DK2069476T3 - Metabolic MODIFICATION of arabinose-fermenting YEAST CELLS - Google Patents

Description

DESCRIPTION

Field of the invention [0001] The invention relates to an yeast cell having the ability to use L-arabinose and/or to convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product and to a process for producing a fermentation product wherein this cell is used.

Background of the invention [0002] Fuel ethanol is acknowledged as a valuable alternative to fossil fuels. Economically viable ethanol production from the hemicellulose fraction of plant biomass requires the simultaneous fermentative conversion of both pentoses and hexoses at comparable rates and with high yields. Yeasts, in particular Saccharomyces spp., are the most appropriate candidates for this process since they can grow and ferment fast on hexoses, both aerobically and anaerobically. Furthermore they are much more resistant to the toxic environment of lignocellulose hydrolysates than (genetically modified) bacteria.

[0003] EP 1 499 708 describes a process for making S. cerevisiae strains able to produce ethanol from L-arabinose. These strains were modified by introducing the araA (L-arabinose isomerase) gene from Bacillus subtilis, the araB (L-ribulokinase) and araD (L-ribulose-5-P4-epimerase) genes from Escherichia coli. Furthermore, these strains were either carrying additional mutations in their genome or overexpressing a TAL1 (transaldolase) gene. However, these strains have several drawbacks. They ferment arabinose in oxygen limited conditions. In addition, they have a low ethanol production rate of 0.05 g.g'1.h'1 (Becker and Boles, 2003). Furthermore, these strains are not able to use L-arabinose under anaerobic conditions. Finally, these S. cerevisiae strains have a wild type background, therefore they can not be used to co-ferment several C5 sugars.

[0004] WO 03/062430 and WO 06/009434 disclose yeast strains able to convert xylose into ethanol. These yeast strains are able to directly isomerise xylose into xylulose.

[0005] Still, there is a need for alternative strains for producing ethanol, which perform better and are more robust and resistant to relatively harsh production conditions.

Description of the figures [0006]

Figure 1. Plasmid maps of pRW231 and pRW243.

Figure 2. Growth pattern of shake flask cultivations of strain RWB219 (o) and IMS0001 (·) in synthetic medium containing 0.5% galactose (A) and 0.1% galactose + 2% L-arabinose (B). Cultures were grown for 72 hours in synthetic medium with galactose (A) and then transferred to synthetic medium with galactose and arabinose (B). Growth was determined by measuring the ODø60·

Figure 3. Growth rate during serial transfers of S. cerevisiae IMS0001 in shake flask cultures containing synthetic medium with 2% (w/v) L-arabinose. Each datapoint represents the growth rate estimated from the Οϋββο measured during (exponential) growth. The closed and open 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 datapoint represents the growth rate estimated from the CO2 profile (solid line) during exponential growth.

Figure 5. Sugar consumption and product formation during anaerobic batch fermentations of strain IMS0002. The fermentations were performed in 1 synthetic medium supplemented with: 20 g I"1 arabinose (A); 20 g Γ1 glucose and 20 g I"1 arabinose (B); 30 g I"1 glucose, 15 g I'1 xylose, and 15 g I"1 arabinose (C);.Sugar consumption and product formation during anaerobic batch fermentations with a mixture of strains IMS0002 and RWB218. The fermentations were performed in 1 liter of synthetic medium supplemented with 30 g M glucose, 15 g M xylose, and 15 g M arabinose (D). Symbols: glucose (·); xylose (o); arabinose (); ethanol calculated from cumulative CO2 production (□); ethanol measured by HPLC (A); cumulative CO2 production (Δ); xylitol(T)

Figure 6. Sugar consumption and product formation during an anaerobic batch fermentation of strain IMS0002 cells selected for anaerobic growth on xylose. The fermentation was performed in 1 liter of synthetic medium supplemented with 20 g I"1 xylose and 20 g I"1 arabinose. Symbols: xylose (o); arabinose (); ethanol measured by HPLC (A); cumulative CO2 production (Δ); xylitol(T).

Figure 7. Sugar consumption and product formation during an anaerobic batch fermentation of strain IMS0003. The fermentation was performed in 1 liter of synthetic medium supplemented with: 30 g I'1 glucose, 15 g H xylose, and 15 g I'1 arabinose. Symbols: glucose (·); xylose (o); arabinose (); ethanol calculated from cumulative CO2 production (□); ethanol measured by HPLC (A); cumulative CO2 production (Δ);

Description of the invention

Yeast cell [0007] In a first aspect, the invention relates to a yeast cell capable of expressing the following nucleotide sequences, whereby the expression of these nucleotide sequences confers on the yeast cell the ability to use L-arabinose and/or to convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into ethanol: 1. (a) a nucleotide sequence encoding an arabinose isomerase (araA), wherein said nucleotide sequence is selected from the group consisting of: 1. (i) nucleotide sequences encoding an araA, said araA comprising an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:1. 2. (ii) nucleotide sequences comprising a nucleotide sequence that has at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:2. 3. (iii) nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i) or(ii); 2. (b) a nucleotide sequence encoding a L-ribulokinase (araB), wherein said nucleotide sequence is selected from the group consisting of: 1. (i) nucleotide sequences encoding an araB, said araB comprising an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:3. 2. (ii) nucleotide sequences comprising a nucleotide sequence that has at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:4. 3. (iii) nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i) or (ii); 3. (c) a nucleotide sequence encoding an L-ribulose-5-P-4-epimerase (araD), wherein said nucleotide sequence is selected from the group consisting of: 1. (i) nucleotide sequences encoding an araD, said araD comprising an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:5. 2. (ii) nucleotide sequences comprising a nucleotide sequence that has at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:6. 3. (iii) nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i) or(ii); 4. (iv) wherein in the items (iii). hybridisation is determined under hybridization conditions that allow a nucleic acid sequence of 200 nucleotides to hybridise at a temperature of 65 °C in a solution comprising about 1M salt, and washing at 65 °C in a solution of about 0.1M, where the hybridisation is performed for 10 hours and washing is performed one hour with two changes of the washing solution.

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Sequence identity and similarity [0008] Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of 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.

[0009] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the BestFit, BLASTP, BLASTN, and FASTA( Altschul, S. F. 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 most preferred algorithm used is EMBOSS (http://www.ebi.ac.uk/emboss/alinnV Preferred parameters for amino acid sequences comparison using EMBOSS are gap open 10.0, gap extend 0.5, Blosum 62 matrix Preferred parameters for nucleic acid sequences comparison using EMBOSS are gap open 10.0, gap extend 0.5, DNAfull matrix (DNA identity matrix).

