WO2023208762A2 - Cellule de levure mutante et procédé de production d'éthanol - Google Patents

Cellule de levure mutante et procédé de production d'éthanol Download PDF

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WO2023208762A2
WO2023208762A2 PCT/EP2023/060423 EP2023060423W WO2023208762A2 WO 2023208762 A2 WO2023208762 A2 WO 2023208762A2 EP 2023060423 W EP2023060423 W EP 2023060423W WO 2023208762 A2 WO2023208762 A2 WO 2023208762A2
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yeast cell
protein
acid sequence
nucleic acid
activity
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PCT/EP2023/060423
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WO2023208762A3 (fr
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Aafke Cornelie Albertine VAN AALST
Robert MANS
Jacobus Thomas Pronk
Mickel Leonardus August Jansen
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Dsm Ip Assets B.V.
Technische Universiteit Delft
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Publication of WO2023208762A2 publication Critical patent/WO2023208762A2/fr
Publication of WO2023208762A3 publication Critical patent/WO2023208762A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01014—L-Iditol 2-dehydrogenase (1.1.1.14), i.e. sorbitol-dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01255—Mannitol dehydrogenase (1.1.1.255)

Definitions

  • the invention relates to a mutant yeast cell and to a process for the production of ethanol wherein said yeast cell is used.
  • Yeast-based fermentation processes are applied for industrial production of a broad and rapidly expanding range of chemical compounds from conventional and renewable carbohydrate feedstocks.
  • redox balancing of the cofactor couple NADH/NAD + can cause important challenges for product yields.
  • a major challenge relating to the stoichiometry of yeast-based ethanol production is that growing anaerobic cultures invariably produce glycerol as byproduct . It has been estimated that, in typical industrial ethanol processes, up to about 4 wt.% of the sugar feedstock is converted into glycerol (as described in the article by Nissen et al, 2000).
  • Glycerol production under anaerobic conditions is primarily linked to the redox balancing mechanisms in the yeast cell.
  • sugar dissimilation occurs via so- called alcoholic fermentation.
  • NADH formed via the NAD+-dependent glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate, to ethanol via NADH-dependent alcohol dehydrogenase.
  • the fixed stoichiometry of this redox-neutral dissimilatory pathway causes problems when a net reduction of NAD+ to NADH occurs elsewhere in the metabolism.
  • Glycerol formation is initiated by reduction of the glycolytic intermediate dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (glycerol-3P), a reaction catalyzed by NADH-dependent glycerol 3-phosphate dehydrogenase. Subsequently, the glycerol 3-phosphate formed in this reaction is hydrolysed by glycerol- 3-phosphatase to yield glycerol. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae. The production of glycerol is undesired as it reduces overall conversion of sugar to a desired fermentation product such as ethanol. Further, the presence of glycerol in effluents of fermentation plants may impose costs for waste-water treatment.
  • DHAP glycolytic intermediate dihydroxyacetone phosphate
  • glycerol-3P glycerol 3-phosphate
  • a recombinant yeast cell comprising one or more recombinant nucleic acid sequences encoding an NAD+ dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) activity.
  • the cell may for example lack enzymatic activity needed for the NADH dependent glycerol synthesis or the cell may have a reduced enzymatic activity with respect to the NADH dependent glycerol synthesis compared to its corresponding wild-type yeast cell.
  • WO2014/129898 describes a recombinant cell functionally expressing heterologous nucleic acid sequences encoding for ribulose-1 ,5-phosphate carboxylase/oxygenase (EC 4.1 .1 .39; herein abbreviated as “Rubisco”), and optionally molecular chaperones for Rubisco, and phosphoribulokinase (EC 2.7.1.19; herein abbreviated as “PRK”).
  • Rubisco ribulose-1 ,5-phosphate carboxylase/oxygenase
  • PRK phosphoribulokinase
  • HXT13 and HXT17 genes encoding hexose transporter-like proteins, as well as annotated mannitol dehydrogenase (MDH) genes DSF1 and YNR073C were found to be upregulated when yeast was adapted to grow on mannitol.
  • MDH mannitol dehydrogenase
  • mutant yeast cell comprising :
  • the invention provides a process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell as described herein, wherein the carbon source composition comprises at least a sugar alcohol and wherein the process is carried out under oxygenlimited conditions or anaerobic conditions.
  • the above mutant yeast and process advantageously allow for an improved conversion of sugar alcohols and/or an increased amount of ethanol being retrieved from a carbon source composition comprising at least a sugar alcohol.
  • each of the above protein / amino acid sequences is preferably encoded by a DNA / nucleic acid sequence that is codon-pair optimized for expression in a yeast, more preferably for expression in a Saccharomyces cerevisiae yeast.
  • a DNA / nucleic acid sequence that is codon-pair optimized for expression in a yeast, more preferably for expression in a Saccharomyces cerevisiae yeast.
  • Promoters may be regulated from strong to weak and may include one or more of TDH3, FBA1 , ENO2, PGK1 , TEF1 , HTA1 , HHF2, RPL8A, CHO1 , RPS3, EFT2, HTA2, ACT1 , PFY1 , CUP1 , ZUO1 , VMA6 and/or ANB1 , HEM13, YHK8, FET4, TIR4, AAC3. Description of the Figures
  • Figure 1 provides an illustration of the construction of plasmid pUDE885 of Example 1
  • Figure 2 provides an illustration of the construction of plasmid pUDE941 of Example 2
  • Figure 3 provides an illustration of the construction of yeast strain IMX2506 of Example 9
  • Figure 4 provides a graphic of results of the Example 11. :residual sorbitol concentration measured during pre-steady state sampling of anaerobic bioreactor chemostat cultures of S. cerevisiae strains IMX2506 (GPD1 Agpd2 pDAN1-prk cbbm Hxt15"f Sor2"f) (circles) and IME324 (GPD1 GPD2) (squares) at a dilution rate of 0.025 h -1 on 10 g L -1 of glucose and 10 g L -1 of sorbitol. For IMX2506 (circles) the average sorbitol concentration and standard deviation are based on four chemostat cultures.
  • the compound in principle includes all enantiomers, diastereomers and cis/trans isomers of that compound that may be used in the particular aspect of the invention; in particular when referring to such as compound, it includes the natural isomer(s).
  • carbon source refers to a source of carbon, preferably a compound or molecule comprising carbon.
  • the carbon source is a carbohydrate.
  • a carbohydrate is understood herein to be an organic compound made of carbon, oxygen and hydrogen.
  • the carbon source may be selected from the group consisting of mono-, di- and/or polysaccharides, polyols, acids and acid salts. More preferably the carbon source is a compound selected from the group of glucose, arabinose, xylose, galactose, mannose, rhamnose, fructose, glycerol, sugar alcohols and acetic acid or a salt thereof.
  • sugar alcohol refers to a carbohydrate, suitably derived from a sugar, containing one hydroxyl group attached to each carbon atom. More preferably the sugar alcohol is a sugar alcohol comprising 5 carbon atoms (i.e. a pentose alcohol) or 6 carbon atoms (a hexose alcohol). Suitable examples of sugar alcohols include arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, idotol, inositol, isomalt, erythritol, maltitol and lactitol. Sugar alcohols can suitably be prepared by hydrogenation of sugars. For the avoidance of doubt, glycerol is not a sugar alcohol.
  • the term “ferment”, and variations thereof such as “fermenting”, “fermentation” and/or “fermentative”, is used herein in a classical sense, i.e. to indicate that a process is or has been carried out under anaerobic conditions.
  • An anaerobic fermentation is herein defined to be a fermentation carried out under anaerobic conditions.
  • Anaerobic conditions are herein defined as conditions without any oxygen or in which essentially no oxygen is consumed by the yeast cell. Conditions in which essentially no oxygen is consumed suitably corresponds to an oxygen consumption of less than 5 mmol/l.h -1 , in particular to an oxygen consumption of less than 2.5 mmol/l.h -1 , or less than 1 mmol/l.h -1 .
  • 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable).
  • This suitably corresponds to a dissolved oxygen concentration in a culture broth of less than 5 % of air saturation, more suitably to a dissolved oxygen concentration of less than 1 % of air saturation, or less than 0.2 % of air saturation.
  • the term “fermentation process” refers to a process for the preparation or production of a fermentation product.
  • cell refers to a eukaryotic or prokaryotic organism, preferably occurring as a single cell.
  • the cell is a yeast cell. That is, the mutant cell is selected from the group of genera consisting of yeast.
  • yeast and “yeast cell” are used herein interchangeably and refer to a phylogenetically diverse group of single-celled fungi, most of which are in the division of Ascomycota and Basidiomycota.
  • the budding yeasts ("true yeasts") are classified in the order Saccharomycetales.
  • the yeast cell according to the invention is a yeast cell and is preferably a yeast cell derived from the genus of Saccharomyces. More preferably the yeast cell is a yeast cell of the species Saccharomyces cerevisiae.
  • mutant for example referring to a “mutant yeast”, a “mutant cell”, a “mutant micro-organism” and/or a “mutant strain”, as used herein, refers to a yeast, cell, micro-organism or strain, respectively, which in comparison to its parent, wild-type, counterpart has undergone a genetic modification, i.e. a “mutation”.
  • the genetic modification can for example be the result of a laboratory evolutionary process or recombinant DNA technique(s).
  • An example of a laboratory evolutionary process is adaptive evolution. Adaptive evolution is an evolutionary process whereby a population becomes better suited (adapted) to its habitat or habitats. After applying evolutionary pressure, via natural selection, appropriate mutants can be obtained.
  • the mutant yeast cell is a recombinant yeast cell. Further preferences for such recombinant yeast cell are as described herein.
  • mutated as used herein regarding proteins or polypeptides means that at least one amino acid in the wild-type or naturally occurring protein or polypeptide sequence has been replaced with a different amino acid, inserted or deleted from the sequence via mutagenesis of nucleic acids encoding these amino acids.
  • Mutagenesis is a well-known method in the art, and includes, for example, site-directed mutagenesis by means of PCR or via oligonucleotide-mediated mutagenesis as described in Sambrook et al., Molecular Cloning-A Laboratory Manual, 2nd ed., Vol. 1-3 (1989).
  • mutated means that at least one nucleotide in the nucleic acid sequence of that gene or a regulatory sequence thereof, has been replaced with a different nucleotide, or has been deleted from the sequence via mutagenesis, resulting in the transcription of a protein sequence with a qualitatively of quantitatively altered function or the knock-out of that gene.
  • an “altered gene” has the same meaning as a mutated gene.
  • recombinant for example referring to a “recombinant yeast”, a “recombinant cell”, “recombinant micro-organism” and/or “recombinant strain” as used herein, refers to a yeast, cell, microorganism or strain, respectively, containing nucleic acid which is the result of one or more genetic modifications. Simply put the yeast, cell, micro-organism or strain contains a different combination of nucleic acid from (either of) its parent(s). To construe a recombinant yeast, cell, micro-organism or strain, recombinant DNA technique(s) and/or another mutagenic technique(s) can be used.
  • a mutant yeast and/or a mutant yeast cell may comprise nucleic acid not present in the corresponding wild-type yeast and/or cell, which nucleic acid has been introduced into that yeast and/or yeast cell using recombinant DNA techniques (i.e.
  • a transgenic yeast and/or cell which nucleic acid not present in said wild-type yeast and/or cell is the result of one or more mutations - for example using recombinant DNA techniques or another mutagenesis technique such as UV-irradiation - in a nucleic acid sequence present in said wildtype yeast and/or yeast cell (such as a gene encoding a wild-type polypeptide) or wherein the nucleic acid sequence of a gene has been modified to target the polypeptide product (encoding it) towards another cellular compartment.
  • the term “recombinant” may suitably relate to a yeast, cell, micro-organism or strain from which nucleic acid sequences have been removed, for example using recombinant DNA techniques.
  • the mutant yeast is a recombinant yeast, respectively a recombinant yeast cell. That is, preferably the mutant yeast, respectively the mutant yeast cell, is a transgenic or transformed yeast, respectively a transgenic or transformed yeast cell.
