Glycerol free ethanol production

Abstract

The invention relates to a recombinant yeast comprising a recombinant yeast comprising a nucleotide sequence coding for a glycerol dehydrogenase, a nucleotide sequence coding for a ribulose-1,5-biphosphate carboxylase oxygenase (EC 4.1.1.39); a nucleotide sequence coding for a phosphoribulokinasey (EC 2.7.1.19); a nucleotide sequence allowing the expression of a glucoamylase (EC 3.2.1.20 or 3.2.1.3); and optionally a nucleotide sequence coding for a glycerol transporter. This cell can be used for the production of ethanol and advantageously produces little or no glycerol.

Claims

1. A recombinant yeast comprising: a nucleotide sequence coding for a glycerol dehydrogenase, a nucleotide sequence coding for a ribulose-1,5-biphosphate carboxylase oxygenase (EC 4.1.1.39); a nucleotide sequence coding for a phosphoribulokinase (EC 2.7.1.19); a nucleotide sequence allowing the expression of a glucoamylase (EC 3.2.1.20 or 3.2.1.3), the glucoamylase comprising the amino acid sequence according to SEQ ID NO: 17 or a functional homologue thereof having a sequence identity of at least 70% to SEQ ID NO: 17; and optionally a nucleotide sequence coding for a glycerol transporter.

2. The recombinant yeast according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol exporter.

3. The recombinant yeast according to claim 2, wherein the deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol exporter comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a FPS1 glycerol exporter.

4. The recombinant yeast according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol kinase (EC 2.7.1.30).

5. The recombinant yeast which according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol-3-phosphate dehydrogenase (GPD1/2).

6. The recombinant yeast according to claim 1 which comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol 3-phosphate phosphohydrolase (GPP1/2).

7. The recombinant yeast according to claim 1 which is a Saccharomyces, optionally Saccharomyces cerevisiae.

8. The recombinant yeast according to claim 1 further comprising one or more nucleotide sequences, coding for molecular chaperones from E. coli which are selected from the group consisting of GroEL, GroES, functional homologues of GroEL, and functional homologues of GroES.

9. A product comprising the recombinant yeast according to claim 1 for preparation of ethanol and/or succinic acid.

10. A process for production of ethanol comprising: fermenting a composition comprising a fermentable carbohydrate under anaerobic conditions in the presence of a recombinant yeast according to claim 1; and recovering the ethanol.

11. The process according to claim 10, wherein the fermentable carbohydrate is obtained from starch, lignocellulose, and/or pectin.

12. The process according to claim 10, wherein said composition comprises an amount of undissociated acetic acid of 10 mM or less.

13. The process according to claim 10 wherein said composition comprises an amount of undissociated acetic acid of between 50 μM and 10 mM.

14. The process according to claim 10, wherein the fermentable carbohydrate is selected from the group of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose, and mannose.

Description

(1) In one aspect the invention provides a recombinant yeast comprising: a nucleotide sequence coding for a glycerol dehydrogenase, a nucleotide sequence coding for a ribulose-1,5-biphosphate carboxylase oxygenase (EC 4.1.1.39); a nucleotide sequence coding for a phosphoribulokinase (EC 2.7.1.19); a nucleotide sequence allowing the expression of a glucoamylase (EC 3.2.1.20 or 3.2.1.3); and optionally a nucleotide sequence coding for a glycerol transporter.

(2) Glucoamylase, also referred to as amyloglucosidase, alpha-glucosidase, glucan 1,4-alpha glucosidase, maltase glucoamylase, and maltase-glucoamylase, catalyses at least the hydrolysis of terminal 1,4-linked alpha-D-glucose residues from non-reducing ends of amylose chains to release free D-glucose.