[0010] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; lie to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

Hybridising nucleic acid sequences [0011] Nucleotide sequences encoding the enzymes expressed in the cell of the invention may also be defined by their capability to hybridise with the nucleotide sequences of SEQ ID NO.'s 2, 4, 6, 8, 16, 18, 20, 22, 24, 26, 28, 30 respectively, under stringent hybridisation conditions. Stringent hybridisation conditions are herein defined as conditions that allow a nucleic acid sequence of 200 nucleotides, to hybridise at a temperature of 65°C in a solution comprising 1 M saltand washing at 65°C in a solution comprising about 0.1 M salt, The hybridisation is performed for 10 hours and washing is performed for one hour with two changes of the washing solution.

AraA

[0012] A preferred nucleotide sequence encoding a arabinose isomerase (araA) expressed in the cell of the invention is selected from the group consisting of: 1. (a) nucleotide sequences encoding an araA polypeptide said araA comprising an amino acid sequence that has at least 80, 85, 90, 95, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO. 1; 2. (b) nucleotide sequences comprising a nucleotide sequence that has at least 80, 90, 95, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO. 2; 3. (c) nucleotide sequences the complementary strand of which hybridises to a nucleic acid molecule sequence of (a) or (b); [0013] The nucleotide sequence encoding an araAmay encode either a prokaryotic or an eukaryotic araA, i.e. an araAwith an amino acid sequence that is identical to that of an araA that naturally occurs in the prokaryotic or eukaryotic organism. The present inventors have found that the ability of a particular araA to confer to a eukaryotic host cell the ability to use arabinose and/or to convert arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product such as ethanol when co-expressed with araB and araD does not depend so much on whether the araA is of prokaryotic or eukaryotic origin. Rather this depends on the relatedness of the araA’s amino acid sequence to that of the sequence SEQ ID NO. 1.

AraB

[0014] A preferred nucleotide sequence encoding a L-ribulokinase (AraB) expressed in the cell of the invention is selected from the group consisting of: 1. (a) nucleotide sequences encoding a polypeptide comprising an amino acid sequence that has at least 80, 85, 90, 95, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO. 3; 2. (b) nucleotide sequences comprising a nucleotide sequence that has at least 80, 90, 95, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO.4; 3. (c) nucleotide sequences the complementary strand of which hybridises to a nucleic acid molecule sequence of (a) or (b); [0015] The nucleotide sequence encoding an araB may encode either a prokaryotic or an eukaryotic araB, i.e. an araB vwth an amino acid sequence that is identical to that of a araB that naturally occurs in the prokaryotic or eukaryotic organism. The present inventors have found that the ability of a particular araB to confer to a eukaryotic host cell the ability to use arabinose and/or to convert arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product vtfien co-expressed with araA and araD does not depend so much on whether the araB is of prokaryotic or eukaryotic origin. Rather this depends on the relatedness of the araB's amino acid sequence to that of the sequence SEQ ID NO. 3.

AraD

[0016] A preferred nucleotide sequence encoding a L-ribulose-5-P-4-epimerase (araD) expressed in the cell of the invention is selected from the group consisting of: (d) nucleotide sequences encoding a polypeptide comprising an amino acid sequence that has at least 80, 85, 90, 95, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO. 5; (e) nucleotide sequences comprising a nucleotide sequence that has at least 80, 85, 90, 95, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO.6; (f) nucleotide sequences the complementary strand of which hybridises to a nucleic acid molecule sequence of (a) or (b); (g) nucleotide sequences the sequence of which differs from the sequence of a nucleic acid molecule of (c) due to the degeneracy of the genetic code.

[0017] The nucleotide sequence encoding an araD may encode either a prokaryotic or an eukaryotic araD, i.e. an araD with an amino acid sequence that is identical to that of a araD that naturally occurs in the prokaryotic or eukaryotic organism. The present inventors have found that the ability of a particular araD to confer to a eukaryotic host cell the ability to use arabinose and/or to convert arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product when coexpressed with araA and araB does not depend so much on whether the araD is of prokaryotic or eukaryotic origin. Rather this depends on the relatedness of the araD's amino acid sequence to that of the sequence SEQ ID NO. 5.

[0018] Surprisingly, the codon bias index indicated that expression of the Lactobacillus plantarum araA, araB and araD genes were more favorable for expression in yeast than the prokaryolic araA, araB and araD genes described in EP 1 499 708.

[0019] It is to be noted that L. plantarum is a Generally Regarded As Safe (GRAS) organism, which is recognized as safe by 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 a fungal araA, araB or araD respectively (e.g. from a Basidiomaycete), more preferably an araA, araB or araD respectively from an anaerobic fungus, e.g. an anaerobic fungus that belongs to the families 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, most preferably from Lactobacillus plantarum species. Preferably, one, two or three or the araA, araB and araD nucleotide sequences originate from a Lactobacillus genus, more preferably a Lactobacillus plantarum species.The bacterial araA expressed in the cell of the invention is not the Bacillus subtilis araA disclosed in EP 1 499 708 and given as SEQ ID NO:9. SEQ ID NO: 10 represents the nucleotide acid sequence coding for SEQ ID NO: 9. The bacterial araB and araD expressed in the cell of the invention are not the ones of Escherichia coli (E. coli) as disclosed in EP 1 499 708 and given as SEQ ID NO: 11 and SEQ ID NO: 13. SEQ ID NO: 12 represents the nucleotide acid sequence coding for SEQ ID NO:11. SEQ ID NO: 14 represents the nucleotide acid sequence coding for SEQ ID NO: 13.

[0020] To increase the likelihood that the (bacterial) araA, araB and araD enzymes respectively are expressed in active form in a eukaryotic host cell of the invention such as yeast, the corresponding encoding nucleotide sequence may be adapted to optimise its codon usage to that of the chosen eukaryotic host cell. The adaptiveness of a nucleotide sequence encoding the araA, araB, and araD enzymes (or other enzymes of the invention, see below) to the codon usage of the chosen host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li , 1987, Nucleic Acids Research 15: 1281-1295; also see: Jansen et al., 2003, Nucleic Acids Res. 31(8):2242-51). An adapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3,0.4,0.5,0.6or0.7.

In a preferred embodiment, expression of the nucleotide sequences encoding an ara A, an ara B and an ara D as defined earlier herein confers to the cell the ability to use L-arabinose and/or to convert it into L-ribulose, and/or xylulose 5-phosphate. Without wishing to be bound by any theory, L-arabinose is expected to be first converted into L-ribulose, which is subsequently converted into xylulose 5-phosphate which is the main molecule entering the pentose phosphate pathway. In the context of the invention, "using L-arabinose" preferably means that the optical density measured at 660 nm (Οϋρρο) °f transformed cells cultured under aerobic or anaerobic conditions in the presence of at least 0.5 % L-arabinose during at least 20 days is increased from approximately 0.5 till 1.0 or more. More preferably, the ODes60 is increased from 0.5 till 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. Most preferably, the cells are cultured in the presence of approximately 2% L-arabinose.