  • transgenic refers to a yeast and/or cell, respectively, containing nucleic acid not naturally occurring in that yeast and/or cell and which has been introduced into that yeast and/or cell using for example recombinant DNA techniques, such as a recombinant yeast and/or cell.
  • gene refers to a nucleic acid sequence that can be transcribed into mRNAs that are then translated into protein.
  • a gene encoding for a certain protein refers to the one or more nucleic acid sequence(s) encoding for such a protein.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e. g., peptide nucleic acids).
  • a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene.
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
  • polypeptide polypeptide
  • peptide protein
  • modifications including, but not limited to, glycosylation, lipid attachment, sulphation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • enzyme refers herein to a protein having a catalytic function. Where a protein catalyzes a certain biological reaction, the terms “protein” and “enzyme” may be used interchangeable herein.
  • EC enzyme class
  • the enzyme class is a class wherein the enzyme is classified or may be classified, on the basis of the Enzyme Nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), which nomenclature may be found at http://www.chem.qmul.ac.uk/iubmb/enzyme/.
  • Every nucleic acid sequence herein that encodes a polypeptide also includes any conservatively modified variants thereof. This includes that, by reference to the genetic code, it describes every possible silent variation of the nucleic acid.
  • the term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences due to the degeneracy of the genetic code.
  • degeneracy of the genetic code refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are "silent variations" and represent one species of conservatively modified variation.
  • nucleotide sequence and “nucleic acid sequence” are used interchangeably herein.
  • the term “functional homologue” (or in short “homologue”) of a polypeptide and/or amino acid sequence having a specific sequence (e.g. “SEQ ID NO: X”), as used herein, refers to a polypeptide and/or amino acid sequence comprising said specific sequence with the proviso that one or more amino acids are substituted, deleted, added, and/or inserted, and which polypeptide has (qualitatively) the same enzymatic functionality for substrate conversion.
  • the term functional homologue is meant to include nucleic acid sequences which differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.
  • Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • Amino acid or nucleotide sequences are said to be homologous when exhibiting a certain level of similarity.
  • Two sequences being homologous indicate a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by “percent identity” or “percent similarity”, which is high or low respectively.
  • percent identity or “percent similarity”
  • level of homology or “percent homology” are frequently used interchangeably.
  • a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the homology between two sequences (Kruskal, J. B.
  • the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice,P. Longden.l. and Bleasby.A. Trends in Genetics 16, (6) pp276 — 277, http://emboss.bioinformatics.nl/).
  • EBLOSUM62 is used for the substitution matrix.
  • EDNAFULL is used for nucleotide sequences.
  • Other matrices can be specified.
  • the optional parameters used for alignment of amino acid sequences are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • the homology or identity is the percentage of identical matches between the two full sequences over the total aligned region including any gaps or extensions.
  • the homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment including the gaps.
  • the identity defined as herein can be obtained from NEEDLE and is labelled in the output of the program as “IDENTITY”.
  • the homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.
  • the identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as “longest-identity”.
  • a variant of a nucleotide or amino acid sequence disclosed herein may also be defined as a nucleotide or amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the nucleotide or amino acid sequence specifically disclosed herein (e.g. in de the sequence listing).
  • amino acid similarity the skilled person may also take into account so-called “conservative” amino acid substitutions, as will be clear to the skilled person.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagineglutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; lie to Leu or Vai; Leu to lie or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.
  • Nucleotide sequences of the invention may also be defined by their capability to hybridise with parts of specific nucleotide sequences disclosed herein, respectively, under moderate, or preferably under stringent hybridisation conditions.
  • Stringent hybridisation conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridise at a temperature of about 65°C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at 65°C in a solution comprising about 0.1 M salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength.
  • the hybridisation is performed overnight, i.e. at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution.
  • Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridise at a temperature of about 45°C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength.
  • the hybridisation is performed overnight, i.e. at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution.
  • These conditions will usually allow the specific hybridisation of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridisation conditions in order to specifically identify sequences varying in identity between 50% and 90%.
  • “Expression” refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.
  • “Overexpression” refers to expression of a gene by a recombinant cell in excess to its expression in a corresponding wild-type cell. Such overexpression can for example be arranged for by: increasing the frequency of transcription of one or more nucleic acid sequences, for example by operational linking of the nucleic acid sequence to a promoter functional within the recombinant cell; and/or by increasing the number of copies of a certain nucleic acid sequence.
  • upregulate refers to a process by which a cell increases the quantity of a cellular component, such as RNA or protein. Such an upregulation may be in response to or caused by a genetic modification.
  • pathway or “metabolic pathway” is herein understood a series of chemical reactions in a cell that build and breakdown molecules.
  • Nucleic acid sequences i.e. polynucleotides
  • proteins i.e. polypeptides
  • “Homologous” with respect to a host cell means that the nucleic acid sequence does naturally occur in the genome of the host cell or that the protein is naturally produced by that cell. Homologous protein expression may e.g. be an overexpression or expression under control of a different promoter.
  • the host cell is a yeast.
  • heterologous with respect to the host cell, means that the polynucleotide does not naturally occur in that way in the genome of the host cell or that the polypeptide is not naturally produced by that cell.
  • Heterologous protein expression involves expression of a protein that is not naturally produced in that way in the host cell.
  • heterologous may refer to a nucleic acid or protein is a nucleic acid or protein that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form.
  • a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
  • heterologous expression refers to the expression of heterologous nucleic acids in a host cell.
  • the expression of heterologous proteins in eukaryotic host cell systems such as yeast are well known to those of skill in the art.
  • a polynucleotide comprising a nucleic acid sequence of a gene encoding a certain protein or enzyme with a specific activity can be expressed in such a eukaryotic system.
  • transformed/transfected cells may be employed as expression systems for the expression of the enzymes. Expression of heterologous proteins in yeast is well known.
  • yeasts Two widely utilized yeasts are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
  • promoters including 3-phosphoglycerate kinase or alcohol oxidase
  • promoter is a DNA sequence that directs the transcription of a (structural) gene. Typically, a promoter is located in the 5'-region of a gene, proximal to the transcriptional start site of a (structural) gene. Promoter sequences may be constitutive, inducible or repressible. In an embodiment there is no (external) inducer needed.
  • vector includes reference to an autosomal expression vector and to an integration vector used for integration into the chromosome.
  • expression vector refers to a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest under the control of (i.e. operably linked to) additional nucleic acid segments that provide for its transcription.
  • additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • an expression vector comprises a nucleic acid sequence that comprises in the 5' to 3' direction and operably linked: (a) a yeast-recognized transcription and translation initiation region, (b) a coding sequence for a polypeptide of interest, and (c) a yeast- recognized transcription and translation termination region.
  • “Plasmid” refers to autonomously replicating extrachromosoma I DNA which is not integrated into a microorganism's genome and is usually circular in nature.
  • an “integration vector” refers to a DNA molecule, linear or circular, that can be incorporated in a microorganism's genome and provides for stable inheritance of a gene encoding a polypeptide of interest.
  • the integration vector generally comprises one or more segments comprising a gene sequence encoding a polypeptide of interest under the control of (i.e. operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and one or more segments that drive the incorporation of the gene of interest into the genome of the target cell, usually by the process of homologous recombination.
  • the integration vector will be one which can be transferred into the target cell, but which has a replicon which is nonfunctional in that organism. Integration of the segment comprising the gene of interest may be selected if an appropriate marker is included within that segment.
  • host cell a cell, such as a yeast cell, that is to be transformed with one or more nucleic acid sequences encoding for one or more heterologous proteins, to construe a transformed cell, also referred to as a recombinant cell.
  • the transformed cell may contain a vector and may support the replication and/or expression of the vector.
  • Transformation and “transforming”, as used herein, refers to the insertion of an exogenous polynucleotide (i.e. an exogenous nucleic acid sequence) into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.
  • anaerobic constitutive expression is herein understood that nucleic acid sequence is constitutively expressed in an organism under anaerobic conditions. That is, under anaerobic conditions the nucleic acid sequence is transcribed in an ongoing manner, i.e. under such anaerobic conditions the genes are always “on”.
  • disruption is herein understood any disruption of activity, including, but not limited to, deletion, mutation and reduction of the affinity of the disrupted gene and expression of RNA complementary to such disrupted gene. It includes all nucleic acid modifications such as nucleotide deletions or substitutions, gene knock-outs, and other actions which affect the translation or transcription of the corresponding polypeptide and/or which affect the enzymatic (specific) activity, its substrate specificity, and/or or stability. It also includes modifications that may be targeted on the coding sequence or on the promotor of the gene.
  • a gene disruptant is a cell that has one or more disruptions of the respective gene. Native to yeast herein is understood as that the gene is present in the yeast cell before the disruption.
  • encoding has the same meaning as “coding for”.
  • coding for has the same meaning as “one or more genes coding for a sorbitol dehydrogenase”.
  • nucleic acid sequences encoding a protein or an enzyme As far as genes or nucleic acid sequences encoding a protein or an enzyme are concerned, the phrase “one or more nucleic acid sequences encoding a X”, wherein X denotes a protein, has the same meaning as “one or more nucleic acid sequences encoding a protein having X activity”. Thus, by way of example, “one or more nucleic acid sequences encoding a sorbitol dehydrogenase” has the same meaning as “one or more nucleic acid sequences encoding a protein having sorbitol dehydrogenase activity”.
  • NADH refers to reduced, hydrogenated form of nicotinamide adenine dinucleotide.
  • NAD+ refers to the oxidized form of nicotinamide adenine dinucleotide. Nicotinamide adenine dinucleotide may act as a so-called cofactor, assisting in biochemical reactions and/or transformations in a cell.
  • redox reaction The conversion of NADH into NAD+ and vice-versa is a so-called redox reaction.
  • redox reductionoxidation
  • electrons are transferred from a donor (i.e. a reducing agent that is being oxidized) to an acceptor (i.e. an oxidizing agent that is being reduced).
  • Electron-transfer reactions proceed in the direction in which electrons flow from sources (reducing agents) to sinks (oxidizing agents).
  • sources reducing agents
  • sinks oxidizing agents
  • the NAD+ ions can serve as an electron sink to NADH.
  • a “redox sink” is herein understood a metabolic pathway that, overall, consumes or oxidizes NADH into NAD+ and/or prevents or reduces the consumption or reduction of NAD+ into NADH.
  • a nonnative metabolic pathway is a metabolic pathway that does not occur in the corresponding wild-type cell.
  • a non-native metabolic pathway forming a redox sink is preferably a non-native metabolic pathway that, as compared to a corresponding wild-type yeast cell, increases NADH consumption and/or decreases NAD+ consumption.
  • NADH dependent is herein equivalent to NADH specific and NADH dependency is herein equivalent to NADH specificity.
  • NADH dependent enzyme an enzyme that is exclusively depended on NADH as a co-factor or that is predominantly dependent on NADH as a cofactor.
  • exclusive NADH dependent an enzyme that has an absolute requirement for NADH over NADPH. That is, it is only active when NADH is applied as cofactor.
  • predominantly NADH-dependent an enzyme that has a higher specificity and/or a higher catalytic efficiency for NADH as a cofactor than for NADPH as a cofactor.
  • K m NADP + / K m NAD + is between 1 and 1000, between 1 and 500, between 1 and 200, between 1 and 100, between 1 and 50, between 1 and 10, between 5 and 100, between 5 and 50, between 5 and 20 or between 5 and 10.
  • the Km’s for the enzymes herein can be determined as enzyme specific, for NAD + and NADP + respectively, using know analysis techniques, calculations and protocols. These are described for instance in Lodish et al., Molecular Cell Biology 6 th Edition, Ed. Freeman, pages 80 and 81 , e.g. Figure 3-22.
  • the ratio of the catalytic efficiency for NADPH/NADP+ as a cofactor (fcat/K m ) NADP+ to NADH/NAD+ as cofactor (k C at/K m ) NAD+ i.e.
  • the catalytic efficiency ratio (kcat/K m ) NADP+ : (kcat/K m ) NAD+ is more than 1 :1 , more preferably equal to or more than 2:1 , still more preferably equal to or more than 5:1 , even more preferably equal to or more than 10:1 , yet even more preferably equal to or more than 20:1 , even still more preferably equal to or more than 100:1 , and most preferably equal to or more than 1000:1 .