(3) In an embodiment the glucoamylase has an amino acid sequence according to SEQ ID NO: 17 or is a functional homologue thereof having a sequence identity of at least 50%, preferably at least 60%, 70%, 75%, 80%, 85%, 90%, 95, 98%, or 99%. The polypeptide of SEQ ID NO: 17 encodes a “mature glucoamylase”, referring to the enzyme in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

(4) In an embodiment the nucleotide sequence allowing the expression of a glucoamylase encodes a polypeptide having an amino acid sequence of SEQ ID NO: 18 or a variant thereof having a sequence identity of at least 50%, preferably at least 60%, 70%, 75%, 80%, 85%, 90%, 95, 98%, or 99%. Amino acids 1-17 of the SEQ ID NO: 18 may encode for a signal sequence.

(5) In another embodiment the nucleotide sequence allowing the expression of a glucoamylase encodes a polypeptide having an amino acid sequence of SEQ ID NO: 19 or a variant thereof having a sequence identity of at least 50%, preferably at least 60%, 70%, 75%, 80%, 85%, 90%, 95, 98%, or 99%. Amino acids 1-19 of the SEQ ID NO: 19 may encode for a signal sequence.

(6) A signal sequence (also referred to as signal peptide, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) can be present at the N-terminus of a polypeptide (here, the glucoamylase) where it signals that the polypeptide is to be excreted, for example outside the cell and into the media.

(7) In an embodiment the glycerol dehydrogenase is a NAD.sup.+ linked glycerol dehydrogenase (EC 1.1.1.6). Such enzyme may be from bacterial origin or for instance from fungal origin. An example is gldA from E. coli.

(8) Alternatively, the enzyme having glycerol dehydrogenase activity is a NADP.sup.+ linked glycerol dehydrogenase (EC 1.1.1.72).

(9) When the recombinant yeast is used for ethanol production, which typically takes place under anaerobic conditions, NAD.sup.+ linked glycerol dehydrogenase are preferred.

(10) In an embodiment the recombinant yeast comprises one or more nucleotide sequences encoding a heterologous glycerol dehydrogenase represented by amino acid sequence SEQ ID NO: 1, 2, 3 or 13 or a functional homologue thereof a having sequence identity of at least 50%, preferably at least 60%, 70%, 75%, 80% 85%, 90%, 95%, 98% or 99%.

(11) It is understood that the recombinant yeast has an endogenous nucleotide sequence coding a dihydroxy acetone kinase, such as a DAK1 gene. Such nucleotide sequence is preferably placed under control of a constitutive promoter. In an embodiment the recombinant yeast comprises one or more nucleic acid sequences encoding a dihydroxy acetone kinase represented by amino acid sequence according to SEQ ID NO: 4, 5, 6, or 14 or by a functional homologue thereof having a sequence identity of at least 50%, preferably at least 60%, 70%, 75%, 80%, 85%, 90%, 95, 98%, or 99%, which nucleotide sequence is preferably placed under control of a constitutive promoter. The dihydroxy acetone kinase may also have glyceraldehyde kinase activity.

(12) The recombinant yeast optionally comprises a nucleotide sequence coding for a glycerol transporter. In this embodiment any glycerol that is externally available in the medium (e.g. from the backset in corn mash) or secreted after internal cellular synthesis may be transported into the cell and converted to ethanol. In an embodiment the recombinant yeast comprises one or more nucleic acid sequences encoding a heterologous glycerol transporter represented by amino acid sequence SEQ ID NO:7 or a functional homologue thereof having a sequence identity of at least 50%, preferably at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

(13) In an embodiment the recombinant yeast comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol exporter (e.g FPS1). In an embodiment the recombinant yeast comprises one or more nucleic acid sequences encoding a heterologous glycerol transporter represented by amino acid sequence SEQ ID NO: 8 or a functional homologue thereof having a sequence identity of at least 50%, preferably at least 60%, 70%, 75%, 80% 85%, 90%, 95%, 98% or 99%.

(14) In another embodiment the recombinant yeast comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol kinase (EC 2.7.1.30). An example of such an enzyme is Gut1p.

