BRPI0719262B1 - SACCHAROMYCES CELL AND PROCESS FOR ETHANOL PRODUCTION - Google Patents

Description

Field of the Invention The invention relates to a eukaryotic cell having the ability to use L-arabinose and / or convert L-arabinose to L-ribulose, and / or xylulose. -phosphate and / or a desired fermentation product and a process for producing a fermentation product whereby this cell is used. Background of the Invention Ethanol fuel is known as a valuable alternative to fossil fuels. Economically viable ethanol production from the hemicellulose fraction yielding plant biomass requires simultaneous fermentative conversion of both pentoses and hexoses at comparable rates and with high yields. Yeasts, specifically Saccharomyces spp., Are the most appropriate candidates for this process, as they can develop and ferment rapidly in hexoses, both aerobically and anaerobically. Additionally, they are much more resistant to the toxic environment of lignocellulose hydrolysates than bacteria (genetically modified).

EP 1 499 708 describes a process for manufacturing S. c & rèvlsiâè strains capable of producing ethanol from L-arabinose. These strains were modified by introduction of the Bacillus subtilis ara A (L-arabinose isomerase) gene, the araB (L-ribulokinase) and araD (L-ribulose-5-P4-epimerase) genes. Additionally, these strains carried both additional mutations in their genome and overexpressing a TAL1 (transaldolase) gene. However, these strains have several disadvantages. They ferment arabinose under limited oxygen conditions. In addition, they have a low tannin production rate of 0.05 g.g_1.h_1 (Becker and Boles, 2003). Additionally, these strains are not able to use L-arabinose under anaerobic conditions. Finally, these S. cerevisiae strains have a wild-type base, so they cannot be used to co-ferment various C5 sugars.

WO 03/062430 and WO 06/009434 disclose yeast strains capable of converting xylose to ethanol. These yeast strains are capable of directly isomerizing xylose into xylulose.

Still, there is a need for alternative strains for ethanol production that work better and are more robust and resistant to relatively harsh production conditions.

Description of the Drawings Figure 1. Plasmid maps of pRW231 and pRW243.

[007] Figure 2. Growth pattern of RWB219 (O) and IMS0001 (·) shake flask cultures in synthetic medium containing 0.5% galactose (A) and 0.1% galactose + 2% L -arabinosis (B). Cultures were grown for 72 hours in galactose (A) synthetic medium and then transferred to galactose and arabinose (B) synthetic medium. Growth was determined by ODeeo measurement. Figure 3. Growth rate during serial transfer of S. cerevisiae IMS0001 in shake flask cultures containing 2% (weight / volume) L-arabinose synthetic medium. Each data point represents the estimated OÜ660 growth rate measured during (exponential) growth. Open and closed circles represent duplicate serial transfer experiments.

Figure 4. Growth rate during an anaerobic SBR fermentation of S. cerevisiae IMS0001 in synthetic medium with 2% (w / v) L-arabinose. Each data point represents the estimated growth rate of the CO2 profile (full line) during exponential growth.

[010] Figure 5. Sugar consumption and product formation during anaerobic batch fermentations of the IMS0002 strain. Fermentations were performed in a synthetic medium supplemented with: 20 g L-1 arabinose (A); 20 g L_1 glucose and 20 g L_1 arabinose (B); 30 g L-1 glucose, 15 g L_1 xylose, and 15 g L_1 arabinose (C);

Sugar consumption and product formation during anaerobic batch fermentations with a mixture of IMS0002 and RWB218 strains. Fermentations were performed in 1 liter of synthetic medium supplemented with 30 g L_1 glucose, 15 g L_1 xylose, and 15 g L_1 arabinose (D). Symbols: glucose (·); xylose (O); arabinose (I); ethanol calculated from cumulative CO2 production (□); HPLC-measured ethanol (A); cumulative CO2 production (Δ); xylitol (T).

[011] Figure 6. Sugar consumption and product formation during anaerobic batch fermentation of cells of strain IMS0002 selected for anaerobic xylose growth. Fermentation was performed in 1 liter of synthetic medium supplemented with 20 g L_1 xylose and 20 g L_1 arabinose. Symbols: xylose (O); arabinose (■); HPLC-measured ethanol (▲); cumulative CO2 production (Δ); xylitol (T).

[012] Figure 7. Sugar consumption and product formation during an anaerobic batch fermentation of the IMS0003 strain. Fermentation was carried out in 1 liter of synthetic medium supplemented with: 30 g L-1 glucose, 15 g L_1 xylose and 15 g L-1 arabinose. Symbols: glucose (·); xylose (O); arabinose (■); calculated ethanol from cumulative CO2 production (□); HPLC-measured ethanol (A); cumulative CO2 production (Δ).

Description of the Invention Eukaryotic Cell In a first aspect, the invention relates to a eukaryotic cell capable of expressing the following nucleotide sequences, whereby expression of these nucleotide sequences gives the cell the ability to use L-arabinose and / or converting L-arabinose to L-ribulose, and / or 5-phosphate xylulose and / or a desired fermentation product such as ethanol: (a) a nucleotide sequence encoding an arabinose isomerase (araA), whereby a said nucleotide sequence is selected from the group consisting of: (i) nucleotide sequences encoding an araA, said araA comprising an amino acid sequence having at least 55% sequence identity to the amino acid sequence of SEQ ID NO: 1. (ii) nucleotide sequences comprising a nucleotide sequence that has at least 60% sequence identity to the nucleotide sequence of SEQ ID NO: 2. (iii) nucleotide sequences wherein the complementary strand thereof hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); (iv) nucleotide sequences the sequences thereof differ from the sequence of a nucleic acid molecule of (iii) due to the degeneration of the genetic code, (b) a nucleotide sequence encoding an L-ribulokinase (araB), whereby the said nucleotide sequence is selected from the group consisting of: (i) nucleotide sequences encoding an araB, said araB comprising an amino acid sequence having at least 20% sequence identity with the amino acid sequence of SEQ ID NO: 3. (ii) nucleotide sequences comprising a nucleotide sequence that has at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 4. (iii) nucleotide sequences wherein the complementary strand thereof hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); (iv) nucleotide sequences sequences thereof differ from the sequence of a nucleic acid molecule of (iii) due to the degeneration of the genetic code, (c) a nucleotide sequence encoding an L-ribulose-5-P-4- whereby said nucleotide sequence is selected from the group consisting of: (i) nucleotide sequences encoding an araD, said araD comprising an amino acid sequence having at least 60% sequence identity to the nucleotide sequence. amino acid of SEQ ID NO: 5. (ii) nucleotide sequences comprising a nucleotide sequence that has at least 60% sequence identity to the nucleotide sequence of SEQ ID NO: 6. (iii) nucleotide sequences where the complementary strand thereof hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); (iv) nucleotide sequences sequences thereof differ from the sequence of a nucleic acid molecule of (iii) by reason of the degeneration of the genetic code.

A preferred embodiment relates to a eukaryotic cell capable of expressing the following nucleotide sequences, so expression of these nucleotide sequences gives the cell the ability to use L-arabinose and / or convert L-arabinose to L -ribulose, and / or 5-phosphate xylulose and / or a desired fermentation product such as ethanol: (a) a nucleotide sequence encoding an arabinose isomerase (araA), whereby said nucleotide sequence is selected from the group consisting of in: (i) nucleotide sequences comprising a nucleotide sequence having at least 60% sequence identity to the nucleotide sequence of SEQ ID NO: 2, (ii) nucleotide sequences where the complementary strand hybridizes to each other. a nucleic acid molecule of the sequence of (i); (iii) nucleotide sequences the sequences thereof differ from the sequence of a nucleic acid molecule from (ii) due to the degeneration of the genetic code, (b) a nucleotide sequence encoding L-ribulokinase (araB), whereby the said nucleotide sequence is selected from the group consisting of: (i) nucleotide sequences encoding an araB, said araB comprising an amino acid sequence having at least 20% sequence identity with the amino acid sequence of SEQ ID NO: 3. (ii) nucleotide sequences comprising a nucleotide sequence that has at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 4. (iii) nucleotide sequences wherein the complementary strand thereof hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); (iv) nucleotide sequences sequences thereof differ from the sequence of a nucleic acid molecule of (iii) due to the degeneration of the genetic code, (c) a nucleotide sequence encoding an L-ribulose-5-P-4- wherein said nucleotide sequence is selected from the group consisting of: (i) nucleotide sequences encoding an araD, said araD comprising an amino acid sequence having at least 60% sequence identity to the amino acid of SEQ ID NO: 5. (ii) nucleotide sequences comprising a nucleotide sequence that has at least 60% sequence identity to the nucleotide sequence of SEQ ID NO: 6. (iii) nucleotide sequences where the complementary strand thereof hybridizes to a nucleic acid molecule of the sequence of (i) or (ii); (iv) nucleotide sequences sequences thereof differ from the sequence of a nucleic acid molecule of (iii) by reason of the degeneration of the genetic code.

Sequence Identity and Similarity Sequence identity is defined herein as the relationship between two amino acid sequences (polypeptide or protein) or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. Generally, sequence identities or similarities are compared relative to the total length of the compared sequences. In the art, "identity" also means the degree of sequence relationship between amino acid or nucleic acid sequences, as the case may be, as determined by the combination of strings of such sequences. The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes from one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by various methods known to those skilled in the art.

Preferred methods for determining identity are designed to provide the greatest match between the sequences tested. Methods for determining identity and similarity are coded in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, BestFit, BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Mol. Biol. 215: 403-410 (1990)). , publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894.) A more preferred algorithm used is EMBOSS (http: // www. ebi .ac.uk / Preferred parameters for comparison of amino acid sequences using EMBOSS are open slit 10.0, extended slit 0.5, Blosum matrix 62. Preferred parameters for comparison of nucleic acid sequence using EMBOSS are open slit 10.0, extended slit 0.5, full DNA matrix (DMA identity matrix).

Optionally, in determining the degree of amino acid similarity, one skilled in the art may take into account amino acid substitutions termed "conservative" as will be apparent to one skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, an amino acid group having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; an amino acid group having aliphatic hydroxyl side chains is serine and threonine; an amino acid group having amide-containing side chains is asparagine and glutamine; an amino acid group having aromatic side chains is phenylalanine, tyrosine and tiptophan; an amino acid group having basic side chains is lysine, arginine and histidine; and an amino acid group having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. Amino acid sequence substitution variants 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.

Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala in ser; Arg in lys; Asn in gin or his; Asp in glu; Cys in being or ward; Gin in asn; Glu in asp; Gly in pro; His is asn or gin; Ile in read or go; Read it in ile or go; Lys in arg;

Gin in glu; Met in leu or ile; Phe in met, leu or tyr; Be in thr; Thr in being; Trp in tyr; Tyr in trp or phe; e, Go in ile or read.

Nucleic Acid Sequence Hybridization Nucleotide sequences encoding the enzymes expressed in the cell of the invention can also be defined by their ability to hybridize to the nucleotide sequences of SEQ ID NOs 2, 4, 6, 8, 16, 18 , 20, 22, 24, 26, 28, 30 respectively, under conditions of moderate or preferably stringent hybridization. Stringent hybridization conditions are defined herein as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100, and more preferably about 200 or more nucleotides, to hybridize at a temperature of about 100 ° C. about 65 ° C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having comparable ionic strength, and washing at 65 ° C in a solution comprising about 0.1 M salt, or least preferably 0.2 x SSC or any other solution having comparable ion resistance. Preferably, the hybridization is performed overnight, i.e. for at least 10 hours and preferably the wash is performed for at least one hour with at least two changes of the wash solution. These conditions will allow sequence specific hybridization having about 90% or more of sequence identity.