In the context of the invention, a cell is able “to convert L-arabinose into L-ribulose” when detectable amounts of L-ribulose are detected in cells cultured under aerobic or anaerobic conditions in the presence of L-arabinose (same preferred concentrations as in previous paragraph) during at least 20 days using a suitable assay. Preferably the assay is HPLC for L-ribulose.

In the context of the invention, a cell is able "to convert L-arabinose into xylulose 5-phosphate" when an increase of at least 2% of xylulose 5-phosphate is detected in cells cultured under aerobic or anaerobic conditions in the presence of L-arabinose (same preferred concentrations as in previous paragraph) during at least 20 days using a suitable 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 of at least 5%, 10%, 15%, 20%, 25% or more. In another preferred embodiment, expression of the nucleotide sequences encoding an ara A, ara B and ara D as defined earlier herein confers to the cell the ability to convert L-arabinose into a desired fermentation product when cultured under aerobic or anaerobic conditions in the presence of L-arabinose (same preferred concentrations as in previous paragraph) during at least one month till one year. More preferably, a cell is able to convert L-arabinose into a desired fermentation product when detectable amounts of a desired fermentation product are detected using a suitable assay and wfnen the cells are cultured under the conditions given in previous sentence. Even more preferably, the assay is HPLC. Even more preferably, the fermentation product is ethanol.

[0021] A cell for transformation with the nucleotide sequences encoding the araA, araB, and araD enzymes respectively as described above, preferably is a host cell capable of active or passive xylose transport into and xylose isomerisation within the cell. The cell preferably is capable of active glycolysis. The cell may further contain an endogenous pentose phosphate pathway and may contain endogenous xylulose kinase activity so that xylulose isomerised from xylose may be metabolised to pyruvate. The cell further preferably contains enzymes for conversion of pyruvate to a desired fermentation product such as ethanol, lactic acid, 3-hydroxy-propionic 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 or a cephalosporin. The cell may be made capable of producing butanol by introduction of one or more genes of the butanol pathway as disclosed in W02007/041269.

[0022] A preferred cell is naturally capable of alcoholic fermentation, preferably, anaerobic alcoholic fermentation. The host cell further preferably has a high tolerance to ethanol, a high tolerance to low pH (i.e. capable of growth at a pH lower than 5, 4, 3, or 2,5) and towards organic acids like lactic acid, acetic acid or formic acid and sugar degradation products such as furfural and hydroxy-methylfurfural, and a 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 modified through genetic selection or by genetic modification. A suitable host cell is a eukaryotic microorganism like e.g. a fungus, however, most suitable as host cell are yeasts or filamentous fungi.

[0023] Yeasts are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In: Introductory Mycology, John Wiley & Sons, Inc., New York) that predominantly grow in unicellular form. Yeasts may either grow by budding of a unicellular thallus or may grow by fission of the organism. Preferred yeasts as host cells belong to one of the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hanzsenula, Kloeckera, Schwanniomyces, or Yarrowia. Preferably the yeast is capable of anaerobic fermentation, more preferably anaerobic alcoholic fermentation.

[0024] Filamentous fungi are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina. These fungi are characterized by a vegetative mycelium composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism of most filamentous fungi is obligately aerobic. Preferred filamentous fungi as host cells belong to one of the genera Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium, or Penicillium.

[0025] Over the years suggestions have been made for the introduction of various organisms for the production of bio-ethanol from crop sugars. In practice, however, all major bio-ethanol production processes have continued to use the yeasts of the genus Saccharomyces as ethanol producer. This is due to the many attractive features of Saccharomyces species for industrial processes, i.e., a high acid-, ethanol- and osmo-tolerance, capability of anaerobic growth, and of course its high alcoholic fermentative capacity. Preferred yeast species as host cells include S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. mandanus, K. fragilis.

[0026] In a preferred embodiment, the host cell of the invention is a host cell that has been transformed vwth a nucleic acid construct comprising the nucleotide sequence encoding the araA, araB, and araD enzymes as defined above. In one more preferred embodiment, 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 is capable of expression of the araA, araB, and/or araD enzymes in the host cell. To this end the nucleic acid construct may be constructed as described in e.g. WO 03/0624430. The host cell may comprise a single but preferably comprises multiple copies of each nucleic acid construct. The nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2μ or pKD1 (Fleer et al., 1991, Biotechnology 9:968-975) plasmids. Preferably, however, each nucleic acid construct is integrated in one or more copies into the genome of the host cell. Integration into the host cell's genome may occur at random by illegitimate recombination but preferably nucleic acid construct is integrated into the host cell's genome by homologous recombination as is well known in the art of fungal molecular genetics (see e.g. 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 each operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequences in a cell to confer to the cell the ability to use L-arabinose, and/or to convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate. Preferably the cell is a yeast cell. Accordingly, in a further aspect, the invention also encompasses a nucleic acid construct as earlier outlined herein. 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 been all earlier defined herein.

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

[0027] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the nucleic acid sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

[0028] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. 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.

[0029] The promoter that could be used to achieve the expression of the nucleotide sequences coding for araA, araB and/or araD may be not native to the nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. Although the promoter preferably is heterologous to the coding sequence to which it is operably linked, it is also preferred that the promoter is homologous, i.e. endogenous to the host cell. Preferably the heterologous promoter (to the nucleotide sequence) is capable of producing a higher steady state level of the transcript comprising the coding sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is the promoter that is native to the coding 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. more than 50% of the available carbon source consists of arabinose, or arabinose and glucose, or xylose and arabinose or xylose and arabinose and glucose), most preferably as sole carbon sources. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters. A preferred promoter for use in the present invention will in addition be insensitive to catabolite (glucose) repression and/or will preferably not require arabinose and/or xylose for induction.

[0030] Promotors having these characteristics are widely available and known to the skilled person. Suitable examples of such promoters include e g. promoters from glycolytic genes, such as the phosphofructokinase (PPK), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK) promoters from yeasts or filamentous fungi; more details about such promoters from yeast may be found in (WO 93/03159). Other useful promoters are ribosomal protein 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 promoter (ΗΧΓ7) or the glyceraldehyde-3-phosphate dehydrogenase (TDH3). The sequence of the PGI1 promoter is given in SEQ ID NO:51. The sequence of the HXT7 promoter is given in SEQ ID NO:52. The sequence of the TDH3 promoter is given in SEQ ID NO:49. Other promoters, both constitutive and inducible, and enhancers or upstream activating sequences will be known to those of skill in the art. The 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 araA, araB and araD genes of L. plantarum. More preferably, the eukaryotic cell is a yeast cell, even more preferably a S. cerevisiae strain transformed with the araA, araB and araD genes of L. plantarum. Most preferably, the cell is either CBS 120327 or CBS 120328 both deposited at the CBS Institute (The Netherlands) on September 27th, 2006.

[0031] The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically be operably linked to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. When used to indicate the relatedness of two nucleic acid sequences the term "homologous" means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as earlier presented. Preferably the region of identity is greater than about 5 bp, more preferably the region of identity is greater than 10 bp.