(15) In a further embodiment, the recombinant yeast naturally lacks enzymatic activity needed for the NADH-dependent glycerol synthesis or has reduced enzymatic activity needed for NADH-dependent glycerol synthesis compared to its corresponding wild type yeast, for example yeast belonging to the species Brettanomyces intermedius.

(16) In an embodiment the recombinant yeast comprises a deletion or disruption of one or more endogenous nucleotide sequences encoding a glycerol 3-phosphate phosphohydrolase and/or encoding a glycerol-3-phosphate dehydrogenase. Such a deletion or disruption may result in decrease or removal of enzymatic activity. A deleted or disrupted glycerol-3-phosphate dehydrogenase preferably may belong to EC 1.1.5.3, such as GUT2, or to EC 1.1.1.8, such as PDP1 and or PDP2.

(17) In embodiment the recombinant yeast is free of nucleotide sequences encoding NADH-dependent glycerol-3-phosphate dehydrogenase.

(18) A reduced enzymatic activity can be achieved by modifying one or more nucleotide sequences encoding a NAD-dependent glycerol 3-phosphate dehydrogenase activity (GPD) or one or more nucleotide sequences encoding a glycerol phosphate phosphatase activity (GPP), such that the enzyme is expressed considerably less than in the wild-type or such that the nucleotide sequence encodes a polypeptide with reduced activity. 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. Alternatively, 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. Examples of genes in S. cerevisiae encoding GPD-activity are GPD1, GPD2, and GPP-activity are GPP1 and GPP2.

(19) GPD and/or GPP may be entirely deleted, or at least a part is deleted which encodes a part of the enzyme that is essential for its activity. In particular, 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. In particular, good results have been obtained with the inactivation of the GPD1 and GPD2 genes in Saccharomyces cerevisiae by integration of the marker genes kanMX and hphMX4. Subsequently this DNA fragment is transformed into a host cell. Transformed cells that express the dominant marker gene are checked for correct replacement of the region that was designed to be deleted, for example by a diagnostic polymerase chain reaction or Southern hybridization.

(20) The Rubisco may be a single-subunit Rubisco or a Rubisco having more than one subunit. In particular, good results have been achieved with a single-subunit Rubisco. In particular, good results have been achieved with a form-II Rubisco, more in particular CbbM.

(21) SEQ ID NO: 11 shows a suitable sequence of a suitable Rubisco. It is encoded by the cbbM gene from Thiobacillus denitrificans. An alternative to this Rubisco is a functional homologue of this Rubisco, in particular such functional homologue comprising an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 11. Suitable natural Rubisco polypeptides are given in Table 1 of WO2014/129898.

(22) The Rubisco is preferably functionally expressed in the microorganism, at least during use in an industrial process for preparing a compound of interest.

(23) In an embodiment the functionally expressed Rubisco has an activity, defined by the rate of ribulose-1,5-bisphosphate-dependent .sup.14C-bicarbonate incorporation by cell extracts of at least 1 nmol.Math.min.sup.−1.Math.(mg protein).sup.−1, in particular an activity of at least 2 nmol.Math.min.sup.−1.Math.(mg protein).sup.−1, more in particular an activity of at least 4 nmol.Math.min.sup.−1.Math.(mg protein).sup.−1. The upper limit for the activity is not critical. In practice, the activity may be about 200 nmol.Math.min.sup.−1.Math.(mg protein).sup.−1 or less, in particular 25 nmol.Math.min.sup.−1.Math.(mg protein).sup.−1, more in particular 15 nmol.Math.min.sup.−1.Math.(mg protein).sup.−1 or less, e.g. about 10 nmol.Math.min.sup.−1.Math.(mg protein).sup.−1 or less. The conditions for an assay for determining this Rubisco activity are as found in Example 4 of WO2014/129898.