Moderate conditions are defined herein as conditions that allow nucleic acid sequences of at least 50 nucleotides, preferably about 200 or more nucleotides, to hybridize at a temperature of about 45 ° C in a solution comprising about 1 µM. M salt, preferably 6 x SSC or any other solution having 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 comparable ionic strength. Preferably, hybridization is performed overnight, i.e. for at least 10 hours, and preferably washing is performed for at least one hour with at least two changes of the wash solution. These conditions will allow sequence specific hybridization having up to 50% sequence identity. One of skill in the art will be able to modify these hybridization conditions to specifically identify sequences ranging in identity from 50% to 90%.

AraA

A preferred nucleotide sequence encoding an arabinose isomerase (araA) expressed in the cell of the invention is selected from the group consisting of: (a) nucleotide sequences encoding an araA polypeptide comprising an amino acid sequence having at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1; (b) nucleotide sequences comprising a nucleotide sequence having at least 60, 70, 80, 90, 95, 97, 98, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 2; (c) nucleotide sequences wherein the complementary strand thereof hybridizes to a nucleic acid molecule sequence of (a) or (b); (d) nucleotide sequences, where the sequence thereof differs from the sequence of a nucleic acid molecule from (c) due to the degeneration of the genetic code.

The nucleotide sequence encoding an araA can encode both a prokaryotic and eukaryotic araA, that is, an araA with an amino acid sequence that is identical to that of a naturally occurring araA in the prokaryotic or eukaryotic organism. The present inventors have found that the ability of a specific araA to confer on a eukaryotic host cell the ability to use arabinose and / or convert arabinose to L-ribulose, and / or 5-phosphate xylulose and / or to a desired fermentation product such. as ethanol when coexpressed with araB and araD does not much depend on whether araA is of prokaryotic or eukaryotic origin. Instead, it depends on the relationship of the araA amino acid sequence to that sequence SEQ ID NO: 1.

AraB

A preferred nucleotide sequence encoding an L-ribulokinase (AraB) expressed in the cell of the invention is selected from the group consisting of: (a) nucleotide sequences encoding a polypeptide comprising an amino acid sequence having at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 ; (b) nucleotide sequences comprising a nucleotide sequence having at least 50, 60, 70, 80, 90, 95, 97, 98, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4; (c) nucleotide sequences wherein the complementary strand thereof hybridizes to a nucleic acid molecule sequence of (a) or (b); (d) nucleotide sequences, where the sequence thereof differs from the sequence of a nucleic acid molecule from (c) by degeneration of the genetic code.

The nucleotide sequence encoding an araB can encode both a prokaryotic and eukaryotic araB, that is, an araB with an amino acid sequence that is identical to that of a naturally occurring araB in the prokaryotic or eukaryotic organism. The present inventors have found that the ability of a specific araB to confer on a eukaryotic host cell the ability to use arabinose and / or convert arabinose to L-ribulose and / or 5-phosphate xylulose and / or a desired fermentation product when coexpressed. with araA and araD not much depends on whether araB is of prokaryotic or eukaryotic origin. Instead, it depends on the ratio of the amino acid sequence of araB to that sequence SEQ ID NO: 3.

AraD

A preferred nucleotide sequence encoding an L-ribulose-5-P-4-epimerase (araD) expressed in the cell of the invention is selected from the group consisting of: (e) nucleotide sequences encoding a polypeptide comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 5; (f) nucleotide sequences comprising a nucleotide sequence having at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99% sequence identity to the nucleotide sequence of SEQ ID. N06; (q) nucleotide sequences where the complementary strand thereof hybridizes to a nucleic acid molecule sequence of (a) or (b); (h) nucleotide sequences, where the sequence thereof differs from the sequence of a nucleic acid molecule from (c) by degeneration of the genetic code.

The nucleotide sequence encoding an araD can be either a prokaryotic or eukaryotic araD, that is, an araD with an amino acid sequence that is identical to that of a naturally occurring araD in the prokaryotic or eukaryotic organism. The present inventors have found that the ability of a specific araD to give a eukaryotic host cell the ability to use arabinose and / or convert arabinose to L-ribulose, and / or 5-phosphate xylulose and / or a desired fermentation product when coexpressed with araA and araB does not much depend on whether araD is of prokaryotic or eukaryotic origin. Instead, it depends on the ratio of the araD amino acid sequence to that sequence SEQ ID NO: 5.

Surprisingly, the codon tilt index indicated that the expression of the Lactobacillus plantarum araA, araB and araD genes was more favorable for yeast expression than that of the prokaryotic araA, araB and araD genes described in EP 1 499 708.

[027] It should be noted that L. plantarum is a Generally Considered Safe (GRAS) organism, which is recognized as safe by the Food Registration Authorities. Therefore, a preferred nucleotide sequence encodes an araA, araB or araD respectively having an amino acid sequence that is related to the sequences SEQ ID NO: 1, 3, or 5 respectively, as defined above. A preferred nucleotide sequence encodes an araA, araB or araD fungus respectively (e.g., from a Basidiomycete), more preferably an araA, araB or araD respectively of an anaerobic fungus, for example an anaerobic fungus belonging to the Neocallimastix, Caecomyces, Piromyces, Orpinomyces or Ruminomyces. Alternatively, a preferred nucleotide sequence encodes a bacterial araA, araB or araD, respectively, preferably from a gram positive bacterium, more preferably from the genus Lactobacillus, more preferably from the Lactobacillus plantarum species. Preferably one, two or three of the araA, araB and araD nucleotide sequences originate from a genus Lactobacillus, more preferably from the species Lactobacillus plantarum. The bacterial araA expressed in the cell of the invention is not the Bacillus subtilis araA disclosed in EP 1 499 708 and provided as SEQ ID NO: 9. SEQ ID NO: 10 represents the nucleic acid sequence encoding SEQ ID NO: 9. The bacterial araB and araD expressed in the cell of the invention are not those of Escherichia coli (E. coli) as disclosed in EP 1 499 708 and provided as SEQ ID NO: 11 and SEQ ID NO: 13. SEQ ID NO: 12 represents the nucleic acid sequence encoding SEQ ID NO: 11. SEQ ID NO: 14 represents the nucleic acid sequence encoding SEQ ID NO: 13.

In order to increase the likelihood that the araA, araB and araD (bacterial) enzymes respectively will be expressed in active form in a eukaryotic host cell of the invention, such as yeast, the corresponding coding nucleotide sequence may be adapted to optimize its codon usage for that of the chosen eukaryotic host cell. Adaptation of a nucleotide sequence encoding the araA, araB and araD enzymes (or other enzymes of the invention, see below) to the use of the chosen host cell codon may be expressed as codon adaptation index (CAI). The codon adaptation index is defined herein as the measurement of the relative adaptation of a gene's codon usage towards the codon usage of highly expressed genes. The relative adaptation (w) of each codon is the reason for the use of each codon in relation to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptation values. Non-synonymous codons and termination codons (dependent on the genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of more abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 1281-1295; see also: Jansen et al., 2003, Nucleic Acids Res. (8): 2242-51). An adapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7.

In a preferred embodiment, expression of nucleotide sequences encoding an ara A, ara B and ara D, as defined hereinabove, gives the cell the ability to use L-arabinose and / or convert it to L-ribulose, and / or 5-phosphate xylulose. Without wishing to be bound by any theory, it is expected that L-arabinose is converted first to L-ribulose, which is subsequently converted to 5-phosphate xylulose which is the major molecule that enters the pentose phosphate pathway. In the context of the invention, "use of L-arabinose" preferably means that the optical density measured at 660 nm (OD660) of transformed cells grown under aerobic or anaerobic conditions in the presence of at least 0.5% L-arabinose for at least 20 days is increased from approximately 0.5 to 1.0 or more. More preferably, the OÜ660 is increased from 0.5 to 1.5 or more. More preferably, the cells are cultured in the presence of at least 1%, at least 1.5%, at least 2% L-arabinose. More preferably, the cells are cultured in the presence of approximately 2% L-arabinose.

[030] In the context of the invention, a cell is capable of "converting L-arabinose to L-ribulose" when detectable amounts of L-ribulose are detected in cells grown under aerobic or anaerobic conditions in the presence of L-arabinose (same preferred concentrations. as in the previous paragraph) for at least 20 days using an appropriate assay. Preferably the assay is HPLC for L-ribulose.

[031] In the context of the invention, a cell is capable of "converting L-arabinose to 5-phosphate xylulose" when an increase of at least 2% of 5-phosphate xylulose is detected in cells grown under aerobic or anaerobic conditions in the presence L-arabinose (same preferred concentrations as in the previous paragraph) for at least 20 days using an appropriate assay. Preferably, an HPCL-based assay for xylulose 5-phosphate has been described in Zaldivar J., et al. ((2002), Appl. Microbiol. Biotechnol., 59: 436-442). This assay is briefly described in the experimental part. More preferably, the increase is at least 5%, 10%, 15%, 20%, 25% or more.

In another preferred embodiment, expression of nucleotide sequences encoding an ara A, ara B and ara D, as defined hereinabove, gives the cell the ability to convert L-arabinose into a desired fermentation product when grown under aerobic or anaerobic conditions in the presence of L-arabinose (same preferred concentrations as in the previous paragraph) for at least one month to one year. More preferably, a cell is capable of converting L-arabinose into a desired fermentation product when desirable amounts of a desired fermentation product are detected using an appropriate assay and when the cells are cultured under the conditions provided in the previous sentence. Even more preferably, the assay is HPLC. Even more preferably, the fermentation product is ethanol.

A cell for transformation with nucleotide sequences encoding the araA, araB and araD enzymes respectively, as described above, is preferably a host cell capable of active or passive transport of xylose in a xylose isomerization within the cell. The cell is preferably capable of active glycolysis. The cell may additionally contain an endogenous pentose phosphate pathway and may contain endogenous xylulose kinase activity so that the xylose isomerized xylulose can be metabolised to pyruvate. The cell additionally and preferably contains enzymes for converting pyruvate into the desired fermentation product, such as ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, acid fumaric acid, an amino acid, 1,3-propane diol, ethylene, glycerol, butanol, a β-lactam antibiotic or a cephalosporin. The cell may be transformed to be capable of producing butanol by introducing one or more butanol pathway genes as disclosed in WO2007 / 041269.

A preferred cell is naturally capable of alcoholic fermentation, preferably anaerobic alcoholic fermentation. The additional host cell preferably has a high tolerance to ethanol, a high tolerance to low pH (i.e. capable of growth at a pH below 5, 4, 3 or 2.5) and towards organic acids such as, lactic acid, acetic acid or formic acid and sugar degradation products such as furfural and hydroxy methylfurfural and high tolerance to elevated temperatures. Any of these characteristics or activities of a host cell may be naturally present in a host cell or may be introduced or modified through genetic selection or genetic modification. An appropriate host cell is a eukaryotic microorganism such as, for example, a fungus, however, more appropriately as a host cell is yeast or filamentous fungi.

Yeasts are defined herein as eukaryotic microorganisms and include all species of the Eumycotina subdivision (Alexopoulos, C.J., 1962, In: Introductory Mycology, John Wiley & Sons, Inc., New York) that grow predominantly in unicellular form. Yeast can either grow by budding from a unicellular stalk or they can grow by cracking of the organism. Preferred yeast host cells belong to one of the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia. Preferably the yeast is capable of anaerobic fermentation, more preferably anaerobic alcoholic fermentation.

Filamentous fungi are defined herein as eukaryotic microorganisms which include all filamentous forms of the Eumycotina subdivision. These fungi are characterized by a vegetative mycelium composed of chitin, cellulose and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically and genetically distinct from yeast. Vegetative growth by filamentous fungi is accomplished by hyphal elongation and carbon catabolism of the most filamentous fungi is obligatorily aerobic. Preferred filamentous fungi as host cells belong to one of the genera Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium or Penicillium.

