WO2018073107A1 - Eukaryotic cell comprising xylose isomerase - Google Patents

Abstract

Eukaryotic cell comprising a heterologous xylose isomerase, wherein the xylose isomerase is loaded with Mn and wherein the Mn metal loading is at least 0.10 mol Mn (mol XylA subunit)^-1, preferably about 0.30 mol Mn (mol XylA subunit)^-1.

Description

EUKARYOTIC CELL COMPRISING XYLOSE ISOMERASE

Field

The present application relates to a eukaryotic cell comprising xylose isomerase.

Background

In conventional feedstocks for fermentative production of fuel ethanol, such as corn starch and cane sugar, sugars predominantly occur as dimers or polymers of hexose sugars, which can be efficiently and rapidly fermented by Saccharomyces cerevisiae. Economically feasible ethanol production from non-food lignocellulosic feedstocks additionally requires efficient, anaerobic fermentation of D-xylose and optionally L-arabinose. Although wild-type strains of S. cerevisiae cannot ferment these pentose sugars, they can slowly convert D-xylulose. A eukaryotic cell comprising xylose isomerase gene (xylA or XI) is known from WO2003062430. A eukaryotic cell comprising genes encoding L-arabinose conversion enzymes is known from WO2008051480.

Description of the Figures

Fig.1 Anaerobic growth of S. cerevisiae IMX696 (xylA, PPP†, XKS7†) on xylose requires a prolonged adaptation period, (a) (top) Growth, xylose consumption and product formation after inoculation of aerobically pre-grown cells in anaerobic bioreactors containing synthetic medium with xylose (20 g I^"1). Symbols: ·, xylose,■, glycerol, o, biomass,□, glycerol, (b) (second row) Colony-forming units (CFU) on anaerobically incubated xylose medium reflect adaptation to xylose without oxygen, (c) (third row). CFU on aerobically incubated xylose medium reflect trade-off between aerobic and anaerobic growth on xylose, (d) (fourth row) and (e) (bottom) CFU on anaerobically and aerobically incubated glucose medium, respectively, showing that oxygen sensitivity of cells adapted to anaerobic growth on xylose is not carbon-source dependent. Data shown in Figure 1 are from one of two independent replicates.

Fig. 2 Deletion of PMR1 enables anaerobic growth on xylose of engineered S. cerevisiae without prior adaptation phase. Growth and product formation of S. cerevisiae strain IMX906 (xylA, PPP†, XKS1 , pmr1 on xylose (20 g Γ¹) in anaerobic bioreactors. Symbols: ·, xylose, ■, ethanol, o, biomass,□, glycerol. The data shown are from one of two independent replicates.

Fig. 3 S. cerevisiae strains carrying mutations in PMR1 show impaired aerobic growth on xylose. Aerobic growth curves in shake-flask cultures grown on synthetic medium with 20 g I^" xylose. Symbols indicate the following S. cerevisiae strains: ·, IMX696 (xylA, PPP†, XKS1 ), u, IMX906 (xylA, PPP†, XKS1†, pmrIA), o, IMX979 (xylA, PPP†, XKS1†, PMR1), ♦, IMS0488 (isolate from IMX696 culture adapted to anaerobic growth on xylose carrying PMR1^G249V mutation) and□ IMS0489 (isolate from IMX696 culture adapted to anaerobic growth on xylose PMR1^W387* mutation). Data shown are from a single flask experiment for each strain. For all strains, data obtained from independent duplicate experiments differed by less than 5%.

Fig. 4 Off-gas CO2 profiles of anaerobic bioreactor cultures on synthetic medium with xylose (20 g I^"1). Black and grey lines indicate results from independent duplicate cultures of strain S. cerevisiae IMX696 (xylA, PPP†, XKS7†) and strain IMX979 (xylA, PPP†, XKS1†, PMR1), respectively.

Fig. 5 Growth of S. cerevisiae strains on xylose in aerobic shake-flask cultures; (a) IMX696 (xylA, PPP†, XKS7†). (b) IMS0488 (isolated from culture of strain IMX696 adapted to anaerobic growth on xylose). Both strains were grown in aerobic shake flasks on synthetic medium containing 20 g I^"1 xylose. Symbols: ·, xylose,■, ethanol and o, biomass. The data shown in the Figure are from a single shake-flask experiment of each strain. Data from duplicate experiments with each strain differed by less than 5%.

Fig. 6 Schematic overview of the integrated construct that enables xylose consumption in IMX696 (xylA, PPP†, XKS7†). The construct consists of 15 cassettes containing 60bp homologous sequences named A to Q. The fragments were transformed with pUD335 allowing for a Cas9-induced double-strand break in GRE3. Correct integration of all the fragments in GRE3 was verified by diagnostic PCR.

Fig. 7: integration of manganese transporter overexpression cassettes on INT1 locus in

IMX696.

Summary

An object of the present application is to improve the xylose conversion anaerobically of the eukaryotic cell comprising xylose isomerase. Another object of the present application is to provide a eukaryotic cell comprising xylose isomerase that has the xylose conversion without a lag phase, or without the requirement of additional mutations to start xylose conversion, in anaerobic growth on xylose. Another object is to provide a eukaryotic cell comprising xylose isomerase that has more active xylose isomerase protein. Another object is to provide a eukaryotic cell comprising xylose isomerase wherein the copy number of xylose isomerase is reduced. One or more of the above objects are attained in the present application that provides a eukaryotic cell comprising a heterologous xylose isomerase, wherein xylose isomerase is loaded with Mn2+ and wherein the Mn metal loading is at least 0.10 mol Mn (mol XylA subunit)^-1 , preferably about 0.30 mol Mn (mol XylA subunit)^-1.

Detailed description

Throughout the present specification and the accompanying claims the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. All embodiments herein may be cross-combined with eachother.

The present application provides a eukaryotic cell comprising a heterologous xylose isomerase, wherein xylose isomerase is loaded with Mn²⁺ and wherein the Mn metal loading is at least 0.10 mol Mn (mol XylA subunit)^-1 , preferably about 0.30 mol Mn (mol XylA subunit)^-1 or at least 0.20 mol Mn (mol XylA subunit) ¹ , preferably about 0.30 mol Mn (mol XylA subunit)^"1.

The "xylose isomerase is loaded with Mn" herein means that Mn²⁺ (divalent Mn²⁺ ion) is offered to the xylose isomerase (herein abbreviated as XI or XylA) in the eukaryotic cell so that it functions as the metal cofactor for the XI. Manganese is herein abbreviated as Mn, which can herein also mean Mn²⁺ or Mn²⁺. Mn metal loading is herein measured as shown in the examples, with glucose as carbon source. See for the results table 1 1.

In an embodiment, the eukaryotic cell comprising a heterologous xylose isomerase is a cell wherein one or more manganese homeostasis related gene is disrupted, or overexpressed and wherein the disruption or overexpression causes and increased cytosolic and/or intracellular Mn²⁺ concentration.

The cytosol or cytoplasmic matrix is the liquid found inside cells. It constitutes most of the intracellular fluid (ICF). It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into many compartments. In the eukaryotic cell, the cytosol is within the cell membrane and is part of the cytoplasm, which also comprises the mitochondria, plastids, and other organelles (but not their internal fluids and structures); the cell nucleus is separate. Intracellular herein means "inside the cell". It is used in contrast to extracellular (outside the cell). The cell membrane is the barrier between the two.

Manganese homeostasis: Homeostasis or homoeostasis is the property of a system in which a variable (for example, the concentration of a substance (herein Manganese) is actively regulated to remain very nearly constant. This regulation occurs inside a defined environment (mostly within a living organism's body). Manganese homeostasis related gene that code for proteins that have an influence in the regulation of the Manganese concentration in de cell. Examples of Manganese homeostasis related genes are given hereinafter.

In an embodiment, the one or more disrupted or overexpressed manganese homeostasis related gene is chosen from the group consisting of: Golgi Ca²7Mn²⁺ ATPase (PMR1 ) gene, divalent metal transporter SMF1 gene (SMF1 ), Mn transporter PH084 gene, Mn transporter ATX1 gene; divalent metal transporter SMF2 gene (SMF2); and a regulator gene which is involved in the localization of the Mn²⁺ transporters SMF1 and PH084 (such as BSD2, ECM21 , CSR2, PHO80, and PH085).

These genes are herein collectively designated as "manganese homeostasis related genes" and the corresponding proteins as "manganese homeostasis related proteins".

Mutations which may result in disruption of PMR1 include Q783A, Q783V, V335G, V335A, V335Q, Q783V, V335Q, Q783L, V335A, Q783L, V335T, Q783E, and V335A, corresponding to SEQ ID NO: 136. These mutations are preferred because they impede Mn transport but not affect Ca transport, or to a lesser extent.

In an embodiment the eukaryotic cell comprises a disruption or deletion of a regulator which is involved in the localization of the Mn²⁺ transporters SMF1 and PH084.

In an embodiment, the one or more of Golgi Ca²7Mn²⁺ ATPase (PMR1) gene is disrupted or deleted.

In an embodiment in the eukaryotic cell, one or more divalent metal transporter SMF1 gene (SMF1), and/or divalent metal transporter SMF2 gene (SMF2) is overexpressed. In an embodiment, in the eukaryotic cell PMR1 gene is deleted. In an embodiment in the eukaryotic cell PMR1 gene is disrupted and wherein the disruption is chosen from the group consisting of:

a) introduction of a premature stop codon in the PMR1 gene;

b) mutation in the PMR1 gene corresponding to amino acid change G746T or W387* in PMR1 protein;

c) expression of the PMR1 gene with a promoter that has promoter activity under aerobic condition and has substantially no promoter activity under anaerobic condition.

These manganese homeostasis related genes are now described in more detail.

High affinity Ca²7 Mn²⁺ P-type ATPase; required for Ca²⁺ and Mn²⁺ transport into Golgi; involved in Ca²⁺ dependent protein sorting, processing; D53A mutant (Mn²⁺ transporting) is rapamycin sensitive, Q783A mutant (Ca²⁺ transporting) is rapamycin resistant; Mn²⁺ transport into Golgi lumen required for rapamycin sensitivity; mutations in human homolog ATP2C1 cause acantholytic skin condition Hailey-Hailey disease; human ATP2C1 can complement yeast null mutant, e.g. YGL167C. In an embodiment Golgi Ca²7Mn²⁺ ATPase (PMR1 ) is disrupted or deleted in the eukaryotic cell.

Divalent metal ion transporter; broad specificity for di-valent and tri-valent metals; post- translationally regulated by levels of metal ions; member of the Nramp family of metal transport proteins. In an embodiment the divalent metal transporter SMF1 is overexpressed in the eukaryotic cell.

Divalent metal ion transporter involved in manganese homeostasis; has broad specificity for di-valent and tri-valent metals; post-translationally regulated by levels of metal ions; member of the Nramp family of metal transport proteins. In an embodiment the divalent metal transporter SMF2 is overexpressed in the eukaryotic cell.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences known to one of skilled in the art. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The promoter that could be used to achieve the expression of a nucleotide sequence coding for an enzyme according to the present application, may be not native to the nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. The promoter may, however, be homologous, i.e. endogenous, to the host cell.

Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art.

In an embodiment, suitable promoters in eukaryotic host cells are those having promoter activity under aerobic condition and has substantially no promoter activity under anaerobic condition, also designated herein as aerobic promoters. These promoters in herein are used as promoter for manganese homeostasis genes, for example one or more of Golgi Ca²7Mn²⁺ ATPase (PMR1) gene. Examples of such aerobic promoters are shown in table 1.

Table 1 : Suitable aerobic promoters

ORF Gene name Ratio aerobic/anaerobic

YOR388C FDH1

YPL275W

YPL276W

YDR256C CTA1

YHR096C HXT5

YNL195C

YGR1 10W

YCR010C

YDL218W

YPL223C GRE1 64

YJR095W ACR1 57

YMR303C ADH2 47

YGR236C 40

YHR139C SPS100 36

YPR151 C 31

YMR107W 23

YMR1 18C 22

YLR174W IDP2 21

YPL201 C 16

YDR380W 16

YMR058W FET3 13

YBR047W 13

YML054C CYB2 12

YLR205C 12

YPL147W PXA1 12

YDR070C 1 1

YPR001W CIT3 1 1

YER065C ICL1 1 1 ORF Gene name Ratio aerobic/anaerobic

YKR009C FOX2 1 1

YLL053C 1 1

YGR256W GND2 10

The higher the ratio aerobic/anaerobic is the better suited for this application is the promoter. Suitable other promoters than those given in table 1 , may be selected by the skilled person based on the (high) ratio aerobic/anaerobic.