(24) The inventors have found that the recombinant yeast cell of the invention having genes coding for a glycerol dehydrogenase, a RuBisCO, and a PRK may have suboptimal ethanol yield, as compared to similar cells without a glucoamylase.

(25) In an embodiment the recombinant yeast comprises one or more nucleotide sequences, preferably a heterologous nucleotide sequences, coding for molecular chaperones, said chaperones preferably originating from a prokaryote, more preferably a bacterium, even more preferably E. coli.

(26) Chaperones—when expressed—are preferably capable of functionally interacting with an enzyme in the microorganism, in particular with at least one of Rubisco and PRK. Chaperones are proteins that provide favourable 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). A review article about chaperones that is useful herein is written by Yébenes (2001); “Chaperonins: two rings for folding”; Hugo Yebenes et al. Trends in Biochemical Sciences, August 2011, Vol. 36, No. 8.

(27) In an embodiment, the one or more chaperone is from a bacterium, more preferably from Escherichia, in particular E. coli GroEL and GroES from E. coli may in particular encoded in a microorganism according to the invention. Other preferred chaperones are chaperones from Saccharomyces, in particular Saccharomyces cerevisiae Hsp10 and Hsp60. If 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.

(28) In eukaryotes the proteins Hsp60 and Hsp10 are structurally and functionally nearly identical to GroEL and GroES, respectively. Thus, it is contemplated that Hsp60 and Hsp10 from any eukaryotic cell may serve as a chaperone for the Rubisco. See Zeilstra-Ryalls J, Fayet O, Georgopoulos C (1991). “The universally conserved GroE (Hsp60) chaperonins”. Annu Rev Microbiol. 45: 301-25. doi:10.1146/annurev.mi.45.100191.001505. PMID 1683763 and Horwich A L, Fenton W A, Chapman E, Farr G W (2007). “Two Families of Chaperonin: Physiology and Mechanism”. Annu Rev Cell Dev Biol. 23: 115-45. doi:10.1146/annurev.cellbio.23.090506.123555. PMID 17489689.

(29) As an alternative to GroEL a functional homologue of GroEL may be present, in particular a functional homologue comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 10. Suitable natural chaperones polypeptides homologous to SEQ ID NO: 10 are given in Table 4 of WO2014/129898.

(30) As an alternative to GroES a functional homologue of GroES may be present, in particular a functional homologue comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO: 9. Suitable natural chaperones polypeptides homologous to SEQ ID NO: 9 are given in Table 3 of WO2014/129898.

(31) In an embodiment, a 10 kDa chaperone from Table 3 of WO2014/129898 is combined with a matching 60 kDa chaperone from Table 4 from WO2014/129898 of the same organism genus or species for expression in the host. For instance: >gi|189189366|ref|XP_001931022.1|:71-168 10 kDa chaperonin [Pyrenophora tritici-repentis] expressed together with matching >gi|189190432|ref|XP_001931555.1| heat shock protein 60, mitochondrial precursor [Pyrenophora tritici-repentis Pt-1C-BFP].

(32) All other combinations from Table 3 and 4 of WO2014/129898 similarly made with same organism source are also available to the skilled person for expression.

(33) In an embodiment the PRK is originating from a plant selected from Caryophyllales, in particular from Amaranthaceae, in particular from Spinacia.

(34) In an embodiment the recombinant yeast comprises one or more nucleic acid sequences encoding a PRK represented by amino acid sequence represented by SEQ ID NO: 12 or by a functional homologue thereof having sequence identity of at least 50%, preferably at least 60%, 70%, 75%, 80% 85%, 90%, 95%, 98%, or 99%.

(35) A functionally expressed phosphoribulokinase (PRK, EC 2.7.1.19) is capable of catalysing the chemical reaction:
ATP+D-ribulose 5-phosphate ADP+D-ribulose 1,5-bisphosphate  (I)

(36) Thus, the two substrates of this enzyme are ATP and D-ribulose 5-phosphate, whereas its two products are ADP and D-ribulose 1,5-bisphosphate.