  • the predominantly NADH-dependent enzyme may have a catalytic efficiency ratio (fcat/K m ) NADP+ : (fcat/K m ) NAD+ of equal to or less than 1 .000.000.000:1 (i.e. 1 .10 9 : 1 ) .
  • the mutant yeast cell is preferably a yeast cell, or derived from a yeast cell, from the genus of Saccharomycetaceae or the genus of Schizosaccharomycetaceae.
  • yeast cells include Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces jurei, Saccharomyces pastorianus, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus.
  • Saccharomyces such as Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces jurei, Saccharomyces pastorianus, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus.
  • yeast cells further include Schizosaccharomyces, such as Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus;.
  • Schizosaccharomyces such as Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus;.
  • Other exemplary yeasts include Torulaspora such as Torulaspora delbrueckii; Kluyveromyces such as Kluyveromyces marxianus; Pichia such as Pichia stipitis, Pichia pastoris or pichia angusta; Zygosaccharomyces such as Zygosaccharomyces bailii; Brettanomyces such as Brettanomyces inter minims; Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis and Dekkera anomala; Metschmkowia, Issatchenkia, such as Issatchenkia orientalis, KJoeckera such as KJoeckera apiculata; and Aureobasidium such as Aureobasidium pullulans.
  • Torulaspora such
  • the yeast cell is preferably a yeast cell of the genus Schizosaccharomyces, herein also referred to as a Schizosaccharomyces yeast cell, or a yeast cell of the genus Saccharomyces, herein also referred to as a Saccharomyces yeast cell. More preferably the yeast cell is a yeast cell derived from a yeast cell of the species Saccharomyces cerevisiae, herein also referred to as a Saccharomyces cerevisae yeast cell.
  • the yeast cell is an industrial yeast cell.
  • the living environments of yeast cells in industrial processes are significantly different from that in the laboratory.
  • Industrial yeast cells must be able to perform well under multiple environmental conditions which may vary during the process. Such variations include changes in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have potential impact on the cellular growth and ethanol production of the yeast cell.
  • An industrial yeast cell can be understood to refer to a yeast cell that, when compared to a laboratory counterpart, has a more robust performance. That is, when compared to a laboratory counterpart, the industrial yeast cell shows less variation in performance when one or more environmental conditions selected from the group of nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, are varied during fermentation.
  • the yeast cell is constructed on the basis of an industrial yeast cell as a parent or a host, wherein the construction is conducted as described hereinafter.
  • industrial yeast cells are Ethanol Red® (Fermentis) Fermiol® (DSM) and Thermosacc® (Lallemand).
  • the mutant yeast cell described herein may be derived from a parent yeast cell capable of producing a fermentation product.
  • the parent yeast cell is an industrial yeast cell as described herein above.
  • the yeast cell described herein is derived from a parent yeast cell having the ability to produce ethanol.
  • the mutant yeast cell is a recombinant yeast cell.
  • This recombinant yeast cell may be derived from any host cell capable of producing a fermentation product.
  • the host cell is an industrial yeast cell as described herein above.
  • the mutant yeast cell is derived from a host cell having the ability to produce ethanol.
  • the yeast cell described herein may be derived from the parent or host cell through any technique known by one skilled in the art to be suitable therefore. Such techniques may include any one or more of adaptive evolution, mutagenesis, recombinant DNA technology (including, but not limited to, CRISPR-CAS techniques), selective and/or adaptive evolution, mating, cell fusion, and/or cytoduction between yeast strains. Suitably the one or more desired genes are incorporated in the yeast cell by a combination of one or more of the above techniques.
  • the mutant yeast cell suitably comprises a first genetic modification for, preferably constitutive, expression of a NAD+ dependent protein that functions in a first metabolic pathway converting a sugar alcohol into a fermentation product.
  • one or more first genetic modifications may result in the, preferably constitutive, expression of merely one NAD+ dependent protein, or more than one NAD+ dependent protein, such as two or more NAD+ dependent proteins, that function in a first metabolic pathway converting a sugar alcohol into a fermentation product.
  • the mutant yeast cell comprises one or more first genetic modifications for anaerobic constitutive expression (i.e. constitutive expression under anaerobic conditions) of one or more NAD+ dependent proteins that function in a first metabolic pathway converting a sugar alcohol into a fermentation product.
  • the "one or more first genetic modifications for, preferably constitutive, expression of a NAD+ dependent protein that functions in a first metabolic pathway converting a sugar alcohol into a fermentation product are chosen from the group consisting of: a) one or more first genetic modifications comprising or consisting of a genetic modification to constitutively express and/or upregulate the activity of one or more proteins having NAD+ dependent sugar alcohol dehydrogenase activity, preferably a NAD+ dependent sorbitol dehydrogenase and/or a NAD+ dependent mannitol dehydrogenase; and/or b) one or more first genetic modifications comprising or consisting of a genetic modification to downregulate the activity of one or more proteins that play a role in the glucose repression of the yeast.
  • the NAD+ dependent protein is an enzyme having the ability to convert a sugar alcohol as described herein.
  • Preferred sugar alcohols include sorbitol and mannitol. More preferably the NAD+ dependent protein is a protein having NAD+ dependent sugar alcohol dehydrogenase activity. Most preferably the protein having NAD+ dependent sugar alcohol dehydrogenase activity is a NAD+ dependent sorbitol dehydrogenase or a NAD+ dependent mannitol dehydrogenase.
  • Such sorbitol dehydrogenase and mannitol dehydrogenase are preferable derived from Saccharomyces cerevisiae.
  • the first metabolic pathway to convert a sugar alcohol preferably comprises a NAD+ dependent sugar alcohol dehydrogenase. More preferably the NAD+ dependent protein that functions in the first metabolic pathway converting a sugar alcohol into a fermentation product is therefore such a NAD+ dependent sugar alcohol dehydrogenase.
  • the mutant yeast cell is a mutant yeast cell comprising one or more first genetic modifications for, preferably constitutive, expression of a NAD+ dependent sugar alcohol dehydrogenase.
  • the mutant yeast cell can be a mutant yeast cell, comprising :
  • the first genetic modifications may result in the constitutive expression of merely one NAD+ dependent sugar alcohol dehydrogenase, or more than one NAD+ dependent sugar alcohol dehydrogenase, such as two or more NAD+ dependent sugar alcohol dehydrogenases.
  • the first metabolic pathway further comprises a sugar alcohol transporter and more preferably the mutant yeast cell comprises one or more genetic modifications to upregulate the activity of one or more sugar alcohol transporters.
  • the first genetic modifications may therefore suitably include modifications for the, preferably constitutive, expression of merely one sugar alcohol transporter, or more than one sugar alcohol transporter, such as two or more sugar alcohol transporters, that function in the first metabolic pathway converting a sugar alcohol into a fermentation product.
  • the mutant yeast cell is therefore a mutant yeast cell comprising one or more first genetic modifications for constitutive expression of a NAD+ dependent sugar alcohol dehydrogenase and optionally for constitutive expression of a sugar alcohol transporter.
  • the one or more first genetic modifications allow for an increase of the activity, as compared to the non-modified yeast cell, of the NAD+ dependent protein(s) that function(s) in the first metabolic pathway to convert a sugar alcohol into a fermentation product.
  • the mutant yeast cell is a mutant yeast cell comprising one or more first genetic modifications for increasing, as compared to the non-modified yeast cell, the activity of a NAD+ dependent sugar alcohol dehydrogenase and/or for increasing, as compared to the non-modified yeast cell, the activity of a sugar alcohol transporter.
  • the mutant yeast cell can be a mutant yeast cell, comprising :
  • the activity of the NAD+ dependent sugar alcohol dehydrogenases and/or the sugar alcohol transporter can be increased in any manner known to be suitable therefore by the person skilled in the art.
  • the mutant yeast cell can be prepared with known recombination techniques.
  • the manners to increase activity may for example include:
  • nucleic acid sequence encoding for the protein or enzyme is herein also referred to as the “coding sequence” or the “coding nucleic acid sequence”.
  • coding sequence or the “coding nucleic acid sequence”.
  • a protein or enzyme may be overexpressed by increasing the copy number of the gene coding for the protein or enzyme in the host cell, for example by integrating additional copies of the gene in the host cell's genome, by expressing the gene from an episomal multicopy expression vector or by introducing a episomal expression vector that comprises multiple copies of the gene.
  • overexpression of a protein or enzyme in the mutant yeast cell may be achieved by using a, preferably heterologous, promoter.
  • a promotor may suitably be nonnative to the nucleic acid sequence coding for the protein or enzyme to be overexpressed, i.e. a promoter that is heterologous to the coding nucleic acid sequence to which it is operably linked.
  • a promoter preferably is heterologous to the coding nucleic acid sequence to which it is operably linked, it is still possible for the promoter itself to be homologous, i.e. endogenous to the host cell.
  • the promoter may be a promoter that is normally operably linked to another nucleic acid sequence within the cell.
  • the heterologous promoter is capable of producing a higher steady state level of the transcript comprising the coding nucleic acid sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is the promoter that is native to the coding nucleic acid sequence.
  • Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters.
  • the coding nucleic acid sequence used for overexpression of the proteins or enzymes mentioned above may preferably be homologous to the host cell. However, coding nucleic acid sequences that are heterologous to the host may also be used.
  • the mutant yeast may comprises a genetic modification to downregulate one or more genes that have a role in glucose repression. More preferably, the mutant yeast may comprise a genetic modification to downregulate the activity of a homologous gene encoding a TUP1 protein and/or CYC8 protein of the yeast.
  • TUP1 protein examples include:
  • a functional homologue thereof comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of respectively SEQ ID NO: 47; or
  • a functional homologue comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 47; wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 47.
  • Examples of such a CYC8 protein include:
  • a functional homologue thereof comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of respectively SEQ ID NO: 48; or
  • a functional homologue comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 48; wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 48.
  • Overexpression of a protein or enzyme when referring to the production of such a protein or enzyme in a mutant yeast cell, suitably means that the protein or enzyme is produced at a higher level of specific enzymatic activity as compared to the unmodified host cell under identical conditions.
  • the enzymatically active protein (or proteins in case of multi-subunit enzymes) is produced in greater amounts, or rather at a higher steady state level as compared to the unmodified host cell under identical conditions.
  • the mRNA coding for the enzymatically active protein is produced in greater amounts, or again rather at a higher steady state level as compared to the unmodified host cell under identical conditions.
  • the above NAD+ dependent sugar alcohol dehydrogenase is preferably overexpressed by a factor of at least 1 .1 , at least 1 .2, at least 1 .5, at least 2, at least 5, at least 10 or at least 20 as compared to a strain which is genetically identical except for the genetic modification causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.
  • sugar alcohol dehydrogenase a protein having sugar alcohol dehydrogenase activity
  • NAD+ dependent sugar alcohol dehydrogenase a NAD+ dependent protein having sugar alcohol dehydrogenase activity
  • sorbitol dehydrogenase any protein having sorbitol dehydrogenase activity
  • mannitol dehydrogenase any protein having mannitol dehydrogenase activity.
  • a "NAD+ dependent protein having sugar alcohol dehydrogenase activity” can herein also be referred to as a "protein having NAD+ dependent sugar alcohol dehydrogenase activity", a “sugar alcohol dehydrogenase protein”, a “sugar alcohol dehydrogenase enzyme”, or simply a "NAD+ dependent sugar alcohol dehydrogenase” or "sugar alcohol dehydrogenase”.
  • the sugar alcohol is a sugar alcohol having equal to or more than 5 carbon atoms, more preferably equal to or more than 6 carbon atoms.
  • suitable sugar alcohols include arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, idotol, inositol, isomalt, maltitol and lactitol.
  • sugar alcohol dehydrogenases examples include arabitol dehydrogenase, xylitol dehydrogenase, ribitol dehydrogenase, mannitol dehydrogenase, sorbitol dehydrogenase, galactitol dehydrogenase, fucitol dehydrogenase, idotol dehydrogenase, inositol dehydrogenase, isomalt dehydrogenase, maltitol dehydrogenase and lactitol dehydrogenase.