The application further relates to the use of Mn²⁺ as a cofactor for xylose isomerase protein in a cell, wherein the cytosolic concentration of Mn²⁺ in the cell is from 2 to 100 nmol/10⁹ (1 ,000,000,000) cells, 2 to 50 nmol/10⁹ cells or 2 to 40 nmol /10⁹ cells, 3 to 100 nmol/10⁹ cells, 4 to 100 nmol/10⁹ cells, 5 to 100 nmol/10⁹ cells,.6 to 100 nmol/10⁹ cells, 3 to 50 nmol/10⁹ cells, 4 to 50 nmol/10⁹ cells, 5 to 50 nmol/10⁹ cells, 6 to 50 nmol/10⁹ cells, 3 to 40 nmol/10⁹ cells, 4 to 40 nmol/10⁹ cells, 5 to 40 nmol/10⁹ cells, 6 to 40 nmol/10⁹ cells. Cells may be counted with a cell counter.

The application further relates to a process for the fermentation of a substrate to produce a fermentation product with an eukaryotic cell as described herein, wherein the xylose consumption is at least 10%, at least 20%, or at least 25% increased relative to the corresponding fermentation with wild-type eukaryotic cell. In an embodiment of the process, the fermentation product is ethanol and the ethanol yield is at least about 0.5 %, or at least 1 % higher than that of a process with the corresponding wild-type eukaryotic cell. In an embodiment of the process, pentose and glucose are co-fermented. In an embodiment of the process, a hydrolysate of lignocellulosic material is fermented.

Preferably the eukaryotic cell is capable of anaerobic fermentation, more preferably anaerobic alcoholic fermentation. Hydrolysate is an enzymatic hydrolysate of lignocellulosic material. The hydrolysate may comprise Mn²⁺ or a source of Mn²⁺.

Embodiments of the application are described herein in detail.

"Eukaryotic cell" is herein defined as any eukaryotic microorganism. Eukaryotes belong to the taxon Eukarya or Eukaryota. The defining feature that sets eukaryotic cells apart from prokaryotic cells (Bacteria and Archaea) is that they have membrane-bound organelles, especially the nucleus, which contains the genetic material, and is enclosed by the nuclear envelope. The presence of a nucleus gives eukaryotes their name, which comes from the Greek ευ (EU, "well") and κάρυον (karyon, "nut" or "kernel"). Eukaryotic cells also contain other membrane-bound organelles such as mitochondria and the Golgi apparatus. Many unicellular organisms are eukaryotes, such as protozoa and fungi. All multicellular organisms are eukaryotes. Unicellular eukaryotes consist of a single cell throughout their life cycle. Microbial eukaryotes can be either haploid or diploid. The eukaryotic cell herein may be any eukaryotic microorganism, for example a yeast or a filamentous fungus.

Yeasts are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina that predominantly grow in unicellular form. Yeasts may either grow by budding of a unicellular thallus or may grow by fission of the organism. A preferred yeast as a cell of the application may belong to the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces Issatchenkia, Spathapora, Debaryomyces, Zygosaccharomyces, or Yarrowia. Preferably the yeast is one capable of anaerobic fermentation, more preferably one capable of anaerobic alcoholic fermentation.

Filamentous fungi are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina. These fungi are characterized by a vegetative mycelium composed of chitin, cellulose, and other complex polysaccharides.

The filamentous fungi of the suitable for use as a cell of the present application are morphologically, physiologically, and genetically distinct from yeasts. Filamentous fungal cells may be advantageously used since most fungi do not require sterile conditions for propagation and are insensitive to bacteriophage infections. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism of most filamentous fungi is obligately aerobic. Preferred filamentous fungi as a host cell of the application may belong to the genus Aspergillus, Trichoderma, Humicola, Acremoniurra, Fusarium or Penicillium. More preferably, the filamentous fungal cell may be a Aspergillus niger, Aspergillus oryzae, a Penicillium chrysogenum, or Rhizopus oryzae cell.

Over the years suggestions have been made for the introduction of various organisms for the production of bio-ethanol from crop sugars. In practice, however, all major bio-ethanol production processes have continued to use the yeasts of the genus Saccharomyces as ethanol producer. This is due to the many attractive features of Saccharomyces species for industrial processes, i.e. a high acid-, ethanol-and osmo- tolerance, capability of anaerobic growth, and of course its high alcoholic fermentative capacity. Preferred yeast species as host cells include S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus or K fragilis Z. rouxii, Z. baillii, S. pallisidarum, S. stipitis or Issatchenkia orientalis..

The eukaryotic cell may be able to convert plant biomass, celluloses, hemicelluloses, pectins, rhamnose, galactose, fucose, maltose, maltodextrins, ribose, ribulose, or starch, starch derivatives, sucrose, lactose and glycerol, for example into fermentable sugars. Accordingly, a cell of the application may express one or more enzymes such as a cellulase (an endocellulase or an exocellulase), a hemicellulase (an endo- or exo-xylanase or arabinase) necessary for the conversion of cellulose into glucose monomers and hemicellulose into xylose and arabinose monomers, a pectinase able to convert pectins into glucuronic acid and galacturonic acid or an amylase to convert starch into glucose monomers.

The term "gene" means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region and a 3'nontranslated sequence (3'end) comprising a polyadenylation site. "Expression of a gene" refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.

The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloned" genetically modified organisms (GMO's) (self-cloning is defined herein as in European Directive 98/81 /EC Annex II). When used to indicate the relatedness of two nucleic acid sequences the term "homologous" means that one single-stranded nucleic acid sequence may hybridize to a complementary single- stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later.

The terms "heterologous" and "exogenous" when used with respect to a nucleic acid

(DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but have been obtained from another cell or synthetically or recombinantly produced. In an embodiment, a heterologous gene may replace a homologous gene, in particular a corresponding homologous gene (expression enzyme with same function, but herein with a different co-factor, i.e. NAD⁺ dependent). Alternatively the homologous proteins, i.e. exogenous proteins, that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in gene may be modified in the cell to become NAD⁺ dependent, e.g. by one or more point mutations in the genome, e.g. with CRISPR CAS technology. Generally, though not necessarily, such nucleic acids encode the cell in which the exogenous RNA is present. Heterologous/exogenous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is herein encompassed by the term heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other. The "specific activity" of an enzyme is herein understood to mean the amount of activity of a particular enzyme per amount of total host cell protein, usually expressed in units of enzyme activity per mg total host cell protein. In the context of the present application, the specific activity of a particular enzyme may be increased or decreased as compared to the specific activity of that enzyme in an (otherwise identical) wild type host cell.

"Anaerobic conditions" or an anaerobic fermentation process is herein defined as conditions or a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors.

"Disruption" is herein understood to mean any disruption of activity, and includes, but is not limited to deletion, mutation, reduction of the affinity of the disrupted gene and expression of antisense RNA complementary to corresponding mRNA. Native in eukaryotic cell herein is understood as that the gene is present in the eukaryotic cell before the disruption. It includes the situation that the gene native in eukaryotic cell is present in a wild-type eukaryotic cell, a laboratory eukaryotic cell or an industrial eukaryotic cell. It further includes situations wherein the activity of the gene is reduced under specific condition(s) and is maintained at other specific conditions. This is herein accomplished by aerobic promoter used to promote the manganese homeostasis related gene(s).

A "xylose isomerase" (EC 5.3.1.5) is herein defined as an enzyme that catalyses the direct isomerisation of D-xylose into D-xylulose and/or vice versa. The enzyme is also known as a D-xylose ketoisomerase. A xylose isomerase herein may also be capable of catalysing the conversion between D-glucose and D-fructose (and accordingly may therefore be referred to as a glucose isomerase). A xylose isomerase herein may require a bivalent cation, such as magnesium, manganese or cobalt as a cofactor.

Accordingly, a cell of the application is capable of isomerising xylose to xylulose. The ability of isomerising xylose to xylulose is conferred on the host cell by transformation of the host cell with a nucleic acid construct comprising a nucleotide sequence encoding a defined xylose isomerase. A cell of the application isomerises xylose into xylulose by the direct isomerisation of xylose to xylulose. This is understood to mean that xylose is isomerised into xylulose in a single reaction catalysed by a xylose isomerase, as opposed to two step conversion of xylose into xylulose via a xylitol intermediate as catalysed by xylose reductase and xylitol dehydrogenase, respectively.

A (U) of xylose isomerase activity may herein be defined as the amount of enzyme producing 1 unit nmol of xylulose per minute, under conditions as described in the examples and/or by Kuyper 2003 .

In an embodiment, the cell comprised genes that express enzymes of an L-arabinose fermentation pathway. EP 49 708 discloses the construction of a L-arabinose-fermenting strain by overexpression of the L-arabinose pathway. In the pathway, the enzymes L-arabinose isomerase (araA), L-ribulokinase (araB), and L-ribulose-5-phosphate 4-epimerase (araD) are involved converting L-arabinose to L-ribulose, -L-ribulose-5-P, and D-xylulose-5-P, respectively.

In an embodiment in the eukaryotic cell PPP enzymes and xylulokinase are overexpressed.

The eukaryotic cell may contain genes of a pentose metabolic pathway non-native to the eukaryotic cell and/or that allow the eukaryotic cell to convert pentose(s). In one embodiment, the eukaryotic cell may comprise one or two or more copies of one or more xylose isomerases and/or one or two or more copies of one or more xylose reductase and xylitol dehydrogenase genes, allowing the eukaryotic cell to convert xylose. In an embodiment thereof, these genes may be integrated into the eukaryotic cell genome. In another embodiment, the eukaryotic cell comprises the genes araA, araB and araD. It is then able to ferment arabinose. In one embodiment of the application the eukaryotic cell comprises xy/A-gene, XYL1 gene and XYL2 gene and/or XKS1- gene, to allow the eukaryotic cell to ferment xylose; deletion of the aldose reductase (GRE3) gene; overexpression of PPP-genes, TALI, TKL1, RPE1 and RKI1 to allow the increase of the flux through the pentose phosphate path-way in the cell, and/or overexpression of GAL2 and/or deletion of GAL80. Thus though inclusion of the above genes, suitable pentose or other metabolic pathway(s) may be introduced in the eukaryotic cell that were non-native in the (wild type) eukaryotic cell. According to an embodiment, the following genes may be introduced in the eukaryotic cell by introduction into a host cell:

1 ) a set consisting of PPP-genes TALI, TKL1, RPE1 and RKI1, optionally under control of strong constitutive promoter;

2) a set consisting of a xy/A-gene under control of strong constitutive promoter;

3) a set comprising a XKS7-gene under control of strong constitutive promoter,

4) a set consisting of the genes araA, araB and araD under control of a strong constitutive promoter

5) deletion of an aldose reductase gene

The above cells may be constructed using known recombinant expression techniques. The co-factor modification may be effected before, simultaneous or after any of the modifications 1 )-5).

The eukaryotic cell according to the application may be subjected to evolutionary engineering to improve its properties. Evolutionary engineering processes are known processes. Evolutionary engineering is a process wherein industrially relevant phenotypes of a microorganism, herein the eukaryotic cell, can be coupled to the specific growth rate and/or the affinity for a nutrient, by a process of rationally set-up natural selection. Evolutionary Engineering is for instance described in detail in Kuijper, M, et al, FEMS Eukaryotic cell Research 5(2005) 925-934, WO2008041840 and WO20091 12472. After the evolutionary engineering the resulting pentose fermenting eukaryotic cell is isolated. The isolation may be executed in any known manner, e.g. by separation of cells from a eukaryotic cell broth used in the evolutionary engineering, for instance by taking a cell sample or by filtration or centrifugation. In an embodiment, the eukaryotic cell is marker-free. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. Marker-free means that markers are essentially absent in the eukaryotic cell. Being marker-free is particularly advantageous when antibiotic markers have been used in construction of the eukaryotic cell and are removed thereafter. Removal of markers may be done using any suitable prior art technique, e.g. intramolecular recombination.

In one embodiment, the industrial eukaryotic cell is constructed on the basis of an inhibitor tolerant host cell, wherein the construction is conducted as described hereinafter. Inhibitor tolerant host cells may be selected by screening strains for growth on inhibitors containing materials.

The eukaryotic cell further may comprise those enzymatic activities required for conversion of pyruvate to a desired fermentation product, such as ethanol, butanol (e.g. n- butanol, 2-butanol and isobutanol), lactic acid, 3 -hydroxy- propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid, itaconic acid, an amino acid, 1 ,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic or a cephalosporin.