(37) PRK 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.

(38) The PRK can be from a prokaryote or a eukaryote. Good results have been achieved with a PRK originating from a eukaryote. Preferably the eukaryotic PRK originates from a plant selected from Caryophyllales, in particular from Amaranthaceae, more in particular from Spinacia.

(39) As an alternative to PRK from Spinacia a functional homologue of PRK from Spinacia may be present, in particular a functional homologue comprising a sequence having at least 70%, 75%, 80%. 85%, 90% or 95% sequence identity with the PRK from Spinacia.

(40) The one or more PRK nucleotide sequences may be under the control of a promoter (the “PRK promoter”) that enables higher expression under anaerobic conditions than under aerobic conditions.

(41) In an embodiment the PRK promoter is ROX1 repressed. ROX1 is herein haeme-dependent repressor of hypoxic gene(s); that mediates aerobic transcriptional repression of hypoxia induced genes such as COX5b and CYC7; the repressor function is regulated through decreased promoter occupancy in response to oxidative stress; and contains an HMG domain that is responsible for DNA bending activity; involved in the hyperosmotic stress resistance. ROX1 is regulated by oxygen.

(42) According to Kwast et al. (in: Genomic Analysis of Anaerobically induced genes in Saccharomyces cerevisiae: Functional roles of ROX1 and other factors in mediating the anoxic response, 2002, Journal of bacteriology vol 184, no 1 p 250-265): “Although Rox1 functions in an O.sub.2-independent manner, its expression is oxygen (haeme) dependent, activated by the haeme-dependent transcription factor Hap1 [Keng, T. 1992. HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 12: 2616-2623]. Thus, as oxygen levels fall to those that limit haeme biosynthesis [Labbe-Bois, R., and P. Labbe. 1990. Tetrapyrrole and heme biosynthesis in the yeast Saccharomyces cerevisiae, p. 235-285. In H. A. Dailey (ed.), Biosynthesis of heme and chlorophylls. McGraw-Hill, New York, N.Y.], ROX1 is no longer transcribed [Zitomer, R. S., and C. V. Lowry. 1992. Regulation of gene expression by oxygen in Saccharomyces cerevisiae. Microbiol. Rev. 56:1-11], its protein levels fall [Zitomer, R. S., P. Carrico, and J. Deckert. 1997. Regulation of hypoxic gene expression in yeast. Kidney Int 51:507-513], and the genes it regulates are de-repressed.”

(43) In an embodiment, the PRK promoter is ROX1-repressed. In an embodiment, the PRK promoter has one or more ROX1 binding motif.

(44) In an embodiment, the PRK promoter comprises in its sequence one or more of the motif according to SEQ ID NO: 15.

(45) In an embodiment, the PRK promoter is the native promoter of a nucleotide sequence selected from the list consisting of: FET4, ANB1, YHR048W, DAN1, AAC3, TIR2, DIPS, HEM13, YNR014W, YAR028W, FUN 57, COX5B, OYE2, SUR2, FRDS1, PIS1, LAC1, YGR035C, YAL028W, EUG1, HEM14, ISU2, ERG26, YMR252C and SML1, in particular FET4, ANB1, YHR048W, DAN1, AAC3, TIR2, DIPS and HEM13.

(46) In an embodiment, the PRK promoter comprises in its sequence one or more of the motif according to TCGTTYAG and/or according to SEQ ID NO: 16.

(47) In particular such PRK promoter is native promoter of a DAN, TIR or PAU gene. In an embodiment, the PRK promoter is the native promoter of a gene selected from the list consisting of: TIR2, DAN1, TIR4, TIR3, PAU7, PAU5, YLL064C, YGR294W, DAN3, YIL176C, YGL261C, YOL161C, PAU1, PAU6, DAN2, YDR542W, YIR041W, YKL224C, PAU3, YLL025W, YOR394W, YHL046C, YMR325W, YAL068C, YPL282C, PAU2, PAU4, in particular the PRK promoter is the native promoter of a gene selected from the list consisting of: TIR2, DAN1, TIR4, TIR3, PAU7, PAU5, YLL064C, YGR294W, DAN3, YIL176C, YGL261C, YOL161C, PAU1, PAU6, DAN2, YDR542W, YIR041W, YKL224C, PAU3, YLL025W.

