cell cultures, such as by measuring the amount of the corresponding gene expression product in recombinant cells. Specifically, transcription rate can be determined by microarrays, transcriptional intensity on Northern blotting, or by using quantitative real-time PCR (qRT-PCR) or RNA sequencing (RNA-seq), where data show differences in expression levels between conditions with high and low growth rates, or between conditions using different culture medium compositions, and a high signal intensity compared to a reference promoter.

The expression rate can be determined, for example, by the amount of reporter gene (such as eGFP) expressed.

As described herein, the ECP exhibits relatively high transcriptional strength, for example, reflected by at least 15% higher transcription rate or intensity compared to the natural pGAP promoter (also known as the "homologous pGAP promoter") in the host cell. Preferably, the transcription rate or intensity is at least any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or even higher than that of the natural pGAP promoter, such as at least any one of 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%, as determined in host cells (e.g., eukaryotic) selected as host cells for recombination purposes to generate POIs.

The natural pGAP promoter typically initiates the expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutive promoter present in most living organisms. GAPDH (EC 1, 2, 1, 12) (a key enzyme in glycolysis and gluconeogenesis) plays a crucial role in catabolism and anabolism of carbohydrates.

Natural pGAP promoters exhibit specific activity in recombinant eukaryotic cells similar to their activity in native eukaryotic cells of the same species or strain, including unmodified (non-recombinant) or recombinant eukaryotic cells. These natural pGAP promoters are generally understood as endogenous promoters, thus homologous to host cells, and can be used as standard or reference promoters for comparative purposes. The relative expression or transcriptional strength of promoters as described herein is typically compared to natural pGAP promoters used in cells of the same species or strain used to generate POIs.

Regarding inducible or repressible regulatory elements, such as promoters as described herein, the term "regulatory" refers to an element that is inhibited in host cells in the presence of excess substrate (such as nutrients in cell culture medium), for example, during the growth phase of batch culture, and deinhibited to induce strong activity according to a fed-batch strategy, such as during the production phase (e.g., when the amount of nutrients is reduced, or when substrate is added during feeding). Regulatory elements can also be designed to be regulatorable such that the element is inactive without the addition of cell culture additives and is active in the presence of such additives. Therefore, the expression of POIs under the control of such regulatory elements can be induced upon the addition of such additives.

As described in this article, ECP is a relatively strong tunable promoter that is typically silenced or inhibited under cell growth conditions (growth phase) and activated or de-inhibited under production conditions (production phase), and is therefore well-suited for inducing POI production in production cell lines by limiting carbon sources.

Specifically, the promoters described herein are carbon sources that can be modulated by differential promoter strength, as determined in tests comparing their strength in the presence and restriction of glucose, showing that they are still repressed at relatively high glucose concentrations (preferably at a concentration of at least 10 g/L, more preferably at a concentration of at least 20 g/L). Specifically, the promoters described herein are fully induced at limited glucose concentrations, taking into account a glucose threshold concentration that fully induces the promoter, typically less than 20 g/L, preferably less than 10 g/L, less than or at most 1 g/L, even less than 0.1 g/L or less than 50 mg/L, and preferably having, for example, at least 50% of the full transcriptional strength of the natural homologous pGAP promoter at glucose concentrations less than 40 mg/L.

As used herein in the context of this disclosure (e.g., to characterize the carbon-source-regulated promoters described herein), the term "repressed" or "repressed" refers to interference with the transcription of a target gene (encoding a target protein) under the transcriptional control of a promoter that is understood to be repressible, resulting in reduced expression of the target protein in the cell.

The inhibition of ECP described herein occurs specifically when an inhibitor is present in the cell culture medium. An inhibitor can be a specific carbon source or an inhibitory amount of carbon source, such as above a specific threshold. When the inhibitor is removed from the culture medium, or reduced to below the threshold at which inhibition ceases, the expression of the target gene or protein is termed "de-inhibited." In de-inhibition, ECP is understood to be fully induced, and the expression of the target protein is typically at least 1.5 times the basal level expressed by the cell under promoter inhibition conditions.

Specifically, compared to the transcription of the gene when the ECP is de-repressed or fully induced, the transcription of the target gene under the control of the ECP described herein can be suppressed by at least any one of 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95%, or completely suppressed (100% suppressed), or completely suppressed.

Comparing promoter strength under repressed and depressed conditions determines the tunable nature of the promoter and the corresponding induction ratio. According to some embodiments, the induction ratio is understood as the differential promoter strength, determined by the initiation of POI production upon switching to induction conditions below a predetermined carbon source threshold, and compared to the strength in the repressed state. Transcriptional strength is generally understood as the strength in the fully induced state, i.e., exhibiting approximately maximum activity under depressed conditions. Differential promoter strength is determined, for example, based on the efficiency or yield of POI production in recombinant host cell lines under depressed conditions compared to repressed conditions, or by the amount of transcript. Tunerable promoters as described herein have preferred differential promoter strengths (induction ratios) that are at least 1.5-fold or at least 2-fold, more preferably at least 5-fold, even more preferably at least 10-fold, more preferably at least 20-fold, more preferably at least 30-fold, 40-fold, 50-fold, or 100-fold in the depressed (fully induced) state compared to the repressed state, also understood as fold induction.

As used herein, the term "mutation" refers to a method of providing a mutant nucleotide sequence, such as by inserting, deleting, and/or substituting one or more nucleotides to obtain a variant with at least one change in a non-coding or coding region. Mutagenesis can be performed by random, semi-random, or site-directed mutagenesis. The specific ECP and corresponding nucleotide sequences described herein can be used to generate variants that are also tunable promoters suitable for the purposes described herein. Such variants can be generated by appropriate mutagenesis methods using the ECP nucleotide sequences provided herein as parental sequences. Such mutagenesis methods include those that use the corresponding parental promoter sequence information as a template to engineer nucleic acids or to synthesize nucleotide sequences de novo. Specific mutagenesis methods employ appropriate promoter engineering.

The exemplary ECP described herein can, for example, be modified to produce promoter variants with altered expression levels and regulatory properties. For instance, promoter libraries can be prepared by mutagenesis of selected promoter sequences, which can then be used as parental molecules to fine-tune gene expression in eukaryotic cells, for example, by analyzing variants expressed under different fermentation strategies and selecting appropriate variants. Synthetic libraries of variants can be used, for example, to select promoters that match the requirements for generating a selected POI. Such variants can exhibit increased expression efficiency and differential expression under carbon-rich and restrictive conditions in (e.g., eukaryotic) host cells. Typically, this results in large, randomized gene libraries with high genetic diversity, which can be selected based on specific desired genotypes or phenotypes.

Some ECP variants may be size variations of the ECP nucleotide sequence provided herein, and/or include more than one element or region of the promoter described herein (such as the core regulatory region, master regulatory region, or T motif), and/or include one or more (identical or different) segments of the ECP nucleotide sequence.

Specific mutagenesis methods provide point mutations, particularly tandem point mutations, of one or more nucleotides in the sequence, such as altering at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or even more consecutive nucleotides in the promoter nucleotide sequence. Point mutations are typically at least one of deletions, insertions, and/or substitutions of one or more nucleotides. The promoter sequence can be mutated distally, particularly within the 5' region, reaching up to 50% of the full-length promoter sequence. This 5' region can be highly variable without substantially losing promoter activity. The promoter sequence can be specifically mutated within the master regulatory region; however, it is preferable to have high sequence identity with the exemplary master regulatory region and, particularly, with the exemplary core regulatory region, such as, for example, at least any one of 80%, 85%, 90%, or 95%. Outside any core or master regulatory region, sequence variability may be higher, and the ECP may remain functional, for example, where sequence identity is less than 80% or less than 85%.

Any mutations within the core or main regulatory region are generally conserved, meaning they can maintain (or even improve) recognition by specific transcription factors.

Specifically, the ECP described herein may include a heterozygous nucleotide sequence, such as the core or master regulatory region described herein, and further include one or more regions or alternative (natural or artificial) promoter sequences, such as in the 3' region (specifically the 3' end), which includes at least 10 or 15 3'-terminal nucleotide sequences, including the translation initiation site at the 3' end (e.g., up to 20 nt, 25 nt, or 30 nt) of different promoters (e.g., any constitutive or regulated (or other inducible) promoters), thereby replacing the translation initiation site of the ECP promoter.

As used in this article, the terms "nucleotide sequence" or "nucleic acid sequence" refer to DNA or RNA. "Nucleic acid sequence" or "polynucleotide sequence," or simply "polynucleotide," refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to 3' end. It includes expression cassettes, self-replicating plasmids, infectious polymers of DNA or RNA, and non-functional DNA or RNA.

