EA046260B1 - OBTAINING OGM - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the field of recombinant production of biological molecules in host cells. More specifically, it relates to a method for recombinantly producing human milk oligosaccharides (HMOs) using genetically modified cells expressing the basic messenger superfamily (MFS) protein.

State of the art

Human milk oligosaccharides (HMOs) constitute the third largest solid component of human milk and are highly resistant to enzymatic hydrolysis. As a consequence, a significant portion of the HMOs remains largely undigested and unabsorbed, allowing them to penetrate into the colon. In the colon, HMOs can serve as substrates for the formation of the intestinal ecosystem by selectively stimulating the growth of specific saccharolytic bacteria. This selectivity is thought to be beneficial for both infants and adults, as strains of Bifidobacterium species are thought to have positive effects on gut health. (Chichiowski M. et al. (2012) J. Pediatr. Gastroenterol. Nutr. 5:251-258; Elison E. et al. (2016) Brit J. Nutr, 116: 1356-1368).

In addition to their prebiotic properties, HMOs are associated with additional beneficial effects, which expands the scope of their application. (Kunz S. et al. (2014) Food Oligosaccharides: Production, Analysis and Bioactivity, 1st Edition, p 5-20, Eds. Moreno J. and Luz Sanz M., John Wiley & Sons, Ltd).

The obvious health benefits of HGMs have led to approval of their use in food products such as infant formula and food products, as well as in consumer health products. Biotechnological production of OGM represents a valuable, cost-effective and large-scale method for the production of OGM. It is based on genetically modified bacteria engineered to express the glycosyltransferases required to synthesize the desired oligosaccharides, and utilizes the bacteria's innate pool of nucleotide sugars as OGM precursors. Recent developments in the biotechnological production of HGM have overcome some of the inherent limitations of bacterial expression systems. For example, bacterial cells that produce OGM can be genetically modified to increase the limited intracellular pool of nucleotide sugars in bacteria (WO 2012112777), to increase the activity of enzymes involved in the production of OGM (WO 2016040531), or to facilitate the secretion of synthesized OGM into the extracellular environment (WO 2012112777). WO 2010142305, WO 2017042382). In addition, the expression of genes of interest in recombinant cells can be regulated by specific promoters or other gene expression regulators, such as those recently described in WO 2019123324.

The approach described in WO 2010142305 and WO 2017042382 has the advantage that it reduces the metabolic burden on the producing cell due to high levels of recombinant gene expression, i.e. using the methods of WO 2012112777, WO 2016040531 or WO 2019123324. This approach is attracting increasing attention to recombinant cells producing HGM, for example, fermentation procedures have recently been described, as well as several new sugar transporter genes encoding proteins that can facilitate the clearance of recombinantly produced 2'-fucosyllactose (2'-FL), the most common HGM in human milk (WO 2018077892, US 201900323053, US 201900323052). However, there is currently no algorithm that can accurately identify the correct transporter protein for the efflux of various recombinantly produced OGM structures among the numerous bacterial proteins with predicted transporter function in several protein databases, such as UniProt, since the structures/factors , which determine the substrate specificity of sugar transporters, are still insufficiently studied and remain extremely unpredictable.

Disclosure of the Invention

Efficient sugar efflux transporter proteins for various recombinantly produced OGMs and the development of recombinant cells expressing these proteins are advantageous for large-scale industrial production of OHMs.

The present invention relates to recombinant cells capable of producing human milk oligosaccharide (HMO), wherein the cells express a heterologous gene encoding a putative MFS (major facilitator superfamily) transport protein derived from the bacterium Pantoea vagans. More specifically, the invention relates to a genetically modified cell optimized for the production of oligosaccharides, especially OGM, containing a recombinant nucleic acid encoding a protein whose sequence is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 (Fig. 4). The amino acid sequence designated herein as SEQ ID NO: 1 is an amino acid sequence that is 100% identical to the amino acid sequence having GenBank accession number IDWP_048785139.1 (https://www.ncbi.nlm.nih.goV/ protein/WP_048785139.1). The MFS transporter protein having the amino acid sequence of SEQ ID NO: 1 is identified herein as Vag protein, or

- 1 046260 Vag conveyor, or Vag, interchangeable; a nucleic acid sequence encoding a Vag protein is identified herein as a vag encoding nucleic acid/DNA, or vag gene, or vag.

The present invention shows that the use of recombinant OGM-producing cells that express the Vag protein leads to very distinct improvements in the OGM production process, both in terms of OGM fermentation and purification. The recombinant cells and methods for producing HMOs described herein provide both higher yields of total HMOs produced, lower byproduct formation or byproduct to product ratio, lower biomass production per fermentation, and easier recovery of HMOs during downstream processing of the fermentation broth.

Surprisingly, it was found that the expression of the DNA sequence encoding Vag in various OGM-producing cells is associated with the accumulation of some OGMs in the extracellular environment and the accumulation of other OGMs within the producing cells, as well as with an increase in the overall production of OGMs. Unexpectedly, it turned out that an increase in the yield of the resulting OHMs is characteristic of OHMs consisting of either three or four monosaccharide units, i.e. 2'-fucosyllactose (2TL), 3-fucosyllactose (3FL), 3-sialyllactose (3'SL), 6'-sialyllactose (6'SL), lacto-H-triose 2, (LNT-2), lacto-H -neotetraose (LNnT) and lacto-H-tetraose (LNT), but not for larger oligosaccharide structures such as pentasaccharides and hexasaccharides, which accumulate inside producing cells. Surprisingly, it was also found that the total production of OGM lacto-H-neotetraose (LNnT) and lacto-H-tetraose (LNT) by the corresponding OGM-producing Vag-expressing cells is also increased, while the formation of by-products, e.g. -H-neohexaoses (pLNnH) and para-lactoH-hexaoses II (LNH-II) are, respectively, often reduced in these cells, and these by-product oligosaccharides usually accumulate inside the cells in the OGM production system. Further, quite unexpectedly, it was found that the expression of Vag protein in HGM-producing cells leads to reduced biomass production during high cell density fermentations and healthier cell cultures, which is reflected, for example, in a decrease in the number of dead cells at the end of fermentation. making the production process more efficient as more product is produced per unit of biomass.

Accordingly, the first aspect of the invention relates to a genetically modified cell capable of producing one or more HGMs, wherein said cell includes a recombinant nucleic acid encoding a protein of SEQ ID HO: 1, or a functional homolog thereof with an amino acid sequence identical to at least 80 %, preferably at least 85% identical, more preferably at least 90% identical to the sequence of SEQ ID BUT: 1.

A second aspect of the invention relates to a nucleic acid construct comprising nucleic acid sequence(s) encoding an MFS transporter protein, wherein the nucleic acid sequence encoding the protein has at least 70% identity to the sequence of SEQ ID BUT: 2. for example at least 80%, for example at least 85%, for example at least 95%, for example at least 99%, and also to a genetically modified cell containing a nucleic acid construct that is Escherichia coli.

In one aspect, the nucleic acid construct includes nucleic acid sequence(s) encoding an MFS transporter protein, wherein the nucleic acid sequence is at least 70% identical to SEQ ID HO: 2.

The third aspect of the invention relates to a method for producing one or more oligosaccharides, comprising the following steps:

(i) obtaining a genetically modified cell capable of producing HGM, wherein said cell includes a recombinant nucleic acid encoding a protein of SEQ ID HO: 1, or a functional homologue thereof with an amino acid sequence that is at least 80% identical, preferably at least identical 85%, more preferably at least 90% identical to the sequence of SEQ ID HO: 1;

(ii) culturing cells in accordance with (i) in a suitable cell culture medium to express said recombinant nucleic acid;

(iii) collecting one or more OGM obtained in step (ii).

The invention also relates to the use of a genetically modified cell or nucleic acid construct comprising a heterologous nucleic acid sequence encoding a major messenger superfamily (MFS) protein, said nucleic acid sequence having at least 70% sequence identity to SEQ ID BUT: 2 for the production of one or several human milk oligosaccharides (HMOs).

As mentioned above, when cultivating genetically modified cells capable of producing one or more OGMs and containing a nucleic acid encoding the Vag transporter protein, it was unexpectedly discovered that the corresponding one or more OGMs produced

- 2 046260 are produced in high yields, while the formation of by-products and biomass is reduced. This makes it easier to recover these HMOs in downstream processes because the overall recovery and purification procedure involves fewer steps, the overall procedure is simplified, the overall purification time is reduced, and hence product recovery becomes less labor intensive and more cost effective.

These effects of increased product yield and easier product recovery make the present invention superior to the disclosures of the prior art.

Other aspects and useful features of the present invention are described in detail and illustrated by the following non-limiting worked examples.

Brief description of drawings

Fig. 1 shows (A) the percentage (%) of relative LNnT concentrations in total samples for seven strains that express the GlcNacT and Gal4T genes and a heterologous transporter gene, yberC0001_9420, bad, fred, marc, vag, dog, or yabM, respectively. Strain MP3672 expresses glycosyltransferase genes and does not express transporter genes; (B) Percentage (%) of relative concentrations of LNnT in the supernatant of cells expressing vag or yabM.

In fig. 2 shows the percentage (%) of relative concentrations of LNnT and byproducts for strains MP3020, MP4065 and MP4064 in the total sample, supernatant samples and sediment samples. Although all three strains demonstrate optimal expression of the essential glycosyltransferase genes, it is MP4065 and MP4064 that express the heterologous vag transporter gene under the control of the Plac or PglpF element, respectively.

Fig. 3 shows the percentage (%) of relative concentrations of LNT and byproducts for strains MP4473 and MP4546 in the total, supernatant and sediment samples. Although both strains demonstrate optimal expression of the essential glycosyltransferase genes, only MP4546 expresses the heterologous vag transporter gene under the control of the PglpF element.

In fig. 4 shows the sequence SEQ ID NO: 1.

Carrying out the invention

In the following, embodiments of the invention will be described in more detail. Each specific feature variation may be applied to other embodiments of the invention unless specifically stated otherwise.

In general, all terms used herein should be interpreted in accordance with their ordinary meaning in the technical field and as applicable to all aspects and embodiments of the invention, unless expressly defined or indicated otherwise. All references to a/an/the [cell, sequence, gene, transporter, stage, etc.] should be interpreted explicitly as referring to at least one instance of the specified cell, sequence, gene, transporter, stage, etc. unless expressly stated otherwise. The steps of any method disclosed herein do not have to be performed in the exact order disclosed unless explicitly stated.