[037] Over the years, suggestions have been made for the introduction of various organisms for the production of bioethanol from harvest sugars. In practice, however, all major bioethanol production processes continued to use Saccharomyces yeasts as ethanol producers. This is due to the many attractive aspects of Saccharomyces species for industrial processes, ie, tolerance to acid, ethanol and high osmosis, anaerobic growth capacity and of course their high alcoholic fermentation capacity. Preferred yeast species as host cells include S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus, K. fragilis.

In a preferred embodiment, the host cell of the invention is a host cell that has been transformed with a nucleic acid construct comprising the nucleotide sequence encoding the araA, araB and araD enzymes as defined above. In one or more preferred embodiments, the host cell is co-transformed with three nucleic acid constructs each nucleic acid construct comprising the nucleotide sequence encoding araA, araB or araD. The nucleic acid construct comprising the araA, araB, and / or araD coding sequence being capable of expression of the araA, araB, and / or araD enzymes in the host cell. For this purpose, the nucleic acid construct may be constituted as described, for example, in WO 03/0624430. The host cell may comprise a single copy, but preferably comprises multiple copies of each nucleic acid construct. The nucleic acid construct may be maintained episomically and thus comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may be based, for example, on the 2μ yeast or pKD1 plasmids (Fleer et al., 1991, Biotechnology 9: 968-975). Preferably, however, each nucleic acid construct is integrated into one or more copies within the host cell genome. Integration within the host cell genome may occur randomly by illegitimate recombination, but preferably the nucleic acid construct is integrated into the host cell genome by homologous recombination, as is well known in the art of molecular fungal genetics (see, for example, WO 90/14423, EP-A-0 481 008, EP-A-0 635 574 and US 6,265,186).

Accordingly, in a more preferred embodiment, the cell of the invention comprises a nucleic acid construct comprising the araA, araB, and / or araD coding sequence and is capable of expression of the araA, araB and / or araD enzymes. In an even more preferred embodiment, the araA, araB, and / or araD coding sequences are operably linked to a promoter that provides sufficient expression of the corresponding nucleotide sequences in the cell to give the cell the ability to use L-arabinose, and / or converting L-arabinose to L-ribulose, and / or 5-phosphate xylulose. Preferably the cell is a yeast cell. Accordingly, in a further aspect, the invention also encompasses a nucleic acid construct as noted hereinbefore. Preferably, a nucleic acid construct comprises a nucleic acid sequence encoding an araA, araB and / or araD. Nucleic acid sequences encoding an araA, araB or araD have all been defined hereinabove.

Even more preferably, expression of the corresponding nucleotide sequences in a cell gives the cell the ability to convert L-arabinose to a desired fermentation product as defined hereinbelow. In an even more preferred embodiment, the fermentation product is ethanol. Even more preferably, the cell is a yeast cell.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements (or coding or nucleic acid sequences) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed in functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the coding sequence. Operatively linked means that the nucleic acid sequences being linked are typically continuous and, when necessary, join two protein coding regions, continuous and in the reading frame.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the transcription direction of the gene transcription initiation site. and being structurally identified by the presence of a DNA-dependent RNA polymerase binding site, transcription initiation sites and any other DNA sequences, including but not limited to transcription factor binding sites, repressor protein binding sites and activator and any other nucleotide sequences known to one of skill in the art to act directly or indirectly to regulate the amount of promoter transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.

The promoter that would be used to obtain expression of the araA, araB and / or araD encoding nucleotide sequences may not be native to the nucleotide sequence encoding the enzyme to be expressed, that is, a promoter that is heterologous to nucleotide sequence (coding sequence) to which it is operably linked. While the promoter preferably is heterologous to the coding sequence to which it should be operably linked, it is also preferred that the promoter is homologous, i.e. endogenous to the host cell. Preferably, the heterologous promoter (with respect to the nucleotide sequence) is capable of producing a higher steady-state level of transcription comprising the coding sequence (or is capable of producing more transcriptional molecules, i.e. mRNA molecules, per unit). with respect to the promoter that is native to the coding sequence, preferably under conditions where arabinose or arabinose and glucose or xylose and arabinose or xylose and arabinose and glucose are available as carbon sources, more preferably as major carbon sources (i.e. that is, more than 50% carbon source availability consists of arabinose or arabinose and glucose or xylose and arabinose or xylose and arabinose and glucose), more preferably as the sole carbon sources. Appropriate promoters in the context include both constitutive and inducible natural promoters as well as genetically transformed promoters. A preferred promoter for use in the present invention will also be sensitive to catabolite (glucose) repression and / or preferably will not require arabinose and / or xylose for induction.

Promoters having these characteristics are widely available and known to the person skilled in the art. Suitable examples of such promoters include, for example, glycolytic gene promoters such as phosphofrutokinase (PPK) promoters, triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK) ), phosphoglycerate kinase (PGK) from yeast or filamentous fungi; Further details of these yeast promoters can be found in (WO 93/03159). Other useful promoters are ribosomal proteins encoding gene promoters, the lactase gene promoter (LAC4), alcohol dehydrogenase promoters (ADH1, ADH4 and the like), the enolase promoter (ENO), the glucose-6-phosphate isomerase promoter. (PGI1, Hauf et al., 2000) or the hexose (glucose) transporter (HXT7) or glyceraldehyde-3-phosphate dehydrogenase (TDH3) promoter. The PGII promoter sequence is provided in SEQ ID NO: 51. The HXT7 promoter sequence is provided in SEQ ID NO: 52. The TDH3 promoter sequence is provided in SEQ ID NO: 49. Other promoters, both constitutive and inducible and enhancers or upstream activation sequences will be known to those skilled in the art. Promoters used in the host cells of the invention may be modified, if desired, to affect their control characteristics.

A preferred cell of the invention is a eukaryotic cell transformed with the L. plantarum araA, araB and araD genes. More preferably, the eukaryotic cell is a yeast cell, even more preferably a S. cerevisiae strain transformed with the L. plantarum araA, araB and araD genes. More preferably, the cell is both CBS 120327 and CBS 120328 both filed with the CBS Institute (Netherlands) on September 27, 2006.

The term "homologous" when used to indicate the relationship between a given (recombinant) nucleic acid molecule or polypeptide and a given host organism or host cell means that in nature said nucleic acid molecule or polypeptide is produced by a host cell or organisms of the same species, preferably of the same strain variety. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically be operably linked to another promoter sequence, or, if applicable, another secretory signal sequence and / or terminator sequence relative to its natural environment. When used to indicate the relationship of the two nucleic acid sequences, the term "homologous" means that a single stranded nucleic acid sequence can hybridize to the complementary single stranded nucleic acid sequence. The degree of hybridization will depend on a number of factors including the amount of sequence identity and the hybridization conditions such as temperature and salt concentration as set forth above. Preferably, the identity region is greater than 5 base pairs, more preferably, the identity region is greater than 10 base pairs.

[047] The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to non-naturally occurring nucleic acid or protein as part of the organism, cell, genome or sequence of DNA or RNA where it is present. is present or found in a cell or location or locations in the genome or sequence of DNA or RNA that differ from that in which it is found in nature. Nucleic acids or heterologous proteins are not endogenous to the cell into which it is introduced, but were obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which DNA is transcribed or expressed. Similarly, exogenous RNA encodes proteins that are not normally expressed in the cell in which exogenous RNA is present. Nucleic acids and heterologous proteins may also be referred to as nucleic acids or tin proteins. Any nucleic acid or protein that one skilled in the art recognizes as being heterologous or tin to the cell in which it is expressed is encompassed herein by the term nucleic acid or heterologous protein. The term heterologous also applies to unnatural combinations of nucleic acid or amino acid sequences, that is, combinations where at least two of the combined sequences are foreign to each other. Preferred Eukaryotic Cell Able to Use and / or Convert L-arabinose and xylose In a more preferred embodiment, the cell of the invention expressing araA, araB and araD is capable of using L-arabinose and / or converting it to L -ribulose, and / or 5-phosphate xylulose and / or a desired fermentation product as defined hereinbefore and additionally exhibit the ability to use xylose and / or convert xylose to xylulose. The conversion of xylose to xylulose is preferably an isomerization step (direct isomerization of xylose to xylulose). This type of cell is therefore capable of using both L-arabinose and xylose. "Use" of xylene preferably has the same meaning as "use" of L-arabinose as described hereinabove.

Enzyme definitions are as used in WO 06/009434, for xylose isomerase (EC 5.3.1.5), xylulose kinase (EC 2.7.1.17), ribulose 5-phosphate epimerase (5.1.3.1), ribulose 5-phosphate isomerase (EC 5.3.1.6), transcetolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2), and aldose reductase "(EC 1.1.1.21).

In a preferred embodiment, the eukaryotic cell of the invention expressing araA, araB and araD as defined hereinabove has the ability to isomerize xylose into xylulose as described, for example, in WO 03/0624430 or WO 06 / 009434. The ability to isomerize xylose into xylulose is conferred to a host cell by transforming a host cell with a nucleic acid construct comprising a nucleotide sequence encoding xylose isomerase. The ability of the transformed host cell to isomerize xylose to xylulose is a direct disomerization of xylose to xylulose. It is understood that xylose isomerized xylose is a simple reaction catalyzed by a xylose isomerase, as opposed to the two-step conversion of xylose to xylulose via a xylitol intermediate as catalyzed by xylose reductase and xylitol dehydrogenase, respectively.

The nucleotide sequence encodes xylose isomerase which is preferably expressed in active form in the transformed host cell of the invention. Thus, expression of the nucleotide sequence in the host cell produces a xylose isomerase with specific activity of at least 10 U of xylose isomerase activity per protein MG at 30 ° C, preferably at least 20, 25, 30, 50, 100, 200, 300 or 500 U per mg at 30 ° C. The specific activity of xylose isomerase expressed in the transformed host cell is defined herein as the amount of units of xylose isomerase activity per MG of protein of the host cell free lysate, for example, a yeast free lysate . The determination of xylose isomerase has already been described herein.

Preferably, expression of the nucleotide sequence encoding xylose isomerase in the host cell produces xylose isomerase with a Km for xylose that is less than 50, 40, 30 or 25 mM, more preferably, Km for xylose is about 20 mM or less.

A preferred nucleotide sequence encoding xylose isomerase may be selected from the group consisting of: (e) nucleotide sequences encoding a polypeptide comprising an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 15; (f) nucleotide sequences comprising a nucleotide sequence having at least 40, 50, 60, 70, 80, 90, 95, 97, 98, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 16; (g) nucleotide sequences wherein the complementary strand thereof hybridizes to a nucleic acid molecule sequence of (a) or (b); (h) nucleotide sequences, where the sequence thereof differs from the sequence of a nucleic acid molecule from (c) by degeneration of the genetic code.

The nucleotide sequence encoding the xylose isomerase may encode both a prokaryotic and eukaryotic xylose isomerase, i.e. a xylose isomerase with an amino acid sequence that is identical to that of a naturally occurring xylose isomerase in the prokaryotic or eukaryotic organism. The present inventors have found that the ability of a specific xylose isomerase to give a eukaryotic host cell the ability to isomerize xylose into xylulose does not depend greatly on whether the isomerase is of prokaryotic or eukaryotic origin. Instead, it depends on the ratio of the isomerase amino acid sequence to the Piromyces sequence (SEQ ID NO: 7). Surprisingly, Piromyces isomerase is more related to prokaryotic isomerases than to other known yeast isomerase. Therefore, a preferred nucleotide sequence encodes xylose isomerase having an amino acid sequence that is related to the Piromyces sequence as defined above. A preferred nucleotide sequence encodes a fungal xylose isomerase (e.g. from a Basidiomycete), more preferably an anaerobic fungal xylose isomerase, for example an anaerobic fungal xylose isomerase belonging to the Neocallimastix, Caecomyces, Piromyces, Oromyomyces or Orcesomyces families . Alternatively, a preferred nucleotide sequence encodes a bacterial xylose isomerase, preferably a gram negative bacterium, more preferably a Bacteroides class or Bacteroides isomerase, more preferably B. thetaiotaomicron (SEQ ID NO: 15).