  • glycerol is not considered to be a sugar alcohol and the sugar alcohol is not glycerol.
  • the sugar alcohol dehydrogenase is therefore not glycerol dehydrogenase.
  • the sugar alcohol is a sugar alcohol comprising 6 carbon atoms (also referred to as a “C6 sugar alcohol”).
  • Most preferred sugar alcohols are sorbitol and mannitol.
  • Most preferred NAD+ dependent sugar alcohol dehydrogenases are NAD+ dependent sorbitol dehydrogenase and NAD+ dependent mannitol dehydrogenase.
  • the sugar alcohol is not xylitol and/or preferably the sugar alcohol dehydrogenase is not xylitol dehydrogenase.
  • Sugar alcohols comprising 5 carbon atoms also referred to as "C5 sugar alcohols”
  • sugar alcohol dehydrogenases for such C5 sugar alcohols are less preferred.
  • sugar alcohol dehydrogenase is:
  • NAD+ dependent sorbitol dehydrogenase chosen from the group consisting of NAD+ dependent sorbitol dehydrogenase 1 (SOR1), preferably having an amino acid sequence of SEQ ID NO: 9, and NAD+ dependent sorbitol dehydrogenase 2 (SOR2), preferably having an amino acid sequence of SEQ ID NO: 11 ; or
  • NAD+ dependent mannitol dehydrogenase chosen from the group consisting of NAD+ dependent mannitol dehydrogenase 1 (MAN1), preferably having an amino acid sequence of SEQ ID NO: 13, and NAD+ dependent mannitol dehydrogenase 2 (MAN2), preferably having an amino acid sequence of SEQ ID NO: 15; or
  • a functional homologue of any of the above preferably a functional homologue comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of respectively SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13 or SEQ ID NO: 15; or a functional homologue comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13 or SEQ ID NO: 15, wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more
  • the nucleic acid sequence encoding the sugar alcohol dehydrogenases as listed above can be an exogenous or heterologous nucleic acid sequence or endogenous or native nucleic acid sequence.
  • the mutant yeast cell comprises a nucleic acid sequence encoding an exogenous or heterologous sugar alcohol dehydrogenase
  • one or more endogenous or native nucleic acid sequence(s) encoding one or more native sugar alcohol dehydrogenases may be deleted or disrupted.
  • mutant yeast cell is a mutant yeast cell comprising:
  • nucleic acid sequence encoding for any of the above mentioned sugar alcohol dehydrogenases
  • nucleic acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the nucleic acid sequence of respectively SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; and/or
  • nucleic acid sequence having one or several substitutions, insertions and/or deletions as compared to the nucleic acid sequence of respectively SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, wherein more preferably the nucleic acid sequence has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 nucleic acid substitutions, insertions and/or deletions as compared to the nucleic acid sequence of respectively SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
  • the first genetic modifications can be present for example in the form of:
  • the mutant yeast cell can thus suitably be a mutant yeast cell, comprising : one or more first genetic modifications for constitutive expression, and preferably overexpression, of a nucleic acid sequence chosen from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16; or a nucleic acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with such nucleic acid sequence; or a nucleic acid sequence having one or several substitutions, insertions and/or deletions as compared to such nucleic acid sequence, wherein more preferably the nucleic acid sequence has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no
  • the NAD+ dependent protein is sorbitol dehydrogenase and/or the mutant yeast cell comprises one or more first genetic modifications for constitutive expression or overexpression of a sorbitol dehydrogenase, for example in the form of:
  • an exogenous promoter operably linked to an endogenous nucleic acid sequence encoding for a sorbitol dehydrogenase
  • mutant yeast cell comprises an exogenous gene or exogenous nucleic acid sequence coding for an NAD+ dependent sorbitol dehydrogenase selected from the group consisting of:
  • NAD+ dependent sorbitol dehydrogenase comprising an amino acid sequence with at least 50 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with one or more of such aforementioned NAD+ dependent sorbitol dehydrogenase;
  • NAD+ dependent sorbitol dehydrogenase comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of one or more of such aforementioned NAD+ dependent sorbitol dehydrogenase, wherein preferably the amino acid sequence of any of the above functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to such aforementioned NAD+ dependent sorbitol dehydrogenase.
  • a sorbitol dehydrogenase having an amino acid sequence as listed in SEQ ID NO: 11 is especially preferred.
  • SEQ ID NO: 11 shows the amino acid sequence of a highly preferred NAD+ dependent sorbitol dehydrogenase protein, i.e. sorbitol dehydrogenase 2 (SOR2), encoded by a nucleic acid sequence from Saccharomyces cerevisiae.
  • SEQ ID NO 12 shows a suitable nucleic acid sequence encoding for this highly preferred amino acid sequence.
  • sorbitol dehydrogenase proteins include functional homologues of this protein, preferably functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 11 or functional homologues comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID NO: 11 , wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ
  • Rhodobacter sphaeroides (as described in the article of Philippsen et al., titled “Structure of zinc- independent sorbitol dehydrogenase from Rhodobacter sphaeroides at 2.4 A resolution", published in Acta Crystallogr D Biol Crystallogr., vol. 61 (2005), pages 374-379, herewith incorporated by reference);
  • the NAD+ dependent protein can be mannitol dehydrogenase and/or the mutant yeast cell can comprise one or more first genetic modifications for constitutive expression or overexpression of a mannitol dehydrogenase, for example in the form of: - an exogenous nucleic acid sequence encoding for a mannitol dehydrogenase, suitably in one or more copies, and/or - an exogenous promoter operably linked to an endogenous nucleic acid sequence encoding for a mannitol dehydrogenase, and/or
  • mutant yeast cell comprises an exogenous gene coding for an NAD+ dependent mannitol dehydrogenase selected from the group consisting of:
  • NAD+ dependent mannitol dehydrogenase comprising an amino acid sequence with at least 50 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with one or more of such aforementioned NAD+ dependent mannitol dehydrogenase;
  • the amino acid sequence of any of the above functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to such aforementioned NAD+ dependent mannitol dehydrogenase.
  • a mannitol dehydrogenase having an amino acid sequence as listed in SEQ ID NO: 15 is especially preferred.
  • SEQ ID NO: 15 shows the amino acid sequence of a NAD+ dependent suitable mannitol dehydrogenase protein, i.e. mannitol dehydrogenase 2 (MAN2) encoded by a nucleic acid sequence from Saccharomyces cerevisiae.
  • MAN2 mannitol dehydrogenase 2
  • SEQ ID NO 16 shows an optimized nucleic acid sequence encoding for this amino acid sequence.
  • mannitol dehydrogenase proteins include functional homologues of this protein, preferably functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 15; or functional homologues comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID NO: 15, wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID
  • the mutant yeast cell may preferably comprise one or more transporters suitable for the transport of a sugar alcohol into the mutant yeast cell.
  • a “sugar alcohol transporter” is thus herein understood to be a protein capable of transporting the sugar alcohol into the mutant yeast cell.
  • sugar alcohols are as described above.
  • Such sugar alcohols may suitably be transported by a pentose transporter or hexose transporter, dependent on the type of sugar alcohol.
  • the sugar alcohol is a sugar alcohol comprising 6 carbon atoms (also referred to as a “C6 sugar alcohol”).
  • C6 sugar alcohol is preferably transported by a hexose transporter.
  • the sugar alcohol transporter is a hexose transporter.
  • the above exemplified sorbitol and/or mannitol are preferably transported by a hexose transporter into the mutant yeast cell.
  • Suitable sugar alcohol transporters include those as mentioned by Jordan et al.
  • the sugar alcohol transporter is a hexose transporter chosen from the group consisting of HXT13, HXT15 and HXT17.
  • hexose transporters include hexose transporters chosen from the group consisting of:
  • - HXT13 preferably having an amino acid sequence of SEQ ID NO: 17;
  • - HXT15 preferably having an amino acid sequence of SEQ ID NO: 19;
  • a functional homologue comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of respectively SEQ ID NO: 17 or SEQ ID NO: 19; and/or a functional homologue comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 17 or SEQ ID NO: 19, wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 17
  • the nucleic acid sequence encoding the hexose transporters as listed above can be an exogenous or heterologous nucleic acid sequence or an endogenous or native nucleic acid sequence.
  • the mutant yeast cell comprises a nucleic acid sequence encoding an exogenous or heterologous hexose transporter, one or more endogenous or native nucleic acid sequence(s) encoding one or more native hexose transporters may be deleted or disrupted.
  • mutant yeast cell is a mutant yeast cell comprising:
  • nucleic acid sequence encoding for any of the above mentioned hexose transporters
  • nucleic acid sequence of SEQ ID NO: 18, or SEQ ID NO: 20 and/or - a nucleic acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the nucleic acid sequence of respectively SEQ ID NO: 18 or SEQ ID NO: 20; and/or
  • nucleic acid sequence having one or several substitutions, insertions and/or deletions as compared to the nucleic acid sequence of respectively SEQ ID NO: 18 or SEQ ID NO: 20, wherein more preferably the nucleic acid sequence has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 nucleic acid substitutions, insertions and/or deletions as compared to the nucleic acid sequence of respectively SEQ ID NO: 18 or SEQ ID NO: 20.
  • HXT13 and HXT15 have moderate affinity for sorbitol, while HXT13, HXT15 and HXT17 exhibit high affinity for mannitol.
  • the inventors of the present invention have found that the hexose transporter HXT13 can advantageously be combined with sorbitol dehydrogenase to render exceptionally good results.
  • SEQ ID NO: 17 shows the amino acid sequence of a hexose transporter HXT13.
  • suitable hexose transporters include functional homologues of this protein, preferably functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 17; or functional homologues comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID NO: 17, wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than
  • hexose transporters are highly conserved amongst the Saccharomyces species, functional homologues are very likely to be found in other Saccharomyces species including for example Saccharomyces paradoxus. Examples of suitable hexose transporters and their origin are given in Table 3 below, with reference to their sequence identity with the amino acid sequence of SEQ ID NO:17.
  • Candida glycerinogenes (as described in the article by Liang et al., titled “Identification and characterization from Candida glycerinogenes of hexose transporters having high efficiency at high glucose concentrations", published in Applied Microbiology and Biotechnology vol.102, (2016) pages 5557-5567, herewith incorporated by reference).
  • polyol/H + symporters can be used for the transport of sugar alcohols, such as for example sorbitol and/or mannitol.
  • the sugar alcohol transporter can also be a polyol/H + symporter.
  • transport is coupled. Connecting these polyol/proton symporters to the sugar alcohol dehydrogenase can advantageously result in a lower energy use and an increase in yield.
  • the mutant yeast cell comprises one or more heterologous nucleic acid sequences encoding a hexose transporter and/or polyol/H+ symporter, whilst one or more native nucleic acid sequence(s) encoding one or more native hexose transporters and/or polyol/H+ symporter may be deleted or disrupted.
  • Polyol/H + symporters that can be suitable for the transport of sugar alcohols, such as sorbitol and/or mannitol, are those characterized in S. cerevisiae and those identified in other yeasts and/or plants, such as :
  • Polyol/H + symporters include functional homologues of the above proteins, for example functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity therewith.
  • Table 3 alternative hexose transporters (proteins) for expression with % identity as compared to
  • the mutant yeast cell further comprises a second genetic modification for, preferably constitutive, expression of a protein that functions in a second metabolic pathway forming a non-native redox sink.
  • these one or more second genetic modifications are one or more second genetic modifications for the functional expression of one or more heterologous nucleic acid sequences encoding for one or more NADH dependent proteins that function in a second metabolic pathway to convert NADH to NAD+.
  • second metabolic pathways Several examples of such second metabolic pathways exist, as illustrated further below.
  • the mutant yeast cell comprises one or more second genetic modifications for anaerobic constitutive expression of one or more NADH dependent proteins that function in a second metabolic pathway to convert NADH to NAD+.
  • the "one or more second genetic modifications for constitutive expression of a protein that functions in a second metabolic pathway forming a non-native redox sink" are chosen from the group consisting of: a) one or more second genetic modifications comprising or consisting of:
  • a heterologous nucleic acid sequence encoding a protein comprising phosphoketolase activity (EC 4.1 .2.9 or EC 4.1 .2.22, PKL); and/or
  • a heterologous nucleic acid sequence encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity. and/or c) one or more second genetic modifications comprising or consisting of: a heterologous nucleic acid sequence encoding a protein comprising NADH dependent acetylating acetaldehyde dehydrogenase activity.