The present invention generally relates to a genetically modified cell for efficient production of oligosaccharides and to the use of said genetically modified cell in a process for producing oligosaccharides. In particular, the present invention relates to a genetically modified cell capable of synthesizing an oligosaccharide, preferably a heterologous oligosaccharide, especially human milk oligosaccharide (HMO). Accordingly, cells of the invention are modified to express a set of recombinant nucleic acids that are necessary for the cell to synthesize one or more OGMs (which enable the cell to synthesize one or more OGMs), such as genes encoding one or more enzymes with glycosyltransferase activity described below. The oligosaccharide-producing recombinant cell of the invention is further modified to include a heterologous recombinant nucleic acid sequence, preferably a DNA sequence encoding an MFS (major facilitator superfamily) protein derived from the bacterium Pantoea vagans. More specifically, the invention relates to a genetically modified cell optimized for the production of one or more specific oligosaccharides, in particular one or more specific OGMs, comprising a recombinant nucleic acid encoding a protein having at least 80% sequence similarity, preferably at least 85 %, more preferably at least 90% and even more preferably at least 95% sequence similarity to the amino acid sequence of SEQ ID NO: 1 (Fig. 4). The amino acid sequence designated herein as SEQ ID NO: 1 is an amino acid sequence that is 100% identical to the amino acid sequence having GenBank accession number: WP_048785139.1 (https://www.ncbi.nlm.nih.goV /protein/WP_048785139.1).

Accordingly, the first aspect of the invention relates to a genetically modified cell capable of producing one or more HGMs, wherein said cell includes a recombinant nucleic acid encoding a protein of SEQ ID NO: 1, or a functional homologue thereof with at least 80% amino acid sequence identity, preferably at least 85% identical, more preferably at least 90% identical to the sequence of SEQ ID NO: 1.

The MFS transporter protein having the amino acid sequence SEQ ID NO: 1 is identified

- 3 046260 are referred to herein as Vag protein, or Vag transporter, or Vag, interchangeably; a nucleic acid sequence encoding a vag protein is identified herein as a vag nucleic acid/DNA encoding or vag gene or vag.

The term master messenger superfamilies (MFS) refers to a large and extremely diverse family of a class of secondary active transporters that are responsible for the transport of a number of different substrates, including sugars, drugs, hydrophobic molecules, peptides, organic ions, etc. The specificity of sugar transport proteins is highly unpredictable, and the identification of a new transport protein with specificity for, for example, oligosaccharides requires simple laboratory experiments (for more information, see the review by Reddy V. S. et al. (2012), FEBS J. 279(11): 2022-2035). The term MFS transporter in this context means a protein that facilitates the transport of an oligosaccharide, preferably OGM, across the cell membrane, preferably the transport of an OGM/oligosaccharide synthesized by the host cell from the cytosol to the external environment of the cell, preferably an OGM/oligosaccharide containing three or four sugar units , especially 2'FL, 3FL, 6'SL, DFL, LNT, LNT-2 and LNnT. Additionally or alternatively, the MFS transporter may also promote the efflux of molecules that are not considered OGM or oligosaccharides according to the present invention, such as lactose, glucose, cellular metabolites or toxins.

The term [defined] % sequence identity in the context of two or more nucleic acid or amino acid sequences means that two or more sequences share a given percentage of nucleotides or amino acid residues when compared and aligned for maximum agreement in the comparison window or designated nucleic acid or amino acid sequences (i.e., the sequences have at least 90 percent (%) identity). Percent identity of nucleic acid or amino acid sequences can be determined using the BLAST 2.0 sequence comparison algorithm with default parameters or by manual alignment and visual inspection (see, for example, http://www.ncbi.nlm.nih.gov/BLAST/) . This definition also applies to the complement of the test sequence and to sequences that have deletions and/or additions, as well as those that have substitutions. An example of an algorithm suitable for determining percent identity, sequence similarity and alignment is the BLAST 2.2.20 algorithm described in Altschul et al., Nucl. Acids Res. 25, 3389 (1997). BLAST 2.2.20 is used to determine the percentage identity of nucleic acid and protein sequences according to the invention. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information, (http://www.ncbi.nlm.nih.gov/). CLUSTAL Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/), EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss_needle/), MAFFT (http: //mafft.cbrc.jp/alignment/server/) or MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) are examples of sequence alignment algorithms.

In the context of the invention, the term oligosaccharide means a saccharide polymer containing a number of monosaccharide units. In some embodiments, preferred oligosaccharides are polymers of saccharides consisting of three or four monosaccharide units, i.e. trisaccharides or tetrasaccharides. Preferred oligosaccharides of the invention are human milk oligosaccharides (HMOs).

The term human milk oligosaccharide or HMO in this context means the complex carbohydrate found in human breast milk (for reference, see Urashima et al.: Milk Oligosaccharides. Nova Science Publisher (2011); or Chen, Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). HMOs have a core structure comprising a lactose unit at the reducing end, which may be extended by one or more e-N-acetyllactosaminyl units and/or one or more β-lacto-N-biosyl units, and this core structure may be substituted with α-L-fucopyranosyl and/or or an a-N-acetylneuraminyl (sialyl) moiety. In this regard, non-acidic (or neutral) OGMs are devoid of a sialyl residue, while acidic OHMs have at least one sialyl residue in their structure. Non-acidic (or neutral) OGM can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-triose 2 (LNT2), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-Nneohexaose (pLNnH ), para-lacto-M-hexaose (pLNH) and lacto-M-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-fucosyllactose I (LNDFH-I), 3-fucosyllactose (3FL), difucosyllactose (DFL), lacto -N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lactoN-hexaose II (FLNH-II), lacto-N -fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-p-lacto-N-hexaose I (FpLNH-I) , fucosyl-p-lacto-Nneohexaose II (F-pLNnH II) and fucosyl-lacto-K-neohexaose (FLNnH). Examples of acidic HMOs include 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), 3-fucosyl-3'-sialyllactose (FSL), 3'-Osialyllacto-N-tetraose a (LST a), fucosyl-LST a (FLST a), 6'-O-sialyllacto-N-tetraose b (LST b), fu

- 4 046260 cosyl-LST b (FLST b), 6'-O-sialyllacto-M-neotetraose (LST c), fucosyl-LST c (FLST c), 3'-Osialyllacto-M-neotetraose (LST d), fucosyl -LST d (FLST d), sialyl-lacto-M-hexaose (SLNH), sialyl-lacto-M-neohexaose I (SLNH-I), sialyl-lacto-M-neohexaose II (SLNH-II) and disiallyl-lacto-M -tetraose (DSLNT). In the context of the present invention, lactose is not considered as a type of HGM.

In some embodiments of the invention, tri-OHM and tetra-OHM may be preferred, for example, trisaccharides 2'FL, 3FL, 6'SL, LNT-2 and tetrasaccharides DFL, LNT, LNnT.

To be able to synthesize one or more OGMs, the recombinant cell of the invention includes at least one recombinant nucleic acid that encodes a functional enzyme with glycosyltransferase activity. The galactosyltransferase gene may be integrated into the genome (via chromosomal integration) of the host cell, or alternatively, it may be incorporated into plasmid DNA and expressed as plasmid-borne. If two or more glycosyltransferases, such as LNT or LNnT, are required for the production of OGM, then two or more recombinant nucleic acids encoding different enzymes with glycosyltransferase activity can be integrated into the genome and/or expressed from a plasmid, for example β-1,3N- acetylglucosaminyltransferase (the first recombinant nucleic acid encoding the first glycosyltransferase) in combination with β-1,4-galactosyltransferase (the second recombinant nucleic acid encoding the second glycosyltransferase) to produce LNnT, where the first and second recombinant nucleic acids can be independently integrated into chromosome or cloned on a plasmid. In one preferred embodiment, both the first and second recombinant nucleic acids are integrated into the chromosome of the producing cell; in another embodiment, at least one of the first and second glycosyltransferases is plasmid-borne. The protein/enzyme with glycosyltransferase activity (glycosyltransferase) can be selected in various embodiments from enzymes having α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3/4-fucosyltransferase, α-1,4- fucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, β-1,3-M-acetylglucosaminyltransferase, β-1,6-M-acetylglucosaminyltransferase, β-1,3-galactosyltransferase and β-1, 4-galactosyltransferases. For example, production of LNnT requires that the modified cell express an active β-1,3N-acetylglucosaminyltransferase enzyme and an active β-1,4-galactosyltransferase enzyme. Certain non-limiting embodiments of proteins having glycosyltransferase activity that may be encoded by recombinant genes contained in the producer cell may be selected from the non-limiting examples in Table 1. 1.

Table 1

Gene Protein ID sequences (GenBank) Description OGM example IgtANm WP 002248149.1 ββ-1,3-Ν- acetylglucosamine transferase LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, FpLNH I, pLNnH lgtA_Nm_MC58 AAF42258.1 β-1,3-Ν- acetylglucosamine transferase LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, FpLNH I, pLNnH IgtAHd AAN05638.1 β-1,3-Ν- acetylglucosamine transferase LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, FpLNH I, pLNnH IgtA Ng PID2 AAK70338.1 β-1,3-Ν- acetylglucosamine transferase LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, FpLNH I, pLNnH