As used herein, the term "target protein (POI)" refers to a polypeptide or protein produced in a host cell via recombinant technology. More specifically, the protein may be a polypeptide that is not naturally present in the host cell, i.e., a heterologous protein, or it may be native to the host cell, i.e., a protein homologous to the host cell, but produced, for example, by transformation with a self-replicating vector containing a nucleic acid sequence encoding the POI, or by integration of one or more copies of a nucleic acid sequence encoding the POI into the genome of the host cell via recombinant technology, or by recombination modification of one or more regulatory sequences controlling the expression of the gene encoding the POI, such as recombination modification of promoter sequences. In some cases, as used herein, the term POI may also refer to any metabolite of the host cell mediated by a protein such as that expressed via recombinant technology.

As used in this paper, the term "scaffold" describes a group of multifaceted, compact, and stably folded proteins (differing in size, structure, and origin) that serve as starting points for the generation of antigen-binding molecules. Inspired by the structure-function relationship of antibodies (immunoglobulins), such alternative protein scaffolds provide a robust, conserved structural framework that supports interaction sites that can be remodeled for tight and specific recognition of a given (biological) molecular target.

The term "sequence identity" of variants, homologs, or orthologs, compared to the parental nucleotide or amino acid sequence, indicates the degree of identity between two or more sequences. Two or more amino acid sequences may have identical or conserved amino acid residues at corresponding positions, up to a certain extent of 100%. Two or more nucleotide sequences may have identical or conserved base pairs at corresponding positions, up to a certain extent of 100%.

Sequence similarity searching is an effective and reliable strategy for identifying homologs with an excessive (e.g., at least 50%) sequence identity. Commonly used sequence similarity searching tools include, for example, BLAST, FASTA, and HMMER.

Sequence similarity searches can identify homologous proteins or genes by detecting excessive similarity and statistically significant similarity reflecting a common ancestor. Homologous proteins can include orthologous proteins, which are understood herein as identical proteins in different organisms, such as variants of such proteins in different organisms or species.

Orthologous sequences of the same protein in different organisms or species are generally homologous to the protein sequence, especially orthologs originating from the same genus. Typically, orthologs have at least about 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity, up to 100%. Specifically, orthologs can be identified when an orthologous sequence replaces the FLO8 protein or the gene encoding the FLO8 protein in a host cell, said orthologous sequence being modified to knock out the endogenous FLO8 protein. For example, if a presumed FLO8 protein is functional in *Pichia pastoris* or *Saccharomyces cerevisiae* host cells, replacing the endogenous FLO8 protein encoded by a gene that has been knocked out in such *Pichia pastoris* and *Saccharomyces cerevisiae* host cells, then for the purposes described herein, such a presumed FLO8 protein can be considered an FLO8 protein homolog.

The FLO8 protein, including or composed of the amino acid sequence identified as SEQ ID NO:1, is derived from *Phomormus frenulum*. Homologous sequences are known to exist in other eukaryotic or prokaryotic host cells. For example, yeast cells contain corresponding homologous sequences, particularly in *Pichia pastoris*, which has been reclassified as a new genus *Phomormus* and divided into three species: *Phomormus pastoris*, *Phomormus frenulum*, and *Phomormus pseudopasteurus*. Specific homologous sequences are found, for example, in *Pasteurella multocida* (e.g., SEQ ID NO:3, such as those encoded by nucleotide sequences including or consisting of SEQ ID NO:4), *Saccharomyces cerevisiae* (e.g., SEQ ID NO:5 or SEQ ID NO:6), *Yersinia lipolytica* (e.g., SEQ ID NO:7), *Henrynori* polymorpha (e.g., SEQ ID NO:8), or *Aspergillus niger* (e.g., SEQ ID NO:9).

Any homologous sequence of FLO8 protein having the specific sequence identity described herein, particularly any FLO8 protein that is an ortholog of the Pichia pastoris FLO8 protein, is included in the definition of FLO8 protein described herein.

The "percentage of amino acid sequence identity (%)" for amino acid sequences, homologs, and orthologs described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

For the purposes described herein, the sequence identity between two amino acid sequences was determined using the following exemplary parameters with NCBI BLAST program version BLASTP 2.8.1: program: blastp, word length: 6, expected value: 10, hitlist size: 100, gapcosts: 11.1, matrix: BLOSUM62, filter string: F, composition adjustment: conditional composition score matrix adjustment.

The "percentage of identity (%)" for a nucleotide sequence (e.g., a promoter or gene) is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to nucleotides in the DNA sequence, after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignments used to determine the percentage of nucleotide sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

As used herein with respect to POIs, the terms “isolated” or “isolated” should refer to compounds that have been adequately isolated from their naturally associated environment (particularly cell culture supernatants) and thus exist in a “purified” or “substantially pure” form. However, “isolated” does not necessarily mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the essential activity and may be present (e.g., due to incomplete purification). Isolated compounds can be further formulated to prepare their formulations, and can still be isolated for practical purposes—for example, when used for diagnostics or therapy, POIs can be mixed with pharmaceutically acceptable carriers or excipients.

As used herein, the term "purified" refers to a formulation comprising at least 50% (mol/mol), preferably at least 60%, 70%, 80%, 90%, or 95% of a compound (e.g., POI). Purity is measured by a method suitable for the compound (e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.). The isolated and purified POIs described herein can be obtained by purifying cell culture supernatants to reduce impurities.

Various methods can be used for the separation and purification of recombinant peptide or protein products, such as methods utilizing differences in solubility, such as salting out and solvent precipitation; methods utilizing differences in molecular weight, such as ultrafiltration and gel electrophoresis; methods utilizing differences in charge, such as ion exchange chromatography; methods utilizing specific affinity, such as affinity chromatography; methods utilizing differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and methods utilizing differences in isoelectric point, such as isoelectric focusing.

The following standard methods are preferred: cell (fragment) separation and washing by microfiltration or tangential flow filtration (TFF) or centrifugation; POI purification by precipitation or heat treatment; POI activation by enzymatic digestion; POI purification by chromatography (such as ion exchange (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography, size exclusion chromatography (SEC), or HPLC); and POI precipitation concentration and washing by ultrafiltration step.

The highly purified product is substantially free of contaminating proteins and preferably has a purity of at least 90%, more preferably at least 95%, or even at least 98%, and at most 100%. The purified product can be obtained by purifying cell culture supernatant or from cell debris.

Isolated and purified POIs can be identified using conventional methods such as Western blotting, HPLC, activity assays, or ELISA.

As used herein, the term “recombinant” should mean “prepared by or resulting from genetic engineering.” A recombinant host can be engineered to remove and/or inactivate one or more nucleotides or nucleotide sequences, and can specifically include expression vectors or cloning vectors containing recombinant nucleic acid sequences (particularly using host-exogenous nucleotide sequences). Recombinant proteins are produced by expressing the corresponding recombinant nucleic acid in the host. As used herein, the term “recombinant” with respect to POIs includes POIs prepared, expressed, produced, or isolated by recombinant means, such as POIs isolated from host cells transformed to express POIs. According to the invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the scope of the art can be used. Such techniques are well explained in the literature. See, for example, Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor, (1982).

Some recombinant host cells are “engineered” host cells, which are understood to be host cells that have been genetically engineered, i.e., manipulated through human intervention. When a host cell is engineered to reduce or downexpress a given gene or corresponding protein, the host cell is manipulated such that it no longer has the ability to express such a gene and protein, respectively, compared to a host cell under the same conditions before manipulation, or compared to a host cell that has not been engineered to downexpress the gene or protein.

According to specific embodiments, it has been surprisingly found that reduced expression of genes encoding FLO8 in host cells, particularly the deletion of such genes, has a positive effect on the expression levels of genes controlled by methanol-free inducible promoters (ECPs), thus allowing higher expression levels without loss of carbon source promoter regulation. Therefore, cells with and without deletions of the corresponding endogenous gene (flo8 gene) encoding the FLO8 protein were generated, and the strength and regulation of the gene under ECP control were tested. For several exemplary POIs (which are intracellular or secreted model proteins), increased expression was shown in cells lacking the corresponding flo8 gene, which can be traced back to increased transcriptional levels. Increased expression was observed in both small-scale screening cultures and controlled production processes in bioreactors. Therefore, a novel expression system has been developed that allows for the formation of much higher yields of the product in methanol-free media, such as in yeasts like Pichia pastoris, compared to previously existing systems.