In order to increase the likelihood that xylose isomerase is actively expressed in a eukaryotic host cell, such as yeast, the nucleotide sequence encoding xylose isomerase may be adapted to optimize its use of the eukaryotic host cell codon as appropriate. defined in this document earlier.

[056] A host cell for transformation with the nucleotide sequence encoding xylose isomerase as described above is preferably a host capable of activating or inactivating xylose transport within the cell. The host cell preferably contains active glycolysis. The host cell may additionally contain an endogenous pentose phosphate pathway and may contain endogenous xylulose kinase activity, so that the xylose isomerized xylulose may be metabolised to pyruvate. The host preferably contains enzymes for converting pyruvate into a desired fermentation product, such as ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, a amino acid, 1,3-propane diol, ethylene, glycerol, butanol, a β-lactam antibiotic or a cephalosporin. A suitable host cell is a host cell that is naturally capable of alcoholic fermentation, preferably anaerobic alcoholic fermentation. The host cell additionally and preferably has a high tolerance to ethanol, a high tolerance to low pH (i.e. capable of developing at a pH of less than 5, 4, 3 or 2.5) and towards organic acids such as as lactic acid, acetic acid or formic acid and sugar degradation products such as furfural and hydroxymethylfurfural and high tolerance to elevated temperatures. Any of these characteristics or activities of the host cell may be naturally present in the host cell or may be introduced or altered by genetic modification. An appropriate cell is a eukaryotic microorganism similar to, for example, a fungus, but more suitable as host cells are yeast or filamentous fungi. Preferred yeasts and filamentous fungi have already been defined herein.

[057] As used herein, the word host cell has the same meaning as the cell.

The cell of the invention is preferably transformed with a nucleic acid construct comprising the nucleotide sequence encoding xylose isomerase. The nucleic acid construct which is preferably used is the same as that used comprising the nucleotide sequence encoding araA, araB or araD.

[059] In another preferred embodiment of the invention, the cell of the invention: expressing araA, araB and araD and exhibiting the ability to directly isomerize xylose into xylulose as defined hereinbefore comprises a genetic modification that increases pentose pathway flow phosphate as described in WO 06/009434. Specifically, genetic modification causes an increase in the flow of the non-oxidative pentose phosphate pathway. A genetic modification that causes increased flow of the non-oxidative part of the pentose phosphate pathway is understood herein to mean a modification that increases flow by a factor of at least 1.1, 1.2, 1.5, 2, 5. , 10 or 20, compared to flow in a strain that is genetically identical, except for genetic modification causing increased flow. The flow of the non-oxidative part of the pentose phosphate pathway can be measured by growth of the xylose-modified host as a single carbon source, determining the specific xylitol production rate from the specific xylose consumption rate if any xylitol is produced. However, the flow of the non-oxidative part of the pentose phosphate pathway is proportional to the growth rate of xylose as the sole carbon source, preferably with the anaerobic rate of growth in xylose as the sole carbon source. There is a linear relationship between the rate of growth in xylose as a single carbon source (pmax) and the flow of the non-oxidative part of the pentose phosphate pathway. The specific xylose consumption rate (Qs) is equal to the growth rate (μ) divided by the sugar biomass yield (Yxs) since the sugar biomass yield is constant (under a given set of conditions: anaerobic growth medium, pH, strain genetic history, etc., ie Qs = μ / Yxs). Therefore, the increased flow of the non-oxidative part of the pentose phosphate pathway can be deduced from the increase in the maximum growth rate under these conditions. In a preferred embodiment, the cell comprises a genetic modification that increases the flow of the pentose phosphate pathway and which has a specific xylose consumption rate of at least 346 mg xylose / g biomass / hr.

Genetic modifications that increase the flow of the pentose phosphate pathway can be introduced into the host cell in various ways. These include, for example, obtaining higher steady-state activity levels of xylose kinase and / or one or more of the enzymes of the non-oxidative part of the pentose phosphate pathway and / or a reduced steady-state level of non-aldose reductase activity. specific. These changes in steady-state activity levels may be effected by mutant selection (spontaneous or chemical- or radiation-induced) and / or recombinant DNA technology, for example by overexpression or inactivation, respectively of genes encoding the enzymes or factors regulating these genes.

[061] In a more preferred host cell, genetic modification comprises overexpression of at least one pentose phosphate pathway enzyme (non-oxidative part). Preferably, the enzyme is selected from the group consisting of enzymes encoding ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transcetolase and transaldolase as described in WO 06/009434.

[062] Various enzyme combinations of the pentose phosphate pathway (non-oxidative part) may be overexpressed. For example the enzymes that are overexpressed may be at least ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase enzymes; or at least the enzymes ribulose-5-phosphate isomerase and transcetolase; or at least the enzymes ribulose-5-phosphate isomerase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase and transcetolase; or at least the enzymes ribulose-5-phosphate epimerase and transaldolase; or at least the transketolase and transaldolase enzymes; or at least the enzymes ribulose-5-phosphate epimerase, transcetolase and transaldolase; or at least ribulose-5-phosphate isomerase, transcetolase and transaldolase enzymes; or at least the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, and transaldolase; or at least the ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, and transcetolase enzymes. In one embodiment of the invention each enzyme ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transcetolase and transaldolase are overexpressed in the host cell. Most preferred is a host cell in which the genetic modification comprises at least overexpression of both transketolase and transaldolase enzymes as such, a host cell already capable of anaerobic growth in xylose. In fact, under some conditions, we have found that host cells overexpressing only transcetolase and transaldolase already have the same anaerobic growth rate in xylose as host cells expressing all four enzymes, ie ribulose-5-phosphate isomerase. , ribulose-5-phosphate epimerase, transcetolase and transaldolase. In addition, host cells overexpressing both ribulose-5-phosphate isomerase enzymes 3 ribulose-5-phosphate epimerase are preferred over host cells overexpressing only isomerase or epimerase as overexpression of only one of these enzymes may produce metabolic imbalances.

There are various means available in the art for enzyme overexpression in the cells of the invention. Specifically, an enzyme may be overexpressed by increasing the copy number of the genetic coding for the enzyme in a host cell, for example by integrating additional copies of the gene into the host cell genome, by expressing the gene from a vector of episomal expression or by introducing a episomal expression vector comprising multiple copies of the gene.

Alternatively, overexpression of enzymes in the host cells of the invention may be accomplished by employing a promoter that is not native to the sequence encoding the enzyme to be overexpressed, that is, a promoter that is heterologous to the sequence of coding to which it is operatively linked. Appropriate promoters for this purpose have already been defined in this document.

The coding sequence used for enzyme overexpression preferably is homologous to the host cell of the invention. However, coding sequences that are heterologous with respect to the host cell of the invention may similarly be applied as mentioned in WO 06/009434.

A nucleotide sequence used for ribulose-5-phosphate isomerase overexpression in the host cell of the invention is a nucleotide sequence encoding a polypeptide with ribulose-5-phosphate isomerase activity, so preferably the polypeptide has an amino acid sequence. having at least 50, 60, 70, 80, 90 or 95% identity to SEQ ID NO: 17 or whereby the nucleotide sequence is capable of hybridizing to the nucleotide sequence of SEQ ID NO: 18 under moderate conditions. preferably under stringent conditions.

[067] A nucleotide sequence used for ribulose-5-phosphate epimerase overexpression in the host cell of the invention is a nucleotide sequence encoding a polypeptide with ribulose-5-phosphate epimerase activity, so preferably the polypeptide has an amino acid sequence. having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO: 19 or whereby the nucleotide sequence is capable of hybridizing to the nucleotide sequence of SEQ ID NO: 20 under moderate conditions, preferably under stringent conditions.

[068] A nucleotide sequence used for transcetolase overexpression in the host cell of the invention is a nucleotide sequence encoding a transcetolase activity polypeptide, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity to SEQ ID NO: 21 or whereby the nucleotide sequence is capable of hybridizing to the nucleotide sequence of SEQ ID NO: 22 under moderate conditions, preferably under stringent conditions.

[069] A nucleotide sequence used for transaldolase overexpression in the host cell of the invention is a nucleotide sequence encoding a transaldolase activity polypeptide, so preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity to SEQ ID NO: 23 or whereby the nucleotide sequence is capable of hybridizing to the nucleotide sequence of SEQ ID NO: 24 under moderate conditions, preferably under stringent conditions.

Overexpression of an enzyme, when referring to enzyme production in a genetically modified host cell, means that the enzyme is produced at a higher level of specific enzyme activity compared to the unmodified host cell under identical conditions. This usually means that enzymatically active protein (or proteins in the case of multi-subunit enzymes) is produced in larger quantities or rather at a higher steady state level compared to the unmodified host cell under identical conditions. Similarly, this generally means that the mRNA coding for the enzymatically active protein is produced in larger quantities or again instead at a higher steady state level compared to the unmodified host cell under identical conditions. Overexpression of an enzyme is thus preferably determined by measuring the level of specific enzyme activity in a host cell using appropriate enzyme assays as described herein. Alternatively, enzyme overexpression may be indirectly determined by quantitation of the enzyme-specific firm-level level of the enzyme, for example, by using enzyme-specific antibodies or by quantitation of the enzyme-specific firm-level mRNA coding. The latter may specifically be suitable for pentose phosphate pathway enzymes for which enzyme assays are not readily practicable as substrates for the enzymes are not commercially available. Preferably in the host cells of the invention, an enzyme to be overexpressed is overexpressed by a factor of at least 1.1, 1.2, 1.5, 2, 5, 10, or 20 compared to a genetically identical strain except by genetic modification causing overexpression. It should be understood that these overexpression levels can be applied to the steady state level of enzyme activity, the steady state level of enzyme protein, as well as the steady state level of transcription encoding the enzyme.

[071] In a further preferred embodiment, the host cell of the invention: expressing araA, araB and araD, and exhibiting the ability to directly isomerize xylose into xylulose and optionally comprising a genetic modification that increases pentose pathway flow as defined herein. The above document further comprises a genetic modification that enhances the specific activity of xylulose kinase. Preferably the genetic modification causes overexpression of a xylulose kinase, for example by overexpression of a nucleotide sequence encoding a xylulose kinase. The gene encoding xylulose kinase may be endogenous to the host cell or may be a xylulose kinase that is heterologous to the host cell. A nucleotide sequence used for overexpression of xylulose kinase in the host cell of the invention is a nucleotide sequence encoding a xylulose kinase activity polypeptide, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity to SEQ ID NO: 25 or whereby the nucleotide sequence is capable of hybridizing to the nucleotide sequence of SEQ ID NO: 26 under moderate conditions, preferably under stringent conditions.

A specifically preferred xylulose kinase is a xylose kinase that is related to Piromyces xylBase kinase as mentioned in WO 03/0624430. A more preferred nucleotide sequence for use in overexpressing xylulose kinase in the host cell of the invention is a nucleotide sequence encoding a xylulose kinase activity polypeptide, whereby preferably the polypeptide has an amino acid sequence having at least 45, 50, 55, 60, 65, 70, 80, 90 or 95% identity with SEQ ID NO: 27 or whereby the nucleotide sequence is capable of hybridizing to the nucleotide sequence of SEQ ID NO: 28 under moderate conditions, preferably under conditions stringent.