- 5 046260

IgtANgNCCP 1 1945 ACF31229.1 β-1,3-Ν- acetylglucosamine transferase LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, FpLNH I, pLNnH IgtAPast AAKO2595.1 β-1,3-Ν- acetylglucosamine transferase LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, FpLNH I, pLNnH IgtANc EEZ72046.1 β-1,3-Ν- acetylglucosamine transferase LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, FpLNH I, pLNnH lgtA_Nm_87255 ELK60643.1 β-1,3-Ν- acetylglucosamine transferase LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, pLNH, FpLNH I, pLNnH galT Hp/HP0826 NP 207619.1 ββ-1,4-galactosyltransferase LNnT, LNFP-III, LNFP-VI, pLNH I, F-pLNH I, pLNnH galTNm/ IgtB AAF42257.1 β-1,4-galactosyltransferase LNnT, LNFP-III, LNFP-VI, pLNH I, F-pLNH I, pLNnH wbgO WP 000582563.1 β-1,3-galactosyltransferase LNT, LNFP-I, LNFP-II, LNFP-V, LNDFH-I, LNDFH-II, pLNH, FpLNHI cpsIBJ ABO50723.1 ββ-1,3-galactosyltransferase LNT, LNFP-I, LNFP-II, LNFP-V, LNDFH-I, LNDFH-II, pLNH, FpLNHI Jhp0563 AEZ55696.1 β-1,3-galactosyltransferase LNT, LNFP-I, LNFP-II, LNFP-V, LNDFH-I, LNDFH-II, pLNH, FpLNHI galTK homologous to BD182026 β-1,3-galactosyltransferase LNT, LNFP-I, LNFP-II, LNFP-V, LNDFH-I, LNDFH-II, pLNH, FpLNHI fiitC WP 080473865.1 a-1,2-fucosyltransferase 2'-FL, DFL, LNFP-I, LNDFH-I FucT2_HpUA802 AAC99764.1 a-1,2-fucosyltransferase 2'-FL, DFL, LNFP-I, LNDFH-I FucT2_EcO126t ABE98421.1 a-1,2-fucosyltransferase 2'-FL, DFL, LNFP-I, LNDFH-I FucT2_Hml2198 CBG40460.1 a-1,2-fucosyltransferase 2'-FL, DFL, LNFP-I, LNDFH-I FucT2_Pm9515 ABM71599.1 a-1,2-fucosyltransferase 2'-FL, DFL, LNFP-I, LNDFH-I FucT2 HpF57 BAJ59215.1 a-1,2-fucosyltransferase 2'-FL, DFL, LNFP-I, LNDFH-I FucT6 3 Bf CAH09151.1 a-1,3-fucosyltransferase 2'-FL, З'-FL, DFL, LNFP-I, LNFPIII, LNFP-V, LNFP-VI, LNDFH-II, F-pLNH I FucT7_3_Bf CAH09495.1 a-1,3-fucosyltransferase 2'-FL, З'-FL, DFL, LNFP-I, LNFPIII, LNFP-V, LNFP-VI, LNDFH-II, F-pLNH I FucT_3_Am ACD04596.1 a-1,3-fucosyltransferase 2'-FL, З'-FL, DFL, LNFP-I, LNFPIII, LNFP-V, LNFP-VI, LNDFH-II, F-pLNH I

- 6 046260

MAMAR764 AGC02224.1 a-1,3-fucosyltransferase 2'-FL, З'-FL, DFL, LNFP-I, LNFPIII, LNFP-V, LNFP-VI, LNDFH-II, F-pLNH I Mg791 AEQ33441.1 a-1,3-fucosyltransfsraza 2'-FL, З'-FL, DFL, LNFP-I, LNFPIII, LNFP-V, LNFP-VI, LNDFH-II, F-pLNH I Moitoi_00703 YP 007354660 a-1,3-fucosyltransferase 2'-FL, З'-FL, DFL, LNFP-I, LNFPIII, LNFP-V, LNFP-VI, LNDFH-II, F-pLNH I fiitA NP207177.1 a-1,3-fucosyltransferase 2'-FL, З'-FL, DFL, LNFP-I, LNFPIII, LNFP-V, LNFP-VI, LNDFH-II, F-pLNH I fucT AAB81031.1 a-1,3-fucosyltransferase 2'-FL, З'-FL, DFL, LNFP-I, LNFPIII, LNFP-V, LNFP-VI, LNDFH-II, F-pLNH I fucTIII AY450598.1 a-1,4-fucosyltransferase LNDFH-I, LNDFH-II fucTa AFI 94963.1 a-1,3/4-fucosyltransferase LNFP-II, LNDFH-I, LNDFH-II Pd2.6ST BAA25316.1 a-2.6- sialyltransferase 6'-SL PspST6 BAF92026.1 a-2.6- sialyltransferase 6'-SL Pi ST6 145 BAF91416.1 a-2.6- sialyltransferase 6'-SL Pi ST6 119 BAI49484.1 a-2.6- sialyltransferase 6'-SL NST AAC44541.1 a-2,3- sialyltransferase 3'-SL

One aspect of the present invention is to provide a nucleic acid construct comprising heterologous nucleic acid sequence(s) encoding a protein capable of transporting a sugar, which is a major messenger superfamily (MFS) protein as shown in SEQ ID NO: 1, or a functional homolog thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1, and the nucleic acid sequence encoding the MFS protein has at least 70% sequence identity to SEQ ID NO: 2 .

The term heterologous nucleic acid sequence, recombinant gene/nucleic acid/DNA encoding or nucleic acid encoding sequence refers to an artificial nucleic acid sequence (i.e., obtained in vitro using standard laboratory methods for obtaining nucleic acid sequences) that includes a set of sequential, non-overlapping triplets (codons) that are transcribed into mRNA and translated into a polypeptide when they are placed under the control of appropriate control sequences, i.e. promoter The boundaries of the coding sequence are usually defined by the ribosome binding site located just upstream of the open reading frame at the 5' end of the mRNA, the translation start codon (AUG, GUG, or UUG), and the translation stop codon (UAA, UGA, or UAG). The coding sequence may include, but is not limited to, genomic DNA, cDNA, synthetic and recombinant nucleic acid sequences. The term nucleic acid includes RNA, DNA and cDNA molecules. It is clear that as a result of the degeneration of the genetic code, many nucleotide sequences encoding a given protein can be obtained. The term nucleic acid is used interchangeably with the term polynucleotide. An oligonucleotide is a short-chain nucleic acid molecule. A primer is an oligonucleotide, occurring naturally as a purified restriction fragment or produced synthetically, that is capable of acting as an initiation point for synthesis when placed under conditions that induce synthesis of a primer extension product complementary to the nucleic acid strand (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase, and at a suitable temperature and pH). The primer is preferably single-stranded for maximum amplification efficiency, but alternatively it can be double-stranded. If the primer is double-stranded, it is first processed to separate its strands before being used to prepare extension products. Preferably the primer is a deoxyribonucleotide. The primer must be long enough to initiate the synthesis of extension products in the presence of an inducing agent. The exact length of the primers will depend on many factors, including temperature, primer source, and method application.

The recombinant nucleic acid sequence of the invention may be a DNA coding sequence, such as a gene, or a non-coding DNA sequence, such as regulatory DNA, such as a promoter sequence. One aspect of the invention relates to the production of a recombinant cell comprising recombinant DNA sequences encoding enzymes necessary for the production of one or more OGMs and a DNA sequence encoding the Vag transporter. Accordingly, in one embodiment the invention relates to con

- 7 046260 nucleic acid structure, including the coding sequence of the nucleic acid, i.e. a recombinant DNA sequence of a gene of interest, e.g., a glycosyltransferase gene or a vag gene, and a non-coding DNA sequence, e.g., a promoter DNA sequence, e.g., a recombinant promoter sequence derived from a lac operon promoter or a glp operon, or a promoter sequence derived from another DNA sequence genomic promoter, or synthetic promoter sequence, where the coding and promoter sequences are operably linked. The term operably linked refers to a functional relationship between two or more nucleic acid segments (eg, DNA). Typically, this refers to the functional relationship of a transcription regulatory sequence to a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or modulates transcription of the coding sequence in a suitable host cell or other expression system. Typically, promoter transcriptional control sequences that are operably linked to the transcribed sequence are physically adjacent to the transcribed sequence, i.e. they are cis-acting sequences.

In one embodiment, the nucleic acid construct of the invention may be part of a vector DNA; in another embodiment, the construct is an expression cassette/cartridge integrated into the genome of a host cell. Accordingly, the term nucleic acid construct means an artificially engineered segment of nucleic acid, especially a segment of DNA, that is intended to be transplanted into a target cell, such as a target cell, such as a bacterial cell, to modify the expression of a host gene or the expression of a gene sequence. coding DNA that can be included in the construct. In the context of the invention, the nucleic acid construct includes a recombinant DNA sequence comprising two or more recombinant DNA sequences: a substantially non-coding DNA sequence including a promoter DNA sequence, and a coding DNA sequence encoding a protein of interest, for example a Vag protein, a glycosyltransferase or other protein, suitable for the production of OGM in the host cell. Preferably, the construct includes additional non-coding DNA sequences that either regulate transcription or translation of the coding DNA construct, for example, a DNA sequence that facilitates ribosome binding to the transcript, a leader DNA sequence that stabilizes the transcript, or a transcription terminator sequence.

Integration of a recombinant gene of interest included in constructs (i.e., an expression cassette) into the bacterial genome can be achieved by conventional means, for example, using linear cartridges containing flanking sequences homologous to a specific region on the chromosome, as described for the attTn7 site (Waddell C.S. and Craig N.L., Genes Dev. (1988) Feb; 2(2):137-49.); methods of genomic integration of nucleic acid sequences in which recombination is mediated by the Red recombinase function of phage λ or the RecE/RecT recombinase function of Rac prophage (Murphy, J Bacteriol. (1998); 180(8):2063-7; Zhang et al., Nature Genetics (1998)20: 123-128 Muyrers et al., EMBO Rep. (2000) 1(3):239-243); methods based on Red/ET recombination (Wenzel et al., Chem Biol. (2005), 12(3):349-56; Vetcher et al., Appl Environ Microbiol. (2005);71(4):1829- 35); or using positive clones, i.e. clones that carry an expression cassette can be selected, for example, using a marker gene, or a loss or gain of function gene.

One copy of an expression cassette comprising the gene of interest may be sufficient to produce the desired OGM and achieve the desired effects of the invention. Accordingly, in some preferred embodiments, the invention provides a recombinant OGM-producing cell that includes one, two or three copies of a gene of interest integrated into the genomic DNA of the cell. In some embodiments, a single copy of the gene is preferred.

In one preferred embodiment, the recombinant nucleic acid coding sequence of the nucleic acid construct of the invention is a heterologous sequence with respect to the promoter, which means that the equivalent native coding sequence in the genome of the species of origin is transcribed under the control of a different promoter sequence (i.e., not the promoter sequence of the construct ). However, with respect to the host cell, the encoding DNA may be either heterologous (i.e., derived from another species or genus), such as, for example, the DNA sequence encoding the Vag protein expressed in Escherichia coli host cells, or homologous (ie, derived from a host cell), such as, for example, colon operon genes, GMAB genes.

The term regulatory element or promoter or promoter region or promoter element is a nucleic acid sequence that is recognized and bound by DNA-dependent RNA polymerase during transcription initiation. A promoter, together with other nucleic acid sequences that regulate transcription and translation (also called control sequences), is necessary for the expression of a given gene or

- 8 046260 group of genes (i.e. operon). Typically, transcriptional and translational control sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, transcription terminator sequences, and enhancer or activator sequences. The transcription start site refers to the first nucleotide transcribed and is designated +1. Nucleotides downstream of the start site are numbered +2, +3, +4, etc., and nucleotides in the 5' opposite (upstream) direction are numbered -1, -2, -3, etc. The promoter DNA sequence of the construct can be derived from the promoter region of any gene in the genome of the selected species, preferably from the promoter region of the genomic DNA of E. coli. Accordingly, any DNA promoter sequence that can bind to RNA polymerase and initiate transcription is suitable for practicing the invention. In principle, any promoter DNA sequence can be used to control the transcription of a recombinant gene construct of interest, and different or the same promoter sequences can be used to control the transcription of different genes of interest integrated into the genome of a host cell or into the DNA of an expression vector. To ensure optimal expression of the recombinant gene(s) included in the construct, the construct may contain additional regulatory sequences, for example, a leader DNA sequence, such as a DNA sequence derived from the 5' untranslated region (5'UTR) of the glp gene E. coli ribosomal binding sequence. Examples of the latter sequences are described in WO 2019123324 (incorporated herein by reference) and are illustrated herein by non-limiting working examples.