In an embodiment, the eukaryotic cell is derived from an industrial eukaryotic cell. An industrial cell and industrial eukaryotic cell may be defined as follows. The living environments of (eukaryotic cell) cells in industrial processes are significantly different from that in the laboratory. Industrial eukaryotic cells must be able to perform well under multiple environmental conditions which may vary during the process. Such variations include change in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have potential impact on the cellular growth and ethanol production of Saccharomyces cerevisiae. Under adverse industrial conditions, the environmental tolerant strains should allow robust growth and production. Industrial eukaryotic cell strains are generally more robust towards these changes in environmental conditions which may occur in the applications they are used, such as in the baking industry, brewing industry, wine making and the biofuel ethanol industry. In one embodiment, the industrial eukaryotic cell is constructed on the basis of an industrial host cell, wherein the construction is conducted as described hereinafter. Examples of industrial eukaryotic cell (S. cerevisiae) are Ethanol Red® (Fermentis) Fermiol® (DSM) and Thermosacc® (Lallemand).

The eukaryotic cells according to the application are preferably inhibitor tolerant, i.e. they can withstand common inhibitors at the level that they typically have with common pretreatment and hydrolysis conditions, so that the eukaryotic cells can find broad application, i.e. it has high applicability for different feedstock, different pretreatment methods and different hydrolysis conditions. In an embodiment the eukaryotic cell is inhibitor tolerant. Inhibitor tolerance is resistance to inhibiting compounds. The presence and level of inhibitory compounds in lignocellulose may vary widely with variation of feedstock, pretreatment method hydrolysis process. Examples of categories of inhibitors are carboxylic acids, furans and/or phenolic compounds. Examples of carboxylic acids are lactic acid, acetic acid or formic acid. Examples of furans are furfural and hydroxy- methylfurfural. Examples or phenolic compounds are vannilin, syringic acid, ferulic acid and coumaric acid. The typical amounts of inhibitors are for carboxylic acids: several grams per liter, up to 20 grams per liter or more, depending on the feedstock, the pretreatment and the hydrolysis conditions. For furans: several hundreds of milligrams per liter up to several grams per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions. For phenolics: several tens of milligrams per liter, up to a gram per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions.

In an embodiment, the eukaryotic cell is a cell that is naturally capable of alcoholic fermentation, preferably, anaerobic alcoholic fermentation. A eukaryotic cell preferably has a high tolerance to ethanol, a high tolerance to low pH (i.e. capable of growth at a pH lower than about 5, about 4, about 3, or about 2.5) and towards organic and/or a high tolerance to elevated temperatures.

Further the application relates to a process for the fermentation of a substrate to produce a fermentation product with an eukaryotic cell as described herein, in the wine industry, wherein the glycerol yield is at least 5%, at least 10% or at least 10%, at least 20% or at least 30% higher than that of a process with the corresponding wild-type eukaryotic cell. In an embodiment of such process, the ethanol yield is not increased or decreased, compared to that of a process with the corresponding wild-type eukaryotic cell.

Any of the above characteristics or activities of a eukaryotic cell may be naturally present in the cell or may be introduced or modified by genetic modification.

The eukaryotic cell is a recombinant cell. That is to say, a eukaryotic cell comprises, or is transformed with or is genetically modified with a nucleotide sequence that does not naturally occur in the cell in question.

Techniques for the recombinant expression of enzymes in a cell, as well as for the additional genetic modifications of a eukaryotic cell are well known to those skilled in the art. Typically such techniques involve transformation of a cell with nucleic acid construct comprising the relevant sequence. Such methods are, for example, known from standard handbooks. Methods for transformation and genetic modification of fungal host cells are known from e.g. EP- A-0635 574, W098/46772, WO99/60102, WO00/37671 , WO90/14423, EP-A-0481008, EP-A- 0635574 and US6,265, 186.

Over the years suggestions have been made for the introduction of various organisms for the production of bio-ethanol from crop sugars. In practice, however, all major bio-ethanol production processes have continued to use the eukaryotic cells of the genus Saccharomyces as ethanol producer. This is due to the many attractive features of Saccharomyces species for industrial processes, i.e. a high acid, ethanol and osmo tolerance, capability of anaerobic growth, and of course its high alcoholic fermentative capacity. Preferred eukaryotic cell species as host cells include S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus or K. fragilis.

A eukaryotic cell may be a cell suitable for the production of ethanol. A eukaryotic cell may, however, be suitable for the production of fermentation products other than ethanol Such non-ethanolic fermentation products include in principle any bulk or fine chemical that is producible by a eukaryotic microorganism such as a eukaryotic cell or a filamentous fungus.

A preferred eukaryotic cell for production of non-ethanolic fermentation products is a host cell that contains a genetic modification that results in decreased alcohol dehydrogenase activity.

Lignocellulose, which may be considered as a potential renewable feedstock, generally comprises the polysaccharides cellulose (glucans) and hemicelluloses (xylans, heteroxylans and xyloglucans). In addition, some hemicellulose may be present as glucomannans, for example in wood-derived feedstocks. The enzymatic hydrolysis of these polysaccharides to soluble sugars, including both monomers and multimers, for example glucose, cellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, galacturonic acid, glucoronic acid and other hexoses and pentoses occurs under the action of different enzymes acting in concert.

In addition, pectins and other pectic substances such as arabinans may make up considerably proportion of the dry mass of typically cell walls from non-woody plant tissues (about a quarter to half of dry mass may be pectins).

Before enzymatic treatment, the lignocellulosic material may be pre-treated. The pretreatment may comprise exposing the lignocellulosic material to an acid, a base, a solvent, heat, a peroxide, ozone, mechanical shredding, grinding, milling or rapid depressurization, or a combination of any two or more thereof. This chemical pretreatment is often combined with heat- pretreatment, e.g. between 150-220°C for 1 to 30 minutes.

The pre-treated material is commonly subjected to enzymatic hydrolysis to release sugars that may be fermented according to the application. This may be executed with conventional methods, e.g. contacting with cellulases, for instance cellobiohydrolase(s), endoglucanase(s), beta-glucosidase(s) and optionally other enzymes, The conversion with the cellulases may be executed at ambient temperatures or at higher temperatures, at a reaction time to release sufficient amounts of sugar(s). The result of the enzymatic hydrolysis is hydrolysis product comprising C5/C6 sugars, herein designated as the sugar composition.

A sugar composition used according to the application comprises glucose and one or more pentose, e.g. arabinose and/or xylose. Any sugar composition may be used in the application that suffices those criteria. Optional sugars in the sugar composition are galactose and mannose. In a preferred embodiment, the sugar composition is a hydrolysate of one or more lignocellulosic material. Lignocelllulose herein includes hemicellulose and hemicellulose parts of biomass. Also lignocellulose includes lignocellulosic fractions of biomass. Suitable lignocellulosic materials may be found in the following list: orchard primings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, switch grass, miscanthus, sweet sorghum, canola stems, soybean stems, prairie grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, softwood, hardwood, poplar, pine, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn hobs, corn kernel, fiber from kernels, products and byproducts from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid waste, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, an energy crop, forest, a fruit, a flower, a grain, a grass, a herbaceous crop, a leaf, bark, a needle, a log, a root, a sapling, a shrub, switch grass, a tree, a vegetable, fruit peel, a vine, sugar beet pulp, wheat midlings, oat hulls, hard or soft wood, organic waste material generated from an agricultural process, forestry wood waste, or a combination of any two or more thereof.

In these lignocelluloses a high amount of sugar is present in the form of glucose, xylose, arabinose and galactose. The conversion of glucose, xylose, arabinose and galactose to fermentation product is thus of great economic importance. Also mannose is present in some lignocellulose materials be it usually in lower amounts than the previously mentioned sugars. Advantageously therefore also mannose is converted by the eukaryotic cell.

It is expected that eukaryotic cells of the present application can be further manipulated to achieve other desirable characteristics, or even higher overall ethanol yields.

Selection of improved eukaryotic cells by passaging the eukaryotic cells on medium containing hydrolysate has resulted in improved eukaryotic cell with enhanced fermentation rates. Using the teachings of the present application, one could readily such improved strains.

By pentose-containing material, it is meant any medium comprising pentose, whether liquid or solid. Suitable pentose-containing materials include hydrolysates of polysaccharide or lignocellulosic biomass such as corn hulls, wood, paper, agricultural by-products, and the like.

By a "hydrolysate" as used herein, it is meant a polysaccharide that has been depolymerized through the addition of water to form mono and oligosaccharide sugars. Hydrolysates may be produced by enzymatic or acid hydrolysis of the polysaccharide-containing material.

Preferably, the eukaryotic cell is able to grow under conditions similar to those found in industrial sources of pentose. The method of the present application would be most economical when the pentose-containing material can be inoculated with the eukaryotic cell variant without excessive manipulation. By way of example, the pulping industry generates large amounts of cellulosic waste. Saccharification of the cellulose by acid hydrolysis yields hexoses and pentoses that can be used in fermentation reactions. However, the hydrolysate or sulphite liquor contains high concentrations of sulphite and phenolic inhibitors naturally present in the wood which inhibit or prevent the growth of most organisms. The examples below describe the fermentation of pentose in acid hydrolysates (or sulphite waste liquor) of hard woods and soft woods by the eukaryotic cells of the present application. It is reasonably expected that eukaryotic cell strains capable of growing in sulphite waste liquor could grow be expected grow in virtually any other biomass hydrolysate.

The application further relates to a process for aerobic propagation of the acetate consuming eukaryotic cell, in particular aerobic propagation of the eukaryotic cell strain. Propagation is herein any process of eukaryotic cell growth that leads to increase of an initial eukaryotic cell population. Main purpose of propagation is to increase a eukaryotic cell population using the eukaryotic cell's natural reproduction capabilities as living organisms. There may be other reasons for propagation, for instance, in case dry eukaryotic cell is used, propagation is used to rehydrate and condition the eukaryotic cell, before it is grown. Fresh eukaryotic cell, whether active dried eukaryotic cell or wet cake may be added to start the propagation directly.

The conditions of propagation are critical for optimal eukaryotic cell production and subsequent fermentation, such as for example fermentation of lignocellulosic hydrolysate into ethanol. They include adequate carbon source, aeration, temperature and nutrient additions. Tank size for propagation and is normally between 2 percent and 5 percent of the (lignocellulosic hydrolysate to ethanol) fermentor size.

In the propagation the eukaryotic cell needs a source of carbon. The source of carbon may herein comprise glycerol, ethanol, acetate and/or sugars (C6 and C5 sugars). Other carbon sources may also be used. The carbon source is needed for cell wall biosynthesis and protein and energy production.

Propagation is an aerobic process, thus the propagation tank must be properly aerated to maintain a certain level of dissolved oxygen. Adequate aeration is commonly achieved by air inductors installed on the piping going into the propagation tank that pull air into the propagation mix as the tank fills and during recirculation. The capacity for the propagation mix to retain dissolved oxygen is a function of the amount of air added and the consistency of the mix, which is why water is often added at a ratio of between 50:50 to 90: 10 mash to water. "Thick" propagation mixes (80:20 mash-to-water ratio and higher) often require the addition of compressed air to make up for the lowered capacity for retaining dissolved oxygen. The amount of dissolved oxygen in the propagation mix is also a function of bubble size, so some ethanol plants add air through spargers that produce smaller bubbles compared to air inductors. Along with lower glucose, adequate aeration is important to promote aerobic respiration, which differs from the comparably anaerobic environment of fermentation. One sign of inadequate aeration or high glucose concentrations is increased ethanol production in the propagation tank.

Generally during propagation, eukaryotic cell requires a comfortable temperature for growth and metabolism, for instance the temperature in the propagation reactor is between 25- 40 degrees Celcius. Generally lower temperatures result in slower metabolism and reduced reproduction, while higher temperatures can cause production of stress compounds and reduced reproduction. In an embodiment the propagation tanks are indoors and protected from the insult of high summer or low winter temperatures, so that maintaining optimum temperatures of between within the range of 30-35 degrees C is usually not a problem.

Further propagation may be conducted as propagation of eukaryotic cell is normally conducted. The application relates to a process for the fermentation of a eukaryotic cell according to the application, wherein there is an improved yield of glycerol, which is advantageous in the wine industry. It also may result in increased reduction of acetate level and/or increased yield of fermentation product, e.g. ethanol, which is advantageous in the biofuel industry.

In an embodiment, the eukaryotic cell according to the application may be a pentose and glucose fermenting eukaryotic cell, including but not limited to such cells that are capable of anaerobic simultaneous pentose and glucose consumption. In an embodiment of the process the pentose-containing material comprises a hydrolysate of ligno-cellulosic material. The hydrolysate may be an enzymatic hydrolysate of ligno-cellulosic material.

The fermentation process may be an aerobic or an anaerobic fermentation process. An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than about 5, about 2.5 or about 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors. In the absence of oxygen, NADH produced in glycolysis and biomass formation, cannot be oxidised by oxidative phosphorylation. To solve this problem many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor thereby regenerating NAD⁺.

Thus, in a preferred anaerobic fermentation process pyruvate is used as an electron (and hydrogen acceptor) and is reduced to fermentation products such as ethanol, butanol, lactic acid, 3 -hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, malic acid, fumaric acid, an amino acid and ethylene.