According to specific examples, the effect of FLO8 disruption on the expression intensity of non-methanol carbon source-regulated promoters pG1, pG3, pG4, and pG6 (which are regulated by carbon sources other than methanol) was compared with that of the constitutive promoter pGAP or the methanol-induced promoter pAOX1. Surprisingly, all non-methanol carbon source-regulated promoters showed statistically significant increases in transcription in the dFLO8 strain, indicating increased expression under the control of these promoters. Conversely, methanol-induced pAOX1 did not show a significant increase in transcription intensity in the dFLO8 mutant compared to the wild type, and had no significant effect on the transcription intensity of pGAP. Therefore, it can be concluded that the activity of pGAP or pAOX1 promoters is not affected by low expression of FLO8.

Surprisingly, it was found that, compared to the standard pGAP promoter, the expression of heterologous target genes producing the target protein was effectively increased in cell cultures when FLO8 was knocked out or disrupted, by using promoters that could be repressed using a carbon source that was not induced by methanol. For example, increased eGFP fluorescence was observed in each of the promoters pG1, pG1-3, pG3, pG4, pG6, pG7, and pG8 in the dFLO8 strain, with an increase of 1.2 to 3.9 times compared to expression in wild-type host cells (without FLO8 disruption).

The following projects are implementation schemes described in this article:

1. A recombinant host cell comprising an endogenous gene encoding an FLO8 protein, said FLO8 protein comprising an amino acid sequence identified as SEQ ID NO:1 or a homolog thereof, said host cell being engineered by one or more gene modifications to reduce the expression of said gene compared to a host cell prior to said one or more gene modifications, and said host cell comprising a heterologous expression cassette comprising a target gene (GOI) controlled by an expression cassette promoter (ECP), said ECP being modulated by a non-methanol carbon source.

2. The host cell according to Project 1, wherein the homolog has at least 25% sequence identity with SEQ ID NO:1.

3. The host cell according to any one of Items 1 to 2, wherein the one or more gene modifications include (i) one or more endogenous polynucleotides or a portion thereof; or (ii) disruption, substitution, deletion or knockout of expression control sequences.

4. The host cell according to Project 3, wherein the endogenous polynucleotide is a gene encoding the FLO8 protein or the homolog.

5. The host cell according to Project 4, wherein the expression control sequence includes any one of a promoter, a ribosome binding site, a transcription or translation initiation and termination sequence, or an enhancer or activator sequence.

6. The host cell according to any one of items 1 to 4, wherein the ECP is induced in the presence of a growth-limiting amount of a non-methanol carbon source, preferably in the absence of methanol; and is inhibited in the presence of an excess of a non-methanol carbon source above the growth-limiting amount.

7. The host cell according to Project 6, wherein the growth limiting amount of the non-methanol carbon source is at most 1 g/L of cell culture medium.

8. The host cell according to any one of items 1 to 7, wherein the ECP comprises at least one first core regulatory region and at least one second core regulatory region, wherein the first core regulatory region has at least 75% sequence identity with SEQ ID NO:17, and the second core regulatory region has at least 75% sequence identity with SEQ ID NO:18.

9. The host cell according to any one of items 1 to 8, wherein the ECP includes at least one regulatory region having at least 85% sequence identity with SEQ ID NO:35.

10. The host cell according to item 8 or 9, wherein the ECP includes at least two first core regulatory regions and/or second core regulatory regions.

11. A host cell according to any one of items 1 to 10, wherein the ECP comprises at least one T motif consisting of any one of SEQ ID NO: 19-34, optionally wherein the T motif is not extended at its 5' or 3' end by one or more thymines.

12. The host cell according to item 11, wherein the ECP comprises at least two of the T motifs.

13. The host cell according to any one of items 1 to 7, wherein the ECP comprises at least 60% sequence identity of at least 300 nt to any one of sequences SEQ ID NO:10-16 or any one of SEQ ID NO:41-45.

14. The host cell according to item 13, wherein the ECP comprises at least 60% sequence identity with any of the full-length sequences SEQ ID NO:10-16 or any of SEQ ID NO:41-45.

15. The host cell according to item 13 or 14, wherein the ECP comprises or is composed of SEQ ID NO:10 or SEQ ID NO:11.

16. The host cell according to any one of items 1 to 15, wherein the expression cassette is contained in an autonomous replication vector or plasmid, or is contained within the chromosome of the host cell.

17. A host cell according to any one of items 1 to 16, wherein the expression cassette further comprises a nucleotide sequence encoding a signal peptide capable of secreting a target protein (POI) encoded by the GOI, preferably wherein the nucleotide sequence encoding the signal peptide is fused adjacent to the 5' end of the GOI.

18. A host cell according to any one of items 1 to 17, wherein the GOI encodes a target protein (POI), said target protein being a peptide or protein selected from the group consisting of antigen-binding proteins, therapeutic proteins, enzymes, peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccine antigens, growth factors, hormones, cytokines, process enzymes, and metabolic enzymes.

19. A host cell according to any one of items 1 to 18, wherein the antigen-binding protein is selected from the group consisting of:

a) Antibodies or antibody fragments, such as chimeric antibodies, humanized antibodies, bispecific antibodies, Fab, Fd, scFv, dimer, trimer, Fv tetramer, microantibodies, single-domain antibodies such as VH, VHH, IgNAR or V-NAR.

b) Antibody mimics, such as Adnectins, affinity molecules, Affilins, Affimers, Affitins, alpha molecules, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, monomers, or NanoCLAMPs; or

c) Fusion proteins including one or more immunoglobulin folding domains, antibody domains, or antibody mimics.

20. The host cell according to any one of items 1 to 19 is:

a) Select yeast cells from the genera consisting of *Pichia pastoris*, *Hansenula polymorpha*, *Kumaria spp.*, *Saccharomyces cerevisiae*, *Kluyveromyces spp.*, *Candida*, *Henrychotomyces henchiformis*, *Yersinia lipolytica*, or *Diplophora*, such as *Pichia pastoris*, *Kumaria freundii*, *Kumaria pastoris*, *Pseudo-Kumaria pastoris*, *Saccharomyces cerevisiae*, *Henrychotomyces henchiformis*, *Kluyveromyces lactis*, *Kluyveromyces marx*, *Yersinia lipolytica*, or *Hansenula polymorpha*; or

b) Cells of filamentous fungi such as Aspergillus bubomori or Trichoderma reesei.

21. A method for increasing the yield of a target protein (POI) by reducing the expression of a gene encoding the FLO8 protein in a host cell, the target protein (POI) being produced by a host cell expressing a target gene (GOI) encoding the POI under the control of a promoter that can be regulated or inhibited by a non-methanol carbon source, the FLO8 protein comprising an amino acid sequence identified as SEQ ID NO:1 or a homolog thereof.

22. A method for producing the POI encoded by the target gene (GOI) by culturing the host cell of any one of items 1 to 20 under conditions that produce the target protein (POI).

23. The method described according to item 21 or 22 includes the following steps:

a) Culture the host cells under growth conditions; and other steps.

b) Under growth-restricted conditions, the host cells are cultured in the presence of a second non-methanol carbon source of up to 1 g/L to induce the expression of the GOI to produce the POI.

24. The method according to item 23, wherein the first carbon source or the second carbon source is selected from sugars, polyols, alcohols, or any one or more mixtures thereof.

25. The method according to item 23 or 24, wherein step a) cultivation is carried out in a batch stage; and step b) cultivation is carried out in a fed-batch or continuous cultivation stage.

26. A method for producing a target protein (POI) in a host cell, comprising the following steps:

a) Genetically engineer the host cells to reduce the expression of the endogenous gene encoding the FLO8 protein, wherein the FLO8 protein comprises the amino acid sequence identified as SEQ ID NO:1 or its homolog;

b) Introducing a heterologous expression cassette into the host cell, the heterologous expression cassette comprising a non-methanol carbon source regulated promoter (particularly the ECP promoter described herein) operably linked to a target gene (GOI) encoding the POI;

c) Culture the host cells under conditions that generate the POI;

d) Optionally isolate the POI from the cell culture; and

e) Optionally purify the POI.

The foregoing description will be more fully understood by referring to the following embodiments. However, such embodiments are merely representative of methods for implementing one or more embodiments of the invention and should not be construed as limiting the scope of the invention.

Example

The following examples will demonstrate that disruption of the transcriptional regulator FLO8 leads to higher transcriptional activity of carbon-regulated promoters such as pG1, pG3, pG4, pG6, and pG8 and their engineered variants, thereby enabling increased production of recombinant proteins under carbon-restricted culture conditions.

Example 1: Construction of Pichia pastoris dFLO8 strain

Wild-type strains of Pichia pastoris, CBS7435 or CBS2612 (CBS-KNAW Fungal Biodiversity Centre, Skichmilk Cultural Centre, Utrecht, Netherlands), were used as host strains.