In some preferred embodiments, the regulatory element for regulating expression of the recombinant gene included in the construct of the invention is the promoter of the glpFKX operon, PglpF; in other preferred embodiments, the promoter is the promoter of the lac operon, Plac.

In yet another aspect, the regulatory element for regulating the expression of a recombinant gene included in the construct of the invention is mglBAC; the galactose transporter/methylgalactosidase promoter PmglB or variants thereof, such as, but not limited to, PmglB_70UTR of the sequence SEQ ID NO: 27 or PmglB_70UTR_SD4 of the sequence SEQ ID NO: 28.

In yet another aspect, the regulatory element for regulating the expression of a recombinant gene included in the construct of the invention is gatYZABCD; the PgatY tagatose-1,6bisP-aldolase promoter or variants thereof, such as, but not limited to, PgatY_U70UTR of the sequence SEQ ID NO: 29.

The currently preferred regulatory element present in the genetically modified cell and/or nucleic acid construct of the present invention is selected from the group consisting of

PgatY 7OUTR, PglpF,

PglpF SD1, PglpF SD10, PglpF SD 2, PglpF SD3, PglpF SD 4, PglpF SD 5, PglpF SD 6, PglpF SD7, PglpF SD8, PglpF SD9, Plac 16UTR, Plac, PmglB 7OUTR and PmglB 7OUTR SD4.

Particularly preferred regulatory elements present in the genetically modified cell and/or nucleic acid construct of the present invention are selected from the group consisting of PglpF and Plac.

However, any promoter that provides transcription and/or regulation of the transcription level of one or more recombinant nucleic acids encoding one or more proteins (or one or more regulatory nucleic acids) that are either necessary or useful for achieving an optimal level of biosynthetic production of one or more OGMs in the host cell, for example, glucosyltransferases, proteins involved in the transmembrane transport of OGM or OGM precursor, proteins regulating gene expression, etc., and allowing the desired effects of the invention to be achieved are suitable for the practice of the invention.

Preferably, the construct of the present invention, including a gene associated with the biosynthetic production of OGM, a promoter DNA sequence and other regulatory sequences, such as a ribosome binding site sequence (for example, the Shine-Dalgarno sequence) expressed in the host cell, provides an OGM of at least 0.03 g/OD (optical density) per 1 L of fermentation medium containing a suspension of host cells, for example, at a level of about 0.05 g/L/OD, about 0.1 g/L/OD or more. For the purposes of the invention, the final level of OGM production is considered sufficient or optimal, and a host cell capable of producing the desired level of OGM is considered a suitable host cell, i.e. the cell may be further modified to express an OGM transporter protein, such as Vag, to achieve at least one of the effects described herein beneficial for OGM production.

- 9 046260

The genetically modified cell and/or nucleic acid construct of the present invention includes a nucleic acid sequence, such as a heterologous gene encoding a putative MFS (major mediator superfamily) transporter protein.

The Vag protein is an MFS transport protein of particular interest in the present invention. Thus, the nucleic acid construct of the present invention includes a nucleic acid sequence having at least 70% sequence identity with the gene, vag, SEQ ID NO: 2.

The nucleic acid sequence contained in the genetically modified cell or nucleic acid construct encodes a protein of SEQ ID NO: 1 or a functional homolog thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 1.

A functional homolog of the protein of SEQ ID NO: 1 can be obtained by mutagenesis. A functional homolog must have at least 50% remaining functionality, such as 60%, 70%, 80%, 90%, or 100%, compared to the functionality of the amino acid sequence of SEQ ID NO: 1. A functional homologue may have higher functionality than functionality of the amino acid sequence SEQ ID NO: 1. A functional homolog of the sequence SEQ ID NO: 1 should be capable of enhancing the production of OGM by the genetically modified cell of the invention.

The genetically modified cell (host cell or recombinant cell) may be, for example, a bacterial or yeast cell. In one preferred embodiment, the genetically modified cell is a bacterial cell. As far as bacterial host cells are concerned, there are in principle no restrictions; they may be eubacteria (gram-positive or gram-negative) or archaebacteria if they can be genetically manipulated to insert the gene of interest and can be cultured on a commercial scale. Preferably, the host cell has the ability to be cultured to a high cell density. Non-limiting examples of bacterial host cells suitable for the recombinant industrial production of OGM according to the invention may include

Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum or Xanthomonas campe str is.

Bacteria of the genus Bacillus can also be used, including

Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus and Bacillus circulans.

Likewise, bacteria of the genera Lactobacillus and Lactococcus can be modified using the methods of this invention, including, but not limited to,

Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jense nii and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. Also part of this invention are strains modified as described herein from the genera

Enterococcus (for example, Enterococcus faecium and

Enterococcus thermophiles), Bifidobacterium (e.g. Bifidobacterium longum,

Bifidobacterium inf antis and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp. and Pseudomonas (eg Pseudomonas fluorescens and Pseudomonas aeruginosa).

Bacteria having the characteristics described herein are cultured in the presence of lactose, and an oligosaccharide, such as OGM, produced by the cell is isolated either from the bacterium itself or from the culture supernatant of the bacterium. In one preferred embodiment, the genetically modified cell of the invention is an Escherichia coli cell.

In another preferred embodiment, the host cell is a yeast cell, for example

- 10 046260

Saccharomyces cerevisiae, Schizosaccharomyces pombe,

Pichiapastoris, Kluveromyces lactis, Kluveromyces marxianus, etc.

The OGM produced by the recombinant cells of the invention can be purified using a suitable procedure available in the art (for example, such as described in WO 2015188834, WO 2017182965 or WO 2017152918). The genetically modified cells of the invention can be produced using standard methods in the art, for example those described in Sambrook et al., Wilson & Walker, Maniatise et al. and Ausubel et al.

A host cell suitable for the production of OGM, eg E. coli, may include an endogenous β-galactosidase gene, or an exogenous β-galactosidase gene, eg E. coli, include an endogenous lacZ gene (eg, GenBank accession number V00296 (GL41901)). For the purposes of the invention, the HGM-producing cell is genetically manipulated to either include any β-galactosidase gene or include an inactivated version of the gene. A gene may be inactivated by complete or partial deletion of the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence may be mutated as it is transcribed, or, if transcribed, the transcript is not translated, or if translated into a protein (i.e., β-galactosidase) , then the protein does not have the appropriate enzymatic activity. Thus, the OGM-producing cell accumulates an increased intracellular pool of lactose, which is beneficial for OGM production.

In some embodiments, the germ cell includes a deficient sialic acid catabolic pathway. The sialic acid catabolic pathway refers to the sequence of reactions, usually controlled and catalyzed by enzymes, that leads to the degradation of sialic acid. The exemplary sialic acid catabolic pathway described herein is the E. coli pathway. In this pathway, sialic acid (Neu5Ac; N-acetylneuraminic acid) is cleaved by the enzymes NanA (N-acetylneuraminic acid lyase), NanK (N-acetylmannosamine kinase), and NanE (N-acetylmannosamine-6-phosphate epimerase), all encoded from the nanATEK-yhcH operon, and suppressed by NanR (http://ecocyc.org/ECOLI). The deficient sialic acid catabolic pathway is replicated in the E. coli host by introducing mutations into the endogenous nanA (N-acetylneuraminase) (e.g., GenBank accession no. D00067.1 (GL216588)) and/or nanK (N-acetylmannosamine kinase) genes (e.g., no. GenBank access (amino acid) BAE77265.1 (GL85676015)), and/or paPE (Nacetylmannosamine-6-phosphate epimerase, GI: 947745, incorporated herein by reference). Optionally, the nanT (N-acetylneuraminate transporter) gene is also inactivated or mutated. Other intermediates of sialic acid metabolism include: (ManNAc-6-P)N-acetylmannosamine-6phosphate; (GlcNAc-6-P)N acetylglucosamine-6-phosphate; (GlcN-6-P) glucosamine 6-phosphate and (Fruc-6-P) fructose 6-phosphate. In some preferred embodiments, nanA is mutated. In other preferred embodiments, nanA and nanK are mutated while nanE remains functional. In another preferred embodiment, nanA and nanE are mutated, while nanK has not been mutated, inactivated or deleted. A mutation is one or more changes in the nucleic acid sequence encoding the gene product nanA, nanK, nanE and/or nanT. For example, a mutation may represent 1, 2, up to 5, up to 10, up to 25, up to 50, or up to 100 changes in the nucleic acid sequence. For example, the nanA, nanK, nanE and/or nanT genes are mutated by null mutation. Null mutations, as described herein, include amino acid substitutions, additions, deletions or insertions that either cause loss of enzyme function (ie, decreased or absent activity) or loss of enzyme (ie, absence of gene product). By deleted we mean that the coding region has been removed in whole or in part so that no (functional) gene product is generated. By inactivation it is meant that the coding sequence has been altered such that the resulting gene product is functionally inactive or encodes a gene product with less than 100% activity, e.g. 90%, 80%, 70%, 60%, 50%, 40 %, 30% or 20% compared to the activity of the native, naturally occurring, endogenous gene product. An unmutated gene or protein does not differ from the native, naturally occurring, or endogenous coding sequence by 1, 2, up to 5, up to 10, up to 20, up to 50, up to 100, up to 200, or up to 500 or more codons or up to the corresponding encoded amino acid sequence.

In addition, the host cell (for example, E. coH) also has the ability to synthesize sialic acid. For example, a bacterium has the ability to synthesize sialic acid through exogenous UDP-GlcNAc 2-epimerase (for example, neuC from Campyiobacter jejuni (GenBank AAK91727.1; GL15193223) or its equivalents (for example, neuC from E. coli S88 (GenBank YP_002392936.1; GI :218560023), Neu5Ac synthase (e.g., neuB from C. jejuni (GenBank AAK91726.1; GI:15193222) or its equivalents (e.g., sialic acid synthase from Flavobacterium limnosediminis, GenBank GL559220424), and/or CMP-Neu5Ac synthase ( for example, neuA from C. jejuni (GenBank AAK91728.1; GI:15193224) or its equivalents (for example, Vibrio brasiliensis sialic acid CMP synthase, GenBank GI:493937153).