[0032] The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but has been 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 the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.

Preferred eukaryotic cell able to use and/or convert L-arabinose and xylose [0033] In a more preferred embodiment, the cell of the invention that expresses araA, araB and araD is able to use L-arabinose and/or to convert it into L-ribulose, and/or xylulose 5-phosphate and/or a desired fermentation product as earlier defined herein and additionally exhibits the ability to use xylose and/or convert xylose into xylulose. The conversion of xylose into xylulose is preferably a one step isomerisation step (direct isomerisation of xylose into xylulose). This type of cell is therefore able to use both L-arabinose and xylose. "Using" xylose has preferably the same meaning as "using" L-arabinose as earlier defined herein. 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), transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2), and aldose reductase" (EC 1.1.1.21).

[0034] In a preferred embodiment, the eukaryotic cell of the invention expressing araA, araB and araD as earlier defined herein has the ability of isomerising xylose to xylulose as e g. described in WO 03/0624430 or in WO 06/009434. The ability of isomerising xylose to xylulose is conferred to the host cell by transformation of the host cell with a nucleic acid construct comprising a nucleotide sequence encoding a xylose isomerase. The transformed host cell's ability to isomerise xylose into xylulose is the direct isomerisation of xylose to xylulose. This is understood to mean that xylose isomerised into xylulose in a single reaction catalysed by a xylose isomerase, as opposed to the two step conversion of xylose into xylulose via a xylitol intermediate as catalysed by xylose reductase and xylitol dehydrogenase, respectively.

[0035] The nucleotide sequence encodes a xylose isomerase that 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 a specific activity of at least 10 U xylose isomerase activity per mg protein 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 the xylose isomerase expressed in the transformed host cell is herein defined as the amount of xylose isomerase activity units per mg protein of cell free lysate of the host cell, e.g. a yeast cell free lysate. Determination of the xylose isomerase activity has already been described earlier herein.

[0036] Preferably, expression of the nucleotide sequence encoding the xylose isomerase in the host cell produces a xylose isomerase with a Km for xylose that is less than 50, 40, 30 or 25 mM, more preferably, the Km for xylose is about 20 mM or less.

[0037] A preferred nucleotide sequence encoding the xylose isomerase may be selected from the group consisting of: (e) nucleotide sequences encoding a polypeptide comprising an amino acid sequence that has 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. 7 or SEQ ID NO: 15; (f) nucleotide sequences comprising a nucleotide sequence that has at least 40, 50, 60, 70, 80, 90, 95, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO. 8 or SEQ ID NO: 16; (g) nucleotide sequences the complementary strand of which hybridises to a nucleic acid molecule sequence of (a) or (b); (h) nucleotide sequences the sequence of which differs from the sequence of a nucleic acid molecule of (c) due to the degeneracy of the genetic code.

[0038] The nucleotide sequence encoding the xylose isomerase may encode either a prokaryotic or an eukaryotic xylose isomerase, i.e. a xylose isomerase with an amino acid sequence that is identical to that of a xylose isomerase that naturally occurs in the prokaryotic or eukaryotic organism. The present inventors have found that the ability of a particular xylose isomerase to confer to a eukaryotic host cell the ability to isomerise xylose into xylulose does not depend so much on whether the isomerase is of prokaryotic or eukaryotic origin. Rather this depends on the relatedness of the isomerase's amino acid sequence to that of the Piromyces sequence (SEQ ID NO. 7). Surprisingly, the eukaryotic Piromyces isomerase is more related to prokaryotic isomerases than to other known eukaryotic isomerases. Therefore, a preferred nucleotide sequence encodes a 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 a xylose isomerase from an anaerobic fungus, e.g. a xylose isomerase from an anaerobic fungus that belongs to the families Neocallimastix, Caecomyces, Piromyces, Orpinomyces, or Ruminomyces. Alternatively, a preferred nucleotide sequence encodes a bacterial xylose isomerase, preferably a Gram-negative bacterium, more preferably an isomerase from the class Bacteroides, or from the genus Bacteroides, most preferably from B. thetaiotaomicron (SEQ ID NO. 15).

[0039] To increase the likelihood that the xylose isomerase is expressed in active form in a eukaryotic host cell such as yeast, the nucleotide sequence encoding the xylose isomerase may be adapted to optimise its codon usage to that of the eukaryotic host cell as earlier defined herein.

[0040] A host cell for transformation with the nucleotide sequence encoding the xylose isomerase as described above, preferably is a host capable of active or passive xylose transport into the cell. The host cell preferably contains active glycolysis. The host cell may further contain an endogenous pentose phosphate pathway and may contain endogenous xylulose kinase activity so that xylulose isomerised from xylose may be metabolised to pyruvate. The host further preferably contains enzymes for conversion of pyruvate to a desired fermentation product such as ethanol, lactic acid, 3-hydroxy-propionic 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 or a cephalosporin. A preferred host cell is a host cell that is naturally capable of alcoholic fermentation, preferably, anaerobic alcoholic fermentation. The host cell further preferably has a high tolerance to ethanol, a high tolerance to low pH (i.e. capable of growth at a pH lower than 5, 4, 3, or 2,5) and towards organic acids like lactic acid, acetic acid or formic acid and sugar degradation products such as furfural and hydroxy-methylfurfural, and a 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 modified by genetic modification. A suitable cell is a eukaryotic microorganism like e.g. a fungus, however, most suitable as host cell are yeasts or filamentous fungi. Preferred yeasts and filamentous fungi have already been defined herein.

[0041] As used herein the wording host cell has the same meaning as cell.

[0042] The cell of the invention is preferably transformed with a nucleic acid construct comprising the nucleotide sequence encoding the xylose isomerase. The nucleic acid construct that is preferably used is the same as the one used comprising the nucleotide sequence encoding araA, araB or araD.

[0043] In another preferred embodiment of the invention, the cell of the invention: • expressing araA, araB and araD, and exhibiting the ability to directly isomerise xylose into xylulose, as earlier defined herein further comprises a genetic modification that increases the flux of the pentose phosphate pathway, as described in WO 06/009434. In particular, the genetic modification causes an increased flux of the non-oxidative part pentose phosphate pathway. A genetic modification that causes an increased flux of the non-oxidative part of the pentose phosphate pathway is herein understood to mean a modification that increases the flux by at least a factor 1.1, 1.2, 1.5, 2, 5, 10 or 20 as compared to the flux in a strain which is genetically identical except for the genetic modification causing the increased flux. The flux of the non-oxidative part of the pentose phosphate pathway may be measured by growing the modified host on xylose as sole carbon source, determining the specific xylose consumption rate and substracting the specific xylitol production rate from the specific xylose consumption rate, if any xylitol is produced. However, the flux of the non-oxidative part of the pentose phosphate pathway is proportional with the growth rate on xylose as sole carbon source, preferably with the anaerobic growth rate on xylose as sole carbon source. There is a linear relation between the growth rate on xylose as sole carbon source (Umax) and the flux 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 yield of biomass on sugar (Y)e) because the yield of biomass on sugar is constant (under a given set of conditions: anaerobic, growth medium, pH, genetic background of the strain, etc.; i.e. Qs = μ/ Y*)· Therefore the increased flux of the non-oxidative part of the pentose phosphate pathway may be deduced from the increase in maximum growth rate under these conditions. In a preferred embodiment, the cell comprises a genetic modification that increases the flux of the pentose phosphate pathway and has a specific xylose consumption rate of at least 346 mg xylose/g biomass/h.