To generate the dFLO8 mutant strain, the gene PP7435_Chr4-0252 (FLO8) was disrupted using a split marker cassette method suitable for Pichia pastoris (Gasser et al., 2013) and described in WO2015158800A1. Briefly, two 1.5 kb regions approximately 200 bp upstream and downstream of the ORF translation origin were amplified using primers A_fw and A_bw, and D_fw and D_bw, respectively (Table 1). By fusion PCR, fragments A and B, homologous to the 5' and 3' ends of the corresponding B and C portions of the resistance marker cassette, were used to flank two approximately 1 kb long, overlapping portions (435 bp) of the KanMX marker cassette (primers B_fw, B_bw, C_fw, and C_bw). As described in (Gasser et al., 2013), two fused fragments, AB and CD, were simultaneously transformed into electrocompetent Pichia pastoris cells. Successful integration required three distinct recombination events that resulted in the replacement of a 0.4 kb fragment at the 5' end of PP7435_Chr4-0252 and its promoter via a KanMX box.

Positive transformants were selected on selective YPD agar plates containing 500 μg ^mL⁻¹ of genimycin (per liter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar-agar). Correct deletion mutants were verified by PCR (Det_fw and Det_bw, Table 1) and gel electrophoresis using primers located outside the cleavage marker cassette.

Table 1: Primers used for constructing split marker boxes

Primers sequence A_fw CGAACATCCATCACCAAAACAC(SEQ ID NO:72) A_bw GTTGTCGACCTGCAGCGTACGGTGTTGCCGGAAAATG(SEQ ID NO:73) D_fw TAGGTGATATCAGATCCACTGATCAATTTGCCCAAGAGACG(SEQ ID NO:74) D_bw GACTGTTGCGATTGCTGGTG(SEQ ID NO:75) B_fw CATTTCGCGGCAACACCGTACGCTGCAGGTCGACAAC(SEQ ID NO:76) B_bw CGGTGAGAATGGCAAAAGCTTAT(SEQ ID NO:77) C_fw AAGCCCGATGCGCCAGAGTTG(SEQ ID NO:78) C_bw CGTCTCTTGGGCAAATTGATCAGTGGATCTGATATCACCTA(SEQ ID NO:79) Det_fw ATCCAGGACACGCTCATCAAG(SEQ ID NO:80)

Example 2: Effects of FLO8 disruption on pG1 and pG1-3 driven intracellular eGFP production

a) Construction of Pichia pastoris dFLO8 strain

Pichia pastoris CBS2612_pG1_eGFP#8 (described in WO2013050551A1) and CBS2612_pG1-3_eGFP#1 (described in WO2017021541A1 and Prielhofer et al., 2018 as CBS2612_pGTH1-D1240) were used as host strains. These strains were shown to integrate a single copy of the Zeocin resistance cassette and the eGFP expression cassette, which consists of the glucose-regulated promoter pG1 (SEQ ID NO:12) or its engineered variant (pG1-3, SEQ ID NO:10), GOI, and the Saccharomyces cerevisiae CYC1 transcription terminator. The corresponding dFLO8 mutant strains were constructed as described in Example 1.

b) Screening for eGFP productivity

For expression screening, single colonies of dFLO8 strain, along with their corresponding parental strains and non-producing wild-type strains, were inoculated into 2 mL of liquid YP medium (per liter: 20 g peptone, 10 g yeast extract) containing 25 μg ^mL⁻¹ Zeocin and 500 μg ^mL⁻¹ Genimycin (if appropriate). The precultures were grown in 24-DWP at 25°C and 280 rpm for approximately 24 h and subsequently inoculated with 2 mL of synthetic screening medium ASMv6 (composition given below) (containing 50 g ^L⁻¹ polysaccharide and 1.5% glucose release enzyme (to achieve a ^glucose release rate of approximately 0.8 mg ^{mL⁻¹ h⁻¹} ; m2p medium development kit)) to an initial _OD₀ of 5 (induction condition). For inhibition conditions, ASMv6 containing 2% glycerol was used. The master cultures were then incubated at 25°C and 280 rpm for an additional 48 h. To measure eGFP expression, cells were diluted to 0.1 OD ₆₀₀ in phosphate-buffered saline (PBS) and analyzed by flow cytometry as described in Stadlmayr et al., 2010. 15,000 cells were analyzed for each sample. Autofluorescence of *Pichia pastoris* was measured using wild-type cells and subtracted from the signal. Normalized eGFP expression levels (fluorescence intensity relative to cell size) are given as a percentage of pGAP-controlled expression (Table 2).

The synthesized screening medium ASMv6 contains per liter: 22.0 g of citrate monohydrate, _{6.30 g of (NH₄)₂HPO₄ , 0.49} g of _MgSO₄ · _7H₂O , 2.64 g of KCl, 0.0535 g of CaCl₂ _{· 2H₂O} , 1.470 mL of PTMO micro-salt stock solution, and 0.4 mg of biotin; the pH is set to 6.5 with KOH (solid).

Each liter of PTM0 trace salt stock solution contains:

6.0g _CuSO₄ · _5H₂O , 0.08g NaI, _3.36g MnSO₄· _H₂O , 0.2g _Na₂MoO₄ · _2H₂O , 0.02g _H₃BO₃ , 0.82g CoCl₂· _6H₂O , _{20.0g ZnCl₂} , _{65.0g FeSO₄} · _7H₂O , and _{5.0ml H₂SO₄} (95%-98% ₎ .

Table 2: Effect of dFLO8 on eGFP expression under the control of pG1 or pG1-3. The expression levels of eGFP relative to pGAP are shown after 48 hours of culture in 2% glycerol (inhibition) or restrictive glucose (induction), and the increase in eGFP fluorescence in dFLO8 strains under induction conditions compared to wild-type pG1 or pG1-3.

*Prepared by the method described in Example 2.

The deletion of FLO8 had a positive effect on pG1-driven expression, resulting in nearly 4-fold higher eGFP levels under induced (glucose-restricted) conditions (Table 2). Therefore, the effect of disrupting FLO8 on the promoter variant pG1-3, which itself has high intrinsic expression, was also investigated. This also showed a positive effect on the promoter variant, further increasing eGFP levels in dFLO8 strains by 2.5 to 3-fold compared to expression from the same promoter in a wild-type background (Table 2).

Example 3: Effect of FLO8 disruption on _PG1-3 -driven productivity of secreted recombinant proteins

Next, the effect of the dFLO8 mutation on the expression of proteins in a pG1-3-driven secretion model was evaluated. For this purpose, expression cassettes of vHH or scR were converted to CBS2612 and CBS2612_dFLO8.

a) Construction of Pichia pastoris strain dFLO8 and recovery of selection markers

The wild-type strain of Pichia pastoris, CBS2612 (CBS-KNAW Fungal Biodiversity Centre, Schickiemilko Cultural Centre, Utrecht, Netherlands), was used as the host strain.

CBS2612_KanR_dFLO8#2 was constructed as described in Example 1. To excise the KanMX selection marker cassette from the genome based on Cre-loxP recombination, as described by Gasser et al., 2013, CBS2612_KanR_dFLO8#2 was transiently transformed by electroporation using plasmid pTAC_Cre_hphMX4. pTAC_Cre_hphMX4 is a derivative of plasmid pYX022 (R&D system) and consists of an E. coli origin of replication, an expression cassette for the Cre-recombinase gene, a hygromycin resistance cassette, and an ARS/CEN element. For its construction, pTAC_Cre_kanMX was digested with EcoR91I (Marx et al., 2008) to remove the kanamycin resistance cassette. Plasmid pGA26_hphMX4 was digested with the same enzyme to excise the hygromycin resistance cassette, and suitable fragments were fused by ligation.

Positive transformants were selected on YPD agar containing 100 μg ^mL⁻¹ hygromycin. Hygromycin-resistant clones were subsequently rescreened in parallel on YPD agar (without hygromycin to promote the loss of pKTAC-CRE_hygR) and on YPD agar containing 500 μg ^mL⁻¹ genimycin. Strains that lost resistance to genimycin were further examined by PCR and gel electrophoresis using primers Det_bw and Det_fw (Table 1) to confirm the excision of the KanMX resistance cassette from the genome. CBS2612_L_dFLO8#2_4 was selected from the corresponding strain for further use.

b) Pichia pastoris strains that secrete antibody fragments scFv(scR) and vHH under the transcriptional control of _PG1-3 . Build

Expression plasmids pPM2d_pAOX_scR and pPM2d_pAOX_vHH are derivatives of the pPUZZLE plasmid backbone (Stadlmayr et al., 2010). They consist of the *E. coli* origin of replication (pUC19), a Zeocin resistance cassette, a *Saccharomyces cerevisiae* α-mating factor precursor leader, the target gene (vHH or scR), a *Saccharomyces cerevisiae* CYC1 transcription terminator, and a locus (3′AOX1 region) for integration into the *Pichia pastoris* genome. For their construction, the genes encoding scFv (scR) and vHH were codon-optimized using DNA 2.0 and obtained as synthetic DNA (sequences described below). A His6-tag was fused at the C-terminus to the gene used for detection. After restriction digestion with XhoI and BamHI (for scR) or EcoRV (for vHH), each gene was ligated into pPM2d_pAOX digested with XhoI and BamHI or EcoRV. The replacement of P _AOX1 with P _G1-3 was achieved by restriction digestion of these plasmids with AlwNI and SbfI, along with the expression plasmid pPM1aZ10_pG1-3_eGFP (also a pPUZZLE derivative described in WO2017021541A1), and fusion with appropriate plasmid fragments. The fragment derived from pPM1aZ10_pG1-3_eGFP also contains a sequence for the AOX terminator, which can target integration into this locus.