[073] In the host cells of the invention, genetic modification that enhances specific xylulose kinase activity may be combined with any modifications that increase the flow of the pentose phosphate pathway as mentioned above, but this combination is not essential to the invention. Thus, a host cell of the invention comprising a genetic modification that enhances specific xylulose kinase activity in addition to expression of the araA, araB and araD enzymes as defined herein is specifically included in the invention. The various devices available in the art for obtaining and analyzing a xylulose kinase in the host cells of the invention are the same as those described above for the pentose phosphate pathway enzymes. Preferably in the host cells of the invention, the xylulose kinase to be overexpressed is overexpressed by at least one factor 1,1, 1,2, 1,5, 2, 5, 10 or 20 compared to a genetically identical strain except for genetic modification leading to overexpression. It should be understood that these overexpression levels can be applied to the steady state level of enzyme activity, the steady state level of enzyme protein, as well as the steady state level of transcription encoding the enzyme.

In a further preferred embodiment, the host cell of the invention: expressing araA, araB and araD, and exhibiting the ability to directly isomerize xylose into xylulose and optionally comprising a genetic modification that increases pentose pathway flow and / or - further comprising a genetic modification that increases the specific activity of xylulose kinase activity, all as described hereinbefore further comprises, a genetic modification that reduces the non-specific activity of aldose reductase in the host cell. Preferably, non-specific aldose reductase activity is reduced in the host cell by one or more genetic modifications that reduce expression or inactivate a gene encoding a non-specific aldose reductase as described in WO 06/009434. Preferably, genetic modifications reduce or inactivate expression of each endogenous copy of a gene encoding a non-specific aldose reductase in host cells. Host cells may comprise multiple copies of genes encoding nonspecific aldoses reductases as a result of di, poly or aneuploid, and / or the host cell may contain several different (that) enzymes with aldose reductase activity that differ in amino acid sequence and which are cloned by a different gene. Also, at such times, preferably the expression of each gene encoding a non-specific aldose reductase is reduced or inactivated. Preferably, the gene is inactivated by deletion of at least part of the gene or by disruption of the gene, whereby in this context the term gene also includes any non-coding sequence upstream or downstream of the coding sequence, deletion or inactivation. (partial) resulting in reduced expression of non-specific aldose reductase activity in the host cell. A nucleotide sequence encoding an aldose reductase whose activity is to be reduced in the host cell of the invention is a nucleotide sequence encoding an aldose reductase activity polypeptide, whereby the polypeptide preferably has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity to SEQ ID NO: 29 or whereby the nucleotide sequence is capable of hybridizing to the nucleotide sequence of SEQ ID NO: 30 under moderate conditions, preferably under stringent conditions.

[075] In the host cells of the invention, the expression of the araA, araB and araD enzymes as defined herein is combined with genetic modification that reduces non-specific aldose reductase activity. Genetic modification leading to reduction of non-specific aldose reductase activity may be combined with any of the modifications by increasing the flow of the pentose phosphate pathway and / or with any of the modifications by increasing the specific xylulose kinase activity in host cells as described above, however these combinations are not essential to the invention. Thus, a host cell expressing araA, araB, and araD, comprising an additional genetic modification that reduces non-specific aldose reductase activity is specifically included in the invention.

[076] In a preferred embodiment, the host cell is CBS 120327 filed with the CBS Institute (Netherlands) on September 27, 2006.

In a further preferred embodiment, the invention relates to modified host cells which are further adapted to L-arabinose (use of L-arabinose and / or conversion thereof to L-ribulose, and / or 5-phosphate xylulose). and / or in a desired fermentation product and, optionally, use of xylose by selection of both spontaneous and induced mutants (e.g., by radiation or chemicals), for growth in L-arabinose and optionally xylose, preferably in L -arabinosis and optionally xylose as the sole carbon source, and more preferably under anaerobic conditions.Selection of mutants can be performed by serial passage of cultures, for example as described in Kuyper et al. (2004, FEMS Yeast Res. 4_ : 655-664) and / or by selective pressure cultivation in a chemostat culture as described in Example 4 of WO 06/009434 This selection process may continue for as long as necessary. This selection process is preferably carried out for one week to one year. However, the selection process may be carried out for a longer period of time if necessary. During the selection process, cells are preferably cultured in the presence of approximately 20 g / l L-arabinose and / or approximately 20 g / l xylose. The cell obtained at the end of this selection process is expected to be enhanced in its ability to use L-arabinose and / or xylose, and / or to convert L-arabinose to L-ribulose and / or 5-phosphate and / or xylulose. or a desired fermentation product, such as ethanol. In this context, "improved cell" may mean that a cell obtained is capable of using L-arabinose and / or xylose more efficiently than the cell derived therefrom. For example, the cell obtained is expected to have better growth: increasing the specific growth rate by at least 2% over the cell from which it is derived under the same conditions. Preferably, the increase is at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more. Specific growth rate can be calculated from OÜ660 as known to one of skill in the art. Therefore, by monitoring OD660, one can deduce the specific rate of growth. In this context "improved cell" may mean that the obtained cell converts L-arabinose to L-ribulose and / or 5-phosphate xylulose and / or a desired fermentation product, such as ethanol in a more efficient manner than in the cell of which drift. For example, the cell obtained is expected to produce larger amounts of L-ribulose and / or 5-phosphate xylulose and / or a desired fermentation product such as ethanol: increase in at least one of these compounds by at least 2% over to the cell from which it derives under the same conditions. Preferably, the increase is at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more. In this context "improved cell" may also mean that the obtained cell converts xylose to xylulose and / or a desired fermentation product such as ethanol more efficiently than the cell from which it is derived. For example, the cell obtained is expected to produce larger amounts of xylulose and / or a desired fermentation product such as ethanol: at least 2% increase of these compounds over the cell from which it is derived under the same conditions. . Preferably, the increase is at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more.

In a preferred host cell of the invention, at least one of the genetic modifications described above, including modifications obtained by mutant selection, gives the host cell the ability to grow on L-arabinose and optionally xylose as a carbon source, preferably as the sole carbon source, and preferably under anaerobic conditions. Preferably the modified host cell does not essentially produce xylitol, for example the xylitol produced is below the limit of detection or, for example, less than 5, 2, 1, 0.5 or 0.3% of carbon consumed on a molar basis. .

Preferably the modified host cell has the ability to develop into L-arabinose and optionally xylose as the sole carbon source at a rate of at least 0.001, 0.005, 0.01, 0.03, 0.05.0 , 1, 0.2, 0.25 or 0.3 h_1 under aerobic conditions, or if applicable, at a rate of at least 0.001, 0.005, 0.01, 0.03, 0.05, 0.07, 0 .08, 0.09, 0.1, 0.12, 0.15 or 0.2 h -1 under anaerobic conditions. Preferably the modified host cell has the ability to grow in the mixture of glucose and L-arabinose and optionally xylose (in a 1: 1 weight ratio) as the sole carbon source at a rate of at least 0.001, 0.005, 0.01, 0.03, 0.05, 0.1, 0.2, 0, 25 or 0.3 h -1 under aerobic conditions, or if applicable, at a rate of at least 0.001, 0.005, 0.01, 0.03, 0.05, 0.1, 0.12, 0.15 or 0.2 h -1 under anaerobic conditions.

Preferably, the modified host cell has a specific consumption rate of L-arabinose and optionally xylose of at least 346, 350, 400, 500, 600, 650, 700, 750, 800, 900 or 1,000 mg / g. cells / hr. Preferably, the modified host cell has a yield of fermentation product (such as ethanol) in L-arabinose and optionally xylose which is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 70. , 80, 85, 90, 95 or 98% of the host cell yield of the fermentation product (such as ethanol) in glucose. More preferably, the modified host cell yield of the fermentation product (such as ethanol) in L-arabinose and optionally xylose is equal to a yield of the host cell fermentation product (such as ethanol) in glucose. Likewise, the modified host cell biomass yield in L-arabinose and optionally xylose is preferably at least 55, 60, 70, 80, 85, 90, 95 or 98% of the host cell biomass yield in glucose. More preferably, the modified host cell biomass yield and optionally xylose is equal to the host cell biomass yield in glucose. It is understood that in comparing yields in glucose and L-arabinose and optionally xylose both yields are compared under aerobic or anaerobic conditions.

In a more preferred embodiment, the host cell is CBS 120328 filed with the CBS Institute (Netherlands) on September 27, 2006 or CBS 121879 filed with the CBS Institute (Netherlands) on September 20, 2007.

In a preferred embodiment, the cell expresses one or more enzymes which give the cell the ability to produce at least one fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acid acetic, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane diol, ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin. In a more preferred embodiment, the host cell of the invention is a host cell for ethanol production. In another preferred embodiment, the invention relates to the transformed host cell for producing fermentation products other than ethanol. Such non-ethanolic fermentation products in principle include any volume or fine chemicals that may be produced by a eukaryotic microorganism, such as yeast or filamentous fungi. Such fermentation products include, for example, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane diol, ethylene, glycerol, butane, a β-lactam antibiotic and cephalosporin. A preferred host cell for producing non-ethanolic fermentation products is a host cell that contains a genetic modification that results in decreased alcohol dehydrogenase activity. Method [083] In a further aspect, the invention relates to fermentation processes wherein a host cell of the invention is used for the fermentation of a carbon source comprising an L-arabinose source and optionally a xylose source. Preferably, the source of L-arabinose and the source of xylose are L-arabinose and xylose. In addition, the carbon source in the fermentation medium also comprises a glucose source. The source of L-arabinose, xylose or glucose may be L-arabinose, xylose or glucose as such or may be any carbohydrate polymer or oligopolymer comprising L-arabinose, xylose or glucose units, such as, for example, lignocellulose, xylans, cellulose, starch, arabinan and the like. For release of xylose or glucose units from such carbohydrates, appropriate carbohydrates (such as xylanases, glycanases, amylases and the like) may be added to the fermentation medium or may be produced by the modified host cell. In the latter case, the modified host cell may be genetically transformed to produce and excrete such carbohydrates. An additional advantage of using oligo or polymeric glucose sources is that they allow to maintain a low (lower) concentration of free glucose during fermentation, for example by employing amounts limiting the rate of carbohydrolases. This, in turn, will prevent the repression of systems necessary for metabolism and transport of non-glucose sugars such as xylose. In a preferred process, the modified host cell fermentes both L-arabinose (optionally xylose) and glucose, preferably and simultaneously when, preferably, a modified host cell is used being insensitive to glucose repression to prevent deaux development. In addition to a source of L-arabinose, optionally xylose (and glucose) as carbon sources, the fermentation medium will further comprise the appropriate and necessary ingredient for the growth of a modified host cell. Fermentation media compositions for growing microorganisms such as yeast or filamentous fungi are well known in the art.

[084] In a preferred process, a process is provided for producing a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid. , fumaric acid, an amino acid, 1,3-propane diol, ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin whereby the process comprises the steps of: (a) fermenting a medium containing a source of L -arabinosis and optionally xylose with a modified host cell as defined herein, whereby the host cell fermentes L-arabinose and optionally xylose in the fermentation product, and optionally (b) recovering the fermentation product.

[085] The fermentation process is a process for producing a fermentation product such as, for example, ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, acid malic acid, fumaric acid, an amino acid, 1,3-propane diol, ethylene, glycerol, butanol, a β-lactam antibiotic such as Penicillin G or Penicillin V and fermentative derivatives thereof, and / or a cephalosporin. The fermentation process may be an aerobic or anaerobic fermentation process. An anaerobic fermentation process is defined herein as a fermentation process operated in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol / L / hr, more preferably 0 mmol / L / h being consumed (that is, undetectable oxygen uptake) and by what organic molecules serve as electron donors and electron acceptors. In the absence of oxygen, NADH produced in glycolysis and biomass formation cannot be oxidized by oxidative phosphorylation. To solve this problem many microorganisms use pyruvate or one of its derivatives like an electron and hydrogen acceptor reason why regenerating NAD +. Thus, in an anaerobic fermentation process, pyruvate is used as an electron (a hydrogen acceptor) and is reduced to fermentation products such as ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane diol, ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin. In a preferred embodiment, the fermentation process is anaerobic. An anaerobic process is advantageous as it is cheaper than the aerobic process: less special equipment is required. In addition, anaerobic processes are expected to provide superior product yield over aerobic processes. Under aerobic conditions, biomass yield is generally higher than aerobic conditions. As a consequence, generally under aerobic conditions, the expected product yield is lower than under anaerobic conditions. According to the inventors, the process of the invention is the first anaerobic fermentation process with a medium comprising a source of L-arabinose that has been developed so far.