The fermentation process is preferably run at a temperature that is optimal for the cell. Thus, for most eukaryotic cells or fungal host cells, the fermentation process is performed at a temperature which is less than about 50°C, less than about 42°C, or less than about 38°C. For eukaryotic cell or filamentous fungal host cells, the fermentation process is preferably performed at a temperature which is lower than about 35, about 33, about 30 or about 28°C and at a temperature which is higher than about 20, about 22, or about 25°C.

The ethanol yield on xylose and/or glucose in the process preferably is at least about 50, about 60, about 70, about 80, about 90, about 95 or about 98%. The ethanol yield is herein defined as a percentage of the theoretical maximum yield.

The application also relates to a process for producing a fermentation product.

The fermentation process according to the present application may be run under aerobic and anaerobic conditions. In an embodiment, the process is carried out under micro-aerophilic or oxygen limited conditions.

An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than about 5, about 2.5 or about 1 mmol/L/h, and wherein organic molecules serve as both electron donor and electron acceptors. An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gas flow as well as the actual mixing/mass transfer properties of the fermentation equipment used. Preferably, in a process under oxygen-limited conditions, the rate of oxygen consumption is at least about 5.5, more preferably at least about 6, such as at least 7 mmol/L/h. A process of the application may comprise recovery of the fermentation product.

In a preferred process the cell ferments both the xylose and glucose, preferably simultaneously in which case preferably a cell is used which is insensitive to glucose repression to prevent diauxic growth. In addition to a source of xylose (and glucose) as carbon source, the fermentation medium will further comprise the appropriate ingredient required for growth of the cell. Compositions of fermentation media for growth of microorganisms such as eukaryotic cells are well known in the art

The fermentation processes may be carried out in batch, fed-batch or continuous mode. A separate hydrolysis and fermentation (SHF) process or a simultaneous saccharification and fermentation (SSF) process may also be applied. A combination of these fermentation process modes may also be possible for optimal productivity. These processes are described hereafter in more detail.

For Simultaneous Saccharification and Fermentation (SSF) mode, the reaction time for liquefaction/hydrolysis or presaccharification step is dependent on the time to realize a desired yield, i.e. cellulose to glucose conversion yield. Such yield is preferably as high as possible, preferably 60% or more, 65% or more, 70% or more, 75% or more 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, even 99.5% or more or 99.9% or more.

According to the application very high sugar concentrations in SHF mode and very high product concentrations (e.g. ethanol) in SSF mode are realized. In SHF operation the glucose concentration is 25g/L or more, 30 g/L or more, 35g/L or more, 40 g/L or more, 45 g/L or more, 50 g/L or more, 55 g/L or more, 60 g/L or more, 65 g/L or more, 70 g/L or more , 75 g/L or more, 80 g/L or more, 85 g/L or more, 90 g/L or more, 95 g/L or more, 100 g/L or more, 1 10 g/L or more, 120g/L or more or may e.g. be 25g/L-250 g/L, 30gl/L-200g/L, 40g/L-200 g/L, 50g/L-200g/L, 60g/L- 200g/L, 70g/L-200g/L, 80g/L-200g/L, 90 g/L-200g/L.

In SSF operation, the product concentration (g/L) is dependent on the amount of glucose produced, but this is not visible since sugars are converted to product in the SSF, and product concentrations can be related to underlying glucose concentration by multiplication with the theoretical maximum yield (Yps max in gr product per gram glucose)

The theoretical maximum yield (Yps max in gr product per gram glucose) of a fermentation product can be derived from textbook biochemistry. For ethanol, 1 mole of glucose (180 gr) yields according to normal glycolysis fermentation pathway in eukaryotic cell 2 moles of ethanol (=2x46 = 92 gr ethanol. The theoretical maximum yield of ethanol on glucose is therefore 92/180 = 0.51 1 gr ethanol/gr glucose. For Butanol (MW 74 gr/mole) or iso butanol, the theoretical maximum yield is 1 mole of butanol per mole of glucose. So Yps max for (iso-)butanol = 74/180 = 0.41 1 gr (iso-)butanol/gr glucose. For lactic acid the fermentation yield for homolactic fermentation is 2 moles of lactic acid (MW = 90 gr/mole) per mole of glucose. According to this stoichiometry, the Yps max = 1 gr lactic acid/gr glucose. Similar calculation may be made for C5/C6 fermentations, in which in addition to glucose also pentoses are included e.g. xylose and/or arabinose. For other fermentation products a similar calculation may be made.

In SSF operation the product concentration is 25g * Yps g/L /L or more, 30 * Yps g/L or more, 35g * Yps /L or more, 40 * Yps g/L or more, 45 * Yps g/L or more, 50 * Yps g/L or more, 55 * Yps g/L or more, 60 * Yps g/L or more, 65 * Yps g/L or more, 70 * Yps g/L or more , 75 * Yps g/L or more, 80 * Yps g/L or more, 85 * Yps g/L or more, 90 * Yps g/L or more, 95 * Yps g/L or more, 100 * Yps g/L or more, 1 10 * Yps g/L or more, 120g/L * Yps or more or may e.g. be 25 * Yps g/L-250 * Yps g/L, 30 * Yps gl/L-200 * Yps g/L, 40 * Yps g/L-200 * Yps g/L, 50 * Yps g/L-200 * Yps g/L, 60 * Yps g/L-200 * Yps g/L, 70 * Yps g/L-200 * Yps g/L, 80 * Yps g/L-200 * Yps g/L, 90 * Yps g/L , 80 * Yps g/L-200 * Yps g/L

Accordingly, the application provides a method for the preparation of a fermentation product, which method comprises:

a. degrading lignocellulose using a method as described herein; and

b. fermenting the resulting material,

thereby to prepare a fermentation product.

The fermentation product of the application may be any useful product. In one embodiment, it is a product selected from the group consisting of ethanol, n-butanol, 2-butanol, isobutanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, fumaric acid, malic acid, itaconic acid, maleic acid, citric acid, adipic acid, an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid, 1 ,3-propane-diol, ethylene, glycerol, a β- lactam antibiotic and a cephalosporin, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, including biofuels and biogas or organic polymers, and an industrial enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductases, a transferase or a xylanase.

For the recovery of the fermentation product existing technologies are used. For different fermentation products different recovery processes are appropriate. Existing methods of recovering ethanol from aqueous mixtures commonly use fractionation and adsorption techniques. For example, a beer still can be used to process a fermented product, which contains ethanol in an aqueous mixture, to produce an enriched ethanol-containing mixture that is then subjected to fractionation (e.g., fractional distillation or other like techniques). Next, the fractions containing the highest concentrations of ethanol can be passed through an adsorber to remove most, if not all, of the remaining water from the ethanol. In an embodiment in addition to the recovery of fermentation product, the yeast may be recycled.

The following non-limiting examples are intended to be purely illustrative. Examples

All S. cerevisiae strains used in this study (Table 2) originate from the CEN.PK lineage. Frozen stock cultures were stored at -80 °C in 30% (vol/vol) glycerol. Plasmids used in this study are presented in Table 3. Expression cassettes required for xylose fermentation were introduced into the GRE3 locus of S. cerevisiae strain IMX581 by simultaneous in-vivo assembly and integration. Expression cassettes for RPE1 , RKI1 , TAL1 , NQM1 , TKL1 , TKL2 and XKS1 were obtained by fusing constitutive promoter sequences, ORFs and terminator sequences amplified from CEN.PK1 13-7D in a fusion-PCR57 using the primers specified as indicated. Plasmid pYM- N1858 was used as a template for the TEF1 promoter. The resulting fragments were cloned into pJET-1.2 blunt-end vectors. Correct assembly was verified by sequencing as described below. PCR amplification of expression cassettes and plasmids was performed using Phusion Hot Start II High Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA), according to the manufacturer's protocol. Integration in GRE3 locus was mediated by a chimeric CRISPR/Cas9 editing system with gRNA expressed from an episomal plasmid. The plasmid backbone was PCR amplified from pMEL.10 using primers 5792-5980. A plasmid insert containing the 20bp gRNA- targeting sequence was obtained by PCR amplification with primers 5978-5979 using pMEL.10 as template. The resulting fragment was fused to the plasmid backbone with the Gibson Assembly Cloning kit (New England Biolabs, Ipswich, MA), yielding plasmid pUDE335. E. coli DH5a cells were transformed with 1 μΙ_ of the Gibson-assembly mix using a Gene PulserXcell Electroporation System (Biorad, Hercules, CA). Plasmid DNA was isolated from E. coli cultures using a Sigma GenElute Plasmid kit (Sigma-Aldrich, St. Louis, MO). The presence of the GRE3 cutting gRNA was confirmed by PCR-amplification using primer pair 2528-960 followed by digestion with FastDigest Clal (Thermo Scientific). The coding region of the Piromyces sp. E2 xylose isomerase gene [Genbank: CAB76571.1] was codon optimized according to the codon preference of highly expressed glycolytic genes. The codon-optimized sequence, flanked by the constitutive TPI 1 promoter and CYC1 terminator, was synthesized by GeneArt GmbH (Regensburg, Germany). After subsequent transformation of the pMK-RQ (GeneArt) based vector pUDR350 into E. coli, nine different expression cassettes of xylA were made, flanked by 60bp synthetic recombinant sequences (Fig. 6). Yeast transformation was performed using the lithium acetate protocol. Strain IMX696 was obtained by adding 200 pmol of each of the 15 fragments combined with 500 ng of plasmid pUDE335. After one hour of incubation in synthetic medium with glucose (SMD) the cells were plated on SM plates with xylose as the carbon source (SMX). Correct assembly of all fragments in the GRE3 locus was confirmed by diagnostic PCR (Dreamtaq, Thermo Scientific) using primers as indicated. Deletion of PMR1 in S. cerevisiae strains IMX696 and CEN.PK1 13- 7D was done by integrating an amdSYM based deletion cassette, which was derived by PCR amplification from pUG-amdSYM using primers 8638/8639 as template. After transformation, cells were plated on glucose synthetic medium with acetamide as the nitrogen source (SMD-Ac). Gene deletion was confirmed by diagnostic PCR and the resulting strains were named IMX906 and IMK692, respectively. To reintegrate PMR1 , the PMR1 ORF was PCR-amplified from CEN.PK1 13-7D and transformed into strain IMX906. After overnight incubation in SMD-Ac, cells were plated on SMD plates supplemented with 2.3 g 1-1 fluoroacetamide (SMD-Fac). Correct integration of PMR1 in the resulting strain, IMX969, was confirmed by diagnostic PCR. Cultivation and media. Shake-flask cultures were grown at 30°C in an orbital shaker at 200 rpm, using 500- ml flasks containing 100 ml medium. Physiological characterization of aerobic growth was performed in shake flasks containing SMX with urea as sole nitrogen source to prevent acidification. Prior to filter sterilization, media were adjusted to pH 5.0 with 2 M KOH. For pre- cultures, SM adjusted to pH 6.0 was autoclaved at 120°C for 20 min after which a 50 w/v % solution of sterile glucose or xylose was added to obtain a final sugar concentration of 20 g 1-1 , together with filter-sterilized vitamin solution 62. Glucose and xylose solutions were autoclaved separately (20 min at 1 10°C). For plates, 2% agar was added to media prior to autoclaving. Frozen stocks (1 ml aliquots in 30 % glycerol) were inoculated directly into pre-culture shake flasks. In late exponential phase an aliquot was transferred to a second pre-culture to obtain an initial OD660 of 0.1. Flasks or anaerobic bioreactors used for characterization were inoculated from these cultures at an initial OD660 of between 0.1 and 0.2. Anaerobic batch cultures were conducted in 2-I bioreactors (Applikon, Delft, The Netherlands) with a working volume of 1 I. Biomass for metal content analysis was grown in 3-I bioreactors (Applikon) with a working volume of 2 I were used. Bioreactor cultures were grown at 30°C, pH 5.0, and stirred at 800 rpm. To ensure anaerobic conditions, bioreactors were equipped with Viton O-rings and Norprene tubing. During cultivation, nitrogen gas (<10 ppm oxygen) was continuously sparged through the cultures at 0.5 I min-1. After autoclaving, synthetic medium used for anaerobic cultivation was supplemented with 0.2 g 1-1 sterile antifoam C (Sigma-Aldrich), as well as Tween 80 (420 mg 1-1 ) and ergosterol (10 mg 1-1 ) dissolved in ethanol.

Cell dry weight (CDW) measurements were done using pre-weighed nitrocellulose filters

(pore size, 0.45 μιτι; Gelman Laboratory, Ann Arbor, Ml) to filter 10 ml of culture. Before weighing the sample, filters were washed with demineralised water and dried in a microwave oven (Bosch, Stuttgart, Germany) for 20 min at 360 W. Growth was monitored by optical density (OD) measurements at a wavelength of 660 nm using a Libra S1 1 spectrophotometer (Biochrom, Cambridge, United Kingdom). A correlation between OD measurements and CDW was used to estimate CDW in samples for which no direct CDW measurements were taken. This correlation was based on at least six points during the exponential phase.