  • WO2014/081803 describes a recombinant microorganism expressing a heterologous phosphoketolase, phosphotransacetylase or acetate kinase and bifunctional acetaldeyde-alcohol dehydrogenase
  • WO2015/148272 describes a recombinant S. cerevisiae strain expressing a heterologous phosphoketolase, phosphotransacetylase and acetylating acetaldehyde dehydrogenase.
  • WO2018172328A1 describes recombinant cell may comprise one or more (heterologous) genes coding for an enzyme having phosphoketolase activity.
  • PDL phosphoketalase
  • a suitable example of the mutant yeast cell according to the invention is therefore a mutant yeast cell, comprising:
  • one or more second genetic modifications comprising or consisting of: - a heterologous nucleic acid sequence encoding a protein comprising phosphoketolase activity (EC 4.1 .2.9 or EC 4.1 .2.22, PKL); and/or
  • ACK acetate kinase activity
  • mutant yeast cell comprising:
  • WO2014/129898 describes a recombinant cell functionally expressing heterologous nucleic acid sequences encoding for ribulose-1 ,5-phosphate carboxylase/oxygenase (EC 4.1 .1 .39; herein abbreviated as “Rubisco”), and optionally molecular chaperones for Rubisco, and phosphoribulokinase (EC 2.7.1.19; herein abbreviated as “PRK”).
  • Rubisco ribulose-1 ,5-phosphate carboxylase/oxygenase
  • PRK phosphoribulokinase
  • mutant yeast cell according to the invention is therefore a mutant yeast cell, comprising:
  • the mutant yeast cell comprises one or more upregulated heterologous nucleic acid sequences encoding for a protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity; one or more upregulated heterologous nucleic acid sequences encoding for a protein having phosphoribulokinase (PRK) activity; and, optionally, one or more upregulated heterologous nucleic acid sequences encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity
  • NAD+ dependent sugar alcohol dehydrogenase such as NAD+ dependent sorbitol dehydrogenase and/or NAD+ dependent mannitol dehydrogenase can advantageously be used to moderate and/or balance any overactivity of the ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco).
  • the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity is herein also referred to as “Rubisco enzyme”, “Rubisco protein” or simply “Rubisco”.
  • Rubisco enzyme ribulose-1 ,5-biphosphate carboxylase oxygenase
  • the Rubisco protein may suitably be selected from the group of eukaryotic and prokaryotic Rubisco proteins.
  • the Rubisco protein is preferably from a non-phototrophic organism.
  • the Rubisco protein may be from a chemolithoautotrophic microorganism. Good results have been achieved with a bacterial Rubisco protein.
  • the Rubisco protein originates from a Thiobacillus, in particular, Thiobacillus denitrificans, which is chemolithoautotrophic.
  • the Rubisco protein may be a single-subunit Rubisco protein or a Rubisco protein having more than one subunit.
  • the Rubisco protein is a single-subunit Rubisco protein.
  • Good results have been obtained with a Rubisco protein that is a so-called form-ll Rubisco protein.
  • a preferred Rubisco protein is the Rubisco protein encoded by the cbbM gene from Thiobacillus denitrificans.
  • SEQ ID NO: 1 shows the amino acid sequence of a suitable Rubisco protein, encoded by the cbbM gene from Thiobacillus denitrificans.
  • Rubisco proteins include functional homologues of this Rubisco protein encoded by the cbbM gene from Thiobacillus denitrificans, preferably functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 1.
  • suitable Rubisco polypeptides and their origin are given in Table 1 of WO2014/129898, incorporated herein by reference, and in Table 4 below, with reference to the sequence identity with the amino acid sequence of SEQ ID NO:1.
  • Table 4 Natural Rubisco polypeptides suitable for expression
  • Rubisco proteins include the highly active Rubisco proteins as described by Davidi D., et al. in their article titled " Highly active rubiscos discovered by systematic interrogation of natural sequence diversity", published in the The Embo Journal (2020) Vol. 39, e104081 .
  • Such suitable Rubisco proteins may include the form II Rubisco protein of Gallionella sp. (for example with an kcat of 22.2 s-1 and a kM of 276 uM) and the form II Rubisco protein of Hydrogenovibrio marinus (for example with a kcat of 15.6 s-1 and a kM of 162 uM).
  • the Rubisco protein is suitably functionally expressed in the mutant yeast cell, at least during use in a fermentation process.
  • the nucleic acid sequence encoding the Rubisco protein and/or the nucleic acid sequence encoding other proteins as described herein (see below), are preferably adapted to optimise their codon usage to that of the host cell in question.
  • the adaptiveness of a nucleic acid sequence encoding an enzyme to the codon usage of a host cell may be expressed as codon adaptation index (CAI).
  • CAI codon adaptation index
  • the codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes in a particular host cell or organism.
  • the relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid.
  • the CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1 , with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li , 1987, Nucleic Acids Research 15: 1281- 1295; also see: Jansen et al., 2003, Nucleic Acids Res. 31_(8):2242-51).
  • An adapted nucleic acid sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9.
  • the sequences have been codon optimized for expression in the fungal host cell in question, such as for example Saccharomyces cerevisiae cells.
  • the nucleic acid sequence encoding the Rubisco protein and/or the nucleic acid sequence encoding other proteins as described herein may be present in one or more copies.
  • the nucleic acid sequence encoding the Rubisco protein and/or the nucleic acid sequence encoding other proteins as described herein is present in multiple copies, more preferably in the range from equal to or more than 2 to equal to or less than 20 copies, most preferably in the range from equal to or more than 3 to equal to or less than 15 copies.
  • the functionally expressed Rubisco protein has an activity, defined by the rate of ribulose-1 ,5-bisphosphate- dependent 14 C-bicarbonate incorporation by cell extracts of at least 1 nmol.min- 1 .(mg protein) -1 , in particular an activity of at least 2 nmol. min -1 .(mg protein) -1 , more in particular an activity of at least 4 nmol. min -1 . (mg protein) -1 .
  • the upper limit for the activity is not critical. In practice, the activity may be about 200 nmol. min -1 . (mg protein) -1 or less, in particular 25 nmol. min -1 . (mg protein) -1 , more in particular 15 nmol.
  • PRK protein having phosphoribulokinase (PRK) activity is herein also referred to as “PRK enzyme”, “PRK protein” or simply “PRK”.
  • PRK enzyme phosphoribulokinase
  • PRK protein protein having phosphoribulokinase activity
  • PRK protein protein having phosphoribulokinase activity
  • PRK protein protein having phosphoribulokinase activity
  • PRK protein protein having phosphoribulokinase
  • a functionally expressed phosphoribulokinase (PRK, (EC 2.7.1.19)) according to the invention is capable of catalyzing the chemical reaction (I):
  • the two substrates of this enzyme are ATP and D-ribulose 5-phosphate; its two products are ADP and D-ribulose 1 ,5-bisphosphate.
  • the PRK protein belongs to the family of transferases, specifically those transferring phosphorus- containing groups (phosphotransferases) with an alcohol group as acceptor.
  • the systematic name of this enzyme class is ATP:D-ribulose- 5-phosphate 1 -phosphotransferase.
  • Other names in common use include phosphopentokinase, ribulose-5-phosphate kinase, phosphopentokinase, phosphoribulokinase (phosphorylating), 5-phosphoribulose kinase, ribulose phosphate kinase, PKK, PRuK, and PRK. This enzyme participates in carbon fixation.
  • the PRK can be from a prokaryote or a eukaryote. Good results have been achieved with a PRK originating from a eukaryote.
  • the PRK protein originates from a plant selected from Caryophyllales , in particular from Amaranthaceae, more in particular from Spinacia.
  • a preferred PRK protein is the PRK protein from Spinacia.
  • SEQ ID NO: 3 shows the amino acid sequence of such PRK protein from Spinacia.
  • PRK proteins include functional homologues of the PRK protein from Spinacia, preferably functional homologues comprising an amino acid sequence sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:3.
  • Suitable natural PRK polypeptides are given in Table 5.
  • Examples of suitable PRK polypeptides and their origin are given in Table 2 of WO2014/129898, incorporated herein by reference, and in Table 5 below, with reference to the sequence identity with the amino acid sequence of SEQ ID NO:3.
  • the nucleic acid sequences encoding for the PRK protein may be under the control of a promoter (the "PRK promoter") that enables higher expression under anaerobic conditions than under aerobic conditions.
  • a promoter the "PRK promoter”
  • PRK promoters are described in WO2017/216136A1 and WO2018/228836, both herein incorporated by reference. More preferably such promoter has a PRK expression ratio anaerobic/aerobic of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more or 50 or more. Further preferences are as described in WO2018/228836, incorporated herein by reference.
  • the nucleic acid sequence encoding the PRK protein may be present in one or more copies.
  • the nucleic acid sequence encoding the PRK protein is present in multiple copies, more preferably in the range from equal to or more than 2 to equal to or less than 20 copies, most preferably in the range from equal to or more than 3 to equal to or less than 15 copies.
  • the mutant yeast cell further comprises one or more nucleic acid sequences encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity.
  • Rosha ribulose-1 ,5-biphosphate carboxylase oxygenase
  • such molecular chaperones are also referred herein as “chaperone protein”, “chaperonin” or simply “chaperone”.
  • Preferences for the chaperones and the nucleic sequences encoding for such are as described in WO2014/129898, incorporated herein by reference.
  • the mutant yeast cell comprises one or more heterologous nucleic acid sequences encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity.
  • Rosha ribulose-1 ,5-biphosphate carboxylase oxygenase
  • Chaperonins are proteins that provide favorable conditions for the correct folding of other proteins, thus preventing aggregation. Newly made proteins usually must fold from a linear chain of amino acids into a three-dimensional form. Chaperonins belong to a large class of molecules that assist protein folding, called molecular chaperones. The energy to fold proteins is supplied by adenosine triphosphate (ATP).
  • ATP adenosine triphosphate
  • the chaperone or chaperones may be prokaryotic chaperones or eukaryotic chaperones.
  • the chaperones may be homologous or heterologous.
  • the mutant yeast cell may comprises one or more nucleic acid sequence encoding one or more homologous or heterologous, prokaryotic or eukaryotic, molecular chaperones, which - when expressed - are capable of functionally interacting with an enzyme in the mutant yeast cell, in particular with at least one of Rubisco and PRK.
  • the chaperone or chaperones are derived from a bacterium, more preferably from Escherichia, in particular E. coli.
  • Preferred chaperones are GroEL and GroEs from E. coli.
  • Other preferred chaperones are chaperones from Saccharomyces, in particular Saccharomyces cerevisiae Hsp10 and Hsp60.
  • the chaperones are naturally expressed in an organelle such as a mitochondrion (examples are Hsp60 and Hsp10 of Saccharomyces cerevisiae) relocation to the cytosol can be achieved e.g. by modifying the native signal sequence of the chaperonins.
  • the proteins Hsp60 and Hsp10 are structurally and functionally nearly identical to GroEL and GroES, respectively.
  • Hsp60 and Hsp10 from any mutant yeast cell may serve as a chaperone for the Rubisco.
  • a functional homologue of GroES may be present, in particular a functional homologue comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of GroES.
  • SEQ ID NO:7 provides an amino acid sequence of GroES. Examples of suitable natural chaperones polypeptide homologous to GroES are given in Table 6.
  • a functional homologue of GroEL may be present, in particular a functional homologue comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of GroEL.
  • SEQ ID NO:5 provides an amino acid sequence of GroEL.
  • Suitable natural chaperones polypeptides homologous to GroEL are given in Table 7.
  • a 10 kDa chaperone from Table 6 is combined with a matching 60kDa chaperone from Table 7 of the same organism genus or species for expression in the mutant yeast cell.
  • this invention thus also provides a recombinant yeast cell, comprising:
  • the yeast cell may further comprise a deletion or disruption of one or more endogenous nucleotide sequence encoding a glycerol 3-phosphate phosphohydrolase gene and/or encoding a glycerol 3-phosphate dehydrogenase gene.