In another preferred embodiment, the fermentation process is carried out under limited oxygen conditions. More preferably, the fermentation process is aerobic and under limited oxygen conditions. An oxygen-limited fermentation process is a process in which oxygen consumption is limited by transferring oxygen from gas to liquid. The degree of oxygen limitation is determined by the amount and composition of the incoming gas flow, as well as the actual mix / mass transfer properties of the fermentation equipment used. Preferably, in the process under limited oxygen conditions, the oxygen uptake rate is at least 5.5, more preferably at least 6 and even more preferably at least 7 mmol / L / h.

[087] The fermentation process is preferably operated at a temperature that is optimal for the modified cell. Thus, for most yeast or fungal cells, the fermentation process is carried out at a temperature which is below 42 ° C, preferably below 38 ° C. For filamentous fungus or yeast host cells, the fermentation process is preferably carried out at a temperature that is below 35, 33, 30 or 28 ° C and a temperature that is above 20, 22 or 25 ° C.

A preferred process is a process for the production of ethanol, whereby the process comprises the steps of: (a) fermenting a medium containing an L-arabinose source and optionally xylose with a modified host cell as defined herein whereby the host cell fermentes L-arabinose and optionally xylose in ethanol; and optionally (b) ethanol recovery. The fermentation medium may also comprise a glucose source which is also fermented in ethanol. In a preferred embodiment, the fermentation process for ethanol production is anaerobic. Anaerobic has been defined in this document earlier. In another preferred embodiment, the fermentation process for ethanol production is aerobic. In another preferred embodiment, the fermentation process for ethanol production is carried out under oxygen-limited, more preferably aerobic and oxygen-limited conditions. The limited oxygen conditions there have been defined in this document earlier.

In the process, the volumetric productivity of ethanol is preferably at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g of ethanol. per liter per hour. The yield of ethanol on L-arabinose and optionally xylose and / or glucose in the process is preferably at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is defined herein as the percentage of the theoretical maximum yield which for glucose and L-arabinose and optionally xylose is 0.51 g ethanol per g glucose or xylose. In another preferred embodiment, the invention relates to a process for producing a fermentation product selected from the group consisting of lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, acid fumaric acid, an amino acid, 1,3-propane diol, ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin. The process preferably comprises the steps of: (a) fermenting a medium containing an L-arabinose source and optionally xylose with a modified host cell as defined hereinabove, whereby the host cell fermentes L-arabinose and optionally xylose. in the fermentation product, and optionally, (b) recovering the fermentation product. In a preferred process, the medium also contains a glucose source.

In the fermentation process of the invention leading to ethanol production, several advantages may be cited compared to known tanol fermentation processes: - anaerobic processes are possible, limited oxygen conditions are also possible, - yields and rates of Higher ethanol yields can be obtained, - the strain used may be able to use L-arabinose and optionally xylose.

Alternatively with respect to the fermentation processes described above, another fermentation process is provided as an additional aspect of the invention, whereby at least two distinct cells are used for the fermentation of a carbon source comprising at least two carbon sources. carbon selected from the group consisting of but not limited to: a source of L-arabinose, a source of xylose and a source of glucose. In this fermentation process, "at least two distinct cells" means that this process is preferably a fermentation co-process. In a preferred embodiment, two distinct cells are used: one being that of the invention as defined above capable of using L-arabinose, and / or converting it to L-ribulose, and / or 5-phosphate xylulose and / or a product. desired fermentation product such as ethanol and optionally being able to use xylose, the other being, for example, a strain that is capable of using xylose and / or converting it into a desired fermentation product such as ethanol as defined in WO 03 / 062430 and / or WO 06/009434. A cell that is capable of using xylose is preferably a strain that exhibits the ability to directly isomerize xylose into xylulose (in one step) as defined hereinbefore. These two distinct strains are preferably cultured in the presence of an L-arabinose source, a xylose source and optionally a glucose source. Three or more distinct cells may be co-cultured and / or three or more carbon sources may be used, provided that at least one cell is capable of using at least one carbon source present and / or converting it into a product. desired fermentation such as ethanol. The expression "use of at least one carbon source" has the same meaning as the expression "use of L-arabinose". The expression "converts it (ie a carbon source) into a desired fermentation product has the same meaning as the expression" converts L-arabinose into a desired fermentation product ".

In a preferred embodiment, the invention relates to a process for producing a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid. , malic acid, fumaric acid, amino acids, 1,3-propane diol, ethylene, glycerol, butanol, β-lactam antibiotic and cephalosporins, whereby the process comprises the steps of: (a) fermenting a medium containing at least one L-arabinose source and a xylose source with a cell of the invention as mentioned hereinbefore and a cell capable of using xylose and / or exhibiting the ability to directly isomerize xylose into xylulose, whereby each cell fermentes L-arabinose and / or xylose in the fermentation product, and optionally (b) recovering the fermentation product.

[093] All preferred embodiments of the fermentation processes as described above are also preferred embodiments of these additional fermentation processes: fermentation product identity, L-arabinose source and xylose source identity, fermentation conditions (aerobic or anaerobic conditions, oxygen-limited conditions, temperature at which the process is being performed, ethanol productivity, ethanol yield).

Genetic Modifications For overexpression of enzymes in the host cells of the invention as described above, as well as for further genetic modification of host cells, preferably yeast, host cells are transformed with the various nucleic acid constructs of the invention by well known methods. in art. Such methods are, for example, known from standard books such as Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press or F. Ausubel et al. eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for genetic transformation and modification of fungal host cells are known, for example, EP-A-0 635 574, WO 98 / 46772, WO 99/60102 and WO 00/37671.

Promoters for use in constructing nucleic acids for enzyme overexpression in the host cells of the invention have been described above. In constructing nucleic acids for overexpression, the 3 'terminus of the nucleic acid sequence encoding the enzyme (s) is preferably operably linked to a transcription terminator sequence. Preferably the terminator sequence is operable in a host cell of choice, such as, for example, the yeast species of choice. In any case, the choice of terminator is not critical; it may, for example, be from any yeast gene, although terminators may sometimes be from a different eukaryotic gene than yeast. The transcription termination sequence further and preferably comprises a polyadenylation signal. Preferred terminator sequences are alcohol dehydrogenase (ADH1) and PGI1 terminators. More preferably, the ADH1 and PGI1 terminators are both from S. cerevisiae (SEQ ID NO: 50 and SEQ ID NO: 53 respectively).

Optionally, a selectable marker may be present in the nucleic acid construct. As used herein, the term "marker" refers to the gene encoding a trait or phenotype that allows classification selection for a host cell containing the marker. The marker gene can be an antibiotic resistance gene, so the appropriate antibiotic can be used to select transformed cells from untransformed cells. Preferably, however, non-antibiotic resistance markers are used, such as auxotrophic markers (URA3, TRP1, LEU2). In a preferred embodiment host cells transformed with the nucleic acid construct are free of marker gene. Methods for constructing recombinant marker gene-free microbial host cells are disclosed in EP-A-0 635 574 and are based on the use of bidirectional markers. Alternatively, a classifiable marker such as Green Fluorescent Protein, lacZ, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase may be incorporated into the construction of the nucleic acids of the invention, allowing the classification of transformed cells.

Optional additional elements that may be present in the construction of the nucleic acids of the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors and / or reporter genes, intron sequences, centromeres, telomeres and / or matrix attachment sequences (MAR). The construction of the nucleic acids of the invention may also comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may be based, for example, on the 2μ or pKD1 yeast plasmids (Fleer et al., 1991, Biotechnology 9q 968-975). Alternatively the nucleic acid construct may comprise integration sequences, preferably by homologous recombination. Such sequences may thus be sequences homologous to the target site for integration into a host cell genome. The construction of the nucleic acids of the invention may be provided in a known manner, which generally involves techniques such as restriction and binding of nucleic acids / nucleic acid sequence, which are referred to in standard books, such as Sambrook and Russel ( 2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press.

[098] Methods for inactivating and disrupting the gene in yeast or fungi are well known in the art (see, for example, Fincham, 1989, Microbiol Rev. 53 (1): 148-70 and EP-A-0 635 574). .

[099] In this document and its claims, the verb "to understand" and its conjugations are used in their non-limiting sense and mean that items accompanying the words are included, but items not specifically mentioned are not included. Furthermore, reference to an element by the indefinite article "one, one" does not exclude the possibility that more than one element is present unless the context clearly requires that there is only one and only one element. The undefined article "o, a" thus generally means "at least one (a)" [100] The invention will be further described by the following examples, which should not be construed as limiting the scope of the invention.

Plasmid Examples and Strain Construction Strains [101] The L-arabinose that consumes the Sachharomyces cerevisiae strain described in this paper is based on strain RWB220, which is itself a derivative of RWB217. RWB217 is a CEN.PK strain in which four genes encoding enzyme expression in the pentose phosphate pathway were overexpressed, TAL1, TKL1, RPE1, RKI1 (Kuyper et al., 2005a). In addition, the gene encoding aldose reductase [GRE3) was deleted. The RWB217 strain also contains two plasmids, a single copy plasmid with a LEU2 marker for xylokinase overexpression (XKS1) and a multiple copy episomal plasmid with URA3 as the marker for xylose isomerase expression, XylA. RWB217 underwent a selection procedure for improved xylose growth that is described in Kuyper et al. (2005b). The procedure resulted in two pure strains, RWB218 (Kuyper et al., 2005b) and RWB219. The difference between RWB218 and RWB219 is that, after the selection procedure, RWB218 was obtained by plating and reruning with glucose as the carbon source, while for RWB219 xylose was used.

[102] The RWB219 strain grew non-selectively on YP with glucose (YPD) as the carbon source to facilitate loss of both plasmids. After plating, simple YPD colonies were tested for plasmid loss by uracil and leucine auxotrophy monitoring. One strain that had lost both plasmids was transformed with pSH47 containing cre recombinase to remove a KanMX cassette (Guldener et al., 1996), still present after integration of the RKI1 overexpression construct. Colonies with the plasmid were resuspended in Pepton Yeast (YP) medium (10 g / L yeast extract and 20 g / L peptone, both from BD Difco Belgium) with 1% galactose and incubated for 1 hour at 30 ° C. About 200 cells were plated in the YPD. The resulting colonies were checked for the loss of the marker KanMX (G418 resistance) and pSH47 (URA3). The strain that had lost both the KanMX marker and plasmid pSH47 was then called RWB220. In order to obtain the strain tested in this patent, RWB220 was transformed with pRW231 and pRW243 (Table 2), resulting in strain IMS0001.

[103] During construction the strains were kept in complex YP: 10 g L_1 yeast extract (BD Difco), 20 g L-1 peptone (BD Difco) or synthetic medium (MY) (Verduyn et al. 1992 ) supplemented with glucose (2%) as carbon source (YPD or MYD) and 1.5% agar for plates. After transformation with the plasmids, the strains were plated in MYD. Yeast transformations were performed according to Gietz and Woods (2002). Plasmids were amplified in blue Escherichia coli XL-1 strain (Stratagene, La Jolla, CA, USA). The transformation was performed according to Inoue et al. (1990). E. coli was grown in LB (Luria-Bertani) plates or in liquid TB medium (Terrdo Broth) for plasmid isolation (Sambrook et al., 1989).