CO2 and O2 concentrations in bioreactor exhaust gas were measured using an NGA 2000 analyzer (Rosemount Analytical, Orrville, OH) after the gas was cooled by a condenser (2°C) and dried with a Permapure type MD-1 10-48P-4 dryer (Permapure, Toms River, NJ). Metabolite levels in culture supernatants obtained by centrifugation were measured via high-performance liquid chromatography (HPLC) analysis on an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA) fitted with a Bio-Rad HPX 87H column (Bio-Rad, Hercules, CA). The column was eluted at 60°C with 0.5 g 1-1 H2S04 at a flow rate of 0.6 ml min-1. Detection was by means of an Agilent refractive-index detector and an Agilent 1260 WD detector. Correction for ethanol evaporation were done for all bioreactor experiments as described previously.

Viability of strain IMX696 during anaerobic cultivation was assessed by plating culture samples. The number of cells per ml was measured using a Z2 Coulter Counter (Beckman Coulter, Woerden, The Netherlands) after which dilutions were plated in duplicate on SMX and SMG agar plates and incubated at 30°C. To limit exposure to oxygen, cells that were used to determine anaerobic viability measurements were sampled directly into a container flushed with argon and immediately transferred into an anaerobic chamber (5% H2, 6% C02, and 89% N2, Sheldon MFG Inc., Cornelius, OR) for plating and incubation. Colony-forming units (CFU) were counted after incubation at 30 °C for 4 days (aerobic growth) or 8 days (anaerobic growth).

DNA sequencing and sequencing analysis. Genomic DNA of strains IMX696, IMS0488 and IMS0489 was isolated using the QIAGEN Blood & Cell Culture DNA Kit with 100/G Genomics- tips (QIAGEN, Valencia, CA) according to the manufacturer's protocol. From these DNA samples, 350-bp insert libraries were constructed using the Nextera XT DNA kit (lllumina, San Diego, CA). Paired-end sequencing (100-bp reads) of genomic or plasmid DNA was performed with an lllumina HiSeq 2500 sequencer (Baseclear BV, Leiden, The Netherlands). Data were mapped to the CEN.PK1 13-7D genome or to in silico-generated plasmid sequences using the Burrows- Wheeler alignment tool and processed with Pilon. Identified single-nucleotide differences were inspected with the Integrated Genomics Viewer (IGV). The chromosomal copy number variance (CNV) was estimated using the Poisson mixture model based algorithm Magnolya. The copy number of xylA was estimated by comparing the read depth to the average read depth of all chromosomes.

Purification of xylose isomerase. Cell pellets were resuspended in 10 mM MOPS, pH 7.0, containing protease inhibitors (cOmplete ULTRA tablets, Roche) and disrupted using a high pressure homogenizer (Constant Systems Ltd, Low March, United Kingdom). Samples were passed through the apparatus twice at 39 kpsi and cell debris was removed by centrifugation at 35,000 x g for 45 min at 4 ^°C. A single-step purification procedure based on anion-exchange chromatography was applied to minimize the loss of protein-bound metals. Cell-free extracts were loaded on a strong anion-exchange column (Resource Q, GE Healthcare, Chicago, IL) equilibrated with 10 mM MOPS, pH 7.0. A gradient elution was applied using 10 mM MOPS, pH 7.0, containing 0 - 200 mM KCI. XylA eluted at approximately 40 mM KCI. Protein concentrations were determined using the theoretical extinction coefficient at 280 nm (ε280, XI = 73,800 M-1 cm-1 ) calculated by the ProtParam tool (http://web.expasy.org/protparam/).

Metal content analysis. Metal concentrations were analysed with an inductively coupled plasma mass spectrometer (ICP-MS, Varian 820). All measurements were performed 5 times for each sample and yttrium was used as an internal standard. Purified protein samples were lyophilized and analysed for contents of magnesium, calcium, iron and manganese. Prior to measurement, samples were dissolved in 1 % nitric acid solution. All analyses were performed on protein samples isolated from two replicate cultures. For intracellular metal analysis, cells were prepared with a protocol adopted from Eide et al (2005). The harvested cells (wet weight of 320 - 560 mg) were washed three times each with 1 mM EDTA solution and subsequently with deionized water (Milli-Q) and suspended in 1 ml 30 % (w/v) nitric acid and incubated at 60 ^°C for 4 h. Cell lysates were centrifuged at 16,000 x g and supernatants were collected. Pellets were washed with 1 ml deionized water and the supernatants were collected as before. The 2 ml of final sample solution containing approximately 15 % (w/v) nitric acid were then subjected to the measurements. The metal content was determined with samples from two separate batch cultures, averaged and converted to nmoles of metal per 10⁹ cells.

In order to determine the XI activity in the presence of different metal cofactors, samples of apo-XylA were prepared by overnight incubation of the purified enzyme with 10 mM EDTA . Subsequently, EDTA was removed by buffer exchange to 20 mM MOPS, pH 7.0. XI activities in the presence of different metals were measured by a coupled enzyme assay using D-sorbitol dehydrogenase70. D-sorbitol dehydrogenase (SDH) was obtained from Roche Diagnostics GmbH (Mannheim, Germany). The reactions were performed at 30 ^°C and pH 7.0 (20 mM MOPS) in 96-well microtiter plates and the decrease in absorbance at 340 nm was measured for 20 min using Synergy Mx microtiter plate reader (BioTek Instruments, Inc., Winooski, VT). For kinetic measurements, 200 μΙ reaction mixtures contained 1 mM of divalent metal solutions (MgCI2, MnCI2 or CaCI2), 250 μΜ NADH, approximately 1 U ml-1 of SDH and D-xylose at concentrations ranging from 0.5 mM to 1 .50 M. One unit of SDH activity was defined as the amount of enzyme required for the conversion of 1 μιηοΐβ of fructose per minute. SDH was added in excess (-30- fold higher in units) to assure that the xylose isomerase reaction was always the rate determining step. Addition of 0.03 - 1 μΜ (depending on the substrate concentration and the metal added) xylose isomerase into the mixtures initiated the reaction.

Table 2: Saccharomyces cerevisiae strains used herein

Strain Relevant genotype/description Reference

Native gene terminator sequences were used for expression of RPE1 , TKL1 , TAL1 , NQM1 , RKI1, TKL2and XKS1.

Table 3

Table 4. Construction of cassettes containing constitutively expressed-pentose phosphate-pathway and XKS1 genes

Primer Purpose SEQID

5924 PGM promoter fragment CEN.PK113-7D 1

5925 PGM promoter fragment CEN.PK113-7D 2

5926 NQM1 ORF fragment CEN.PK113-7D 3

5927 NQM1 ORF fragment CEN.PK113-7D 4

3847 fusion-PCRof pPGM and NQM1 pPGM + NQM1 fragment 5

3276 fusion-PCRof pPGM and NQM1 pPGM + NQM1 fragment 6

5928 TPI1 promoter fragment CEN.PK113-7D 7

5929 TPI1 promoter fragment CEN.PK113-7D 8

5930 RKI1 ORF fragment CEN.PK113-7D 9

5931 RKI1 ORF fragment CEN.PK113-7D 10

4672 fusion-PCR of pTPM and RKI1 pTPI1+ RKI1 fragment 11

3277 fusion-PCR of pTPM and RKI1 pTPI1+ RKI1 fragment 12

5932 PYK1 promoter fragment CEN.PK113-7D 13

5933 PYK1 promoter fragment CEN.PK113-7D 14

5934 TKL2 ORF fragment CEN.PK113-7D 15

5935 TKL2 ORF fragment CEN.PK113-7D 16

3283 fusion-PCR of pPYK1 and TKL2 pPYK1 + TKL2 fragment 17

3288 fusion-PCR of pPYK1 and TKL2 pPYK1 + TKL2 fragment 18

5912 TDH3 promoter fragment CEN.PK113-7D 19

5913 TDH3 promoter fragment CEN.PK113-7D 20

5914 RPE1 ORF fragment CEN.PK113-7D 21 Primer Purpose SEQ ID

5915 RPE1 ORF fragment CEN.PK1 13-7D 22

4870 fusion-PCR of pTDH3and RPE1 pTDH3 + RPE1 fragment 23

3290 fusion-PCR of pTDH3and RPE1 pTDH3 + RPE1 fragment 24

5916 PGK1 promoter fragment CEN.PK1 13-7D 25

5917 PGK1 promoter fragment CEN.PK1 13-7D 26

5918 TKL1 ORF fragment CEN.PK1 13-7D 27

5919 TKL1 ORF fragment CEN.PK1 13-7D 28

3291 fusion-PCR of pPGK1 and TKL1 pPGK1 + TKL1 fragment 29

4068 fusion-PCR of pPGK1 and TKL1 pPGK1 + TKL1 fragment 30

5920 TEF1 promoter fragment pYM-N18 31

5921 TEF1 promoter fragment pYM-N18 32

5922 TAL1 ORF fragment CEN.PK1 13-7D 33

5923 TAL1 ORF fragment CEN.PK1 13-7D 34

3274 fusion-PCR of pTEF1 and TAL1 pTEF1 + TAL1 fragment 35

3275 fusion-PCR of pTEF1 and TAL1 pTEF1 + TAL1 fragment 36

6278 XKS1 ORF fragment CEN.PK1 13-7D 37

6279 XKS1 ORF fragment CEN.PK1 13-7D 38

Table 5. Primers used for amplification of integration fragments

Primer nr Purpose Template SEQ ID

7133 fl_RPE1_H fragment fw pUD347 39

3290 fl_RPE1_H fragment rv pUD347 40

3291 H_TKL1_I fragment fw pUD348 41

4068 H_TKL1_I fragment rv pUD348 42

3274 l_TAL1_A fragment pUD349 43

3275 l_TAL1_A fragment pUD349 44

3847 A_ NQM1 _B pUD344 45

3276 A_ NQM1_B pUD344 46

4672 B_RKI 1_C fragment pUD345 47

3277 B_RKI 1_C fragment pUD345 48

3283 C_TKL2_F fragment pUD346 49

3288 C_TKL2_F fragment pUD346 50

7138 F_xylA_P fragment pUD350 51

7136 F_xylA_P fragment pUD350 52

7139 P_xylA_Q fragment pUD350 53

7137 P_xylA_Q fragment pUD350 54

7142 Q_xylA_E fragment pUD350 55

7140 Q_xylA_E fragment pUD350 56

7141 E_xylA_G fragment pUD350 57

6285 E_xylA_G fragment pUD350 58

6273 G_xylA_D fragment pUD350 59

6284 G_xylA_D fragment pUD350 60

6283 D_xylA_M fragment pUD350 61

6275 D_xylA_M fragment pUD350 62

6287 M_xylA_N fragment pUD350 63 Primer nr Purpose Template SEQ ID

6276 M_xylA_N fragment pUD350 64

6288 N_xylA_0 fragment pUD350 65

6277 N_xylA_0 fragment pUD350 66

6289 0_xylA_L fragment pUD350 67

6627 0_xylA_L fragment pUD350 68

7135 L_XKS1_fl fragment pUD353 69

7134 L_XKS1_fl fragment pUD353 70

8638 PMR1 KO cassette pUG-AmdSYM 71

8639 PMR1 KO cassette pUG-AmdSYM 72

8640 PMR1 reintegration CEN,PK1 13-7D 73

8641 PMR1 reintegration CEN,PK1 13-7D 74

Table 6. Primers used for construction of plasmid containing the gRNA cutting in G

Primer Description Purpose SEQ ID

5792 pUDE335 backbone pMEL.10 75

5980 pUDE335 backbone pMEL.10 76

5978 PUDE335 gRNA pMEL.10 77

5979 PUDE335 gRNA pMEL.10 78

2528 restriction analysis of gRNA PUDE335 79

960 restriction analysis of gRNA PUDE335 80

Table 7. Primers used for verifying integration of fragments

Primer Purpose SEQ ID

6640 checking PPP integration 81

976 checking PPP integration 82

6717 checking PPP integration 83

5603 checking PPP integration 84

4656 checking PPP integration 85

7056 checking xylA integration 86

6632 checking xylA integration 87

7370 checking xylA integration 88

3293 checking xylA integration 89

7369 checking xylA integration 90

4692 checking xylA integration 91

5231 checking xylA integration 92

3354 checking xylA integration 93

4184 checking xylA integration 94

3843 checking xylA integration 95

3837 checking xylA integration 96

6921 checking xylA integration 97

3286 checking xylA integration 98

8640 checking PMR1 KO/integration 99

8641 checking PMR1 KO/integration 100

9 checking PMR1 KO/integration 101

10 checking PMR1 KO/integration 102 Primer Purpose SEQ ID

8792 checking PMR1 KO/integration 103

8793 checking PMR1 KO/integration 104

One-step construction of a xylose-utilizing Saccharomyces cerevisiae strain

To construct a xylose-metabolizing S. cerevisiae strain, nine copies of an expression cassette containing Piromyces xylA, as well as single expression cassettes for constitutive overexpression of the native yeast genes for xylulokinase (XKS1) and for the enzymes of the non- oxidative branch of the pentose-phosphate pathway (RKI1, RPE1, TKL1, TKL2 and TAL1) were introduced in S. cerevisiae CEN.PK1 13-7D. Additionally, an expression cassette for NQM1, a paralog of TAL1 whose duplication has been shown to enhance pentose fermentation by engineered S. cerevisiae, was introduced. A schematic overview Combination of in vivo assembly and CRISPR/Cas9-mediated chromosomal integration (Maris et al 2015) enabled a one-step introduction of all expression cassettes in the GRE3 locus, thereby inactivating GRE3, which encodes a non-specific aldose reductase that can reduce xylose to xylitol. The nine copies of the xylA cassette were introduced as tandem repeats to facilitate adaptation of the xylA copy number by homologous recombination. Transformants obtained after plating on xylose synthetic medium (SMX) plates were restreaked thrice on the same medium. The genome of the resulting strain IMX696 (Table 1 ), in which correct integration of the cassettes was confirmed by diagnostic PCR using primers listed as indicated, was sequenced to assess whether mutations had occurred during growth on SMX plates. No single-nucleotide polymorphisms (SNPs), insertion/deletions in coding regions or changes in chromosomal copy numbers were observed. However, read-depth analysis revealed the presence of 36 rather than 9 copies of the xylA cassette. In aerobic shake- flask cultures on SMX, strain IMX696 exhibited a specific growth rate of 0.21 h^~ (Fig. 3).