  • enzymatic activity needed for the NADH-dependent glycerol synthesis in the yeast cell is reduced or deleted.
  • the reduction or deletion of the enzymatic activity of glycerol 3-phosphate phosphohydrolase and/or glycerol 3-phosphate dehydrogenase can be achieved by modifying one or more genes encoding a NAD-dependent glycerol 3-phosphate dehydrogenase (GPD) and/or one or more genes encoding a glycerol phosphate phosphatase (GPP), such that the enzyme is expressed considerably less than in the wild-type or such that the gene encodes a polypeptide with reduced activity.
  • GPD NAD-dependent glycerol 3-phosphate dehydrogenase
  • GFP glycerol phosphate phosphatase
  • Such modifications can be carried out using commonly known biotechnological techniques, and may in particular include one or more knock-out mutations or site-directed mutagenesis of promoter regions or coding regions of the structural genes encoding GPD and/or GPP.
  • yeast strains that are defective in glycerol production may be obtained by random mutagenesis followed by selection of strains with reduced or absent activity of GPD and/or GPP.
  • S. cerevisiae GPD1, GPD2, GPP1 and GPP2 genes are shown in WO2011010923, and are disclosed in SEQ ID NO: 24-27 of that application.
  • the mutant yeast cell preferably comprises one or more genetic modifications for decreasing or inhibiting the activity of glycerol-3-phosphate dehydrogenase (GPD) and/or glycerol-3- phosphate phosphatase (GPP).
  • GPD glycerol-3-phosphate dehydrogenase
  • GPP glycerol-3- phosphate phosphatase
  • At least one gene encoding a GPD and/or at least one gene encoding a GPP is entirely deleted, or at least a part of the gene is deleted that encodes a part of the enzyme that is essential for its activity.
  • good results have been achieved with a S. cerevisiae cell, wherein the open reading frames of the GPD1 gene and of the GPD2 gene have been inactivated.
  • Inactivation of a structural gene (target gene) can be accomplished by a person skilled in the art by synthetically synthesizing or otherwise constructing a DNA fragment consisting of a selectable marker gene flanked by DNA sequences that are identical to sequences that flank the region of the host cell's genome that is to be deleted.
  • glycerol 3-phosphate phosphohydrolase activity in the cell and/or glycerol 3-phosphate dehydrogenase activity in the cell is advantageously reduced.
  • the mutant yeast cell may further advantageously comprise one or more genetic modifications that increases the flux of the pentose phosphate pathway.
  • the genetic modification(s) may lead to an increased flux through the non-oxidative part of the pentose phosphate pathway.
  • a genetic modification that causes an increased flux of the non- oxidative part of the pentose phosphate pathway is herein understood to mean a modification that increases the flux by at least a factor of about 1 .1 , about 1 .2, about 1 .5, about 2, about 5, about 10 or about 20 as compared to the flux in a strain which is genetically identical except for the genetic modification causing the increased flux.
  • the flux of the non-oxidative part of the pentose phosphate pathway may be measured by growing the modified host on xylose as sole carbon source, determining the specific xylose consumption rate and subtracting the specific xylitol production rate from the specific xylose consumption rate, if any xylitol is produced.
  • the flux of the non-oxidative part of the pentose phosphate pathway is proportional with the growth rate on xylose as sole carbon source, preferably with the anaerobic growth rate on xylose as sole carbon source. There is a linear relation between the growth rate on xylose as sole carbon source (p ma x) and the flux of the non-oxidative part of the pentose phosphate pathway.
  • One or more genetic modifications that increase the flux of the pentose phosphate pathway may be introduced in the host cell in various ways. These including e.g. achieving higher steady state activity levels of xylulose kinase and/or one or more of the enzymes of the non-oxidative part pentose phosphate pathway and/or a reduced steady state level of unspecific aldose reductase activity. These changes in steady state activity levels may be effected by selection of mutants (spontaneous or induced by chemicals or radiation) and/or by recombinant DNA technology e.g. by overexpression or inactivation, respectively, of genes encoding the enzymes or factors regulating these genes.
  • the genetic modification comprises overexpression of at least one enzyme of the (non-oxidative part) pentose phosphate pathway.
  • the enzyme is selected from the group consisting of the enzymes encoding for ribulose- 5- phosphate isomerase, ribulose- 5-phosphate epimerase, transketolase and transaldolase.
  • Various combinations of enzymes of the (non-oxidative part) pentose phosphate pathway may be overexpressed. E.g.
  • the enzymes that are overexpressed may be at least the enzymes ribulose- 5-phosphate isomerase and ribulose-5-phosphate epimerase; or at least the enzymes ribulose- 5-phosphate isomerase and transketolase; or at least the enzymes ribulose-5-phosphate isomerase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase and transketolase; or at least the enzymes ribulose- 5- phosphate epimerase and transaldolase; or at least the enzymes transketolase and transaldolase; or at least the enzymes ribulose- 5-phosphate epimerase, transketolase and transaldolase; or at least the enzymes ribulose- 5-phosphate isomerase, transketolase and transaldolase; or at least the enzymes ribulose- 5-phosphate isomerase, transketolase and transaldolase; or at least the enzymes ribulose- 5-phosphate isome
  • each of the enzymes ribulose- 5- phosphate isomerase, ribulose- 5-phosphate epimerase, transketolase and transaldolase are overexpressed in the host cell. More preferred is a host cell in which the genetic modification comprises at least overexpression of both the enzymes transketolase and transaldolase as such a host cell is already capable of anaerobic growth on xylose. In fact, under some conditions host cells overexpressing only the transketolase and the transaldolase already have the same anaerobic growth rate on xylose as do host cells that overexpress all four of the enzymes, i.e.
  • ribulose-5-phosphate isomerase ribulose- 5- phosphate epimerase
  • transketolase transaldolase
  • host cells overexpressing both of the enzymes ribulose- 5-phosphate isomerase and ribulose-5- phosphate epimerase are preferred over host cells overexpressing only the isomerase or only the epimerase as overexpression of only one of these enzymes may produce metabolic imbalances.
  • ribulose 5-phosphate epimerase (EC 5.1.3.1) is herein defined as an enzyme that catalyses the epimerisation of D-xylulose 5-phosphate into D-ribulose 5- phosphate and vice versa.
  • the enzyme is also known as phosphoribulose epimerase; erythrose-4-phosphate isomerase; phosphoketopentose 3-epimerase; xylulose phosphate 3-epimerase; phosphoketopentose epimerase; ribulose 5-phosphate 3- epimerase; D-ribulose phosphate-3-epimerase; D-ribulose 5-phosphate epimerase; D- ribulose-5-P 3-epimerase; D-xylulose-5-phosphate 3-epimerase; pentose- 5-phosphate 3- epimerase; or D-ribulose-5-phosphate 3-epimerase.
  • a ribulose 5-phosphate epimerase may be further defined by its amino acid sequence.
  • a ribulose 5-phosphate epimerase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a ribulose 5-phosphate epimerase.
  • the nucleotide sequence encoding for ribulose 5-phosphate epimerase is herein designated RPE1.
  • ribulose 5-phosphate isomerase (EC 5.3.1.6) is herein defined as an enzyme that catalyses direct isomerisation of D-ribose 5-phosphate into D-ribulose 5-phosphate and vice versa.
  • the enzyme is also known as phosphopentosisomerase; phosphoriboisomerase; ribose phosphate isomerase; 5-phosphoribose isomerase; D- ribose 5-phosphate isomerase; D-ribose- 5-phosphate ketol-isomerase; or D-ribose-5- phosphate aldose-ketose-isomerase.
  • a ribulose 5-phosphate isomerase may be further defined by its amino acid sequence.
  • a ribulose 5-phosphate isomerase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a ribulose 5-phosphate isomerase.
  • the nucleotide sequence encoding for ribulose 5- phosphate isomerase is herein designated RKI1.
  • transketolase (EC 2.2.1.1) is herein defined as an enzyme that catalyses the reaction: D-ribose 5-phosphate + D-xylulose 5-phosphate ⁇ -> sedoheptulose 7-phosphate + D- glyceraldehyde 3-phosphate and vice versa.
  • the enzyme is also known as glycolaldehydetransferase or sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycolaldehydetransferase.
  • a transketolase may be further defined by its amino acid.
  • transketolase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a transketolase.
  • the nucleotide sequence encoding for transketolase is herein designated TKL1.
  • transaldolase (EC 2.2.1.2) is herein defined as an enzyme that catalyses the reaction: sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate ⁇ -> D-erythrose 4-phosphate + D- fructose 6-phosphate and vice versa.
  • the enzyme is also known as dihydroxyacetonetransferase; dihydroxyacetone synthase; formaldehyde transketolase; or sedoheptulose-7- phosphate :D- glyceraldehyde-3 -phosphate glyceronetransferase.
  • a transaldolase may be further defined by its amino acid sequence.
  • transaldolase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a transaldolase.
  • the nucleotide sequence encoding for transketolase from is herein designated TAL1.
  • the deletion of the aldose reductase (GRE3) gene; and/or overexpression of GAL2 and/or deletion of GAL80 may be advantageous.
  • Such a deletion and/or overexpression can suitably be carried out as described in WO2011131667A1 , and is incorporated herein by reference.
  • the mutant yeast cell further comprises suitable co-factors to enhance the activity of the above mentioned proteins.
  • the recombinant yeast cell may comprise zinc, zinc ions or zinc salts and/or one or more pathways to include such in the cell.
  • the invention further provides a process for the production of a fermentation product, the process comprising fermenting of a feed with a recombinant yeast cell as described above, wherein the feed comprises a source of NAD+ cofactor.
  • the source of NAD+ cofactor is a sugar alcohol.
  • the process is preferably a process for the production of a fermentation product comprising fermenting of a feed with a mutant yeast cell as described above, wherein the feed comprises a sugar alcohol. More preferably the process is a process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell as described above, wherein the carbon source composition comprises at least a sugar alcohol.
  • the process is carried out under oxygen-limited conditions or anaerobic conditions.
  • the sugar alcohol is mannitol or sorbitol. [193]
  • the sugar alcohol is derived from a sugar by a process comprising a preceding or simultaneous hydrogenation of a sugar-containing feed by an inorganic, organic or biological catalyst.
  • the feed comprises a sugar alcohol, such as mannitol and/or sorbitol, in combination with a sugar, such as glucose, arabinose, xylose and/or galactose.
  • a sugar alcohol such as mannitol and/or sorbitol
  • the process is a process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell as described above, wherein the carbon source composition comprises a sugar and a sugar alcohol and wherein both sugar and sugar alcohol are converted into ethanol.
  • the feed comprises a sugar alcohol and a sugar
  • the feed preferably comprises such sugar alcohol and sugar in a weight ratio of sugar alcohol to sugar in the range of 1000:1 to 1 :1000, more preferably 100:1 to 1 :100.
  • the sugar alcohol is present in a higher weight percentage (wt %) than the sugar.
  • the percentage sugar based on the total weight of sugar and sugar alcohol in the feed, lies in the range from more than 0.001 wt % to less than or equal than 50 wt %, more preferably in the range from more than 0.01 wt % to less than or equal to than 49 wt%, still more preferably in the range from more than 0.1 wt% to less than or equal to 45 wt% and most preferably in the range from more than 1 wt% to less than or equal to 40 wt%.
  • the percentage sugar alcohol based on the total weight of sugar and sugar alcohol in the feed, can be equal to or more than 0.1 wt%, more conveniently equal to or more than 1 wt%, still more conveniently equal to or more than 5 wt%, even more conveniently equal to or more than 10 wt% and most conveniently equal to or more than 20 wt%.
  • the percentage sugar alcohol based on the total weight of sugar and sugar alcohol together, is equal to or more than 50 wt%, more preferably equal to or more than 70 wt%, even more preferably equal to or more than 90 wt% and still more preferably equal to or more than 95 wt%. There is no maximum amount of sugar alcohol.
  • the percentage sugar alcohol based on the total weight of sugar and sugar alcohol in the feed, can be equal to or less than 99.99 wt%, equal to or less than 99.9 wt %, equal to or less than 99 wt%, equal to or less than 95 wt% or equal to or less than 90 wt%.