[0044] Genetic modifications that increase the flux of the pentose phosphate pathway may be introduced in the host cell in various ways. These including e.g. achieving higher steady state activity levels of xylulose kinase and/or one or more of the enzymes of the non-oxidative part pentose phosphate pathway and/or a reduced steady state level of unspecific aldose reductase activity. These changes in steady state activity levels may be effected by selection of mutants (spontaneous or induced by chemicals or radiation) and/or by recombinant DNA technology e.g. by overexpression or inactivation, respectively, of genes encoding the enzymes or factors regulating these genes.

[0045] In a more preferred host cell, the genetic modification comprises overexpression of at least one enzyme of the (non-oxidative part) pentose phosphate pathway. Preferably the enzyme is selected from the group consisting of the enzymes encoding for ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase, as described in WO 061009434.

[0046] Various combinations of enzymes of the (non-oxidative part) pentose phosphate pathway may be overexpressed. E.g. the enzymes that are overexpressed may be at least the enzymes ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase; or at least the enzymes ribulose-5-phosphate isomerase and transketolase; or at least the enzymes ribulose-5-phosphate isomerase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase and transketolase; or at least the enzymes ribulose-5-phosphate epimerase and transaldolase; or at least the enzymes transketolase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase, transketolase and transaldolase; or at least the enzymes ribulose-5-phosphate isomerase, transketolase and transaldolase; or at least the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, and transaldolase; or at least the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, and transketolase. In one embodiment of the invention each of the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase are overexpressed in the host cell. More preferred is a host cell in which the genetic modification comprises at least overexpression of both the enzymes transketolase and transaldolase as such a host cell is already capable of anaerobic growth on xylose. In fact, under some conditions we have found that host cells overexpressing only the transketolase and the transaldolase already have the same anaerobic growth rate on xylose as do host cells that overexpress all four of the enzymes, i.e. the ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase. Moreover, host cells overexpressing both of the enzymes ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase are preferred over host cells overexpressing only the isomerase or only the epimerase as overexpression of only one of these enzymes may produce metabolic imbalances.

[0047] There are various means available in the art for overexpression of enzymes in the cells of the invention. In particular, an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the host cell, e.g. by integrating additional copies of the gene in the host cell's genome, by expressing the gene from an episomal multicopy expression vector or by introducing a episomal expression vector that comprises multiple copies of the gene.

[0048] Alternatively overexpression of enzymes in the host cells of the invention may be achieved by using a promoter that is not native to the sequence coding for the enzyme to be overexpressed, i.e. a promoter that is heterologous to the coding sequence to which it is operably linked. Suitable promoters to this end have already been defined herein.

[0049] The coding sequence used for overexpression of the enzymes preferably is homologous to the host cell of the invention. However, coding sequences that are heterologous to the host cell of the invention may likewise be applied, as mentioned in WO 061009434.

[0050] A nucleotide sequence used for overexpression of ribulose-5-phosphate isomerase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with ribulose-5-phosphate isomerase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 17 or whereby the nucleotide sequence is capable of hybridising with the nucleotide sequence of SEQ ID NO. 18, under moderate conditions, preferably under stringent conditions.

[0051] A nucleotide sequence used for overexpression of ribulose-5-phosphate epimerase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with ribulose-5-phosphate epimerase activity, whereby 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 hybridising with the nucleotide sequence of SEQ ID NO. 20, under moderate conditions, preferably under stringent conditions.

[0052] A nucleotide sequence used for overexpression of transketolase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with transketolase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 21 or whereby the nucleotide sequence is capable of hybridising with the nucleotide sequence of SEQ ID NO. 22, under moderate conditions, preferably under stringent conditions.

[0053] A nucleotide sequence used for overexpression of transaldolase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with transaldolase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 23 or whereby the nucleotide sequence is capable of hybridising with the nucleotide sequence of SEQ ID NO. 24, under moderate conditions, preferably under stringent conditions.

[0054] Overexpression of an enzyme, when referring to the production of the enzyme in a genetically modified host cell, means that the enzyme is produced at a higher level of specific enzymatic activity as compared to the unmodified host cell under identical conditions. Usually this means that the enzymatically active protein (or proteins in case of multi-subunit enzymes) is produced in greater amounts, or rather at a higher steady state level as compared to the unmodified host cell under identical conditions. Similarly this usually means that the mRNA coding for the enzymatically active protein is produced in greater amounts, or again rather at a higher steady state level as compared to the unmodified host cell under identical conditions. Overexpression of an enzyme is thus preferably determined by measuring the level of the enzyme's specific activity in the host cell using appropriate enzyme assays as described herein. Alternatively, overexpression of the enzyme may determined indirectly by quantifying the specific steady state level of enzyme protein, e.g. using antibodies specific for the enzyme, or by quantifying the specific steady level of the mRNA coding for the enzyme. The latter may particularly be suitable for enzymes of the pentose phosphate pathway for which enzymatic assays are not easily feasible 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 at least a factor 1.1, 1.2, 1.5, 2, 5, 10 or 20 as compared to a strain which is genetically identical except for the genetic modification causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.

[0055] In a further preferred embodiment, the host cell of the invention: • expressing araA, araB and araD, and exhibiting the ability to directly isomerise xylose into xylulose, and optionally • comprising a genetic modification that increase the flux of the pentose pathway as earlier defined herein further comprises a genetic modification that increases the specific xylulose kinase activity. Preferably the genetic modification causes overexpression of a xylulose kinase, e.g. by overexpression of a nucleotide sequence encoding a xylulose kinase. The gene encoding the 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 polypeptide with xylulose kinase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 25 or whereby the nucleotide sequence is capable of hybridising with the nucleotide sequence of SEQ ID NO. 26, under moderate conditions, preferably under stringent conditions.

[0056] A particularly preferred xylulose kinase is a xylose kinase that is related to the xylulose kinase xylB from Piromyces as mentioned in WO 03/0624430. A more preferred nucleotide sequence for use in overexpression of xylulose kinase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with xylulose kinase activity, 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 hybridising with the nucleotide sequence of SEQ ID NO. 28, under moderate conditions, preferably under stringent conditions.