Anaerobic growth of the engineered xylose-fermenting strain IMX696 was investigated in nitrogen-sparged bioreactor cultures on SMX, supplemented with the anaerobic growth factors Tween-80 and ergosterol. In duplicate experiments, CO2 production, which was continuously monitored in the off-gas of the bioreactors, was only observed after 12 days of incubation (Fig. 4). To investigate this slow adaptation to anaerobic growth on xylose in more detail, the experiment was repeated, with regular analysis of culture viability, metabolite concentrations and growth (Fig. 1 ). Again, no significant xylose consumption occurred during the first 12 days of the experiment. A subsequent increase in biomass concentration coincided with the conversion of xylose to ethanol and glycerol. The specific growth rate after the onset of anaerobic growth was estimated at 0.1 1 h^~ based on biomass dry weight measurements during the mid-exponential growth phase. Biomass and ethanol yields on xylose were 0.086 ± 0.01 g biomass (g xylose)^-1 and 0.382 ± 0.01 g ethanol (g xylose) ¹ , respectively (Fig.1 ). Adaptation to anaerobic growth were further investigated by plating culture samples on synthetic medium with either glucose (SMD) or xylose (SMX). Colony counts on these plates were determined after aerobic and anaerobic incubation (Fig. 1 (b)-(e)). On anaerobic SMX plates, colonies were first observed after 10 d, at which time they represented a fraction of only 1.8- 10⁴ of the number of cells that were plated. Subsequently, consistent with the exponential growth observed by biomass dry weight measurements, the fraction of cells capable of anaerobic growth of xylose rapidly increased (Fig. 1 (b)). When culture samples were plated on SMD, aerobic and anaerobic plates showed similar trends in colony counts: Fig. 1 (d)-(e). Conversely, plating on SMX revealed a strong trade-off between the ability to grow aerobically and anaerobically on xylose. On aerobically incubated SMX plates cell counts did not increase, not even when exponential growth on xylose took off during the final days of the bioreactor experiments and strongly increasing colony counts were observed on anaerobically incubated SMX plates (Fig. 1 (c)).

The dynamics of colony counts on SMX plates (Fig. 1 b) suggested that adaptation to anaerobic growth might have involved one or more mutations. To test this hypothesis, the genomes of strains IMS0488 and IMS0489, which were isolated from the independent anaerobic adaptation experiments shown in Fig. 1 , and replicate experiment (fig. not shown) were sequenced. Unexpectedly, read-depth analysis of both strains revealed a decrease of the xylA copy number to 24 and 25, respectively, as compared to 36 in the parental strain IMX696. No other changes in chromosomal copy numbers were observed. Both strains carried non- synonymous SNPs in the coding region of PMR1, which encodes a high-affinity Golgi Ca²7Mn²⁺ P-type ATPase³. These mutations caused a single amino acid change (Pmr1^G249V) in strain IMS0488 and introduced a premature stop codon (Pmr1^W38 ) in strain IMS0489. Table 8: Single-nucleotide mutations in engineered S. cerevisiae strains adapted to anaerobic growth on xylose.

Mutations in PMR1 enable anaerobic growth on xylose.

To investigate the role of the PMR1 mutations in the adaptation to anaerobic growth on xylose, the gene was deleted in the parental strain IMX696. In replicate anaerobic bioreactor cultures on xylose, the resulting strain IMX906 grew within 24 h and completely consumed all sugar within 70 h (Fig. 2). The specific growth rate of both cultures was 0.08 h \ while biomass and ethanol yields on xylose were 0.086 g ± 0.01 biomass (g xylose)^-1 and 0.40 g ± 0.01 ethanol (g xylose) ¹ , respectively. To check if the phenotypic difference between IMX696 and IMX906 was solely due to the PMR1 deletion, the wild-type allele was reintegrated in strain IMX906. The resulting strain IMX979 showed a lag phase of over 250 h in duplicate anaerobic bioreactor cultures on xylose (Fig. 1 ). This result confirmed that inactivation of PMR1 is sufficient for acquisition of the ability to grow anaerobically on xylose by engineered, XylA-based S.

cerevisiae. The plate count experiments during the anaerobic adaptation phase on xylose suggested a trade-off between aerobic and anaerobic growth on xylose (Fig. 1 (b) and (c)). This possible trade-off was further explored by growth experiments in aerobic shake flasks on SMX. In these experiments, strains in which PMR1 was mutated or deleted consistently showed a 5 lower specific growth rate than strains that carried a wild-type PMR1 allele (0.10 h^~ and

0.21 h \ respectively; Fig. 3). Furthermore, aerobic shake-flask cultures of strains with mutated PMR1 alleles accumulated ethanol to 3-4 fold higher concentrations than corresponding cultures of strains with wild-type PMR1 alleles (Fig. 5). i o Mutations in PMR1 affect intracellular metal concentrations in xylose-metabolizing S. cerevisiae strains

To explore a possible relation between metal homeostasis and anaerobic growth on xylose, we analysed intracellular concentrations of Ca²⁺, Mn2+, Mg²⁺ and Fe²⁺ in biomass samples from anaerobic mid-exponential phase bioreactor cultures using inductively coupled plasma mass

15 spectrometry. Contents of Mg²⁺, Ca²⁺ and Fe²⁺ were similar in all analysed strains, with Mg²⁺ accounting for over 80% of the analysed divalent metal ions, followed by Ca²⁺, and with Fe²⁺ accounting for less than 1 % of the measured metals. Conversely, large differences were observed for the Mn2+ content. While in strains with a wild-type PMR1 allele, Mn2+ represented less than 0.2% of the measured metal ions, 12- to 29-fold higher Mn²⁺ contents were observed in strains

20 with mutated PMR1 alleles, irrespective of whether they were grown on xylose or glucose (Table 8). The observation that mutations in PMR1 affected cellular contents of Mn²⁺ but not those of Ca²⁺.

Metal content of xylose isomerase purified from S. cerevisiae strains.

25 Laboratory evolution studies have identified XI activity as key factor in fast fermentation of xylose to ethanol. XI enzymes are known to be metal dependent, with pronounced differences in metal binding and impact of metal identity on enzyme kinetics. To examine the impact of mutations in PMR1 on metal loading of XylA we purified the enzyme from the bioreactor cultures that were also used to determine cellular metal contents. Concentrations of Mg²⁺, Ca²⁺, Fe²⁺ and

30 Mn2+ were measured in purified protein samples and the amount of each metal per enzyme active site was calculated (Table 7). Based on the assumption that, in vivo, each functional active site of XI contains two divalent metal ions, the calculated metal loading (mol metal/mol XylA monomer) indicates that metal ions were lost during enzyme purification. This notwithstanding, large and consistent differences were observed in the Mn2+ contents of XylA isolated from strains

35 with wild-type and mutated PMR1 alleles (0.017 and 0.30 mol Mn (mol XylA subunit) , respectively, Table 1 1 ). Enzyme-bound and overall cellular metal amounts correlated well, indicating that binding of Mn²⁺ by XylA is strongly influenced by intracellular divalent metal concentrations (Table 9). Table 9: Impact of the deletion of PMR1 on intracellular metal ion concentrations.

S. cerevisiae strains were grown in anaerobic bioreactors on xylose or glucose (20 g I ^~1). Data represent average and mean deviation calculated from analyses on independent duplicate cultures.

Mn²⁺ binding results in superior catalytic efficiency of XylA.

To determine the effect of different metals on catalytic properties of XylA, activities of apoenzyme, isolated from IMX906 cells grown on xylose, were measured after reconstitution with Mg²⁺, Ca²⁺ and Mn²⁺ (Table 10). XylA apoenzyme was prepared by washing with EDTA, followed by assays in the presence of different divalent metals. The enzyme showed the highest catalytic efficiency in the presence of Mn²⁺, with a kcat/Km ratio that was 4-fold and 1500-fold higher than with Mg²⁺ and Ca²⁺, respectively. Both the highest /(cat and the lowest KM were observed with Mn2+ and contributed to this superior catalytic efficiency with this metal cofactor (Table 10). The measured /(cat with Mn²⁺ was almost 3-fold higher than with Mg²⁺ and even 13- fold higher than with Ca²⁺.

Table 10. Kinetic parameters of XylA measured after reconstituting apo-XylA with different divalent metal ions.

/(cat and KM values represent average and mean deviation of independent duplicate

experiments, calculated for each metal

Table 11 : Impact of PMR1 deletion on metal content of XylA.

S. cerevisiae strain Carbon mol metal/mol xylA monomer

(genotype) source Mg²⁺ Ca²⁺ Fe²⁺ Mn²⁺

IMX906 xylose 0.18 ± 0.01

(xylA, PPP†, XKS1], 0.54 ± 0.1 1 0.06 ± 0.01 0.38 ± 0.04 pmrIA) IMX906 glucose 0.20 ± 0.06

0.064 ±

(xylA, PPP†, XKS1 , 0.66 ± 0.07 0.30 ± 0.03

0.02

pmrIA)

IMX696 glucose 0.22 ± 0.06 0.086 ± 0.017 ±

0.93 ± 0.41

(xylA, PPP†, XKS7†) 0.04 0.004

XylA protein was isolated from S. cerevisiae cultures grown on xylose or glucose (20 g I^"1) in anaerobic bioreactors. Data represent average and mean deviation of analyses on xylA isolated from independent duplicate cultures.

Overexpression of manganese transporters

In this example it is described that reported manganese transporters can be overexpressed to facilitate increasing manganese content in the cytosol in the xylose isomerase-expressing S. cerevisiae strain IMX696 (see previous example). Overexpression of these manganese transporters may result in higher manganese loading per unit of xylose isomerase protein, thereby resulting in an increase of xylose isomerase activity to enable anaerobic growth of IMX696 on xylose as sole carbon source.

Table 12. Manganese transporters for overexpression

Expression cassette construction

The ORFs of the manganese transporters (listed in Table 13; SEQ ID NOs: 105, 107, 109, 1 1 1 , 1 13, 1 15, 1 17), the Saccharomyces cerevisiae TPI 1 promoter (SEQ ID NO: 1 19) and Saccharomyces cerevisiae PGK1 terminator (SEQ ID NO: 120) sequences can be synthesized at DNA2.0 (Menlo Park, CA 94025, USA).

The promoter, ORF and terminator sequences can be recombined by using the Golden Gate technology, as described by Engler et al (201 1 ) and references therein. The expression cassettes are cloned into a standard subcloning vector. The ORFs listed in Table 12 are ligated to Sc_TPI1 promoter and Sc_PGK1 terminator resulting in expression cassettes listed in Table 13.