  • the feed comprises 100% sugar alcohols, such as for example sorbitol and/or mannitol. That is, preferably the process is carried out in the absence of glucose, arabinose, xylose, galactose and/or any other sugars.
  • the feed suitably comprises one or more (additional) fermentable carbon sources.
  • the fermentable carbon source preferably comprises or is consisting of one or more fermentable carbohydrates. More preferably, the fermentable carbon source comprises one or more mono-saccharides, disaccharides and/or polysaccharides.
  • the fermentable carbon source may comprise one or more carbohydrates selected from the group consisting of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose, mannose and trehalose.
  • the fermentable carbon source preferably comprising or consisting of one or more carbohydrates, may suitably be obtained from starch, celulose, hemicellulose lignocellulose, and/or pectin.
  • the fermentable carbon source may be in the form of a, preferably aqueous, slurry, suspension, or a liquid.
  • the concentration of fermentable carbohydrate, such as for example glucose, during fermentation is preferably equal to or more than 80g/L.
  • the initial concentration of glucose at the start of the fermentation is preferably equal to or more than 80 g/L, more preferably equal to or more than 90 g/L, even more preferably equal to or more than 100 g/L, still more preferably equal to or more than 110 g/L, yet even more preferably equal to or more than 120 g/L, equal to or more than 130 g/L, equal to or more than 140 g/L, equal to or more than 150 g/L, equal to or more than 160 g/L, equal to or more than 170 g/L, or equal to or more than 180 g/L.
  • the start of the fermentation may be the moment when the fermentable fermentable carbohydrate is brought into contact with the recombinant cell of the invention.
  • the fermentable carbon source may be prepared by contacting starch, lignocellulose, and/or pectin with an enzyme composition, wherein one or more mono-saccharides, disaccharides and/or polysaccharides are produced, and wherein the produced mono-saccharides, disaccharides and/or polysaccharides are subsequenty fermented to give a fermentation product.
  • the fermentable carbohydrate is, or is comprised by a biomass hydrolysate, such as a corn stover or corn fiber hydrolysate.
  • a biomass hydrolysate such as a corn stover or corn fiber hydrolysate.
  • Such biomass hydrolysate may in its turn comprise, or be derived from corn stover and/or corn fiber.
  • hydrolysate a polysaccharide-comprising material (such as corn stover, corn starch, corn fiber, or lignocellulosic material, which polysaccharides have 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.
  • a biomass hydrolysate may be a lignocellulosic biomass hydrolysate.
  • Lignocellulose herein includes hemicellulose and hemicellulose parts of biomass.
  • 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, algae (including macroalgae and microalgae), trees, softwood, hardwood, poplar, pine, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks
  • Algae such as macroalgae and microalgae have the advantage that they may comprise considerable amounts of sugar alcohols such as sorbitol and/or mannitol.
  • 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 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 fermentation process can be carried out in a continuous mode, a batch mode or in a semibatch or fed-batch mode. Preferably the fermentation process is carried out in a batch mode.
  • the process comprising fermenting of a carbon source composition with a mutant yeast cell as described herein, wherein the carbon source composition comprises at least a sugar alcohol, and more preferably the carbon source composition comprises at least a sugar, such as glucose, and a sugar alcohol, such as sorbitol or mannitol.
  • the yeast overexpresses a transporter, such as HXT15, and a sorbitol dehydrogenase, such as SOR2 or a mannitol dehydrogenase, such as MAN2.
  • the sugar such as glucose
  • the sugar alcohol such as sorbitol or mannitol
  • ethanol can then conveniently be both converted into ethanol.
  • glucose is provided in excess conditions.
  • the process is carried out under oxygen-limited conditions or anaerobic conditions.
  • the process is carried out in a batch mode.
  • a carbon source composition comprising at least a sugar, such as glucose, and a sugar alcohol, such as sorbitol or mannitol, can even be converted without the overexpression of a transporter, such as HXT15, and a sorbitol dehydrogenase, such as SOR2, or a mannitol dehydrogenase, such as MAN2.
  • a transporter such as HXT15
  • a sorbitol dehydrogenase such as SOR2
  • a mannitol dehydrogenase such as MAN2.
  • WO2014/129898 describes a recombinant yeast cell, in particular a transgenic yeast cell, functionally expressing one or more recombinant, in particular heterologous, nucleic acid sequences encoding ribulose-l,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK).
  • WO2014/129898 further describes a method for preparing an alcohol, organic acid or amino acid, comprising fermenting a carbon source, in particular a carbohydrate with such a yeast cell, thereby forming the alcohol, organic acid or amino acid, wherein the yeast cell is present in a reaction medium.
  • WO2014/129898 further describes anaerobic chemostat cultivation with 12.5 g/l glucose and 12.5 g/l galactose as the carbon source.
  • WO2014/129898 describes the optional presence of a heterologous nucleic acid sequence encoding a xylitol dehydrogenase from a (naturally) autotrophic organism, but WO2014/129898 does not describe any actual conversion of any carbon source composition comprising at least a sugar, such as glucose, and a sugar alcohol, such as sorbitol or mannitol, under carbon limited circumstances and does not recognize such a possibility.
  • the invention therefore also provides a process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell, wherein the carbon source composition comprises at least a sugar, such as glucose, and a sugar alcohol, such as sorbitol or mannitol, wherein the process is carried out in a fed-batch mode or otherwise under carbon-limited conditions.
  • the mutant yeast cell preferably comprises one or more, preferably recombinant, more preferably heterologous, nucleic acid sequences encoding ribulose-l,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK).
  • mutant yeast cell may conveniently comprise a sugar alcohol dehydrogenase, such as a sorbitol dehydrogenase, such as SOR2, or a mannitol dehydrogenase, such as MAN2, where such alcohol dehydrogenase may or may not be overexpressed.
  • mutant yeast cell may comprise a transporter, such as HXT15, which may or may not be overexpressed.
  • the process is preferably carried out under carbon-limited conditions. That is, the process is preferably carried out under circumstances where the feed of sugar, preferably the feed of glucose, is preferably limited to equal to or less than 60 grams per liter, more preferably equal to or less than 50 grams per liter, still more preferably equal to or less than 40 grams per liter, even more preferably equal to or less than 30 grams per liter and even still more preferably equal to or less than 20 grams per liter.
  • the feed of sugar alcohol is preferably limited to equal to or less than 60 grams per liter, more preferably equal to or less than 50 grams per liter, still more preferably equal to or less than 40 grams per liter, even more preferably equal to or less than 30 grams per liter, even still more preferably equal to or less than 20 grams per liter and most preferably equal to or less than 10 grams per liter.
  • the alternative process is preferably carried out with a a carbon source composition comprising sugar alcohol and sugar in a weight ratio of sugar alcohol to sugar in the range of 1000:1 to 1 :1000, more preferably 100:1 to 1 :100.
  • CRISPR/Cas9-based genome editing as described by Mans R., van Rossum H.M., Wijsman M., Backx A., Kuijpers N.G.A., van den Broek M., Daran-Lapujade P., Pronk J.T., van Maris A.J.A. and Daran J-M. G., in their article titled "CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae", published in FEMS Yeast Research, vol. 15, (2015), fov004).
  • yeast strains were grown in 2% w/v glucose synthetic medium (3.0 g L -1 KH2PO4, 0.5 g L -1 MgSO4'7H2O, 5.0 g L -1 (NH4)2SO4, 1.0 ml L -1 trace elements, 1.0 mL L -1 vitamin solution (as described in the article by Verduyn, C., Postma, E., Scheffers, W. A., & Van Dijken, J. P.
  • E. coliXL-1 blue stock cultures were grown in LB medium (5 g L-1 Bacto yeast extract, 10 g L-1 Bacto tryptone, 5 g L-1 NaCI), supplemented with 100 pg mL-1 ampicillin. Frozen stocks were prepared by addition of glycerol (30% v/v final concentration).
  • PCR amplification for construction of plasmid fragments and yeast integration cassettes was performed with Phusion High Fidelity DNA Polymerase (commercially obtainable from Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s guidelines (Thermo ScientificPhusion High-Fidelity DNA Polymerase Product Information Sheet, 2018), using 30 cycles.
  • the amplified DNA fragments were purified using a GeneJET PCR purification kit (GeneJET PCR Purification Kit (commercially obtainable from ThermoFisher, Waltmann, USA) according to the manufacturer’s guidelines. All purified DNA fragments were stored at -20°C.
  • GeneJET PCR Purification Kit commercially obtainable from ThermoFisher, Waltmann, USA
  • Table 8 S. cerevisiae strains used in these examples
  • Example 1 Construction of plasmid PUDE885 comprising a pACT1-tCPS1 empty vector
  • Plasmid pUD968 was created from plasmid p426-TEF (commercially obtainable from Addgene). Plasmid p426-TEF was amplified using desalted primer pairs 15514/10901 and 15515/7388 to obtain two DNA fragments (amplification carried out according Phusion High Fidelity DNA Polymerase manufacturer’s guidelines as indicated above) as illustrated in Figure 1.
  • the first DNA fragment comprised a nucleotide sequence for the URA3 marker (a gene derived from chromosome V in Saccharomyces cerevisiae).
  • the second DNA fragment comprised the “2 mu ori” nucleotide sequence (a 2micron origin of replication for propagation in S.
  • the “AmpR” nucleotide sequence i.e. AmpR - Beta lactamase gene encoding ampicillin resistance pBBR322 ori - pBR322 origin of replication for propagation in E. coli. Correct fragment sizes were verified by gel electrophoresis on a TopVision Agrose gel 1% according to manufacturer’s guidelines as indicated above. After verification the DNA-fragments were purified using the GeneJET PCR purification kit according to manufacturer’s guidelines as indicated above.
  • the purified fragments were digested by restriction endonucleases Kpnl and Pfol (both commercially obtainable from Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s guidelines and ligated using T4 DNA ligase (commercially obtainable from Thermo Fisher Scientific, Waltham, MA, USA) to create plasmid pUD968, according to manufacturer’s guidelines.
  • plasmid pUD968 was first transformed into E.coli XL1-Blue cells (commercially obtainable from Agilent, Santa Clara, USA) and plated on LB-ampicillin and incubated overnight at 37°C. A single colony was used to inoculate LB-ampicillin liquid medium and incubated overnight at 37°C.
  • the GeneJET Plasmid Miniprep Kit (commercially available from Thermo Fisher Scientific Inc. , Waltman, MA, USA) was used to isolate the plasmid DNA (of the plasmid pUD968) from the E.coli according to manufacturer’s instructions. Correct assembly of the plasmid can be verified either by diagnostic PCR or restriction assay.
  • the isolated plasmid pUD986 is stored at -20°C.
  • This plasmid pUD968 was linearized with restriction endonuclease Kpnl (commercially obtainable by Thermo Scientific as indicated above).
  • the ACT1 promoter (pACT1) and CPS1 terminator (tCPS1) sequences were amplified using primers 15548/15549 and 15550/15551 respectively, using CEN.PK.113- 7D as template.
  • Genomic DNA of pACT1 and tCPS1 was isolated from CEN.PK.113.7D using the method as described in the protocol by Looke, et al., titled “Extraction of genomic DNA from yeasts for PCR-based applications", published in Biotechniques, vol. 50, ., (2011) pages 325-328, herewith incorporated by reference, (further referred to as Looke et al. (2011))
  • Plasmid pUD885 was first transformed into E.coli XL1-Blue cells (commercially obtainable from Agilent, Santa Clara, USA) and plated on LB-ampicillin and incubated overnight at 37°C. A single colony was used to inoculate LB-ampicillin liquid medium and incubated overnight at 37°C for plasmid propagation.
  • the GeneJET Plasmid Miniprep Kit (commercially available from Thermo Fisher Scientific Inc. , Waltman, MA, USA) was used to isolate the plasmid DNA (of the plasmid pUD885) from the E.coli according to manufacturer’s instructions. Correct assembly of the plasmid can be verified either by diagnostic PCR or restriction digestion.
  • the isolated plasmid pUD885 is stored at -20°C.