The resulting expression plasmids pPM1aZ30_pG1-3_scR and pPM1aZ30_pG1-3_vHH were linearized with AscI and converted to CBS2612 (wt), CBS2612_KanR_dFLO8#2 (dFLO8), or CBS2612_L_dFLO8#2_4 (dFLO8L) by electroporation using the standard protocol described in Gasser et al., 2013.

Positive transformants were selected on selective YPD agar containing 50 μg ^mL⁻¹ Zeocin and 500 μg ^mL⁻¹ Genimycin (if applicable).

c) Screening for antibody fragment productivity

For expression screening, single colonies of the corresponding transformants were inoculated into 2 mL of liquid YP medium (per liter: 20 g peptone, 10 g yeast extract) containing 25 μg ^mL⁻¹ Zeocin and 500 μg ^mL⁻¹ Genimycin (if appropriate) and grown in 24-DWP at 280 rpm at 25°C for approximately 24 h. These cultures were then inoculated into 2 mL of synthetic screening medium ASMv6 (see Example 2 for composition) (this medium contains 50 g ^L⁻¹ polysaccharide and 1.5% glucose-releasing enzyme (to achieve a glucose release rate of approximately 0.8 mg ^{h⁻¹ mL⁻¹} ; m2p medium development kit) to an initial _OD₆₀ of 8. Culture conditions were similar to the pre-culture conditions. After 48 hours, 1 mL of the cell suspension was transferred to a pre-weighed 1.5 mL centrifuge tube and centrifuged at 16100 g for 5 min at room temperature. Carefully transfer the supernatant to a new vial and store at -20°C until further use. Weigh the centrifuge tube containing the precipitate again to determine the wet cell weight (WCW). Quantify the recombinant secreted proteins in the supernatant using microfluidic capillary electrophoresis as described below.

d) Quantitative analysis using microfluidic capillary electrophoresis (mCE)

The LabChip GX/GXII system (PerkinElmer) was used for the quantitative analysis of secreted protein titers in the culture supernatant. Consumable Protein Express Lab Chip (760499, PerkinElmer) and Protein Express Reagent Kit (CLS960008, PerkinElmer) were used. Chip and sample preparation were performed according to the manufacturer's recommendations. A brief description of the procedure is given below.

Chip Preparation: After the reagents had reached room temperature, 520 μL and 280 μL of protein expression gel matrix were transferred to rotary filters. 20 μL of protein expression dye solution was added to the rotary filter containing the 520 μL gel matrix. After briefly vortexing the dye-containing rotary filter in the opposite direction, both rotary filters were centrifuged at 9300 g for 10 minutes. To clean the chips, 120 μL of Milli-water was added to all active chip wells, and the chips were subjected to an instrument cleaning procedure. After two further rinsing steps with Milli-water, the remaining fluid was completely aspirated, and appropriate amounts of filtered gel matrix solution and protein expression lower marker solution were added to the appropriate chip wells.

Sample and Ladder Preparation: For sample preparation, 6 μL of sample was mixed with 21 μL of sample buffer in a 96-well microtiter plate. The sample was denatured at 100 °C for 5 min and centrifuged at 1200 g for 2 min. Subsequently, 150 μL of Milli-water was added. The sample solution was briefly mixed by pipetting and centrifuged again at 1200 g for 2 min before measurement. To prepare the ladder, 12 μL of protein expression ladder was denatured at 100 °C for 5 min in a PCR tube. Subsequently, 120 μL of Milli-water was added, and the ladder solution was briefly vortexed, followed by rotating the tube in a microcentrifuge for 15 seconds.

Quantification was performed using LabChip software provided by the manufacturer and compared with BSA standards.

Table 3 shows that, compared with the wild type, the average scR titer in the supernatant of strain dFLO8 was 1.8-fold higher, while the biomass concentration was not different, resulting in a 1.86-fold higher scR yield in the supernatant of strain dFLO8. A similar increase (1.7-fold) in average titer and vHH yield was also observed in strains expressing vHH (Table 4).

Table 3: Average WCW, product titer, and yield of 24-DWP screening of CBS2612 and CB2612_L_dFLO8#2_4 transformed with pPM1aZ30_pG1-3_scR. 20 clones were screened for each construct.

strains WCW±SD Titer ± SD Production ±SD <![CDATA[[g L^-1]]]> <![CDATA[[mg L^-1]]]> <![CDATA[[mg g^-1]]]> wt+pG1-3_scR#1-20 80.8±3.64 29.3±8.99 0.36±0.107 dFLO8L+pG1-3_scR#1-20 79.3±2.40 53.0±4.53 0.67±0.057

Table 4: Average WCW, product titer, and yield of 24-DWP screening of CBS2612 and CB2612_KanR_dFLO8#2 transformed with pPM1aZ30_pG1-3_vHH. 20 clones were screened for each construct.

strains WCW±SD Titer ± SD Production ±SD <![CDATA[[g L^-1]]]> <![CDATA[[mg L^-1]]]> <![CDATA[[mg g^-1]]]> wt+pG1-3_vHH#1-20 92.4±3.21 56.2±32.70 0.61±0.351 dFLO8+pG1-3_vHH#1-20 90.7±4.48 97.0±31.70 1.04±0.360

Next, FLO8 was disrupted in four different vHH-expressing clones (CBS2612_pG1-3_vHH#4, #5, #13, and #15) selected from the above screening using the split marker cassette method as described in Example 1. Subsequently, the productivity of the dFLO8 mutant clones and the corresponding FLO8 parent clones was screened using the 24-DWP screening protocol. Table 5 shows that vHH titers and product yields were 2 to 3 times higher in the dFLO8 clones. To verify that the increased yield levels were based on higher transcriptional expression, vHH transcript levels were quantified by qPCR at different time points. An average of 2-fold higher vHH expression levels was observed in the dFLO8 clones, indicating that the increased vHH titers were indeed based on the higher transcriptional activity of pG1-3 (see Example 4).

Table 5: Mean WCW and titers of four different vHH-expressing strains and their corresponding dFLO8 strains. Four copies of each parental strain and six corresponding dFLO8 strains were screened. (FC: fold change).

strains WCW±SD Titer ± SD FC <![CDATA[[g L^-1]]]> <![CDATA[[mg L^-1]]]> pG1-3_vHH#4 81.8±1.25 81.4±6.42 pG1-3_vHH#4_dFLO8#1-6 78.9±0.94 171.9±3.90 2.11 pG1-3_vHH#5 86.6±1.23 37.9±2.22 pG1-3_vHH#5_dFLO8#1-6 79.8±1.51 91.2±4.17 2.41 pG1-3_vHH#13 77.6±1.81 33.6±1.30 pG1-3_vHH#13_dFLO8#1-6 78.9±1.52 101.7±2.05 3.03 pG1-3_vHH#15 80.1±1.23 37.7±2.05 pG1-3_vHH#15_dFLO8#1-6 78.7±3.70 101.1±4.21 2.68

Example 4: Effects of FLO8 disruption on pG1-3-controlled vHH transcription

To test whether FLO8 disruption leads to increased transcriptional activity of genes under pG1-3 control, six technical replicas of CBS2612_pG1-3_vHH#4 and #13, and the corresponding dFLO8 mutant strains CBS2612_pG1-3_vHH#4_dFLO8_1 and #13_dFLO8_2 (Table 5; Example 3) were cultured in 24-DWP format as described in Example 3. At 2, 19, and 26 hours, 1 mL of culture from two replicas was harvested, centrifuged at 16100 g and 4°C for 1 minute, and the supernatant was discarded. The cell pellet was then resuspended in 1 mL of TRI reagent (Sigma-Aldrich) and stored at -80°C until further processing.