Preparation of neutral OGM containing N-acetylglucosamine in engineered bacteria

- 11 046260 and yeast are known in the art (see, for example, Gebus C et al. (2012) Carbohydrate Research 363 83-90; US 10519475).

For the production of OGM containing N-acetylglucosamine, such as lacto-N-triose 2 (LNT-2), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP- I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose I (LDFH-I), lacto -N-difucohexaose II (LDFH-II) and lacto-N-neodifucohexaose II (LNDFH-III), the bacterium includes a functional lacY gene and a dysfunctional lacZ gene as described above, and it is engineered to include an exogenous UDP-GlcAc gene: Galα/β-R-β-3-Nacetylglucosaminyltransferase or a functional variant or fragment thereof. This exogenous UPD-GlcNAc:Galα/β-Rβ-3-N-acetylglucosaminyltransferase (OcGlAcT) gene can be obtained from any of a number of sources (see Table 1), for example, from the lgtA gene described in L. meningitidis (GenBank protein accession number AAF42258.1) or S. gonorrhoeae (GenBank protein accession number ACF31229.1). Optionally, an additional exogenous glycosyltransferase gene can be coexpressed in the bacterium including an exogenous UDP-GlcAc:Galα/β-R-β-3-N-acetylglucosaminyltransferase gene. For example, the β-1,4-galactosyltransferase gene (Gal4T) is coexpressed with the UPD-GlcAc:Galα/β-R-ββ-acetylglucosaminyltransferase gene. This exogenous β-1,4-galactosyltransferase gene can be obtained from any source, for example, those described in L. meningitidis, lgtB gene (GenBank protein accession number AAF42257.1) or from H. pylori, HP0826/galT gene (GenBank protein accession number NР207619.1). Optionally, the additional exogenous glycosyltransferase gene coexpressed in the bacterium including the exogenous UDP-GlcAc:Galα/β-R-β-3-N-acetylglucosaminyltransferase gene is a β-1,3-galactosyltransferase (Gal3T) gene, e.g. described from E. coli 055:H7, gene wbgO (GenBank protein accession number WP000582563.1), or from H. pylori, gene jhp0563 (GenBank protein accession number AEZ55696.1), or from Streptococcus agalactiae type Ib OI2 gene cpslBJ g (GenBank protein accession number AB050723.1). The disclosed invention also covers functional variants and fragments of any of the enzymes described above.

The N-acetylglucosaminyltransferase gene and/or the galactosyltransferase gene can also be operably linked to Pglp and expressed from the corresponding cassette integrated into the genome. In one embodiment, the gene integrated into the genome is a gene encoding a galactosyltransferase, for example, the HP0826 gene encoding the GalT enzyme from H. pylori (GenBank Protein Accession Number NIP207619.1); in another embodiment, the gene integrated into the genome is a gene encoding β-1,3-N-acetylglucosaminyltransferase, for example, the lgtA gene from N. meningitidis (GenBank protein accession number AAF42258.1). In these embodiments, the second gene, i.e. the gene encoding β-1,3-Nacetylglucosaminyltransferase or galactosyltransferase, respectively, can be expressed either from a genome-integrated or plasmid-borne cassette. The second gene may optionally be expressed either under the control of a glp promoter or under the control of any other promoter suitable for the expression system, for example, Plac.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons (No. w York) provides one skilled in the art with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described. Much of the nomenclature and general laboratory procedures required for this application can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (2012); Wilson K. and Walker J., Principles and Techniques of Biochemistry and Molecular Biology (2010), Cambridge University Press; or in Maniatis et al., Molecular Cloning A laboratory Manual, Cold Spring Harbor Laboratory (2012); or in Ausubel et al., Current protocols in molecular biology, John Wiley and Sohns (2010). The guidelines are hereafter referred to as Sambrook et al., Wilson & Walker, Maniatis et al., Ausubel et al., respectively.

The second aspect of the invention relates to a method for producing one or more OGM, including the following steps:

(i) providing a genetically modified cell capable of producing HGM, wherein said cell comprises a recombinant nucleic acid encoding a protein of the sequence SEQ ID NO: 1, or a functional homolog thereof, the amino acid sequence of which is at least 80% identical, preferably at least identical 85%, more preferably at least 90% identical to the sequence of SEQ ID NO: 1;

(ii) culturing the cell (i) in a suitable cell culture medium to express said recombinant nucleic acid;

(iii) collection of the GMS obtained in step (ii).

According to the invention, the term cultivation (or cultivation or cultivation,

- 12 046260 also called fermentation) refers to the propagation of bacterial expressing cells in a controlled bioreactor in accordance with methods known in the industry.

To obtain one or more OGM, bacteria producing OGM as described herein are cultured according to procedures known in the art in the presence of a suitable carbon source, for example glucose, glycerol, lactose, etc., and the resulting OGM collected from cultivation media and microbial biomass formed during the cultivation process. Thereafter, the HMO is purified according to procedures known in the art, such as those described in WO 2015188834, WO 2017182965 or WO 2017152918, and the purified HMO is used as nutraceuticals, pharmaceuticals or for any other purpose, e.g. research. The production of OGM is usually carried out through large-scale cultivation. The term production and production scale within the meaning of the invention defines fermentation with a minimum volume of 5 liters of culture broth. Typically, a manufacturing scale process is defined by the ability to process large volumes of a drug containing the HMO or HMO of interest and to produce quantities of the protein of interest that meet, for example in the case of a therapeutic compound or composition, the requirements for clinical testing as well as for marketing . In addition to the large volume, the method on a production scale, in contrast to simple methods on a laboratory scale, such as cultivation in shake flasks, is characterized by the use of a technical bioreactor (fermenter) system equipped with devices for mixing, aeration, supply of nutrients, monitoring of process parameters and control process parameters (pH, temperature, dissolved oxygen pressure, back pressure, etc.). To a large extent, the behavior of an expression system on a laboratory scale, such as in shake flasks, benchtop bioreactors, or the deep well format described in the examples of the present disclosure, allows one to predict the behavior of that system in a complex bioreactor environment.

There are no restrictions regarding the suitable culture medium used in the fermentation process. The culture medium may be semi-defined, that is, containing complex compounds of the medium (eg, yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds.

By the term one or more OGMs, it is meant that a OGM production cell may be capable of producing one OGM structure (first OGM) or multiple OGM structures (second, third, etc. OGM). In some embodiments, a host cell that produces a single OGM may be preferred; in other preferred embodiments, a host cell that produces multiple OGM structures may be preferred. Cells producing 2'FL, 3FL, 3'SL, 6'SL or LNT-2 are non-limiting examples of host cells producing OGM of a single structure. Non-limiting examples of host cells capable of producing multiple OGM structures may include DFL, FSL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, pLNnHor-II producer cells.

The term collection in the context of the invention refers to the collection of the resulting OGM after the cessation of fermentation. In various embodiments, this may involve collecting OGMs included in both the biomass (i.e., host cells) and the culture medium, i.e. before/without separating the fermentation broth from the biomass. In other embodiments, the resulting OGM can be collected separately from the biomass and fermentation broth, i.e. after/following the separation of the biomass from the culture medium (i.e. fermentation broth). Separation of cells from the medium can be accomplished by any of the methods well known to one skilled in the art, for example, any suitable type of centrifugation or filtration. Separation of cells from the medium may immediately follow collection of the fermentation broth or may be carried out at a later stage after the fermentation broth has been stored under appropriate conditions. Extraction of produced HMOs from the remaining biomass (or complete fermentation) involves their extraction from the biomass (i.e., producer cells). This can be accomplished by any suitable methods in the art, for example, sonication, boiling, homogenization, enzymatic lysis using lysozyme, or freezing and grinding.

Once recovered from fermentation, the HMOs are available for further processing and purification.

Purification of OHMs obtained by fermentation can be carried out using a suitable procedure described in WO 2016095924, WO 2015188834, WO 2017152918, WO 2017182965, US 20190119314 (all incorporated by reference).

In some embodiments of the invention, a host cell may produce multiple OGMs, where one OGM is a product OGM and some/all of the other OGMs are by-product OGMs. As a rule, side HMOs are either the main precursors of the HMOs or products of further modification of the main HMOs. In some embodiments, it may be desirable to produce the OGM product in large quantities and the OGM by-product in small quantities. The cells and methods for producing OGM described herein provide controlled production of an OGM product with a desired OGM profile, for example in one embodiment

- 13 046260 the resulting mixture of OGM, in which the product of OGM is the dominant OGM compared to other OGM (i.e. by-products) of the mixture, i.e. The OGM product is produced in greater quantities than other OGM by-products; in other embodiments, a cell producing the same mixture of OGM may be configured to produce one or more OGM by-products in greater quantities than the OGM product. For example, the production of LNnT, an OGM product, often produces significant amounts of pLNnH, an OGM byproduct. In another example, LNT production produces a significant amount of the by-product pLNH-II. With the genetically modified cells of the present invention, the level of pLNnH in the LNnT product can be significantly reduced.

Advantageously, the invention provides both a reduction in the by-product to product ratio and an increase in the overall product yield (and/or OGM in general). This reduced by-product formation relative to product formation promotes increased product formation and improves the efficiency of both the production and product recovery process, providing superior OGM production technology.

In various preferred embodiments, different host cells may be selected producing one/both of 2'FL, 3FL, 3'SL, 6'SL, LNT-2, DFL, FSL, LNT, LNnT, DFL, FSL, LNT, LNnT, LNFPI, LNFP-II, LNFP-III, LNFP-IV, LNFP-V, pLNnH, pLNH-II, as an OGM product or by-product. In one preferred embodiment, the product is LNnT and the by-product is pLNnH. In another preferred embodiment, the product is LNT and the by-product is pLNH-II.

The invention also relates to the use of a genetically modified cell and/or nucleic acid construct according to the invention for the production of one or more oligosaccharides, preferably one or more human milk oligosaccharides. In one embodiment, a genetically modified cell and/or nucleic acid construct of the invention is used to produce a specific HGM selected from the group consisting of 2'-FL, 3-FL, DLF, LNT, LNT-II, LNnT, pLNH-II and pLNnH .

In a particularly preferred embodiment, a genetically modified cell and/or nucleic acid construct of the invention is used to produce a specific HGM selected from the group consisting of LNT, LNT-II, LNnT and pLNH-II and pLNnH.

The invention is further illustrated by the following non-limiting examples and embodiments.