  • This example 2 describes how SOR2, a gene encoding sorbitol dehydrogenase 2 from CEN- PK.113-7D, was cloned between promoter ACT1 and terminator CPS1 on plasmid pUDE885 prepared in example 1 .
  • primers 16709/16710 with nucleotides homologous to the open reading frame of SOR2 and to the flanking regions of pACT1 and tCPS1 were used.
  • the genomic DNA for the SOR2 DNA fragment was isolated from CEN.PK.113.7D using the method as described by Looke et al. (2011).
  • Phusion PCR Phusion High-Fidelity DNA Polymerase (2 U/pL), n.d. was used to amplify the SOR2 DNA fragment, according to manufacturer’s guidelines (Thermo ScientificPhusion High-Fidelity DNA Polymerase Product Information Sheet, 2018) using 30 cycles and an annealing temperature of 57 °C.
  • Plasmid pUDE885 was linearized by restriction endonuclease Kpnl. After linearization of pUD885, the purified SOR2 DNA fragment was assembled onto the backbone of linearized pUD885 by Gibson assembly using NEBuilder 2x HIFI DNA assembly master mix (NEBuilder® HiFi DNA Assembly Master Mix
  • NEB commercially obtainable from New England Biolabs Inc
  • Plasmid pUDE941 was used as PCR template to obtain the cassette of the pACT1-SOR2 CPS1 fragment using primers (16715/16716)
  • Example 3 Preparation repair fragments for pTEF1, ORF of HXT15 and tCYCf
  • HXT15 was integrated flanked by the promoter pTEF1 and the terminator tCYCf. Primers with nucleotides homologous to the ORF of HXT15 and to the flanking regions of pTEF1 and tCYCf were used (16705/16706).
  • the genomic DNA for the HXT15 DNA fragment was isolated from CEN.PK.113.7D using the method as described by Looke et al. (2011).
  • Phusion PCR (Phusion High-Fidelity DNA Polymerase (2 U/gL), n.d.) was used to amplify the HXT 15 DNA fragment, according to manufacturer’s guidelines (Thermo ScientificPhusion High-Fidelity DNA Polymerase Product Information Sheet, 2018) using 30 cycles and an annealing temperature of 57 °C.
  • pTEF1 was amplified using Phusion PCR (Phusion High-Fidelity DNA Polymerase (2 U/gL), n.d.) with p426-TEF as template and with primer sets containing homologous nucleotides to the upstream sequence of the X-2 integration site and the HXT15 ORF (16711/17031).
  • tCYCf was amplified using Phusion PCR (Phusion High-Fidelity DNA Polymerase (2 U/gL), n.d.
  • Plasmid pUDR538 is a pROS12- derived plasmid (Mans et al., 2015). The protocol provided in the supplementary materials of the publication of Mans et al. (2015) was followed for the construction of pUDR538.
  • the pROS12 backbone was amplified using primer combination 5793-5793 (double binding) and the plasmid insert (containing the gRNA sequence for X-2) was amplified with primers 10866/10866 (double binding).
  • Example 5 Construction of strain IMX2411
  • Example 5 describes the construction of yeast strain IMX2411 from IMX581.
  • IMX581 is a CEN.PK113-5D -based, Cas9-expressing strain used for subsequent CRISPR-Cas9-mediated genome modifications as described by Mans et al., 2015. pACT1-SOR2-tCPS (as constructed in example 2), pTEF1, HXT15 and t CYC 7 (as constructed in example 3)
  • the intergenic region X-2 of yeast strain IMX581 was used for integration of pACT1-SOR2-tCPS (as constructed in example 2), pTEF1, HXT15 and tCYC1 (as constructed in example 3). Integration into X-2 was found to lead to stable expression of the integrated gene, without interfering with native genes (Mikkelsen et al. 2012). Plasmid pUDR538 was used to target this integration site (as constructed in example 4).
  • Strain IMX2411 was obtained by co-transformation of pUDR538 together with 4 DNA fragments encoding pACT1-SOR2-tCPS (as constructed in example 2), pTEF1, HXT15 and tCYC7 (as constructed in example 3) into IMX581 , according to the lithium-acetate transformation protocol (Gietz and Woods 2002). Transformants were selected on solid YPD medium (10 gl_ -1 Bacto yeast extract, 20 gl_ -1 Bacto peptone, 20 gL -1 glucose and 20 gL -1 agar) supplemented with 200 mgL -1 hygromycin B. Confirmation of the desired genotype was performed by diagnostic colony PCR using Dreamtaq polymerase (Thermo scientific), following the manufacturer’s instructions.
  • Reference strain IME611 was obtained by transforming p426-TEF(empty) into IMX2411 , according to the lithium-acetate transformation protocol (Gietz and Woods 2002). Transformations were plated on solid synthetic medium (3.0 g L’ 1 KH2PO4, 0.5 g L’ 1 MgSO4'7H 2 O, 5.0 g L’ 1 (NH 4 )2SO4, 1.0 ml L’ 1 trace elements, 1 .0 mL L -1 vitamin solution (Verduyn, Postma, Scheffers, & Van Dijken, 1992), 20 gL -1 agar and 20 gL -1 glucose) . SMD plates were used, as these do not contain uracil.
  • Example 7 preparation repair fragment pTEF1-HXT15-tCYC1
  • Genomic DNA of IMX2411 was used as PCR template to obtain the repair fragment pTEF1-HXT15- tCYC1.
  • Genomic DNA was isolated from IMX2411 using the method described by Looke et al. (2011).
  • pTEF1-HXT15-tCYC1 was amplified with flanks to SHR-A and the upstream sequence of the X-2 integration site using PCR Phusion PCR (Phusion High-Fidelity DNA Polymerase (2 U/pL), n.d.) with genomic DNA of IMX2411 as template and primer pair 16711/16712.
  • DNA fragment pTEF1-HXT15-tCYC1 was purified using a GeneJET PCR purification kit (GeneJET PCR Purification Kit) according to manufacturer’s guidelines as indicated above
  • strain IMX1489 was carried out as described by Papapetridis et al. in their article titled “Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield”, published in Biotechnol Biofuels (2016), pages 1 to 17.
  • the RuBisCO/PRK-expressing strain IMX1489 was obtained by co-transformation of pUDR103, the pDAN1 , prk-ORF, tPGK1 sequences, 9 copies of the expression cassette of cbbm and the expression cassettes of groEL and groES (14 fragments), prepared as described by Papapetridis et al, to strain IMX1472 (integration at the SGA1 locus, GPD2-targeting CRISPR plasmid recycled).
  • Example 9 describes the construction of strain IMX2495 from strain IMX1489 (as constructed in example 8).
  • the intergenic region X-2 of yeast strain IMX1489 was used for integration of pACT1-SOR2- tCPS (as constructed in example 2) and pTEF1-HXT15-tCYC1 (as constructed in example 7). Integration into X-2 was found to lead to stable expression of the integrated gene, without interfering with native genes (Mikkelsen et al. 2012). Plasmid pUDR538 was used to target this integration site (as constructed in example 4).
  • Strain IMX2495 was obtained by co-transformation of pUDR538 (example 4) together with the 2 repair fragments encoding pACT1-SOR2-tCPS1 (example 2) and pTEF1-HXT15 CYC1 (example 7) into IMX1489 according to the lithium-acetate transformation protocol (Gietz and Woods 2002). Transformants were selected on solid YPD medium (10 gL -1 Bacto yeast extract, 20 gL -1 Bacto peptone, 20 gL -1 glucose and 20 gL -1 agar) supplemented with 200 mgl_ -1 hygromycin B. Confirmation of the desired genotype was performed by diagnostic colony PCR using Dreamtaq polymerase (Thermo scientific), following the manufacturer’s instructions.
  • Example 10 construction of strain IMX2506
  • Strain IMX2506 was obtained by transforming p426-TEF(empty) into IMX2495, according to the lithium-acetate transformation protocol (Gietz and Woods 2002). Transformations were plated on solid synthetic medium (3.0 g L -1 KH2PO4, 0.5 g L -1 MgSO4'7H2O, 5.0 g L -1 (NH4)2SO4, 1.0 ml L -1 trace elements, 1.0 mL L -1 vitamin solution (Verduyn, Postma, Scheffers, & Van Dijken, 1992), 20 gL -1 agar and 20 gL -1 glucose) . SMD plates were used, as these do not contain uracil.
  • the inflow of medium was set to a flow rate of 0.025 Ltr 1 .
  • a working volume of 1-L was ensured by a level sensor which controls the effluent pump.
  • the pH was kept constant at 5.0 by automatic addition of 2 M KOH.
  • a gas mixture of N2/CO2 (90/10%) was used to ensure anaerobic conditions and supply CO2 to ensure activity of RuBisCO.
  • the gasflow was set at 0.5 L min -1 and a stirrer speed of 800 rpm was used.
  • the outlet gas was cooled to 4°C to minimize evaporation and the bioreactor was kept at a temperature of 30°C. Oxygen diffusion was minimized by the use of Norprene tubing and Viton O-rings.
  • Bioreactor inocula were generated in 500 ml_ shakeflasks containing 100 ml_ synthetic medium containing 20 gl_ -1 glucose.
  • the cultures were inoculated from frozen stock cultures and grown at 30 °C, 200 rpm, under atmospheric air for 15-18 h. These cultures were used to inoculate pre-cultures flaks, which were grown to mid-exponential phase (ODeeo of 3-5) and used to start the bioreactor with an ODeeo of 0.2-0.3.
  • the initial batch phase preceding the continuous cultivation was performed on synthetic medium supplemented with 20 gl_ -1 glucose, the anaerobic growth factors Tween 80 (420 mgL -1 ) and ergosterol (10 mgL -1 ), and
  • Tablet 2 Metabolite concentrations of anaerobic chemostat cultures measured during steady state sampling in the reactor (OUT) and in the medium inflow (IN) of S. cerevisiae strains IME324 and IMX2506.
  • the pH was kept constant at 5.0 by automatic addition of 2 M KOH.
  • a gas mixture of N2/CO2 (90/10%) was used to ensure anaerobic conditions and supply CO2 to ensure activity of RuBisCO.
  • the gasflow was set at 0.5 L min -1 and a stirrer speed of 800 rpm was used.
  • the outlet gas was cooled to 4°C to minimize evaporation and the bioreactor was kept at a temperature of 30°C. Oxygen diffusion was minimized by the use of Norprene tubing and Viton O-rings.
  • Bioreactor inocula were generated in 500 mL shakeflasks containing 100 mL synthetic medium containing 20 gL -1 glucose.
  • the cultures were inoculated from frozen stock cultures and grown at 30 °C, 200 rpm, under atmospheric air for 15-18 h. These cultures were used to inoculate pre-cultures flaks, which were grown to mid-exponential phase (ODeeo of 3-5) and used to start the bioreactor with an ODeeo of 0.15-0.30.
  • Table 14 Metabolite concentrations and corresponding broth volumes at different timepoints during duplicate batch cultures of IME611 in anaerobic bioreactors, pH 5.
  • Table 15 Metabolite concentrations and corresponding broth volumes at different timepoints during duplicate batch cultures of IMX1489 in anaerobic bioreactors, pH 5
  • Table 16 Metabolite concentrations and corresponding broth volumes at different timepoints during duplicate batch cultures of IMX2506 in anaerobic bioreactors, pH 5.
  • CRISPR/Cas9 a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae", FEMS Yeast Research, vol. 15, fov004.

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Abstract

Cellule de levure mutante, comprenant : (i) une première modification génétique pour l'expression d'une protéine dépendante de NAD+ qui fonctionne dans une première voie métabolique convertissant un alcool de sucre en un produit de fermentation ; et (ii) une seconde modification génétique pour l'expression d'une protéine qui fonctionne dans une seconde voie métabolique formant un puits redox non natif. Et un procédé utilisant une telle cellule de levure.
PCT/EP2023/060423 2022-04-25 2023-04-21 Cellule de levure mutante et procédé de production d'éthanol WO2023208762A2 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO2011131667A1 (fr) 2010-04-21 2011-10-27 Dsm Ip Assets B.V. Cellule adaptée pour la fermentation d'une composition de sucre mélangé
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