Plasmids [104] In order to develop into L-arabinose, yeast must express three different genes, one L-arabinose isomerase (AraA), one L-ribulokinase (AraB), and one L-ribulose-5-Ρ 4- epimerase (AraD) (Becker and Boles, 2003). In this paper we chose to express AraA, AraB and AraD of the lactic acid of Lactobacillus plantarum in S. cerevisiae. Because of the eventual goal of consuming L-arabinose in combination with other sugars such as D-xylose, genes encoding the bacterial L-arabinose pathway were combined on the same plasmid with genes encoding D-xylose consumption.

[105] In order to achieve a high level of expression, L. plantarum AraA and AraD genes were ligated into plasmid pAKX002, the plasmid carrying 2μ of XylA.

[106] The AraA cassette was constructed by extending a truncated version of the TDH3 promoter with SpeI5'Ptdh3 and 5'AraAPtdh3 (SEQ ID NO: 49), the AraA gene with Ptdh5'AraA and Tadh3'AraA and the ADH1 terminator (SEQ). ID NO: 50) with 3'AraATadhl and 3'Tadhl-Spel. The three fragments were extracted from the gel and mixed in roughly equimolar amounts. In this mixture, a PCR was performed using SpeI-5'Ptdh3 and 3'TadhlSpeI oligos. The resulting PTDH3-AraA-TADHi cassette was gel purified, cut at the Spel 5'and 3 'sites and then ligated into the pAKX002 cut with Nhel, resulting in plasmid pRW230.

[107] The AraD construct was first performed by enlarging a truncated version of the HXT7 promoter (SEQ ID NO: 52) with SalI5'Phxt7 and 5'AraDPhxt oligos, the AraD gene with Phxt5'AraD and Tpgi3'AraD and the GPU terminator (SEQ ID NO: 53) with oligos 3'AraDTpgi and 3'TpgiSalI. The resulting fragments were extracted from the gel and mixed in roughly equimolar amounts, after which a PCR was performed using SalI5'Phxt7 and 3'TpgilSalI oligos. The resulting PHXT7-AraD-TpGii cassette was gel purified, cut at the Sall 5'and 3 'sites and then ligated into the pRW230-XhoI cut, resulting in plasmid pRW231 (Figure 1).

[108] Since very high L-ribulokinase expression is detrimental to growth (Becker and Boles, 2003), the AraB gene was combined with the XKS1 gene encoding xylulokinase in an integrating plasmid. For this, p415ADHXKS (Kuyper et al., 2005a) was first altered within pRW229 by cutting off both p415ADHXKS and pRS305 with Pvul and linking the Pvul fragment containing ADHXKS-from p415ADHXKS to the pRS305 vector structure, resulting in pRW229.

[109] The cassette containing the L. plantarum AraB gene between the PGI1 promoter (SEQ ID NO: 51) and the ADH1 terminator (SEQ ID NO: 50) was fabricated by amplifying the PGI1 promoter with the SacI5'Ppgil and 5 oligos. 'AraBPpgil, the AraB gene with the Ppgi5'AraB and Tadh3'AraB oligos and the ADH1 terminator with the 3'AraBTadhl and 3'TadhlSacI oligos. The three fragments were extracted from the gel and mixed in roughly equimolar amounts. In the mixture, a PCR was performed with the SacI-5'Ppgil and 3'TadhlSacI oligos. The resulting PpGii-AraB-Tadhi cassette was gel purified, cut at Saci 5 'and 3' sites and then ligated into the pRW229 cut with Saci, resulting in plasmid pRW243 (Figure 1).

[110] Strain RWB220 was transformed with pRW231 and pRW243 (Table 2), resulting in strain IMS0001.

[111] Restriction endonucleases (New England Biolabs, Beverly, MA, USA and Roche, Basel, Switzerland) and DNA ligase (Roche) were used according to the manufacturers instructions. Isolation of the E. coli plasmid was performed with the Qiaprep miniprep kit (Qiagen, Hilden, Germany). DNA fragments were separated on 1% agarose gel (Sigma, St. Louis, MO, USA) into 1 * TBE (Sambrook et al., 1989). Isolation of the gall fragments was performed with a Qiaquick gel extraction kit (Quiagen). The amplification of the AraA, AraB and AraD cassette elements was performed with VentR DNA polymerase (New England Biolabs) according to the manufacturers instructions. The template was S. cerevisiae CEN.PK113-7D chromosomal DNA for promoters and terminators or Lactobacillus plantarum DSM20205 for Ara genes. The polymerase chain reaction (PCR) was performed in a Biometra TGradient Thermocycler (Biometra, Gottingen, Germany) with the following settings: 30 1 minute saturation cycles at 55 ° C, 60 ° C or 65 ° C, 1 to 3 minutes extension at 75 ° C, depending on expected fragment size and 1 minute denaturation at 94 ° C.

Cultivation and Media [112] Cultivation in shake flasks was performed at 30 ° C in a synthetic medium (Verduyn et al, 1992). The pH of the medium was adjusted to 6.0 with 2 M KOH before sterilization. For solid synthetic medium, 1.5% agar was employed.

[113] Precultures were prepared by inoculating 100 mL of medium containing the appropriate sugar into a 500 mL shake flask with a frozen stock culture. After inoculation at 30 ° C on an orbital shaker (200 rpm), this culture was used to inoculate shake flask cultures or fermenter cultures. The synthetic medium for anaerobic culture was supplemented with 0.01 g L_1 of ergosterol and 0.42 g L_1 of Tween 80 dissolved in ethanol (Andreasen and Stier, 1953; Andreasen and Stier, 1954). The anaerobic batch cultivation (sequencing) was performed at 30 ° C in 2 liter laboratory fermenters (Applikon, Schiedam, Netherlands) with a working volume of 1 liter. The culture pH was maintained at pH 5.0 by automatic addition of 2M KOH. The cultures were shaken at 800 rpm and sprayed with 0.5 L min-1 nitrogen gas (<10 ppm oxygen). To minimize oxygen diffusion, the fermenters were equipped with Norprene tubing (Cole Palmer Instrument company, Vernon Hills, USA). Dissolved oxygen was monitored with an oxygen electrode (Applisens, Schiedam, Netherlands). Oxygen-limited conditions were obtained at the same experimental adjustment by upper space sandblasting at approximately 0.05 L min-1.

Determination of Dry Weight [114] Culture samples (10.0 mL) were filtered on pre-weighed nitrocellulose filters (0.45 µm pore size; Gelman Laboratory, Ann Arbor, USA). After removal of the medium, the filters were washed with demineralized water and dried in a microwave oven (Bosch, Stuttgart, Germany) for 20 minutes at 360 W and weighed. Duplicate determinations ranged by at least 1%.

Gas Analysis [115] Exhaust gas was cooled in a condenser (2 ° C) and dried with a Permapure MD-110-48P-4 dryer (Permapure, Toms River, USA). O2 and COa concentrations were determined with an NGA 2000 analyzer (Rosemount Analytical, Orrville, USA). Exhaust gas flow rate and carbon dioxide production and oxygen consumption rates were determined as previously described (Van Urk et al., 1988; Weusthuis et al., 1994). When calculating these specific biomass rates, changes in volume caused by withdrawal of culture samples were taken into account.

Metafoolite Analysis [116] Glucose, xylose, arabinose, xylitol, organic acids, glycerol and ethanol were analyzed by HPLC using a Waters Alliance 2690 HPLC (Waters, Milford, USA) supplied with a BioRad HPX 8? H column (BioRad, Hercules). , USA), a Waters 2410 refractive index detector, and a Waters 2487 UV detector. The column was eluted at 6 ° C with 0.5 g L-1 sulfuric acid at a flow rate of 0.6 mL min -1. .

5-Phosphate Xylulose Assay (Zaldivar J. et al., Appl. Microbiol. Biotechnol., <2002), 59: 436-442) [117] For analysis of intracellular metabolites, such as 5-phosphate xylulose, were collected. of the reactors 5 mL of broth in duplicate, before glucose exhaustion (at 22 and 26 hours of cultivation) and after glucose exhaustion (42, 79 and 131 hours of cultivation). Procedures for metabolic arrest, solid phase metabolite extraction and analysis have been described in detail by Smits H.P. et al. (Anal. Biochem., 261: 36-42, (1998)). However, the high pressure ion exchange chromatography analysis coupled with the pulsed amperometric detection employed to analyze the cell extracts was slightly modified. The solutions employed were eluent A, 75 mM NaOH and eluent B, 500 mM NaAc. In order to prevent carbonate contamination in the eluent solutions, a low carbonate 50% NaOH solution (Baker Analyzed, Deventer, Netherlands) was used instead of NaOH microspheres. The eluents were degassed with helium (He) for 30 minutes and then kept under He atmosphere. A gradient pump was programmed to generate the following gradients: 100% A and 0% B (0 minutes), a linear decrease of A to 70% and a linear increase of B to 30% (0-30 minutes), a a linear decrease of A to 30% and a linear increase of B to 70% (30-70 minutes), a linear decrease of A to 0% and a linear increase of B to 100% (70-75 minutes), 0% A and 100% B (75-85 minutes), a linear increase of 100% A and a linear decrease of 0% B (85-95 minutes). The mobile phase was operated at a flow rate of 1 mL / min. Other conditions were in agreement with Smits et al. (1998).

Carbon Recovery [118] Carbon recoveries were calculated as carbon in the formed products divided by the total amount of sugar in the carbon consumed and were based on the 48% biomass carbon content. To correct for ethanol evaporation during fermentations, the amount of ethanol produced was assumed to be equal to the measured cumulative CO? minus the CO2 production that occurred due to biomass synthesis (5.85 mmol CO? per gram of biomass (Verduyn et al., 1990)) and CO? associated with acetate formation.

Selection for Growth on L-arabinose [119] The IMS0001 strain (CBS 120327 deposited with CBS on 9/27/06), containing the genes encoding the pathways for metabolism of both xylose (XylA and XKS1) and arabinose (AraA, AraB, AraD) was constructed according to the procedure described above. Although able to develop in xylose (data not shown), the IMSGGG1 strain does not appear to be able to grow in the solid synthetic medium supplemented with 2% L-arabinose. IMS0001 mutants capable of using L-arabinose as a carbon source for development were selected by serial transfer in the shake flasks and by sequencing batch culture in the fermenters (SBR).

[120] For serial transfer experiments, a 500 mL shake flask containing 100 mL of synthetic medium containing 0.51 galactose was inoculated with both the ΙΜΞ0001 strain and the RWB219 reference strain. After 72 hours, at an optical density of 660 nm of 3.0, the cultures were used to inoculate a new shake flask containing 0.1% galactose and 21% arabinose. Based on the determination of HPLC with D-ribulose as calibration standard, it was determined that already in the first cultures of strain IMS0001, in the medium containing a galactose / arabinose mixture, part of the arabinose was converted to ribulose and subsequently excreted from the supernatant. These HPLC analyzes were performed using a Waters Alliance 2690 HPLC (Waters, Milford, USA) provided with a BioRad HPX 87H column (BioRad, Hercules, USA), a Waters 2410 refractive index detector, and a Waters 2487 UV detector. The column was eluted at 60 ° C with 0.5 g L -1 sulfuric acid at a flow rate of 0.6 mL min -1. In contrast to the reference strain RWB219, the OD660 of the IMS0001 strain culture increased after galactose depletion. After approximately 850 hours of arabinose growth of the IMS0001 strain had been observed (Figure 2), this culture was transferred at an OD660 of 1.7 to a shake flask containing 2% arabinose. The cultures were then sequentially transferred to fresh medium containing 2% arabinose at a Οϋβεο of 2-3. The use of arabinose was confirmed by occasional measurement of arabinose concentrations by HPLC (data not shown). The growth rate of these crops increased from 0 to 0.15 h_1 in approximately 3,600 hours (Figure 3).