[0057] In the host cells of the invention, genetic modification that increases the specific xylulose kinase activity may be combined with any of the modifications increasing the flux of the pentose phosphate pathway as described above, but this combination is not essential for the invention. Thus, a host cell of the invention comprising a genetic modification that increases the specific xylulose kinase activity in addition to the expression of the araA, araB and araD enzymes as defined herein is specifically included in the invention. The various means available in the art for achieving and analysing overexpression of a xylulose kinase in the host cells of the invention are the same as described above for enzymes of the pentose phosphate pathway. Preferably in the host cells of the invention, a xylulose kinase to be overexpressed is overexpressed by at least a factor 1.1,1.2,1.5, 2, 5,10 or 20 as compared to a strain which is genetically identical except for the genetic modification causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.

[0058] In a further preferred embodiment, the host cell of the invention: • expressing araA, araB and araD, and exhibiting the ability to directly isomerise xylose into xylulose, and optionally • comprising a genetic modification that increase the flux of the pentose pathway and/or • further comprising a genetic modification that increases the specific xylulose kinase activity all as earlier defined herein further comprises a genetic modification that reduces unspecific aldose reductase activity in the host cell. Preferably, unspecific aldose reductase activity is reduced in the host cell by one or more genetic modifications that reduce the expression of or inactivate a gene encoding an unspecific aldose reductase, as described in WO 06/009434. Preferably, the genetic modifications reduce or inactivate the expression of each endogenous copy of a gene encoding an unspecific aldose reductase in the host cell. Host cells may comprise multiple copies of genes encoding unspecific aldose reductases as a result of di-, poly- or aneu-ploidy, and/or the host cell may contain several different (iso)enzymes with aldose reductase activity that differ in amino acid sequence and that are each encoded by a different gene. Also in such instances preferably the expression of each gene that encodes an unspecific 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 up- or down-stream of the coding sequence, the (partial) deletion or inactivation of wfnich results in a reduction of expression of unspecific 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 a polypeptide with aldose reductase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 29 or whereby the nucleotide sequence is capable of hybridising with the nucleotide sequence of SEQ ID NO. 30 under moderate conditions, preferably under stringent conditions.

[0059] 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 unspecific aldose reductase activity. The genetic modification leading to the reduction of unspecific aldose reductase activity may be combined with any of the modifications increasing the flux of the pentose phosphate pathway and/or with any of the modifications increasing the specific xylulose kinase activity in the host cells as described above, but these combinations are not essential for the invention. Thus, a host cell expressing araA, araB, and araD, comprising an additional genetic modification that reduces unspecific aldose reductase activity is specifically included in the invention.

In a preferred embodiment, the host cell is CBS 120327 deposited at the CBS Institute (The Netherlands) on September 27th, 2006.

[0060] In a further preferred embodiment, the invention relates to modified host cells that are further adapted to L-arabinose (use L-arabinose and/or convert it into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product and optionally xylose utilisation by selection of mutants, either spontaneous or induced (e.g. by radiation or chemicals), for growth on L-arabinose and optionally xylose, preferably on L-arabinose and optionally xylose as sole carbon source, and more preferably under anaerobic conditions. Selection of mutants may be performed by serial passaging of cultures as e.g. described by Kuyper et al. (2004, FEMS Yeast Res. 4: 655-664) and/or by cultivation under selective pressure in a chemostat culture as is described in Example 4 of WO 06/009434. This selection process may be continued as long as necessary. This selection process is preferably carried out during one week till one year. However, the selection process may be carried out for a longer period of time if necessary. During the selection process, the 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 improved as to its capacities of using L-arabinose and/or xylose, and/or converting L-arabinose into L-ribulose and/or xylulose 5-phosphate and/or a desired fermentation product such as ethanol. In this context "improved cell" may mean that the obtained cell is able to use L-arabinose and/or xylose in a more efficient way than the cell it derives from. For example, the obtained cell is expected to better grow: increase of the specific growth rate of at least 2% than the cell it derives from under the same conditions. Preferably, the increase is of at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more. The specific growth rate may be calculated from ODøøo as known to the skilled person. Therefore, by monitoring the ODøøo, one can deduce the specific growth rate. In this context "improved cell" may also mean that the obtained cell converts L-arabinose into L-ribulose and/or xylulose 5-phosphate and/or a desired fermentation product such as ethanol in a more efficient way than the cell it derives from. For example, the obtained cell is expected to produce higher amounts of L-ribulose and/or xylulose 5-phosphate and/or a desired fermentation product such as ethanol: increase of at least one of these compounds of at least 2% than the cell it derives from under the same conditions. Preferably, the increase is of 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 into xylulose and/or a desired fermentation product such as ethanol in a more efficient way than the cell it derives from. For example, the obtained cell is expected to produce higher amounts of xylulose and/or a desired fermentation product such as ethanol: increase of at least one of these compounds of at least 2% than the cell it derives from under the same conditions. Preferably, the increase is of at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more.

[0061] In a preferred host cell of the invention at least one of the genetic modifications described above, including modifications obtained by selection of mutants, confer to the host cell the ability to grow on L-arabinose and optionally xylose as carbon source, preferably as sole carbon source, and preferably under anaerobic conditions. Preferably the modified host cell produce essentially no xylitol, e.g. the xylitol produced is below the detection limit or e.g. less than 5, 2, 1, 0.5, or 0.3 % of the carbon consumed on a molar basis.

[0062] Preferably the modified host cell has the ability to grow on L-arabinose and optionally xylose as 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"^ under anaerobic conditions Preferably the modified host cell has the ability to grow on a mixture of glucose and L-arabinose and optionally xylose (in a 1:1 weight ratio) as 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"^ 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"^ under anaerobic conditions.

[0063] Preferably, the modified host cell has a specific L-arabinose and optionally xylose consumption rate of at least 346, 350, 400, 500, 600, 650, 700, 750, 800, 900 or 1000 mg /g cells/h. Preferably, the modified host cell has a yield of fermentation product (such as ethanol) on L-arabinose and optionally xylose that is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 85, 90, 95 or 98% of the host cell's yield of fermentation product (such as ethanol) on glucose. More preferably, the modified host cell's yield of fermentation product (such as ethanol) on L-arabinose and optionally xylose is equal to the host cell's yield of fermentation product (such as ethanol) on glucose. Likewise, the modified host cell's biomass yield on L-arabinose and optionally xylose is preferably at least 55, 60, 70, 80, 85, 90, 95 or 98% of the host cell's biomass yield on glucose. More preferably, the modified host cell's biomass yield on L-arabinose and optionally xylose is equal to the host cell's biomass yield on glucose. It is understood that in the comparison of yields on glucose and L-arabinose and optionally xylose both yields are compared under aerobic conditions or both under anaerobic conditions.