RNA isolation was performed as described in Example 6. To remove residual DNA, the RNA samples were treated using a DNA-free ^™ kit (Ambion) according to the manufacturer's instructions. Subsequently, the quality, purity, and concentration of the RNA were analyzed by gel electrophoresis and spectrophotometry using a NanoDrop 2000 (Thermo Fisher Scientific).

cDNA synthesis was performed using the Biozym cDNA Synthesis Kit, following the manufacturer's instructions. In short, 500 ng of total RNA was added to the master mixture containing reverse transcriptase, dNTPs, an RNase inhibitor, and synthesis buffer. VN(NEB) was used to initiate oligomeric d(T) ₂₃ . The reaction mixture was incubated at 55°C for 60 minutes. Enzyme inactivation was then achieved by incubating the reaction mixture at 99°C for 5 minutes.

For quantitative real-time PCR (qPCR), vHH-specific primers were used (Table 6). Normalization was performed by comparison with ACT1 expression levels (Table 6). To analyze 1 μL of cDNA, water and primers were mixed with SensiMix SYBR 2x Master Mix (Bioline) and analyzed in a real-time PCR cycler (Rotor-Gene, Qiagen).

Table 6: Primers for quantitative real-time PCR used for vHH transcript analysis

Primers sequence vHH_fw TGTAACGTGAATGTCGGATTTG(SEQ ID NO:81) vHH_bw TAGTGATGGTGGTGGTGATG(SEQ ID NO:82)10 Act1_fw CCTGAGGCTTTGTTCCACCCACT(SEQ ID NO:83) Act1_bw GGAACATAGTAGCAC CGGCATAACGA(SEQ ID NO:84)

All samples were measured in triplicate. Data analysis was performed using Rotor-Gene software and a comparative quantitation (QC) method.

Table 7 shows that in the dFLO8 strain, the mean vHH-transcriptosome level increased during all culture time points analyzed.

Table 7: Mean relative vHH transcript levels at different screening culture time points

Example 5: Effect of FLO8 disruption on pG1-3-driven antibody fragment production in fed-batch culture in a laboratory-scale bioreactor

Prior to fed-batch culture, as described in Example 3, a genetic mycotoxin resistance marker cassette was excised from the genome of strain CBS2612_pG1-3_vHH#4_dFLO8_4 (abbreviated as dFLO8_pG1-3_vHH#4_4) (Table 5, Example 3) via Cre-mediated recombination. The productivity of three copies of the resulting strain CBS2612_pG1-3_vHH#4_dFLO8_4#L1 (abbreviated as L_dFLO8_pG1-3_vHH#4_4_1) was compared with the product levels of three copies of its parent strain using the 24-DPW screening format described in Example 3 (Table 8). The average productivity of CBS2612_pG1-3_vHH#4_dFLO8_4#L1 remained similar to that of its parent strain (p = 0.096).

Table 8: WCW and titers of CBS2612_dFLO8_pG1-3_vHH#4_4 and CBS2612_L_dFLO8_pG1-3_vHH#4_4_1 in 24-DWP screening.

strains WCW±SD Titer ± SD <![CDATA[[g L^-1]]]> <![CDATA[[mg L^-1]]]> dFLO8_pG1-3_vHH#4_4 80.87±1.91 306.34±12.85 L_dFLO8_pG1-3_vHH#4_4_1 80.40±0.57 329.92±8.48

Fed-batch cultures were performed in a 1L benchtop bioreactor (SR0700ODLS; Dasgip, Germany) using strain CBS2612_pG1-3_vHH#4 and the corresponding loxed dFLO8 mutant CBS2612_L_dFLO8_pG1-3_vHH#4_4_1. For the pre-culture, 100 mL of YPG medium containing 50 μg/mL Zeocin was inoculated in a 1L shake flask with 1.0 mL of cryogenic stock solution and incubated at 180 rpm and 25°C for approximately 24 hours. Batch cultures were operated at a working volume of 0.25 L and inoculated to an initial _OD600 of 1.5. The composition of the glycerol batch culture medium is given below. Throughout the process, the temperature was maintained at 30°C, DO was kept at 30% by automatically adjusting the stirrer speed (between 400 rpm and 1200 rpm) and air flow rate (between 9.5 s L ^h⁻¹ and 30 s L ^h⁻¹ ), and the pH was adjusted to 5.0 by automatically adding 12.5% _NH₄OH . Following a sudden increase in DO indicating the end of the batch, linear incremental glucose feed (culture medium composition detailed below) was used, resulting in a rapid initial growth rate, followed by a gradually decreasing μ extension phase, which had been specifically optimized for qP to μ kinetics based on _PG1-3 (Prielhofer et al., 2018).

Each liter of glycerol batch medium contains:

2 _g of citric _acid monohydrate ( _C6H8O7 * _H2O ), 45 g of glycerol, 12.6 g of ( _NH4 ) _2HPO4 , 0.5 _g of ^MgSO4 * _7H2O , 0.9 g of KCl, 0.022 g of CaCl2* _2H2O , _6.6 mL of biotin stock solution (0.2 g ^L⁻¹ ), and 4.6 mL of PTMO trace salt stock solution (as described in Example 2). Concentrated HCl was added to set the pH to 5.

Each liter of glucose feed medium contains:

495 g of glucose monohydrate, 5.2 g of _MgSO₄ · _7H₂O , 8.4 g of KCl, 0.28 g of CaCl₂ _{· 2H₂O} , 11.8 mL of biotin stock solution (0.2 g ^L⁻¹ ), and 10.1 mL of PTMO trace salt stock solution (as described in Example 2).

YDM and secreted recombinant proteins were analyzed at different time points throughout the process (Table 9). For YDM analysis, 1 mL of culture medium was transferred to 2 mL of pre-dried (held at 105 °C for at least 24 hours) and pre-weighed centrifuge tubes. After centrifugation at 16100 g for 5 minutes, the supernatant was carefully transferred to fresh vials and stored at -20 °C until further use. The cell pellet was washed twice with deionized water and dried at 105 °C for at least 24 hours, then weighed again.

As described in Example 3, the supernatant was analyzed by microfluidic capillary electrophoresis (GXII, Perkin-Elmer).

Table 9: YDM and vHH titers during fed-batch culture of CBS2612_pG1-3_vHH#4 and L_CBS2612_dFLO8_pG1-3_vHH#4_4_1 in the bioreactor

*Glucose - Feed Start

As shown in Table 9, the product titer of strain dFLO8 was consistently higher than that of the wt-background strain throughout the entire process. A 5.7-fold increase in productivity was observed at the end of the process. Similarly, in fed-batch cultures, increased vHH transcript levels were observed in strain dFLO8 throughout the entire time period.

Example 6: Effects of FLO8 disruption on expression intensity and regulatory behavior of non-methanol carbon-regulated promoters

In the next step, the effects of FLO8 disruption under induction conditions on the transcriptional intensity of other carbon-regulated promoters (described in WO2013050551A1 and Prielhofer et al., 2013) were investigated. For transcriptome analysis under glucose restriction, CBS7435 wild-type and CBS7436_KanR_dFLO8#2 were used. Precultures and master cultures were cultured in 24-well plates (24-DWP). For the first preculture, single colonies of CBS7435 and CBS7435_KanR_dFLO8#2 were inoculated into 2 mL of YPD (per liter: 10 g yeast extract, 20 g peptone, 20 g glucose, if appropriate) containing 500 μg ^mL⁻¹ genimycin and incubated at 25°C and 280 rpm for approximately 24 hours. For the second pre-culture, 2 mL of M2(D) medium (composition as described below) was inoculated to an initial OD ₆₀₀ of 4. Glucose-releasing polymer beads (12 mm feed beads, Kuhner, CH) were added, releasing glucose at a non-linear rate of 1.63t ^0.74 mg/bead (t = time [h]), and the culture was incubated at 25°C and 280 rpm for approximately 24 h. For the master culture, 2 mL of M2 medium (M2(D), glucose-free) was used. The culture was shaken at 280 rpm and 25°C. The slow release of glucose ensured limited glucose growth. Samples were collected 3 hours after master culture (estimated specific growth rate: 0.1 ^h⁻¹ ), immediately mixed with pre-chilled fixative (5% [v/v] phenol in ethanol [absolute]), aliquoted into sealed tubes, and centrifuged at 16100 g for 1 min. The precipitate was stored at -80°C until further use.

M2(D) per liter contains: 22.0 g glucose monohydrate, 22.0 g citric acid monohydrate, 3.15 g ( _{NH4 )2HPO4 ,} 0.49 g _MgSO4 · _7H2O , 0.80 g KCl, 0.0268 g CaCl2· _2H2O , 1.47 _mL PTM0 trace salt stock solution (as described in Example 1), and 0.4 mg biotin; pH is set to 5 using KOH (solid).