[121] A batch fermentation under limited oxygen conditions was initiated by inoculating 1 L of 2% arabinose supplemented synthetic medium with a 100 ml shake flask culture of IMS0001 cells grown in arabinose with a maximum rate of 2% L-arabinose growth of approximately 0.12 h "1. When growth in arabinose was observed, the culture was subjected to anaerobic conditions by spraying with nitrogen gas. The sequential cycles of anaerobic batch cultivation were initiated by either substitution. 90% of the culture with synthetic medium containing 20 g L_1 of arabinose For each cycle during fermentation with SBR, the exponential growth rate was estimated from the CO2 profile (Figure 4). increased from 0.025 to 0.08 h_1.After 20 cycles the sample was taken and placed in solid synthetic medium supplemented with 2% L-arabin It is incubated at 30 ° C for several days. Separated colonies were rerun twice in the synthetic solid medium with L-arabinose. Finally, the shake flask containing 2% L-L-arabinose synthetic medium was inoculated with a single colony and incubated for 5 days at 30 ° C. This culture was designated as strain IMS0002 (CBS 120328 deposited with Centraal Bureau voor Schimmelculturen (CBS) on 9/27/06). Culture samples were taken, 30% glycerol was added and the samples were stored at -80 ° C.

Mixed Culture Fermentation [122] Biomass hydrolysates, a desired feedstock for industrial biotechnology contain complex mixtures consisting of several sugars among which glucose, xylose and arabinose are generally present in significant fractions. In order to perform the ethanolic fermentation not only of glucose and arabinose but also xylose, an anaerobic batch fermentation was performed with a mixed culture of IMS0002 arabinose fermentation strain and the RWB218 xylose fermentation strain. An anaerobic batch fermenter containing 800 mL of synthetic medium supplied with 30 g L-1 D-glucose, 15 g L-1 π-xylose, and 15 g L-1 L-arabinose was inoculated with 100 mL of -culture of the IMS0002 strain. After 10 hours, 100 mL of RWB218 innocuous was added. In contrast to the mixed sugar fermentation with the IMSG002 strain alone, both xylose and arabinose were consumed after glucose depletion (Figure 5D). The mixed culture consumed all sugars completely and within 80 hours 564.0 ± 6.3 mmol L "1 of ethanol (calculated from CO? Production) were produced with a high total yield of 0.42 g g-1 of Xylitol was produced only in small quantities at a concentration of 4.7 mmol L_1.

IMS0002 strain characterization [123] Growth and product formation of the IMSQQ02 strain were determined during anaerobic batch fermentations in the synthetic medium with either L-arabinose as the sole carbon source or a mixture of glucose, xylose and L-arabinose. The precultures of these anaerobic batch fermentations were prepared in shake flasks containing 100 ml synthetic medium with 2% L-arabinose by inoculation with frozen master solutions at -80 ° C of the ΙΜΞ0002 strain and incubation for 48 hours at 30 ° C. ° C.

[124] Figure 5A shows that the IMS0002 strain is capable of fermenting 20 g L_1 of L-arabinose in ethanol during an anaerobic batch fermentation of approximately 70 hours. The specific growth rate under anaerobic conditions with L-arabinose as the sole carbon source was 0.05 ± 0.001 h-1. Taking into account ethanol evaporation during batch fermentation, ethanol yield of 20 g L_1 arabinose was 0.43 ± 0.003 gg ~ 1. Without evaporation correction, ethanol yield was 0.35 ± 0.01 g g_1 of arabinose. No arabinitol formation was observed during anaerobic growth in arabinose.

[125] In Figure 5B, the ethanolic fermentation of a mixture of 20 g Ir1 glucose and 20 g L-1 L-arabinose by the IMS0002 strain is shown. L-arabinose consumption started after glucose depletion. Within 70 hours, both glucose and L-arabinose were completely depleted. The ethanol yield of total sugars was 0.42 + 0.003 g g_1.

[126] In Figure 5C, the fermentation profile of a mixture of 30 g L_1 glucose, 15 g L-1 D-xylose and 15 g L_1 L-arabinose by the IMS0002 strain is shown. Arabinose consumption was started after glucose depletion. Within 80 hours, both glucose and arabinose were completely depleted. Only 20 mM of 100 mM xylose was consumed by the IMS0002 strain. In addition the formation of 20 mM xylitol was observed. Apparently, xylose was converted to xylitol by strain IMS0002. Consequently, the ethanol yield of total sugars was lower for the fermentations described above: 0.38 ± 0.001 g g_1. The ethanol yield of the total glucose and arabinose was similar to that of other fermentations: 0.43 ± 0.001 g g_1.

[127] Table 1 shows the arabinose consumption rates and ethanol production rates observed in the IMS0002 strain anaerobic batch fermentation. Arabinose was consumed at a rate of 0.23 - 0.75 g h -1 g -1 dry weight of the biomass. The rate of ethanol produced from arabinose ranged from 0.08 - 0.31 g h -1 g -1 of the dry weight of biomass.

[128] Initially, the constructed strain IMS0001 was able to ferment xylose (data not shown). In contrast to our expectations, the selected strain IMS0002 was not able to ferment xylose in ethanol (Figure 5). In order to regain the ability to ferment xylose, a colony of the IMS0002 strain was transferred to 2% D-xylose synthetic solid medium and incubated in an anaerobic jar at 30 ° C for 25 days. Subsequently a colony was again transferred to the synthetic solid medium with 2% arabinose. After 4 days incubation at 30 ° C, the colony was transferred to a shake flask containing 2% arabinose synthetic medium. After incubation at 30 ° C for 6 days, 30% glycerol was added, samples were taken and stored at 80 ° C. A shake flask containing 100 mL of 2% arabinose synthetic medium was inoculated with such a frozen master solution and was used as a preculture for anaerobic batch fermentation in the synthetic medium with 20 g Irl xylose and 20 g L-1. of arabinose. In Figure 6, the fermentation profile of this batch fermentation is shown. Xylose and arabinose were consumed simultaneously. Arabinosis was completed within 70 hours, whereas xylose was completely consumed within 120 hours. At least 250 mM ethanol was produced from total sugars, not taking into account ethanol evaporation. Assuming a final biomass dry weight of 3.2 g L_1 (assuming a biomass yield of 0.08 gg "1 sugar), the estimated ethanol concentration of cumulative production gives CO2 (355 mmol L_ :) was about 330 mmol L_1, corresponding to an ethanol yield of 0.41 g g_1 pentose sugar In addition to ethanol, glycerol and organic acids, a small amount of xylitol was produced (approximately 5 mM).

Strain Selection IM50003 [129] Initially, the constructed strain IMS0001 was able to ferment xylose (data not shown). Contrary to our expectations, the selected strain IM5QQ02 was not able to ferment xylose in ethanol (Figure 5C). In order to regain xylose fermentation capacity, a colony of the IMS0002 strain was transferred to D-xylose 21 synthetic solid medium and incubated in an anaerobic jar at 30 ° C for 25 days. Subsequently, the colony was again transferred to synthetic solid medium with 2% arabinose. After 4 days incubation at 30 ° C, a colony was transferred to a shake flask containing 2% arabinose synthetic medium. After incubation at 30 ° C for 6 days, 30% glycerol was added and samples removed and stored at -80 ° C.

[130] From this frozen master solution, samples were dispersed in solid synthetic medium with 2% L-arabinose and incubated at 30 ° C for several days. Separate colonies were rerun twice in a solid synthetic medium with L-arabinose. Finally, a shake flask containing 2% L-arabinose synthetic medium was inoculated with a single colony and incubated for 4 days at 30 ° C. This culture was designated as IMS0003 (CBS 121879 filed with CBS on 09/20/07). Culture samples were taken, 30% glycerol was added and samples stored at -80 ° C.

Characterization of the IMS0003 strain [131] Development and product formation of the IMS0003 strain were determined during the fermentation of an anaerobic batch in the synthetic medium with a mixture of 30 g L1 glucose, 15 g L "1 D-xylose and 15 g L · -1 of L-arabinose. The preculture for anaerobic batch fermentation was prepared in shake flasks containing 100 mL of 2% L-arabinose synthetic medium by inoculation with a master solution frozen at 80 ° C of the IMS0003 strain and incubated for 48 hours. at 30 ° C.

[132] In Figure 7, the fermentation profile of a mixture of 30 g L_1 glucose, 15 g L_1 D-xylose and 15 g L-1 L-arabinose per IMS0003 strain is shown. Arabinose consumption started after glucose depletion. Within 70 hours, glucose, xylose and arabinose were completely depleted. Xylose and arabinose were consumed simultaneously. At least 406 mM ethanol was produced from total sugars, not taking into account ethanol evaporation. The final calculated ethanol concentration of cumulative CO production; was 572 mmol L_1, corresponding to an ethanol yield of 0.46 g g_1 of total sugar. In contrast to the fermentation of the glucose, xylose and arabinose mixture by the IMS0002 strain (Figure 5C) or a mixed culture of the IMSQ002 and RWB218 strains (Figure 5D), strain ΙΜΞ0003 did not produce desirable amounts of xylitol.

TABLES

Table 1: S, cerevisiae strains employed _________Table 3: Oli strains employed in this work___________ Table 4: [133] Maximum observed glucose and arabinose specific consumption rates and ethanol production rates during anaerobic batch fermentations of S. cerevisiae IMSQQQ2.

Qgiu: cjarà glucose specific consumption rate * cjeth arabinose specific consumption rate, giu * ethanol specific production rate during glucose development: ethanol specific production rate during arabinose development Reference List Andreasen AA, Stier TJ ( 1954) Anaerobic nutrition o Saccharomyces cer & visiae. II. Unsaturated fatty acid requirement for growth in a defined medium. J Cell Physiol 43: 271-281.

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Claims (10)

Saccharomyces cell capable of expressing the following nucleotide sequences introduced therein, characterized in that the expression of these nucleotide sequences confers to the Saccharomyces cell the ability to use L-arabinose and / or convert L-arabinose to L-ribulose, and / or 5-phosphate and / or ethanol xylulose: (a) a nucleotide sequence represented by SEQ ID NO: 2 and its degenerations that generate the same arabinose isomerase (araA) represented by SEQ ID NO: 1 ; (b) a nucleotide sequence represented by SEQ ID NO: 4 and its degenerations that generate the same L-ribulokinase (araB) represented by SEQ ID NO: 3; (c) a nucleotide sequence represented by SEQ ID NO: 6 and its degenerations that generate the same L-ribulose-5-P-4-epimerase (araD) represented by SEQ ID NO: 5; wherein the Saccharomyces cell is further adapted to L-arabinose by mutant selection. Saccharomyces cell according to claim 1, characterized in that one, two or three of the nucleotide sequences of araA, araB and araD originate from a genus Lactobacillus, preferably a species Lactobacillus plantarum. Saccharomyces cell according to claim 1 or 2, characterized in that the Saccharomyces cell belongs to one of the species: 5. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus Saccharomyces cell according to any one of claims 1 to 3, characterized in that the nucleotide sequences encoding araA, araB and / or araD are operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequences in the cell to give the cell the ability to use L-arabinose and / or convert L-arabinose to L-ribulose, and / or 5-phosphate xylulose and / or ethanol. Saccharomyces cell according to any one of claims 1 to 4, characterized in that the fermentation product is ethanol. Process for the production of ethanol, characterized in that it comprises: (a) fermenting a medium containing an arabinose source with a modified Saccharomyces cell as defined in any one of claims 1 to 5, wherein the cell of Saccharomyces fermentes arabinose to ethanol; and (b) ethanol recovery. Process according to Claim 6, characterized in that the medium also contains a source of glucose. Process according to Claim 6 or 7, characterized in that the volumetric ethanol productivity is at least 0.5 g ethanol per liter per hour. Process according to Claim 8, characterized in that the ethanol yield is at least 30%. Process according to any one of claims 6 to 9, characterized in that the process is anaerobic.