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

[0064] In a preferred embodiment, the cell expresses one or more enzymes that confer to the cell the ability to produce at least one fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic 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 more preferred embodiment, the host cell of the invention is a host cell for the production of ethanol. In another preferred embodiment, the invention relates to a transformed host cell for the production of fermentation products other than ethanol. Such non-ethanolic fermentation products include in principle any bulk or fine chemical that is producible by a eukaryotic microorganism such as a yeast or a filamentous fungus. Such fermentation products include e.g. lactic acid, 3-hydroxy-propionic 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.A preferred host cell of the invention for production of non-ethanolic fermentation products is a host cell that contains a genetic modification that results in decreased alcohol dehydrogenase activity.

Method [0065] In a further aspect, the invention relates to fermentation processes in which a host cell of the invention is used for the fermentation of a carbon source comprising a source of L-arabinose and optionally a source of xylose. 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 may also comprise a source of glucose. The source of L-arabinose, xylose or glucose may be L-arabinose, xylose or glucose as such or may be any carbohydrate oligo- or polymer comprising L-arabinose, xylose or glucose units, such as e.g. lignocellulose, xylans, cellulose, starch, arabinan and the like. For release of xylose or glucose units from such carbohydrates, appropriate carbohydrases (such as xylanases, glucanases, 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 engineered to produce and excrete such carbohydrases. An additional advantage of using oligo- or polymeric sources of glucose is that it enables to maintain a low(er) concentration of free glucose during the fermentation, e.g. by using rate-limiting amounts of the carbohydrases. This, in turn, will prevent repression of systems required for metabolism and transport of non-glucose sugars such as xylose. In a preferred process the modified host cell ferments both the L-arabinose (optionally xylose) and glucose, preferably simultaneously in which case preferably a modified host cell is used which is insensitive to glucose repression to prevent diauxic growth. In addition to a source of L-arabinose, optionally xylose (and glucose) as carbon source, the fermentation medium will further comprise the appropriate ingredient required for growth of the modified host cell. Compositions of fermentation media for growth of microorganisms such as yeasts or filamentous fungi are well known in the art.

[0066] In a preferred process, there is provided a process for producing a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic 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: 1. (a) fermenting a medium containing a source of L-arabinose and optionally xylose with a modified host cell as defined herein, whereby the host cell ferments L-arabinose and optionally xylose to the fermentation product, and optionally, 2. (b) recovering the fermentation product.

[0067] The fermentation process is a process for the production of a fermentation product such as e.g. ethanol, lactic acid, 3-hydroxy-propionic 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, such as Penicillin G or Penicillin V and fermentative derivatives thereof, and/or a cephalosporin. The fermentation process may be an aerobic or an anaerobic fermentation process. An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors. In the absence of oxygen, NADH produced in glycolysis and biomass formation, cannot be oxidised by oxidative phosphorylation. To solve this problem many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor thereby regenerating NAD+. Thus, in a preferred anaerobic fermentation process pyruvate is used as an electron (and hydrogen acceptor) and is reduced to fermentation products such as ethanol, lactic acid, 3-hydroxy-propionic 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 antibiotics and a cephalosporin. In a preferred embodiment, the fermentation process is anaerobic. An anaerobic process is advantageous since it is cheaper than aerobic processes: less special equipment is needed. Furthermore, anaerobic processes are expected to give a higher product yield than aerobic processes. Under aerobic conditions, usually the biomass yield is higher than under anaerobic conditions. As a consequence, usually 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 under oxygen-limited conditions. More preferably, the fermentation process is aerobic and under oxygen-limited conditions. An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gasflow as well as the actual mixing/mass transfer properties of the fermentation equipment used. Preferably, in a process under oxygen-limited conditions, the rate of oxygen consumption is at least 5.5, more preferably at least 6 and even more preferably at least 7 mmol/L/h.

[0068] The fermentation process is preferably run at a temperature that is optimal for the modified cell. Thus, for most yeasts or fungal cells, the fermentation process is performed at a temperature which is less than 42°C, preferably less than 38°C. For yeast or filamentous fungal host cells, the fermentation process is preferably performed at a temperature which is lower than 35, 33, 30 or 28°C and at a temperature which is higher than 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 a source of L-arabinose and optionally xylose with a modified host cell as defined herein, whereby the host cell ferments L-arabinose and optionally xylose to ethanol; and optionally, (b) recovery of the ethanol. The fermentation medium may also comprise a source of glucose that is also fermented to ethanol. In a preferred embodiment, the fermentation process for the production of ethanol is anaerobic. Anaerobic has already been defined earlier herein. In another preferred embodiment, the fermentation process for the production of ethanol is aerobic. In another preferred embodiment, the fermentation process for the production of ethanol is under oxygen-limited conditions, more preferably aerobic and under oxygen-limited conditions. Oxygen-limited conditions have already been defined earlier herein.

[0069] In the process, the volumetric ethanol productivity is preferably at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per litre per hour. The ethanol yield on L-arabinose and optionally xylose and/or glucose in the process preferably is at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is herein defined as a 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-hydroxy-propionic 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. The process preferably comprises the steps of (a) fermenting a medium containing a source of L-arabinose and optionally xylose with a modified host cell as defined herein above, whereby the host cell ferments L-arabinose and optionally xylose to the fermentation product, and optionally, (b) recovery of the fermentation product. In a preferred process, the medium also contains a source of glucose.

[0070] In the fermentation process of the invention leading to the production of ethanol, several advantages can be cited by comparison to known ethanol fermentations processes: • anaerobic processes are possible. • oxygen limited conditions are also possible. • higher ethanol yields and ethanol production rates can be obtained. • the strain used may be able to use L-arabinose and optionally xylose.

[0071] Alternatively to the fermentation processes described above, another fermentation process is provided as a further aspect of the invention wherein, at least two distinct cells are used for the fermentation of a carbon source comprising at least two sources of carbon selected from the group consisting of but not limited thereto: a source of L-arabinose, a source of xylose and a source of glucose. In this fermentation process, "at least two distinct cells" means this process is preferably a co-fermentation process. In one preferred embodiment, two distinct cells are used: one being the one of the invention as earlier defined able to use L-arabinose, and/or to convert it into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product such as ethanol and optionally being able to use xylose, the other one being for example a strain which is able to use xylose and/or convert it into a desired fermentation product such as ethanol as defined in WO 03/062430 and/or WO 06/009434. A cell which is able to use xylose is preferably a strain which exhibits the ability of directly isomerising xylose into xylulose (in one step) as earlier defined herein. These two distinct strains are preferably cultived in the presence of a source of L-arabinose, a source of xylose and optionally a source of glucose. Three distinct cells or more may be co-cultivated and/or three or more sources of carbon may be used, provided at least one cell is able to use at least one source of carbon present and/or to convert it into a desired fermentation product such as ethanol. The expression "use at least one source of carbon" has the same meaning as the expression "use of L-arabinose". The expression "convert it (i.e. a source of carbon) into a desired fermentation product has the same meaning as the expression "convert 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-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, amino acids, 1,3-propane-diol, ethylene, glycerol, butanol, β-lactam antibiotics and cephalosporins, whereby the process comprises the steps of: 1. (a) fermenting a medium containing at least a source of L-arabinose and a source of xylose with a cell of the invention as earlier defined herein and a cell able to use xylose and/or exhibiting the ability to directly isomerise xylose into xylulose, whereby each cell ferments L-arabinose and/or xylose to the fermentation product, and optionally, 2. (b) recovering the fermentation product.