For RNA isolation, add 1 mL of TRI reagent (Sigma-Aldrich) and 500 μL of acid-washed glass beads, and lyse cells for 40 seconds at 5.5 m/s in FastPrep-24 (mpbio). Then, add 200 μL of chloroform. Next, shake the sample vigorously and allow it to stand at room temperature for 5–10 minutes. After centrifuging at 16100 g and 4 °C for 10 minutes to promote phase separation, transfer the colorless aqueous phase containing RNA to a new tube and add 500 μL of isopropanol to precipitate the RNA. After incubation for 10 minutes, centrifuge the sample at 16100 g and 4 °C for 10 minutes and discard the supernatant. Wash the RNA precipitate once with 70% ethanol, air dry, and resuspend in RNase-free water.

For transcriptome analysis, a custom-designed Pichia pastoris-specific oligonucleotide array (AMAD-ID034821, 8×15K; Agilent Technologies, USA) was used (Graf et al.; BMCGenomics 2008; 9:390.). cRNA synthesis, hybridization, and scanning were performed according to the Agilent protocol for 2-color expression arrays. Samples were triple-labeled with Cy3 and Cy5 and hybridized to a reference pool generated from cells grown under various culture conditions. Dye exchange experiments were performed on all samples.

The normalization steps and statistical analysis of the microarray data included eliminating color bias using locally weighted MA-scatter plot smoothing (LOESS) followed by inter-array normalization using the Aquantile method. To identify differentially expressed genes and calculate p-values, a linear model with eBayes correction was fitted. Benjamini & Yekutieli, 2001, adjusted p-values for multiple measurements using the false discovery method (FDR). Genes with adjusted p-values < 0.05 were considered statistically significant for differential expression. A fold change cutoff of at least 0.58 > log2 FC < -0.58 was also applied to identify differentially expressed genes.

All steps were performed using R software (Robinson MD, McCarthy DJ, Smyth GK. edgeR: "A Bioconductor package for differential expression analysis of digital gene expression data" 2010. Bioinformatics 26:139–40.) and the limma package. The fold changes in selection of carbon-regulated genes (genes controlled by promoters regulated by carbon sources) are shown in Table 10. Expression of the native FLO8 gene was significantly and substantially reduced in the dFLO8 mutant compared to the wild type. For all carbon-regulated genes, increased transcript levels were observed in the dFLO8 mutant, with transcriptional intensity reaching 3.7 to 11.4-fold higher under induction conditions. All of these genes showed statistically significant higher transcription levels in the dFLO8 mutant strain (adjusted p-value < 0.05). This strongly suggests that disruption of FLO8 enhances the production potential of all non-methanol carbon-regulated promoters as described in Prielhofer et al., 2013 and Prielhofer et al., 2018. Conversely, the expression intensity of the GAP promoter is unaffected by FLO8 disruption.

Table 10: Effects of the dFLO8 mutant on the transcriptional strength of the FLO8 gene and genes controlled by carbon-regulated promoters under glucose restriction induction. Fold change (FC) is shown between the dFLO8 mutant strain and the wild-type strain. Expression changes with adjusted p < 0.05 are considered statistically significant.

Example 7: Effect of FLO8 disruption on transcriptional strength of the methanol-regulated promoter pAOX1

The effect of FLO8 disruption on transcriptional strength was also determined for standard promoters of Pichia pastoris (such as pAOX1 and pGAP) under methanol-induced conditions. Therefore, strain CBS7435, expressing the recombinant Fab fragment under pAOX1 control, and the corresponding dFLO8 mutant strain dFLO8#2 were cultured in methanol-based fed-batch cultures.

Feed-in batches were carried out in a 1.4L DASGIP reactor (Eppendorf, Germany), with a maximum working volume of 1.0L. The incubation temperature was controlled at 25°C, the pH was controlled at 5.0 by adding 25% ammonium hydroxide, and the dissolved oxygen concentration was maintained above 20% saturation by controlling the stirrer speed between 400-1200 rpm and the gas flow rate between 24-72 sL/h.

Inoculum used for fed-batch culture was cultured in shake flasks containing 100 mL of YP medium containing 20 g/L glycerol and 50 μg/mL Zeocin, and incubated at 28 °C and 180 rpm for approximately 24 hours. The culture was used to inoculate a starting volume of 0.4 L into the bioreactor to an initial optical density of 1.0 (600 nm). The batch was completed after approximately 24 hours, and the first (10 mL) salt pellet was provided.

The glycerol feed solution was then fed at a constant rate of 5 mL/h for 5 hours. A methanol pulse (2 g) and salt pellets (10 mL) were then introduced into the culture. After the methanol pulse consumption was indicated by an increase in dissolved oxygen concentration in the culture, a constant feed with a methanol feed solution was initiated at a rate of 1.0 g/h. 10 mL of salt pellets were provided for every 10 g of newly formed biomass, which is equivalent to approximately 43 g of methanol feed medium. As the biomass concentration increased, the methanol feed rate was appropriately increased due to the sudden increase in dissolved oxygen in the culture when the methanol feed was shut off for a short period, once methanol accumulation could be eliminated. The final methanol feed rate was 2.5 g/h.

Samples were collected frequently. The culture was harvested approximately 100 hours later, when the cell density had reached more than 100 g/L of cell dry weight.

The culture medium is as follows:

Each batch culture medium (per liter) contains: 2.0 g citric acid, 12.4 g ( _NH₄ ) _₂HPO₄ , 0.022 g _CaCl₂ · _2H₂O , 0.9 g KCl, 0.5 g _MgSO₄ · _7H₂O , 40 g glycerol, _and 4.6 mL PTM1 microsalt stock solution. The pH is set to 5.0 with 25% HCl.

The glycerol feed batch solution (per liter) contains: 623 g of glycerol, 12 mL of PTM0 microsalt stock solution, and 40 mg of biotin. The composition of PTM0 is given in Example 2.

The methanol feed batch solution (per liter) of pure methanol contains: 12 mL of PTM0 microsalt stock solution and 40 mg of biotin.

Each salt tablet solution contains: 20.8 g of _MgSO₄ · _7H₂O , 41.6 g of KCl, and 1.04 g of _CaCl₂ · _2H₂O .

Intact Fab was quantified by ELISA using an anti-human IgG antibody (ab7497, Abcam) as the coating antibody and a goat anti-human IgG (Fab-specific)-alkaline phosphatase conjugated antibody (Sigma A8542) as the detection antibody. Human Fab/κIgG fragment (Bethyl P80-115) was used as a standard at an initial concentration of 100 ng/mL, and the supernatant sample was diluted accordingly. Detection was performed using pNPP (Sigma S0942). Coating buffer, dilution buffer, and washing buffer were prepared based on PBS ( ₂ mM _KH₂PO₄ , ₁₀ mM _Na₂HPO₄ · _2H₂O , 2.7 mM g KCl, 8 mM NaCl, pH 7.4) and correspondingly with BSA (1% (w/v)) and/or Tween 20 (0.1% (v/v)).

Regarding product titers, the increase in Fab yield under pAOX1 control in the dFLO8 mutant was up to 1.45-fold at the end of the methanol fed batch culture (after 119 hours, as described in WO2015/158800A1), while biomass formation was slightly reduced (by 10-14%).

Microarray samples were collected after 53.5 hours (25 hours methanol feed) and processed as described in Example 6. The fold change in gene selection between the dFLO8 mutant strain and the wild-type strain is shown in Table 11. Similarly, FLO8 transcript levels were significantly lower in the dFLO8 mutant compared to the wild-type. In contrast to the non-methanol-carbon-regulated genes and promoters shown in Table 10, methanol-inducible pAOX1 did not show a significant increase in transcriptional intensity in the dFLO8 mutant under its fully inducible condition compared to the wild-type (because the regulated p-value was greater than 0.05). There was also no significant effect on pGAP in methanol-cultured cells. Therefore, transcription of these standard promoters in Pichia pastoris was not affected by low FLO8 expression.

Table 11: Effects of the dFLO8 mutant on the transcriptional intensity of the FLO8 gene and genes controlled by the methanol-induced fed-batch model under methanol-induced conditions. Fold change (FC) is shown between the dFLO8 mutant strain and the wild-type strain. Expression changes with adjusted p < 0.05 are considered statistically significant.