Examples

Materials and methods

Unless otherwise noted, standard techniques, vectors, control sequence elements, and other expression system elements known in the art of molecular biology are used for nucleic acid manipulation, transformation, and expression of nucleic acids. Such standard techniques, vectors and elements can be found, for example, in the manuals:

Ausubel et al. (eds.), Current

Protocols in Molecular Biology (1995) (John Wiley &Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (eds.), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, J.H. Experiments in molecular genetics (1972.) (Cold spring Harbor Laboratory Press, NY)

The embodiments described below are chosen to illustrate the invention and are not intended to limit the invention in any way.

Strains

The bacterial strain used, MDO, was constructed from Escherichia coli K12 DHL The genotype of E. coli K12 DH1 was: F ^- , λ ^- , gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. The strains used in these examples are described in table. 2.

- 14 046260

table 2

in ta mm o in 11 po 1\ k' ( oo sun k · । in\ louuiii । eno ι nor DH1 - F λ endA 1 recA 1 relA 1 gyrA96thi-1 glnV44hsdRl 7(gk tk) MDO - E coli DH1 AlacZ, AlacA, ΔηαηΚΕΤΑ, AmelA, AwcaJ, AmdoH MP3672 LNnT MDO PglpF-GlcNAcT PglpF-Gal4T (version 1) * MP3708 MDO PglpF-GlcNAcT PglpF-Gal4TPlac-yberC0001 9420 MP3972 MDO PglpF-GlcNAcT PglpF-Gal4TP lac-bad MP3979 MDO PglpF-GlcNAcT PglpF-Gal4TPlac-fred MP4011 MDO PglpF-GlcNAcT PglpF-Gal4TPlac-marc MP3994 MDO PglpF-GlcNAcTPglpF-Gal4TPlac-vag (version 1)** MP3984 MDO PglpF-GlcNAcT PglpF-Gal4TPlac-nec MP4362 MDO PglpF-GlcNAcT PglpF-Gal4T Plac-yabM MP3020 MDO PglpF-GlcNAcTPglpF-Gal4T (version 2) * MP4064 MDO PglpF-GlcNAcT PglpF-Gal4TPglpF-vag MP4065 MDOPglpF-GlcNAcTPglpF-Gal4TPlac-vag (version 2)** MP4473 LNT MDO PglpF-GlcNAcT PglpF-Gal3T MP4546 MDO PglpF-GlcNAcT PglpF-Gal3T PglpF-vag

*Strains MP3672 and MP3020 carry the same heterologous GlcNAcT and Gal4T, but differ in the number of copies of the corresponding gene encoding GlcNAcT.

**Strains MP3994 and MP4065 carry the same heterologous GlcNAcT and Gal4T, but differ in the copy number of the corresponding gene encoding GlcNAcT.

Wednesday

Luria broth (LB) medium was prepared using LB broth powder, Millers (Fisher Scientific), and LB agar plates were prepared using LB agar powder, Millers (Fisher Scientific). If necessary, ampicillin ((100 μg/ml) or any appropriate antibiotic) and/or chloramphenicol (20 μg/ml) was added.

The basic minimal medium had the following composition: NaOH (1 g/l), KOH (2.5 g/l), KH2PO4 (7 g/l), NH4H2PO4 (7 g/l), citric acid (0.5 g/l ), microelements solution (5 ml/l). The initial solution of trace elements contained: ZnSO ₄ -7H ₂ O 0.82 g/l, citric acid 20 g/l, MnSO ₄ -H ₂ O 0.98 g/l, FeSO ₄ -7H ₂ O 3.925 g/l, CuSO ₄ -5H ₂ O 0.2 g/l. The pH of the basic minimal medium was adjusted to 7.0 with 5 N NaOH and autoclaved. Before inoculation, the basal minimal medium was supplemented with 1 mM MgSO ₄ , 4 μg/ml thiamine, 0.5% of the given carbon source (glycerol (Carbosynth)) and, if necessary, added isopropyl-eD-thiogalactoside (IPTG) (0.2 mM). Thiamine, antibiotics and IPTG were sterilized by filtration. All percentage concentrations for glycerol are expressed as v/v and for glucose as w/v.

M9 plates containing 2-deoxygalactose had the following composition: 15 g/L agar (Fisher Scientific), 2.26 g/L 5\\\M9 Minimal Salt (Sigma-Aldrich), 2 mM MgSO ₄ , 4 μg/ml thiamine, 0.2% glycerol and 0.2% 2-deoxy-O-galactose (Carbosynth).

MacConkey indicator plates had the following composition: 40 g/L MacConkey agar base (BD Difco™) and a carbon source at a final concentration of 1%.

Cultivation

Unless otherwise stated, E. coli strains were propagated in Luria-Bertani (LB) medium containing 0.2% glucose at 37°C with agitation. Agar plates were incubated at 37°C overnight.

Chemically competent cells and transformations

E. coli was inoculated from LB plates into 5 ml LB containing 0.2% glucose at 37°C with shaking to CD ₆ oo ~0.4. 2 ml of culture were collected by centrifugation for 25 s at 13000 g. The supernatant was removed, and the cell pellet was resuspended in 600 μl of cold TB solutions (10 mM PIPES, 15 mM CaCl ₂ , 250 mM KCl). Cells were incubated on ice for 20 min followed by sedimentation for 15 s at 13,000 g. The supernatant was removed, and the cell pellet was resuspended in 100 μl of cold TB solution. Plasmid transformation was carried out using 100 μl of competent cells and 1-10 ng of plasmid DNA. Cells and DNA were incubated on ice for 20 min and then heat shocked at 42°C for 45 s. After a 2-minute incubation on ice, 400 μl of SOC (20 g/l tryptone, 5 g/l yeast extract, 0.5 g/l NaCl, 0.186 g/l KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ and 20 mM glucose) and the cell culture was incubated at 37°C with shaking for 1 hour before plating onto selective dishes.

The plasmid was transformed into chemically competent TOP10 cells under conditions recommended by the supplier (ThermoFisher Scientific).

DNA methods

Plasmid DNA from E. coli was isolated using the QIAprep Spin Miniprep kit (Qiagen). Chromosomal DNA from E. coli was isolated using the QIAmp DNA Mini Kit (Qiagen). PCR products were purified using the QIAquick PCR Purification Kit (Qiagen). DreamTaq PCR Master Mix (Thermofisher), Phusion U Hot Start PCR Master Mix (Thermofisher), USER Enzyme (New England Biolab) were used according to the supplier's recommendations. Primers

- 15 046260 were supplied by Eurofins Genomics, Germany. PCR fragments and plasmids were sequenced by Eurofins Genomics. Colony PCR was performed using DreamTaq PCR Master Mix in a T100TM thermal cycler (Bio-Rad).

Table 3. Oligonucleotides used to amplify the plasmid backbone, promoter elements and genes of interest

I nod SEQ II) XO O.pp onukmeo gpdnam pos.tedova g.tyyusg 5' - 3' Description 040 5 ATTAACCCUCCAGGCATCAAATAAAACGAAAGG WITH Frame, straight 068 6 ATGCGCAAAUTGTGAGTTAGCTCACTCATTAG P1 ac. straight 079 7 ATTTGCGCAUCACCAATCAAATTCACCGGGCC Backbone, reverse 0113 8 AGCTGTTUCCTCCTTAGGTACCCAGCTTTTGTTCC C P1ac. back 0261 9 ATGCGCAAAUGCGGCACGCCTTGCAGATTACG Pgl pF. straight 0262 10 AGCTGTTUCCTCCTTGGTTAATGTTTGTTGTATGC G Pgl pF. back kaby745 AND AAACAGCUATGAAGAGCCTGCTGACCCGTAAAC vag .straight kaby746 12 AGGGTTAAUTTAAAACGTTTTTCACACGCGCG vag .reverse kaby721 13 AAACAGCUATGAAGAGCGCGCTGACCTTTAGC yberC0001_9420.n direct kaby722 14 AGGGTTAAUTTACGCCTCACGCCACACGCG yberC0001_9420.o brother kaby733 15 AAACAGCUATGAAGAGCGCGCTGACCTTCAG fred.direct kaby734 16 AGGGTTAAUTTACGCTTCACGCACACGCG fred.reverse kaby729 17 AAACAGCUATGAGCAGCCGTCGTCTGAGC bad. straight kaby730 18 AGGGTTAAUTTACACGTTTTTAACACGGGTCATC AG bad. back kaby741 19 AAACAGCUATGCAGAGCTTCACCCCGCC dog. straight kaby742 20 AGGGTTAAUTTACGCCTGCTCTTTAACACGCAGC dog. back kaby737 21 AAACAGCUATGCAGCGTCTGAGCCGTCTGAG marc, straight kaby738 22 AGGGTTAAUTTAAACTTCACGCACTTTCGCGC marc, reverse 048 23 CCCAGCGAGACCTGACCGCAGAAC galK. straight 049 24 CCCCAGTCCATCAGCGTGACTACC galK. back MP1217 25 AAACAGCUATGAAGGCGCTGTGGAGCCGTCG uAM.straight MP1218 26 AGGGTTAAUCGCCAGCGGAACGCTCTTCACG UAM.reverse

Table 4. Heterologous MFS transporter genes tested in the microbial hosts of the present invention

Origin IIIIIIIIIIII Died moose na Fu iik'uiih squirrel yberCOOOl 9420 Yersinia bercovieri EEQ08298.1 MFS Master Mediator Superfamily 1 fred* Yersinia frederiksenii WP 087817556.1 Conveyor MFS dog* Rosenbergiella nectarea WP_092672081.1 Conveyor MFS marc* Serratia marcescens WP 060448169.1 Conveyor MFS bad* Rouxiella badensis WP 017489914.1 Conveyor MFS vag* Pantoea vag ans WP 048785139.1 Conveyor MFS yabM Erwinia pyrifoliae CAY73138.1 Putative efflux transporter sugar MFS

*gene name is given to identify the nucleic acid encoding a protein having an amino acid sequence corresponding to the GenBank accession number for the purposes of the present invention

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Table 5. Synthetic DNA elements used for direct expression of vag