(48) In an embodiment, the 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.

(49) As used herein “promoter” is a DNA sequence that directs the transcription of a (structural) gene, herein in particular one or more phosphoribulokinase gene. The promoter enables higher expression during anaerobic conditions than under aerobic conditions.

(50) In an embodiment, the PRK promoter may be a synthetic oligonucleotide. It may be a product of artificial oligonucleotide synthesis. Artificial oligonucleotide synthesis is a method in synthetic biology that is used to create artificial oligonucleotides, such as genes, in the laboratory. Commercial gene synthesis services are now available from numerous companies worldwide, some of which have built their business model around this task. Current gene synthesis approaches are most often based on a combination of organic chemistry and molecular biological techniques and entire genes may be synthesized “de novo”, without the need for precursor template DNA.

(51) In an embodiment, the promoter is located in the 5′ region of a the PRK gene, In an embodiment it is located proximal to the transcriptional start site of PRK gene.

(52) The recombinant yeast may be selected from Saccharomycetaceae, in particular from the group of Saccharomyces, such as Saccharomyces cerevisiae; Kluyveromyces, such as Kluyveromyces marxianus; Pichia, such as Pichia stipitis or Pichia angusta; Zygosaccharomyces, such as Zygosaccharomyces bailii; and Brettanomyces, such as Brettanomyces intermedius, Issatchenkia, such as Issatchenkia orientalis and Hansenula.

(53) The PRK promoter may have 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.

(54) In an embodiment the PRK promoter is a synthetic oligonucleotide. The PRK promoter preferably enables expression only during anaerobic conditions.

(55) A suitable PRK promotor is ANB1 and/or DAN1 as mentioned in EP16174382.8.

(56) The recombinant yeast may contain genes of a pentose metabolic pathway non-native to the cell and/or that allow the recombinant cell to convert pentose(s). In one embodiment, the recombinant yeast may comprise one or two or more copies of one or more xylose isomerases and/or one or two or more copies of one or more xylose reductase and xylitol dehydrogenase genes, allowing the recombinant yeast convert xylose. In an embodiment thereof, these genes may be integrated into the recombinant cell genome. In another embodiment, the recombinant yeast comprises the genes araA, araB and araD. It is then able to ferment arabinose. In one embodiment the recombinant yeast comprises xylA-gene, XYL1 gene and XYL2 gene and/or XKS1-gene, to allow the recombinant yeast to ferment xylose; deletion of the aldose reductase (GRE3) gene; overexpression of one or more PPP-genes, e.g. TAL1, TAL2, TKL1, TKL2, RPE1 and RKI1 to allow the increase of the flux through the pentose phosphate path-way in the cell, and/or overexpression of GAL2 and/or deletion of GAL80. Thus though inclusion of the above genes, suitable pentose or other metabolic pathway(s) may be introduced in the recombinant yeast that were non-native in the (wild type) recombinant yeast.

(57) In an embodiment, the following genes may be introduced in the recombinant yeast by introduction into a host cell: 1) a set consisting of PPP-genes TAL1, TKL1, RPE1 and RKI1, optionally under control of strong constitutive promoter; 2) a set consisting of a xylA-gene under under control of strong constitutive promoter; 3) a set comprising a XKS1-gene under control of strong constitutive promoter, 4) a set consisting of the bacterial genes araA, araB and araD under control of a strong constitutive promoter, 5) deletion of an aldose reductase gene