Example 8: Effect of FLO8 disruption on intracellular eGFP expression driven by a promoter not regulated by methanol carbon

Since native gene expression driven by most non-methanol-carbon-regulated promoters (WO2013050551A1; Prielhofer et al., 2013) is significantly upregulated under induction conditions in dFLO8 strains (see Example 6), FLO8 disruption was also detected in the corresponding eGFP-reporter strains described in WO2013050551A1 and Prielhofer et al. (2013). For each non-methanol-inducible promoter, strains carrying the pPM1aZ10_pG#_eGFP expression vector (CBS2612_pG3_eGFP#1; CBS2612_pG4_eGFP#6; CBS2612_pG6_eGFP#53; X33_pG7_eGFP#1; CBS2612_pG8_eGFP#8) were selected, and FLO8 was disrupted using the split marker cassette method described in Example 1. Screening-culturing under induced (glucose-restricted) conditions was performed as described in Example 2, except that the polysaccharide and glucose-releasing enzyme were obtained from different suppliers (EnPump 200, Enpresso). Due to the different properties of the new glucose-releasing enzyme, the concentration was adjusted to 0.4%, corresponding to a constant glucose release rate of approximately 0.6 mg ^{mL⁻¹ h⁻¹} . In each case, two copies of the corresponding parent and at least seven corresponding dFLO8 strains were screened. eGFP productivity was determined as described in Example 2, except that the values were not normalized for cell size. Table 12 shows that disruption of FLO8 resulted in an increase in eGFP fluorescence for each promoter, reaching 1.7-fold and 1.2-fold in pG3 and pG8, respectively, and 2.3-fold and 2.2-fold in pG4 and pG7, respectively. This further highlights the potential of FLO8 disruption to enhance other non-methanol-carbon-regulated promoters besides pG1 and pG1 derivatives.

These results confirm that eGFP expression controlled by pG1 or pG1 derivatives (pG1-3) is increased in dFLO8 host cells compared to wild-type host cells. Table 2 shows that disruption of FLO8 leads to an increase in eGFP fluorescence, which is 3.8 (or 3.9) times in the pG1 case and 2.8 (or 2.9) times in the pG1 case and the pG1-3 case, respectively.

Table 12: Effect of dFLO8 on eGFP expression under the control of a non-methanol carbon-regulated promoter. The table shows the eGFP expression level (as a percentage of pGAP expression) and the fold change of the corresponding dFLO8_pG#_eGFP strain compared to the parental wild-type strain after 48 hours of culture under glucose-restricted (inducible) conditions.

<![CDATA[<u>starter</u>]]> <![CDATA[<u>host cell</u>]]> <![CDATA[<u>eGFP expression relative to pGAP±SD</u>]]> <![CDATA[<u>FC dFLO8/wt</u>]]> pGAP wt 100±2.2% pG3 wt 20±0.7% pG3 dFLO8 33±4.5% 1.7 pG4 wt 42±1.1% pG4 dFLO8 96±9.0% 2.3 pG6 wt 76±0.0% pG6 dFLO8 164±1.4% 2.2 pG7 wt 697±1.0% pG7 dFLO8 1591±2% 2.3 pG8 wt 20±3.1% pG8 dFLO8 24±3.4% 1.2

See references:

Benjamini Y & Yekutieli D (2001) “The Control of the False Discovery Rate in Multiple Testing under Dependency”, The Annals of Statistics 29:1165-1188.

Gasser B, Prielhofer R, Marx H, Maurer M, Nocon J, Steiger M, Puxbaum V, Sauer M & Mattanovich D (2013) “Pichia pastoris: protein production host and model organism for biomedical research” Future Microbiol 8:191-208.

Marx H, Mattanovich D & Sauer M (2008) “Overexpression of the riboflavin biosynthetic pathway in Pichiapastoris” Microb Cell Fact 7:23.

Prielhofer R, Reichinger M, Wagner N, Claes K, Kiziak C, Gasser B & Mattanovich D (2018) "Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose-regulated GTH1 promoter of Pichia pastoris" Biotechnol Bioeng.

Stadlmayr G, Mecklenbrauker A, Rothmuller M, Maurer M, Sauer M, Mattanovich D & Gasser B (2010) “Identification and characterization of novel Pichia pastoris promoters for heterologous protein production” J Biotechnol 150:519-529.

Claims (15)

1. A recombinant host cell comprising an endogenous gene encoding an FLO8 protein, said FLO8 protein comprising an amino acid sequence identified as SEQ ID NO: 1 or a homolog thereof, said host cell being engineered by one or more gene modifications to reduce the expression of said gene compared to a host cell prior to said one or more gene modifications, and said host cell comprising a heterologous expression cassette comprising a target gene (GOI) controlled by an expression cassette promoter (ECP), said ECP being repressible by a non-methanol carbon source. 2. The host cell, wherein the endogenous polynucleotide is a gene encoding the FLO8 protein, wherein the one or more gene modifications include (i) one or more endogenous polynucleotides or a portion thereof; or (ii) disruption, substitution, deletion or knockout of an expression control sequence, preferably wherein the expression control sequence is selected from the group consisting of promoters, ribosome binding sites, transcription or translation initiation and termination sequences, enhancer and activator sequences. 3. The host cell according to claim 1 or 2, wherein the endogenous gene or homolog encoding the FLO8 protein is knocked out by one or more gene modifications. 4. The host cell according to any one of claims 1 to 3, wherein the ECP is induced in the presence of a growth-limiting amount of a non-methanol carbon source, preferably in the absence of methanol; and is inhibited in the presence of an excess of a non-methanol carbon source above the growth-limiting amount. 5. The host cell according to any one of claims 1 to 4, wherein the ECP comprises at least 60% sequence identity of at least 300 nt to any one of sequences SEQ ID NO:10-16 or any one of SEQ ID NO:41-45. 6. The host cell according to claim 5, wherein the ECP comprises or is composed of SEQ ID NO:10 or SEQ ID NO:11. 7. The host cell according to any one of claims 1 to 6, wherein the expression cassette further comprises a nucleotide sequence encoding a signal peptide capable of secreting a target protein (POI) encoded by the GOI, preferably wherein the nucleotide sequence encoding the signal peptide is fused adjacent to the 5' end of the GOI. 8. The host cell according to any one of claims 1 to 7, wherein the GOI encodes a target protein (POI), and the target protein is a peptide or protein selected from the group consisting of antigen-binding proteins, therapeutic proteins, enzymes, peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccine antigens, growth factors, hormones, cytokines, process enzymes, and metabolic enzymes. 9. The host cell according to any one of claims 1 to 8, wherein: a) Yeast cells selected from genera belonging to the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogataea, Yarrowia, and Geotrichum, such as Pichia pastoris, Komagataella phaffii, and Komagataella phaffii. Komagataella pastoris), Komagata ellapseudopastoris, Saccharomyces cerevisiae, Ogataeaminuta, Kluyveromces lactis, Kluyveromes marxianus, Yarrowia lipolytica, or Hansenula polymorpha; or b) Cells of filamentous fungi such as Aspergillus awamori or Trichoderma reesei. 10. A method for increasing the production of a target protein (POI) by reducing the expression of a gene encoding the FLO8 protein in a host cell, the target protein (POI) being produced by a host cell expressing a target gene (GOI) encoding the POI under the control of a promoter that can be repressed by a non-methanol carbon source, the FLO8 protein comprising an amino acid sequence identified as SEQ ID NO:1 or a homolog thereof. 11. A method for producing the POI encoded by the target gene (GOI) by culturing the host cell of any one of claims 1 to 9 under conditions that produce the target protein (POI). 12. The method according to claim 10 or 11, comprising the following steps: a) Culture the host cells under growth conditions; and b) The host cells are cultured under growth-restricted conditions in the presence of a second non-methanol carbon source of up to 1 g/L to induce the expression of the GOI to produce the POI, preferably wherein step a) is performed in a batch culture phase; and step b) is performed in a fed-batch or continuous culture phase. 13. The method of claim 12, wherein the first carbon source or the second carbon source is selected from sugars, polyols, alcohols, or any one or more mixtures thereof. 14. A method for producing a target protein (POI) in a host cell, comprising the following steps: a) Genetically engineer the host cells to reduce the expression of the endogenous gene encoding the FLO8 protein, wherein the FLO8 protein comprises the amino acid sequence identified as SEQ ID NO:1 or its homolog; b) Introducing a heterologous expression cassette into the host cell, the heterologous expression cassette comprising the expression of the target gene (GOI) of the POI under the control of an expression cassette promoter (ECP), the expression cassette promoter being operatively linked to the GOI, the ECP being repressible by a non-methanol carbon source; c) Culture the host cells under conditions that generate the POI; d) Optionally isolate the POI from the cell culture; and e) Optionally purify the POI. 15. The method of claim 14, wherein the ECP comprises at least 60% sequence identity of at least 300 nt to any one of sequences SEQ ID NO:10-16 or any one of SEQ ID NO:41-45.