I nod after 1ovaic. ii.hoc in SEQ II)\() Sequence (5'-3') Description PglpF 3 GCGGCACGCCTTGCAGATTACGGTTTGCC ACACTTTTCATCTTCTCCTGGTGACATAA TCCACATCAATCGAAAATGTTAATAAATTT GTTGCGCGAATGATCTAACAAACATGCAT CATGTACAATCAGATGGAATAAATGGCGC GATAACGCTCATTTTATGACGAGGCACAC ACATTTTAAGTTCGATATTTCTCGTTTTTTGC TCGTTAACGATAAGTTTACAGCATGCCTAC AAGCATCG TGGAGGTCCGTGACTTTCACG CATACAACAAACATTAACCAAGGAGGAAA CAGCT 300 nucleotide DNA expression element Plac 4 ATGCGCAAATTGTGAGTTAGCTCACTCATT AGGCACCCAGGCTTTACACTTTATGCTTC CGGCTCGTATGTTGTGTGGAATTGTGAGCG GATAACAATTTCACACAGGAAACAGCTAT GACCATGATTACGCCAAGCGCGCAATTAA CCCTCACTAAAGGGAACAAAAGCTGGGTA CCTAAGGAGGAAACAGCT 195 nucleotide DNA expression element vag 2 ATGAAGAGCCTGCTGACCCGTAAACGTCG TATTAACCCGGTGTTCCTGGCGTTTATGGC GGCGAGCTTCATGATCGGTGTTGCGGGTG CGCTGCAGGCGCCGACCCTGAGCCTGTTTC TGACCCGTGAGGTGCAAGCGCGTCCGCTG TGGGTGGGCCTGTTCTTTACCGTTAACGCG ATCGCGGGTATTGTGGTTAGCATGCTGGTT GCGAAGCGTAGCGACAGCCGTGGCGA TCG TCG TCGTACCCTGATTCTGTTCTGCTGCGCGAT GGCGTTTTGCAACGCGCTGCTGTTCGCGTT TACCCGTCACTACCTGACCCTGATTACCCT GGGTGTGCTGCTGAGCGCGCTGGCGAGCG TTAGCATGCCGCAGATTTTCGCGCTGGCGC GTGAGTATGCGGACCAAAGCGCGCGTGAA GCGGTGATGTTTAGCAGCGTTATGCGTGC GCAGCTGAGCCTGGCGTGGGT GATTGGCC CGCCGCTGAGCTTCGCGCTGGCGCTGAAC TTCGGTTTTGTGACCCTGTTCCTGGTTGCT GCGGCGCTGTTTCTGGTGTGCATCCTGCTG ATTAAGTTTACCCTGCCGAGCGTTCCGCGT GCGGAACCGCTGATGCGTAGCGGTGGCAT GCCGCTGAGCGGTTGGCGTGACCGTGATG TGCGTCTGCTGTTCATTGCGAGCGTTACCA TGTGGACCTGCAACACCAT GTACATCATTG ACATGCCGCTGTATATCAGCGTTACCCCTGG GTCTGCCGGAGAAACTGGCGGGTCTGCTG ATGGGCACCGCGGCGGGTCTGGAAATTCC GGTGATGCTGCTGGCGGGTCACTATGCGA AGCGTGTTGGTAAACGTAACCTGATGCTG ATTGCGGTGGCGGCGGGCGTTCTGTTCTAT GCGGGTCTGGCGATGTTTGCGAGCCAGAC CGCGCTGATGGCGCTGCAACTG TTCAACG CGGTGTTTATTGGCATCATTGCGGGTATCG GCATGCTGTGGTTCCAGGATCTGATGCCG GGTCGTCCGGGTGCGGCGACCACCATGTT TACCAACAGCATCAGCACCGGTATGATTC TGGCGGGCGTTATCCAAGGCACCCTGAGC GAGCGTTTCGGCCACATTGCGGTGTATTGG CTGGCGCTGGGTCTGGCGGTTGCGGCGTTT GCGATGAGCGCGCTGTGAAAAAACG TTTA A DNA encoding the MFS transporter

Plasmid construction

Plasmid backbones were synthesized containing two I-SceI endonuclease sites separated by two DNA fragments adapted for homologous recombination into the E. coli genome and a T1 transcription terminator sequence. For example, the gal operon (required for homologous recombination in galK) and the T1 transcription terminator sequence (pUC57::garl) were synthesized in one plasmid backbone (GeneScript). The DNA sequences used for homologous recombination in the gal operon span base pairs 3.628.621-3.628.720 and 3.627.5723.627.671 in the Escherichia coli K-12 MG155 sequence, GenBank complete genome: ID: CP014225.1.

Insertion by homologous recombination would result in a 949 bp deletion of galK and a galK- phenotype. In a similar manner, scaffolds can be synthesized from pUC57 (GeneScript) or any other suitable vector containing two endonuclease I-SceI sites separated by two DNA fragments adapted for homologous recombination into the E. coli genome and a T1 transcription terminator sequence. Standard techniques well known in the field of molecular biology were used to design primers and amplify specific DNA sequences from Escherichia coli K-12 DH1 chromosomal DNA. Such standard techniques, vectors and elements can be found, for example, in the manuals: Ausubel et al. (eds.), Current Protocols in Molecular Biology

- 17 046260 (1995) (John Wiley &Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (eds.).

Chromosomal DNA obtained from E. coli DH1 was used to amplify a 300 bp DNA fragment containing the PglpF promoter using oligonucleotides 0261 and 0262, and a 195 bp DNA fragment containing Plac using oligonucleotides 068 and 0113 (Table 3).

A DNA fragment of 1179 bp in length containing a codon-optimized version of the vag gene originating from Pantoea vagans was synthesized using GeneScript (Table 5). The vag gene was amplified by the PNR method using oligonucleotides KABY745 and KABY746.

All PCR fragments (plasmid backbones, promoter-containing elements, and vag gene) were purified and the plasmid backbones, promoter elements (PglpF or Plac), and vag-containing DNA fragment were assembled. Plasmids were cloned using USER standard cloning. Cloning into any suitable plasmid can be accomplished using any standard DNA cloning techniques. Plasmids were transformed into TOP10 cells and selected on LB plates containing 100 μg/ml ampicillin (or any suitable antibiotic) and 0.2% glucose. The constructed plasmids were purified, and the promoter sequence and 5′ end of the vag gene were confirmed by DNA sequencing (MWG Eurofins Genomics). In this way, a genetic cassette was constructed containing any promoter of interest associated with the vag gene.

Table 6. Examples of helper and donor plasmids used to construct strains

(ooi itcici louuiii i enoimi Marker gene pACBSR Para-I-SceI-λ Red, pl5A ori, cam * cam pUC57 pMBl, bla Bla pUC57::#a7 pUC57::galTK’ / Tl-galKM’ Bla

DNA sequences of heterologous genes encoding transporters or glycosyltransferases of interest were codon optimized and synthesized using GenScript. Genes of interest encoding transporter proteins, as shown in Table. 4 were amplified by PCR using suitable primers covering the ATG start codon and the TAA stop codon of the gene (Table 3). To construct donor plasmids with any heterologous gene of interest, standard USER cloning was used to combine purified PCR fragments of the corresponding plasmid backbone, promoter element, and gene of interest. Cloning into the appropriate plasmid can be accomplished using any standard DNA cloning technique. After cloning, DNA was transformed into TOP10 cells and selected on LB plates containing 100 μg/ml ampicillin (or 50 mg/ml kanamycin depending on the base used) and 0.2% glucose. The constructed plasmids were purified, and the promoter sequence and 5′ end of the gene of interest were confirmed by DNA sequencing (MWG Eurofins Genomics).

Strain construction

The bacterial strain used, MDO, was constructed from Escherichia coli K-12 DHL The genotype of E. coli K-12 DH1 is F-, ^λ- , gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. In addition to the E. coli K-12 genotype, DH1 MDO has the following modifications: lacZ: 1.5 kb deletion, lacA: 0.5 kb deletion, nanKETA: deletion, 3.3 kb, melA: 0.9 kb deletion, wcaJ: 0.5 kb deletion, mdoH: 0.5 k deletion .bp, and insertion of the Plac promoter upstream of the gmd gene.

Insertion of an expression cassette containing a promoter linked to the vag gene and a T1 transcription terminator sequence was accomplished using Gene Gorging essentially as described by Herring et al. (Herring, C. D., Glasner, J. D. and Blattner, F. R. (2003). 311). 153-163), and examples of helper and donor plasmids used to construct the strains presented in this application are given in table. 6. Briefly, donor plasmid and helper plasmid were co-transformed into MDO and selected on LB plates containing 0.2% glucose, ampicillin (100 μg/ml) or kanamycin (50 mg/ml) and chloramphenicol (20 μg/ml ). A single colony was inoculated in 1 ml of LB containing chloramphenicol (20 μg/ml) and 10 μl of 20% L-arabinose and incubated at 37°C with shaking for 7-8 hours. For integration into the galK loci of E. coli cells, were plated on M9-DOG plates and incubated at 37°C for 48 hours. Individual colonies formed on MM-DOG plates were streaked again onto LB plates containing 0.2% glucose and incubated for 24 hours at 37 °C. The colonies, which appeared white on MacConkey-galactose agar plates and were sensitive to both ampicillin and chloramphenicol, were expected to have lost the donor and helper plasmid and contained an insertion at the galK loci. Insertions into the galK site were identified by colony PCR using primers 048 (SEQ ID NO: 23) and 049 (SEQ ID NO: 24), and the inserted DNA was verified by sequencing (Eurofins Genomics, Germany).

The integration of genetic cassettes into other loci of E. coli chromosomal DNA was carried out in a similar way using various selection marker genes.

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Deep Well Assay (DWA)

DWA was performed as originally described by Lv et al. (Bioprocess BiosystEng (2016) 39:1737–1747) and optimized for the purposes of the present invention.

More specifically, the strains disclosed in the examples were screened in 24 deep well plates using a 4 day protocol. During the first 24 h, cells were grown to high density, and for the next 72 h, cells were transferred to a medium that allowed gene expression and product formation to be induced. Specifically, during day 1, fresh inocula were prepared using basic minimal medium supplemented with magnesium sulfate, thiamine, and glucose. After 24-hour incubation of the prepared cultures at 34°C with shaking at 700 rpm, the cells were transferred to new basal minimal medium (2 ml) supplemented with magnesium sulfate and thiamine, to which an initial bolus of 20% glucose solution (1 μl) was introduced. and 10% lactose solution (0.1 ml), then 50% sucrose solution was supplied to the cells as a carbon source. After inoculation of the new medium, the cells were shaken at 700 rpm at 28°C for 72 h. After denaturation and subsequent centrifugation, the supernatants were analyzed by HPLC.

For analysis of total samples, cell lysate prepared by boiling was pelleted by centrifugation for 10 min at 4700 rpm. The concentration of OGM in the supernatant was determined using HPLC or HPAC.

Sequence alignment

Heuristic pairwise sequence alignment implemented in BLAST 2.1.2 (Basic Local Alignment Search Tool) (Altschul et al., 1990) in the NCBI database (http://www.ncbi.nlm.nih.gov) was performed to check homology sequences of the transporter proteins mentioned in the present invention. All parameters were kept at default values for each BLAST alignment.

results

Example 1: The Vag Transporter is a New LNT-2 Core Transporter with High Selectivity for LNnT

In the present invention, we tested the ability of selected heterologous MFS transporters to export LNnT from the host cell using a reference strain, namely MP3672. The strain has one PglpF-driven copy of each heterologous glycosyltransferase required for product formation.