Table 13: Expression (prom-ORF-term) cassettes from Golden Gate Cloning composed of the S. cerevisiae promoter TPI1, the different manganese transporter ORFs (listed in table 12) and the S. cerevisiae PGK1 terminator; on the 5' end and 3' end connector sequences compatible to neighbouring pathway brick are listed. Cassette 5' - promoter ORF Terminator 3'- connector connector cSMF1 C Sc_TPI 1 Sc_SMF1 Sc_PGK1 D cSMF2 C Sc_TPI 1 Sc_SMF2 Sc_PGK1 D cSMF1(-68) c Sc_TPI 1 Sc_SMF1(-68) Sc_PGK1 D cSMF7(K33,34R) c Sc_TPI 1 Sc_SMF7(K33,34R) Sc_PGK1 D cSMF1(4xKR) c Sc_TPI 1 Sc_SMF7(4xKR) Sc_PGK1 D

CPH084 c Sc_TPI 1 Sc_PH084 Sc_PGK1 D cATX2 c Sc_TPI 1 Sc_ATX2 Sc_PGK1 D

Strain construction

Strains can be constructed as described in WO2013/144257 and WO2016/1 10512. WO2013/144257describes the techniques enabling the construction of expression cassettes from various genes of interest in such a way, that these cassettes are combined into a pathway and integrated in a specific locus of the yeast genome upon transformation of this yeast. WO2016/1 10512 describes the use of a CRISPR-Cas9 system for integration of expression cassettes into the genome of a host cell, in this case S. cerevisiae. In the construction of IMX696 a S. pyogenes Cas9 expression cassette was already integrated at the CAN1 locus. Upon introduction of an in vivo assembled gRNA-expressing plasmid and repair DNA fragments the intended modifications were made. Firstly, an integration site in the yeast genome is selected. DNA fragments of approximately 500 bp of the up- and downstream parts of the integration locus were amplified by PCR using primers introducing connectors to the generated PCR products. These connectors (50 bp in size) allow for correct in vivo recombination of the pathway upon transformation in yeast. Secondly, the genes of interest, are amplified by PCR, incorporating a different connector (compatible with the connector on the of the neighbouring biobrick) at each flank. Upon transformation of yeast cells with the DNA fragments, in vivo recombination and integration into the genome takes place at the desired location. This technique facilitates parallel testing of multiple genetic designs, as one or more genes from the pathway can be replaced with (an)other gene(s) or genetic element(s), as long as that the connectors that allow for homologous recombination remain constant and compatible with the preceding and following biobrick in the design (WO2013/144257). gRNA expression plasmid

The expression cassettes can be targeted to the INT1 locus. The INT1 integration site is a non-coding region between NTR1 (YOR071 c) and GYP1 (YOR070c) located on chromosome XV of S. cerevisiae. The guide sequence to target INT1 can be designed with a gRNA designer tool (https://www.dna20.com/eCommerce/cas9/input). The gRNA expression cassette (as described by DiCarlo et al., Nucleic Acids Res. 2013; pp.1-8) can be ordered as synthetic DNA cassette (gBLOCK) at Integrated DNA Technologies (Leuven, Belgium) (INT1 gBLOCK; SEQ ID NO: 121 ). In vivo assembly of the gRNA expression plasmid can then be completed by co- transforming a linear fragment derived from yeast vector pRN599. pRN599 is a multi-copy yeast shuttling vector that contains a functional kanMX marker cassette conferring resistance against G418. The backbone of this plasmid is based on pRS305 (Sikorski and Hieter, Genetics 1989, vol. 122, pp.19-27), including a functional 2 micron ORI sequence and a functional kanMX marker cassette (SEQ ID NO: 122).

Transformation of IMX696 with overexpression cassettes of manganese transporters

Strain IMX696 can be transformed with the following fragments resulting in the assembly of overexpression cassettes for the manganese transporters as depicted in Figure 7:

1 ) a PCR fragment (5 -INT1 ) generated with primers BoZ-783 (SEQ ID NO: 123) and DBC- 19944 (SEQ ID NO: 124) with genomic DNA of strain CEN.PK1 13-7D as template;

2) a PCR fragment (P_Tpn-[Mn-Trans.]-TpGKi ) generated with primers DBC-5799 (SEQ ID NO:

125 and DBC-5800 (SEQ ID NO: 126) using either one of the plasmids listed in Table 13 as template (resulting in expression cassette manganese transporter flanked by connectors C and D).

3) a PCR fragment generated with primers DBC-19947 (SEQ ID NO: 127) and DBC-19949 (SEQ ID NO: 128) using genomic DNA of strain CEN.PK1 13-7D as template; this PCR resulted in the 1.2 kb marker cassette conD-l//¾A3-conE (URA3 marker flanked by connectors D and E).

4) a PCR fragment (3 -INT1 ) generated with primers DBC-19946 (SEQ ID NO: 129) and BoZ- 788 (SEQ ID NO: 130) using genomic DNA of strain CEN.PK1 13-7D as template;

5) a PCR fragment (BB-599) generated with primers DBC-13775 (SEQ ID NO: 131 ) and DBC- 13776 (SEQ ID NO: 132) using pRN599 (SEQ ID NO: 122) as template;

6) a PCR fragment (gRNA-INT1 ) generated with primers DBC-13773 (SEQ ID NO: 133) and DBC-13774 (SEQ ID NO: 134) using INT1 gRNA (SEQ ID NO: 121 ) as template.

Transformants are selected on mineral medium (according to recipe Luttik et al., 2000, Journal of Bacteriology 182, 24: 501-517) supplemented with 1.5% bactoagar supplemented with 20 g/L glucose and 0.2 mg G418 m/L. Diagnostic PCR is performed to confirm the correct assembly and integration at the INT1 locus of the manganese transporter overexpression cassettes. The manganese transporter overexpression strains thus constructed are listed in Table 14:

Table 14. Strains constructed in this example

Overexpression of manganese transporters enable anaerobic growth on xylose The constructed strains with manganese transporter overexpression cassettes can be cultivated for growth under anaerobic conditions for 70h. Whereas IMX696 does not show growth within 70 hrs of anaerobic cultivation, the constructed manganese transporter overexpression strains do show growth (as indicated in Table 15).

Table 15. Growth profile on 20 g/L xylose as sole carbon source supplemented to mineral medium and cultivated under anaerobic conditions as in previous example

Table 16. Intracellular manganese concentrations in IMX696 derived strains overex ressin man anese trans orters

In an embodiment, overexpression of SMF1, SMF2, PH084 or ATX2 results in an increase of intracellular Mn²⁺ concentration as compared to cells without overexpression of SMF1, SMF2, PH084 orATX2.

In one embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is at least 2 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is at least 4 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is at least 6 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2. In another embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is at least 8 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2\s at least 10 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is at least 12 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is at least 15 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is at least 20 times higher as compared to a cell without overexpression of SMF1 , SMF2, PH084 or A TX2.

In another embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is at least 30 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In one embodiment, the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is between 2-5 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is between 5-10 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is between 10-15 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment the intracellular Mn²⁺ concentration of a cell with overexpression of SMF1, SMF2, PH084 or ATX2 is between 15-20 times higher as compared to a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In an embodiment, the xylose isomerase activity in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is at least at least 2 times higher than xylose isomerase activity in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the xylose isomerase activity in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is at least at least 4 times higher than xylose isomerase activity in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the xylose isomerase activity in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is at least at least 6 times higher than xylose isomerase activity in a cell without overexpression of SMF1, SMF2, PH084 or ATX2. In another embodiment, the xylose isomerase activity in a cell having SMF1, SMF2, PH084 orATX2 overexpressed is at least at least 8 times higher than xylose isomerase activity in a cell without overexpression of SMF1, SMF2, PH084 orATX2.

In another embodiment, the xylose isomerase activity in a cell having SMF1, SMF2, PH084 or ATX2 overexpression of is at least at least 10 times higher than xylose isomerase activity in a cell without overexpressed SMF1, SMF2, PH084 or ATX2.

In one embodiment, xylose isomerase activity in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 2 and 4 times higher as compared to cells without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, xylose isomerase activity in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 4 and 6 times higher as compared to cells without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, xylose isomerase activity in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 6 and 8 times higher as compared to cells without overexpression of SMF1 or SMF2.

In another embodiment, xylose isomerase activity in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 8 and 10 times higher as compared to cells without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, xylose isomerase activity in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 10 and 20 times higher as compared to cells without overexpression of SMF1, SMF2, PH084 or ATX2.

Manganese loading in xylose isomerase in IMX696 derived strains with manganese transporter overexpression cassettes integrated

Table 17: Impact of manganese transporter overexpression on metal content of XylA.

In an embodiment, the Mn loading in xylose isomerase in a cell having SMF1 or SMF2 overexpressed is at least at least 2 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2. In another embodiment, the Mn loading in xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is at least at least 4 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the Mn loading in xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is at least at least 6 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the Mn loading in xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is at least at least 8 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the Mn loading in xylose isomerase in a cell having SMF1, SMF2,

PH084 or ATX2 overexpressed is at least at least 10 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment, the Mn loading in xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is at least at least 15 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In one embodiment the Mn loading in xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 2 and 4 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment the Mn loading in xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 4 and 6 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment the Mn loading in xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 6 and 8 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment the Mn loading in xylose isomerase in a cell having SMF1, SMF2,

PH084 or ATX2 overexpressed is between 8 and 10 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment the Mn loading in xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 10 and 15 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In another embodiment the Mn loading in xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 15 and 200 times higher than the Mn loading in xylose isomerase in a cell without overexpression of SMF1, SMF2, PH084 or ATX2.

In an embodiment the Mn loading of xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 0.01 and 0.1.

In another embodiment the Mn loading of xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 0.1 and 0.2.

In another embodiment the Mn loading of xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 0.2 and 0.3. In another embodiment the Mn loading of xylose isomerase in a cell having SMF1, SMF2, PH084 orATX2 overexpressed is between 0.3 and 0.4.

In another embodiment the Mn loading of xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 0.4 and 0.5.

In another embodiment the Mn loading of xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 0.5 and 0.6.

In another embodiment the Mn loading of xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 0.6 and 0.7.

In another embodiment the Mn loading of xylose isomerase in a cell having SMF1, SMF2, PH084 or ATX2 overexpressed is between 0.7 and 0.8.

Introduction of mutations in PMR1 selectively abrogating Mn²⁺ transport in IMX696

PMR1 encodes a Ca²7Mn²⁺ transporting P-type ATP-ase localized on the Golgi membrane. PMR1 function is pivotal not only in Mn²⁺- but also in Ca²⁺ cellular homeostasis. PMR7-defective yeast strains are sensitive to both low levels of divalent cations and high levels of calcium in the extracellular environment. Golgi Ca²⁺ cellular homeostasis is involved in protein processing through the secretory pathway. For xylose isomerase activity, Mn²⁺ is shown in previous examples to be the most effective activating metal. Mandal et al., (2003, J Biol Chem, vol. 278, pp. 3592-3598) described PMR1 variants selectively abrogated in Mn²⁺ transport, whereas Ca²⁺ transport has been left (largely) unaffected. Mutations in positions Gln-783 and Val- 335 in Pmrl p have been reported to be instrumental in Mn-transport deficient variants.

This example describes mutations to PMR1 (coding DNA, SEQ ID NO: 135; protein, SEQ ID NO: 136) in a xylose isomerase-expressing Saccharomyces cerevisiae strain to selectively disrupt/decrease Mn²⁺ transport to the Golgi; thereby Mn²⁺ availability in the cytosol can be increased, resulting in higher Mn²⁺ loading in xylose isomerase and in higher xylose isomerase activity, whereas Ca²⁺ homeostasis is left unaffected.

Table 18. Mutations abbrogating Mn²⁺ transport in Saccharomyces cerevisiae PMR1 reported by Mandal et al., 2003

0, no growth; - intermediate growth; +, growth; ND, not detectable

The different mutations resulting in the different PMR1 variants listed in table in Table 18 are made in refence to coding DNA sequence of CEN.PK1 13-7D PMR1 (SEQ ID NO: 135) and to protein sequence of CEN.PK1 13-7D Pmrl p (SEQ ID NO: 136). Similar as with the reintegration construct used in the construction of IMX969, flanking sequences to the PMR1 locus are included. PMR1 integration constructs are typically synthesized at DNA2.0 (Menlo Park, CA 94025, USA). As example, the wildtype PMR1 integration construct is given as SEQ ID NO: 137. The SEQ ID NOs for the PMR1 variant integration constructs are listed in Table 18.

To apply the specific mutations in the PMR1 locus in pmr1 deletion mutant IMX906, the same approach can be used as in the construction of IMX969 (reintegration of wild type PMR1 sequence in IMX906). IMX906 contains the negative selection marker amdSYM at the pmr1 deletion locus ; amdSYM can be selected against with fluoroacetamide.