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

(59) The recombinant yeast may be subjected to evolutionary engineering to improve its properties. Evolutionary engineering processes are known processes. Evolutionary engineering is a process wherein industrially relevant phenotypes of a microorganism, herein the recombinant yeast, can be coupled to the specific growth rate and/or the affinity for a nutrient, by a process of rationally set-up natural selection. Evolutionary Engineering is for instance described in detail in Kuijper, M, et al, FEMS, Eukaryotic cell Research 5(2005) 925-934, WO2008041840 and WO2009112472. After the evolutionary engineering the resulting pentose fermenting recombinant cell is isolated. The isolation may be executed in any known manner, e.g. by separation of cells from a recombinant cell broth used in the evolutionary engineering, for instance by taking a cell sample or by filtration or centrifugation.

(60) In an embodiment, the recombinant yeast is marker-free. As used herein, the term “marker” refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. Marker-free means that markers are essentially absent in the recombinant yeast. Being marker-free is particularly advantageous when antibiotic markers have been used in construction of the recombinant yeast and are removed thereafter. Removal of markers may be done using any suitable prior art technique, e.g. intramolecular recombination.

(61) In one embodiment, the recombinant yeast is constructed on the basis of an inhibitor tolerant host cell, wherein the construction is conducted as described hereinafter. Inhibitor tolerant host cells may be selected by screening strains for growth on inhibitors containing materials, such as illustrated in Kadar et al, Appl. Biochem. Biotechnol. (2007), Vol. 136-140, 847-858, wherein an inhibitor tolerant S. cerevisiae strain ATCC 26602 was selected.

(62) To increase the likelihood that enzyme activity is expressed at sufficient levels and in active form in the recombinant yeast, the nucleotide sequence encoding these enzymes, as well as the Rubisco enzyme and other enzymes of the disclosure are preferably adapted to optimise their codon usage to that of the recombinant yeast in question.

(63) The adaptiveness of a nucleotide sequence encoding an enzyme to the codon usage of a cell may be expressed as codon adaptation index (CAI). 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 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 nucleotide 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. Most preferred are the sequences which have been codon optimised for expression in the host cell in question such as e.g. S. cerevisiae cells.

(64) The invention further provides the use of a recombinant yeast according to the invention for preparation of ethanol.

(65) The present invention also provides a process to produce a fermentation product comprising: fermenting a composition comprising a fermentable carbohydrate, in particular selected from the group of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose and mannose under anaerobic conditions in the presence of a recombinant yeast according to the invention; and recovering the ethanol.

(66) In an embodiment one such composition is a biomass hydrolysate. Such biomass hydrolysate may be a lignocellulosic biomass hydrolysate. Lignocellulose herein includes hemicellulose and hemicellulose parts of biomass. Also lignocellulose includes lignocellulosic fractions of biomass. Suitable lignocellulosic materials may be found in the following list: orchard primings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, switch grass, miscanthus, sweet sorghum, canola stems, soybean stems, prairie grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, softwood, hardwood, poplar, pine, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn hobs, corn kernel, fiber from kernels, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid waste, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, an energy crop, forest, a fruit, a flower, a grain, a grass, a herbaceous crop, a leaf, bark, a needle, a log, a root, a sapling, a shrub, switch grass, a tree, a vegetable, fruit peel, a vine, sugar beet pulp, wheat midlings, oat hulls, hard or soft wood, organic waste material generated from an agricultural process, forestry wood waste, or a combination of any two or more thereof. Lignocellulose, which may be considered as a potential renewable feedstock, generally comprises the polysaccharides cellulose (glucans) and hemicelluloses (xylans, heteroxylans and xyloglucans). In addition, some hemicellulose may be present as glucomannans, for example in wood-derived feedstocks. The enzymatic hydrolysis of these polysaccharides to soluble sugars, including both monomers and multimers, for example glucose, cellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, galacturonic acid, glucuronic acid and other hexoses and pentoses occurs under the action of different enzymes acting in concert. In addition, pectins and other pectic substances such as arabinans may make up considerably proportion of the dry mass of typically cell walls from non-woody plant tissues (about a quarter to half of dry mass may be pectins). Lignocellulosic material may be pretreated. 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.