One copy of each of the selected heterologous transporter genes, i.e. vag, yberC0001_9420, marc, bad, dog, yabM and fred (Table 4), was individually integrated into the genome of strain MP3672 under the control of the Plac promoter, which is known to provide moderate levels of transcripts. The ribosome binding site of the Plac promoter (i.e., 16 bp upstream of the translation start site) was modified (see Table 5) to enhance ribosome binding to synthesized transcripts and thus positively regulate Plac-driven expression at the translational level .

Following the strain construction approach described above and testing strain performance in DWA, we report that only Vag-expressing LNnT-producing cells exhibit a relative increase in both the efflux of LNnT produced and total LNnT production compared to the reference strain.

As shown in FIG. 1A, Plac-driven expression of most transporter genes (marc, fred, bad, dog, yabM, yberC0001_9420) either does not have a significant effect or reduces LNnT production (up to 45%). In contrast, introduction of the vag gene into the genetic background of strain MP3672 leads to a marked increase in LNnT titer (Fig. 1A).

Amino acid sequence alignment of the seven putative MSF transporters tested showed that the Vag transporter has very high sequence coverage (99 to 100%) compared to the other six transporters tested in the present invention, with sequence identity ranging from 65 to 75%. The highest sequence identity (75%) was observed for the Vag sequence and the MFS transporter sequence encoded by the yabM gene (Table 7). Interestingly, the MFS transporter YabM, described in WO 2017042382 as a putative efficient LNT exporter, had no significant effect on either the final total LNnT titer or LNnT efflux (Figures 1A, 1B). Thus, contrary to its relatively high protein sequence similarity to Vag, YabM transporters appear to be ineffective at transporting LNnT, as clearly reflected in the product concentrations found in the extracellular fraction of the corresponding host cell cultures (Fig. 1B). In particular, as shown by analysis of supernatants of bacterial culture fractions, extracellular concentrations of LNnT were significantly higher for strain MP3994 (cells expressing vag) than for strains MP4362 (cells expressing yabM) and MP3672 (comparison cells not expressing any heterologous transporter ) (Fig. 1B).

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Table 7. Homology between various heterologous transporters ___________________of the present invention___________________

Protein name Identification From the body Identical to Vag WP_048785139J (coating) YberC0001_9420 EEQ08298.1 MFS Master Mediator Superfamily 1 Yersinia bercovieri 68% (99%) Fred WP_087817556.1 conveyor MFS Yersinia frederiksenii 69% (99%) Bad WP 017489914.1 conveyor MFS Rouxiella badensis 65% (99%) Nec WP_09267208El conveyor MFS Rosenbergiella nectarea 66% (99%) Marc WP 060448169.1 conveyor MFS Serratia marcescens 71% (99%) YabM CAY73138.1 Putative sugar efflux transporter MFS Erwinia pyrifoliae 75% (99%)

Taken together, these data suggest that the Vag transporter is an efficient transporter of LNnT. This conclusion is based both on the results of a screening procedure for these seven transporter genes, genomically expressed in the same reference strain, under the same culture conditions and under the control of the same promoter, Plac, and on analysis of the transporter protein sequences, which showed that transporter proteins with relatively high homology to Vag, such as YabM and Marc (75 and 71%), either do not export LNnT at all, or, if they do export, it is with very low efficiency.

Example 2: Engineering Escherichia coli to produce LNnT using the vag gene

Escherichia coli K-12 (DH1) MDO strains can be modified to express heterologous genes of interest. For example, strain MP3020 is an LNnT producing strain with overexpression of the β-1,3-M-acetylglucosaminyltransferase gene and the β-1,3-galactosyltransferase gene selected from the non-limiting examples in Table 1. 1.

As HPLC analysis of all samples showed, the insertion of an expression cassette containing the PglpF or Plac promoter element associated with the vag gene into the chromosomal DNA of strain MP3020 in a single copy to obtain strains MP4064 and MP4065, respectively (Table 2), led to i) more high total LNnT titers, ii) similar or slightly higher total LNT-2 concentrations, and iii) similar or significantly lower total pLNnH production (Fig. 2). Apparently, the level of expression of the transporter gene influences the formation of by-products in LNnT-producing strains: Plac-driven expression (relatively moderate) of the vag gene had a positive effect on the total titer of LNnT, which was accompanied by a slight increase in the total titers of pLNnH, but not LNT -2. PglpF-driven expression of the (relatively high) vag gene also resulted in higher LNnT titers than the reference strain, but this strain (MP4064) showed slightly higher total LNT-2 titers and markedly reduced overall pLNnH production (Fig. 2).

However, the significant effect of introducing the vag gene into the LNnT-producing strain is more clearly demonstrated when supernatant samples are analyzed. The concentration of LNnT in the supernatant fraction of cultures of strains MP4064 and MP4065 was increased by more than 2 times compared to that measured in the culture medium of MP3020 (Fig. 2). Although cells expressing the transporter do not exhibit significantly higher total titers of LNT-2, LNT-2 in the supernatant fraction appears to be higher in strains MP4064 and MP4065 than in strain MP3020. As mentioned above, pLNnH production is either slightly increased in strain MP4065 or markedly reduced in strain MP4064. Interestingly, pLNnH was detected only in the cell pellet of both strains MP4064 and MP4065, indicating that expression of the Vag transporter does not affect the export of pLNnH produced (Fig. 2).

Example 3: Engineering Escherichia coli to produce LNT using the vag gene

Escherichia coli K-12 (DH1) MDO strains can be modified to express heterologous genes of interest. For example, strain MP4473 is an LNT-producing strain with overexpression of the β-1,3-M-acetylglucosaminyltransferase gene and the β-1,3-galactosyltransferase gene selected from table. 1.

As HPLC analysis of all samples showed, the insertion of an expression cassette containing a promoter element (PglpF) associated with the vag gene into the chromosomal DNA of strain MP4473 in a single copy to obtain strain MP4546 (Table 2) led to: i) an increase in the total titer LNT (total LNT concentration nearly 50% higher than the reference strain), ii) approximately 6-fold increase in total LNT-2 concentration, and iii) approximately 2.5-fold higher total pLNH-II production (Fig. 3) .

Consistent with the above observations, analysis of supernatant samples showed that the concentration of LNT in the supernatant fraction of cultures of strain MP4546 was markedly increased compared to that measured in the culture medium of MP4473 (Fig. 3). Much higher total titer of LNT-2, from

-

Claims (13)

measured in vag-expressing cells is accompanied by a 5-fold higher concentration of LNT-2 found in the MP4546 culture supernatant fraction. In contrast, the pLNH-II produced appears to be located exclusively in the cell pellet of strain MP4546, possibly because Vag has either low or no substrate specificity for pLNH-II or is unable to transport large sugars such as hexosaccharides (Fig. 3). This lack of capability appears to be advantageous because it allows for easier downstream processing of pLNHII produced by vag-expressing cells, since several different OGMs produced in a single fermentation can be separated from the OHM mixture in the very first step of the purification procedure. CLAIM 1. A genetically modified cell capable of producing one or more human milk oligosaccharides (HMO), where said cell includes a recombinant nucleic acid encoding an MFS transporter protein with the sequence SEQ ID NO: 1 or a functional homolog thereof, the amino acid sequence of which is at least identical at 85% of the sequence SEQ ID NO: 1. 2. A genetically modified cell according to claim 1, where the oligosaccharide is selected from 2'-fucosyllactose (2'FL), 3'-fucosyllactose (3FL), difucosyllactose (DFL), 3'-sialyllactose (3'SL), 6'- sialyllactose (6'SL), lacto-H-triose-2 (LNT-2), lacto-H-neotetraose (LNnT), lacto-H-tetraose (LNT), lacto-Nfucopentaose I (LNFP-I), lacto- N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacTO-N-fucopentaose IV (LNFP-IV) and lacto-N-fucopentaose V (LNFP-V) and/or para- lacto-M-neohexaose (pLNnH). 3. The genetically modified cell as claimed in any one of the preceding claims, wherein the genetically modified cell is Escherichia coli. 4. The genetically modified cell according to any one of the preceding paragraphs, where the genetically modified cell further contains at least one recombinant nucleic acid that encodes a functional enzyme with glycosyltransferase activity selected from the group consisting of a-1,2-fucosyltransferase, a-1 ,3-fucosyltransferases, α-1,3/4fucosyltransferases, α-1,4-fucosyltransferases, α-2,3-sialyltransferases, α-2,6-sialyltransferases, β-1,3-N-acetylglucosaminyltransferases, β-1 ,6-N-acetylglucosaminyltransferase, β-1,3-galactosyltransferase and β-1,4-galactosyltransferase. 5. The genetically modified cell according to any one of the preceding claims, wherein the cell further includes a recombinant DNA sequence containing a regulatory element for regulating the expression of the recombinant nucleic acid. 6. The genetically modified cell according to claim 5, wherein the regulatory element for regulating the expression of the recombinant nucleic acid is a promoter element selected from /aspromoter or glp promoter. 7. A nucleic acid construct comprising a nucleic acid sequence encoding the MFS transporter protein of the sequence SEQ ID NO: 1 or a functional homolog thereof, having more than 85% sequence identity with the sequence SEQ ID NO: 1, where the nucleic acid sequence encoding the protein sequence of SEQ ID NO: 1, has at least 85% sequence identity with SEQ ID NO: 2 and where the construct includes a nucleic acid sequence including a regulatory element. 8. The nucleic acid construct of claim 7, wherein the regulatory element controls the expression of a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 2. 9. The nucleic acid construct according to any one of claims 7, 8, wherein the regulatory element for regulating the expression of the recombinant nucleic acid is an expression element selected from a lac promoter or a glp promoter. 10. The nucleic acid construct according to claim 7 or 8, wherein the nucleic acid construct is part of a vector DNA or expression cassette/cartridge integrated into the genome of the host cell. 11. A method for producing one or more oligosaccharides, including the stages: (i) providing a genetically modified cell capable of producing OGM according to any one of claims 1 to 6; (ii) culturing the cell in accordance with (i) in a suitable cell culture medium to express said recombinant nucleic acid; (iii) collection of one or more OGMs obtained in step (ii). 12. The method according to claim 11, where the resulting OGM is collected from the cultivation medium and microbial biomass formed during the cultivation process. 13. Use of a genetically modified cell according to any of claims 1-6 or a nucleic acid construct according to any of claims 7-10 to obtain one or more breast oligosaccharides -