Firstly, IMX906 is grown on non-selective medium (rich growth medium or medium with uracil supplemented) to grow out plasmid pUDE335 resulting in strain IMX906-ura^~. Strain IMX906-ura^_ is then transformed with a PCR fragment generated with primers PMR1 reintegration forward (SEQ ID NO: 73) and PMR1 reintegration reverse (SEQ ID NO:74) using either one of the DNA sequences listed in Table 18 (SEQ ID Nos: 137 to 146) as template (resulting in PMR1 variant DNA fragment flanked by homologous sequences io mrl deletion locus in IMX906). After overnight incubation in SMD-Ac, transformants can be selected on SMD-FAc agar plates. Correct transformants are identified by Sanger sequencing of PCR fragments of the PMR1 locus confirming the intended mutations are integrated. Constructed strains with PMR1 variants are listed in table 19.

Table 19. Strains constructed in this example

PMR1 variants enable anaerobic growth on xylose

The constructed strains with PMR1 variants can be cultivated for growth under anaerobic conditions for 70 hours. IMX696 serves a negative control (original PMR1 locus), IMX906 serves as positive control for growth, and IMX969 serves as transformation control with wild type PMR1 sequence reintegrated. Whereas IMX696 and IMX969 do not show growth within 70 hours of anaerobic cultivation on xylose, IMX906 and the PMR1 variant strains do show growth (as indicated in Table 20).

Table 20. Growth profile on 20 g/L xylose as sole carbon source supplemented to mineral medium and cultivated under anaerobic conditions as in previous examples

Table 21. Intracellular manganese / calcium concentrations in IMX696 derived strains overexpressing manganese transporters

In an embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of at least 2 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of at least 4 as compared to cells without such mutations. In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of at least 6 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of at least 8 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of at least 10 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of at least 15 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of at least 20 as compared to cells without such mutations.

In an embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of between 2 and 4 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of between 4 and 6 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of between 6 and 8 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of between 8 and 10 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of between 10 and 15 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Mn²⁺ concentration of between 15 and 20 as compared to cells without such mutations.

In an embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Ca²⁺ concentration of between 1 and 2 as compared to cells without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in an increase of intracellular Ca²⁺ concentration of between 1 and 1.5 as compared to cells without such mutations. Manganese loading in xylose isomerase in IMX696 derived strains with manganese transporter overexpression cassettes integrated

Table 22. Im act of man anese trans orter overex ression on metal content of X lA

In an embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading of xylose isomerase which is at least 2 times higher than the Mn loading in xylose isomerase in a cell without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading in xylose isomerase which is at least 4 times higher than the Mn loading in xylose isomerase in a cell without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading in xylose isomerase which is at least 6 times higher than the Mn loading in xylose isomerase in a cell without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading in xylose isomerase which is at least 8 times higher than the Mn loading in xylose isomerase in a cell without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading in xylose isomerase which is at least 10 times higher than the Mn loading in xylose isomerase in a cell without such mutations.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading in xylose isomerase which is at least 15 times higher than the Mn loading in xylose isomerase in a cell without such mutations.

In an embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading of xylose isomerase of between 0.01 and 0.1. In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading of xylose isomerase of between 0.1 and 0.2.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading of xylose isomerase of between 0.2 and 0.3.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading of xylose isomerase of between 0.3 and 0.4.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading of xylose isomerase of between 0.4 and 0.5.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading of xylose isomerase of between 0.5 and 0.6.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading of xylose isomerase of between 0.6 and 0.7.

In another embodiment, mutations in PMR1 selectively abrogating Mn²⁺ transport as indicated above result in a Mn loading of xylose isomerase of between 0.7 and 0.8.

Disruption of SMF1 and PH084 regulators

This example describes mediating functional manganese transport activity by disrupting the regulators that are involved in the localization of the Mn²⁺ transporters Smfl p and Pho84p and as a result increase xylose isomerase activity.

Table 23. Primer sequences used to construct gRNA plasmids

Table 24. Sequences used to construct the double stranded repair fragments

Table 25. Ssequences used for diagnostic PCR to identify successful deletion after transformation.

Strain construction

Disruption of genes involved in regulation of manganese regulation can be performed using a CRISPR-Cas9 based system that is already active in strain IMX696. The gRNA expression can be done by transforming an episomal yeast vector based on pROS13 as described by (DiCarlo et al., 2013; Mans et al., 2015) used primers sequences listed in Table 23 using the yeast transformation method described by (Gietz & Schiestl, 2007). Simultaneous addition of the plasmid based on pROS13, containing the kanMX marker, and the appropriate repair fragments listed in Table 24 will result in the disruption of the targeted gene. Correct transformants are selected by diagnostic PCR using primers listed in Table 25. Double and triple deletion strains 696-ECM21d-CSR1d, 696-ECM21d-CSR1d-BSD2d, 906-ECM21d-CSR1d- BSD2d, 696-PH80d-PHO85d and 696-ECM21d-CSR1d-BSD2d require counterselection of the gRNA expression plasmid in between each transformation round by sequential re-streaking on YPD as described by (Mans et al., 2015), after which the next gene can be disrupted. The disruption of the genes described in this example can also be done in a PMR1 deletion strain, e.g. IMX906. A list of strains is shown in Table 26.

Table 26. Strains

Characterization in anaerobic bioreactors

The constructed strains are characterized in anaerobic batch reactors with 20 g/L glucose. When the cultures reached an OD66o_nm of around 6, biomass is harvested by centrifuging and the cells can be further processed to measure the intracellular metal concentration as described above. Table 27 shows the relative increase in intracellular manganese compared to strain IMX696.

Table 27. Intracellular manganese concentrations in IMX696 derived strains overex ressin man anese trans orters

In an embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an increase of intracellular Mn²⁺ concentration as compared to a cell without such deletion or disruption.

In one embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2,

PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration which is at least 2 times higher as compared to a cell without such deletion or disruption.

In another embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is at least 4 times higher as compared to a cell without such deletion or disruption.

In another embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is at least 6 times higher as compared to a cell without such deletion or disruption.

In another embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is at least 8 times higher as compared to a cell withou such deletion or disruption.

In another embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is at least 10 times higher as compared to a cell such deletion or disruption.

In another embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2,

PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is at least 12 times higher as compared to a cell such deletion or disruption.

In another embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is at least 15 times higher as compared to a cell without such deletion or disruption. In another embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is at least 20 times higher as compared to a cell without such deletion or disruption.

In another embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is at least 30 times higher as compared to a cell without such deletion or disruption.

In one embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration which is between 2-5 times higher as compared to a cell without such deletion or disruption.

In another embodiment a cell having a deletion or disruption of ECM21 , CSR1 , BSD2,

PHO80 and/or PH085results in an intracellular Mn²⁺ concentration is between 5-10 times higher as compared to a cell without such deletion or disruption.

In another embodiment, a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is between 10-15 times higher as compared to a cell without such deletion or disruption.

In another embodiment a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 results in an intracellular Mn²⁺ concentration is between 15-20 times higher as compared to a cell without such deletion or disruption.

In an embodiment, the xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is at least at least 2 times higher than xylose isomerase activity in a cell without such deletion or disruption.

In another embodiment, the xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is at least at least 4 times higher than xylose isomerase activity in a cell without such deletion or disruption.

In another embodiment, the xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is at least at least 6 times higher than xylose isomerase activity in a cell without such deletion or disruption.

In another embodiment, the xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is at least at least 8 times higher than xylose isomerase activity in a cell without such deletion or disruption.

In another embodiment, the xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is at least at least 10 times higher than xylose isomerase activity in a cell without such deletion or disruption.

In one embodiment, xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is between 2 and 4 times higher as compared to a cell without such deletion or disruption.

In another embodiment, xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is between 4 and 6 times higher as compared to a cell without such deletion or disruption. In another embodiment, xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is between 6 and 8 times higher as compared to a cell without such deletion or disruption.

In another embodiment, xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is between 8 and 10 times higher as compared to a cell without such deletion or disruption.

In another embodiment, xylose isomerase activity in a cell having a deletion or disruption of ECM21 , CSR1 , BSD2, PHO80 and/or PH085 is between 10 and 20 times higher as compared to a cell such deletion or disruption.

The increase in intracellular manganese enables anaerobic growth for strains with Mn²⁺ transport deregulated.

The constructed strains with key regulators disrupted can be grown under anaerobic conditions on synthetic medium with 20 g/L xylose as sole carbon source. Whereas IMX696 will not grow within 70 hrs, cultures with strain for which the disruption of protein involved in Mn²⁺ transport do show an increase in biomass (Table 28).

Table 28. Growth profile on 20 g/L xylose as sole carbon source supplemented to mineral medium and cultivated under anaerobic conditions as in previous example

References

DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., & Church, G. M. (2013). Genome

engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res., 41 (7), 4336-4343

Eide et al (2005): Eide DJ, ef al. Characterization of the yeast ionome: a genome-wide analysis of nutrient mineral and trace element homeostasis in Saccharomyces cerevisiae.

Genome Biol 6, 1 (2005).

Entian and Kotter (2007): Entian K-D, Kotter P. 25 Yeast genetic strain and plasmid collections.

Method Microbiol 36, 629-666 (2007).

Gietz, R. D., & SchiestI, R. H. (2007). High-efficiency yeast transformation using the LiAc/SS carrier

DNA/PEG method. Nat Protoc, 2. doi:10.1038/nprot.2007.13 Kuyper et al. (2003) High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae (FEMS Yeast Res. 4: 69-78 (2003).

Mans et al. (2015): Mans R, et al. CRISPR/Cas9: a molecular Swiss army knife for

simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae.

FEMS Yeast Res 15, fov004 (2015).

Maris et al 2015: Gombert AK, van Maris AJ. Improving conversion yield of fermentable sugars into fuel ethanol in 1 st generation yeast-based production processes. Curr Opin

Biotechnol 33, 81-86 (2015).

Claims

1. Eukaryotic cell comprising a heterologous xylose isomerase, wherein xylose isomerase is loaded with Mn²⁺ and wherein the Mn metal loading is at least 0.10 mol Mn (mol XylA subunit)^-1.

2. Eukaryotic cell comprising a heterologous xylose isomerase according to claim 1

wherein one or more manganese homeostasis related gene is disrupted or

overexpressed and wherein the disruption or overexpression causes and increased cytosolic and/or intracellular Mn²⁺ concentration.

3 Eukaryotic cell according to claim 2, wherein the one or more disrupted or

overexpressed manganese homeostasis related gene is chosen from the group consisting of: Golgi Ca²7Mn²⁺ ATPase (PMR1) gene, divalent metal transporter SMF1 gene (SMF1), Mn transporter PH084 gene, Mn transporter A TX1 gene; divalent metal transporter SMF2 gene (SMF2); and a regulator gene which is involved in the localization of the Mn²⁺ transporters SMF1 and PH084.

4. Eukaryotic cell according to any of claims 1 to 3, wherein one or more of Golgi

Ca²7Mn²⁺ ATPase (PMR1) gene is disrupted or deleted.

5. Eukaryotic cell according to any of claims 1 to 4, wherein one or more divalent metal transporter SMF1 gene (SMF1), and/or divalent metal transporter SMF2 gene (SMF2) is overexpressed.

6. Eukaryotic cell according to any of claims 1 to 5 wherein PMR1 gene is deleted.

7. Eukaryotic cell according to any of claims 1 to 6, wherein Golgi Ca2+/Mn²⁺ ATPase (PMR1 ) gene is disrupted and wherein the disruption is chosen from the group consisting of:

a) introduction of a premature stop codon in the Golgi Ca^2+/Mn²⁺ ATPase (PMR1 ) gene; and

b) mutation in the PMR1 gene corresponding to amino acid change G746T or W387* in PMR1 protein.

8. Eukaryotic cell according to any of claims 1 to 7, wherein PPP enzymes and xylulokinase are overexpressed.

9. Use of Mn²⁺ as a cofactor for xylose isomerase protein in a cell, wherein the cytosolic concentration of Mn²⁺ in the cell is from 2 to 100 nmol/10⁹ cells, 2 to 50 nmol/10⁹ cells or 2 to 40 nmol/10⁹ cells.

10. Process for the fermentation of a substrate to produce a fermentation product with an eukaryotic cell according to any of claims 1 to 8, wherein the xylose consumption is at least 10%, at least 20%, or at least 25% increased relative to the corresponding fermentation with wild-type eukaryotic cell. Process according to claim 10, wherein the fermentation product is ethanol and the ethanol yield is at least about 0.5 %, or at least 1 % higher than that of a process with the corresponding wild-type eukaryotic cell.

Process according to claim 10 or 1 1 , wherein pentose and glucose are co-fermented. Process according to any of claims 10 to 12, wherein a hydrolysate of lignocellulosic material is fermented.

Process according to claim 13, wherein the hydrolysate is an enzymatic hydrolysate of lignocellulosic material.

Process according to claim 13 or 14, wherein the hydrolysate comprises Mn²⁺ or a source of Mn²⁺-