(67) In another embodiment such composition is a pre-treated cornstover hydrolysate. Another preferred composition is a corn fiber hydrolysate, which is optionally pre-treated. In yet another embodiment such composition is a starch hydrolysate, such as a corn starch hydrolysate.

(68) In the context of the invention a “hydrolysate” means a polysaccharide that has been depolymerized through the addition of water to form mono and oligosaccharide sugars. Hydrolysates may be produced by enzymatic or acid hydrolysis of the polysaccharide-containing material.

(69) In an embodiment the fermentable carbohydrate is obtained from starch, lignocellulose, and/or pectin.

(70) The starch, lignocellulose, and/or pectin may be contacted with an enzyme composition, wherein one or more sugar is produced, and wherein the produced sugar is fermented to give a fermentation product, wherein the fermentation is conducted with a recombinant yeast of the invention.

(71) The process is particularly useful when glycerol is fed externally to the process, such as crude glycerol from transesterification-based biodiesel production or recirculation of backset, which is then taken up and converted to ethanol by the claimed recombinant yeast.

(72) In an embodiment the composition comprises an amount of undissociated acetic acid of 10 mM or less.

(73) The inventors have found that a recombinant yeast of the invention, specifically a S. cerevisiae cell is particularly sensitive towards acetic acid, as compared to non-recombinant cells. They have surprisingly found that the ethanol yield rapidly decreases when the composition contains more than 10 mM undissociated acetic acid, and that in order to avoid or lessen the negative effect of acetic acid the process should be performed with a composition having an amount of undissociated acetic acid of 10 mM or less, preferably 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, 1 mM or less.

(74) In an embodiment the composition has an initial undissociated acetic acid of 10 mM or less. In another embodiment, the amount of undissociated acetic acid is 10 mM or less throughout the process.

(75) The lower amount of undissociated acetic acid is less important. In one embodiment, the composition is free of undissociated acetic acid.

(76) In an embodiment, the lower limit of the amount of undissociated acetic acid is 50 μM or more, 55 μM or more, 60 μM or more, 70 μM or more, 80 μM or more, 90 μM or more, 100 μM or more.

(77) The skilled person appreciates that the amount of undissociated acetic acid depends inter alia on the total amount of acetic acid in the composition (protonated and dissociated) as well on the pH.

(78) In one embodiment the amount of undissociated acetic acid is maintained at a value of at 10 mM by adjusting the pH, e.g. by adding a base.

(79) The process may comprise the step of monitoring the pH. The pH of the composition is preferably kept between 4.2 and 5.2, preferably between 4.5 and 5.0. The lower pH is preferably such that the amount of undissociated acetic acid is 10 mM or less, which inter alia depends on the total amount of acetic acid in the composition.

(80) The skilled person knows how to provide or select a composition having an amount of undissociated acetic acid 10 mM or less. For example, he/she may measure the amount of undissociated acetic acid in a composition and select only those compositions which have an amount of undissociated acetic acid of 10 mM or less.

(81) Alternatively, if the amount of undissociated acetic acid in a composition exceeds 10 mM, the process may comprise, prior to the fermentation step, adding a base (such as NaOH or KOH) until the amount of undissociated acetic acid in a composition has reached a value of 10 mM or less.

(82) The amount of undissociated acetic acid may be analysed by HPLC. HPLC generally measures all acetic acid (i.e. both undissociated, i.e. protonated form and dissociated form of acetic acid) because the mobile phase is typically acidified. In order to measure the amount of undissociated acetic acid in the composition, a suitable approach is to measure the (total) amount of acetic acid of the composition as-is, measure the pH of the composition, and calculate the amount of undissociated acetic acid using the pKa of acetic acid.