All preferred embodiments of the fermentation processes as described above are also preferred embodiments of this further fermentation processes: identity of the fermentation product, identity of source of L-arabinose and source of xylose, conditions of fermentation (aerobical or anaerobical conditions, oxygen-limited conditions, temperature at which the process is being carried out, productivity of ethanol, yield of ethanol).

Genetic modifications [0072] For overexpression of enzymes in the host cells of the inventions as described above, as well as for additional genetic modification of host cells, preferably yeasts, host cells are transformed with the various nucleic acid constructs of the invention by methods well known in the art. Such methods are e.g. known from standard handbooks, 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 transformation and genetic modification of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671.

[0073] Promoters for use in the nucleic acid constructs for overexpression of enzymes in the host cells of the invention have been described above. In the nucleic acid constructs for overexpression, the 3'-end of the nucleotide acid sequence encoding the enzyme(s) preferably is operably linked to a transcription terminator sequence. Preferably the terminator sequence is operable in a host cell of choice, such as e.g. the yeast species of choice. In any case the choice of the terminator is not critical; it may e.g. be from any yeast gene, although terminators may sometimes work if from a non-yeast, eukaryotic, gene. The transcription termination sequence further preferably comprises a polyadenylation signal. Preferred terminator sequences are the alcohol dehydrogenase (ADH1) and the PGM terminators. More preferably, the ADH1 and the PGM terminators are both from S. cerevisiae (SEQ ID NO:50 and SEQ ID NO:53 respectively).

[0074] Optionally, a selectable marker may be present in the nucleic acid construct. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. The marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Preferably however, non-antibiotic resistance markers are used, such as auxotrophic markers (URA3, TRP1, LEU2). In a preferred embodiment the host cells transformed with the nucleic acid constructs are marker gene free. 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 screenable marker such as Green Fluorescent Protein, lacZ, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase may be incorporated into the nucleic acid constructs of the invention allowing to screen for transformed cells.

[0075] Optional further elements that may be present in the nucleic acid constructs of the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors, and/or reporter genes, intron sequences, centromers, telomers and/or matrix attachment (MAR) sequences. The nucleic acid constructs of the invention may further comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2μ or pKD1 (Fleer et al., 1991, Biotechnology 9:968-975) plasmids. Alternatively the nucleic acid construct may comprise sequences for integration, preferably by homologous recombination. Such sequences may thus be sequences homologous to the target site for integration in the host cell's genome. The nucleic acid constructs of the invention can be provided in a manner known per se, which generally involves techniques such as restricting and linking nucleic acids/nucleic acid sequences, for which reference is made to the standard handbooks, such as Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press.

[0076] Methods for inactivation and gene disruption in yeast or fungi are well known in the art (see e.g. Fincham, 1989, Microbiol Rev. 53(1):148-70 and EP-A-0 635 574).

[0077] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0078] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

Examples

Plasmid and strain construction Strains [0079] The L-arabinose consuming Saccharomyces cerevisiae strain described in this work is based on strain RWB220, which is itself a derivative of RWB217. RWB217 is a CEN.PK strain in which four genes coding for the expression of enzymes in the pentose phosphate pathway have been overexpressed, TAL1, TKL1, RPE1, RKI1 (Kuyper et al., 2005a). In addition the gene coding for an aldose reductase (GRE3), has been deleted. Strain RWB217 also contains two plasmids, a single copy plasmid with a LEU2 marker for overexpression of the xylulokinase (XKS1) and an episomal, multicopy plasmid with URA3 as the marker for the expression of the xylose isomerase, XylA. RWB217 was subjected to a selection procedure for improved growth on xylose which 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 restreaking on mineral medium with glucose as the carbon source, while for RWB219 xylose was used.

Strain RWB219 was grown non-selectively on YP with glucose (YPD) as the carbon source in order to facilitate the loss of both plasmids. After plating on YPD single colonies were tested for plasmid loss by looking at uracil and leucine auxotrophy. A strain that had lost both plasmids was transformed with pSH47, containing the ere recombinase, in order to remove a KanMX cassette (Guldener et al., 1996), still present after integrating the RKI1 overexpression construct. Colonies with the plasmid were resuspended in Yeast Peptone medium (YP) (10g/I yeast extract and 20g/l peptone both from BD Difco Belgium) with 1% galactose and incubated for 1 hour at 30°C. About 200 cells were plated on YPD. The resulting colonies were checked for loss of the KanMX marker (G418 resistance) and pSH47 (URA3). A strain that had lost both the KanMX marker and the pSH47 plasmid was then named RWB220. To obtain the strain tested in this patent, RWB220 was transformed with pRW231 and pRW243 (table 2), resulting in strain IMS0001.

[0080] During construction strains were maintained on complex YP: 10 g I'1 yeast extract (BD Difco), 20 g H 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 in the case of plates. After transformation with plasmids strains were plated on MYD.

[0081] Transformations of yeast were done according to Gietz and Woods (2002). Plasmids were amplified in Escherichia coli strain XL-1 blue (Stratagene, La Jolla, CA, USA). Transformation was performed according to Inoue et al. (1990). E. coli was grown on LB (Luria-Bertani) plates or in liquid TB (Terrific Broth) medium for the isolation of plasmids (Sambrook et al, 1989).

Plasmids [0082] In order to grow on L-arabinose, yeast needs to express three different genes, an L-arabinose isomerase (AraA), a L-ribulokinase (AraB), and a L-ribulose-5-P 4-epimerase (AraD) (Becker and Boles, 2003). In this work we have chosen to express AraA, AraB, and AraD from the lactic acid bacterium Lactobacillus piantarum in S. cerevisiae. Because the eventual aim is to consume L-arabinose in combination with other sugars, like D-xylose, the genes encoding the bacterial L-arabinose pathway were combined on the same plasmid with the genes coding for D-xylose consumption.

[0083] In order to get a high level of expression, the L. piantarum AraA and AraD genes were ligated into plasmid pAKX002, the 2μ XylA bearing plasmid.

The AraA cassette was constructed by amplifying a truncated version of the TDH3 promoter with Spel5'Ptdh3 and 5'AraAPtdh3 (SEQ ID NO: 49), the AraA gene with Ptdh5'AraAand Tadh3'AraAand the ADH1 terminator (SEQ ID NO:50) with 3'AraATadh1 and 3'Tadh1-Spel. The three fragments were extracted from gel and mixed in roughly equimolar amounts. On this mixture a PCR was performed using the Spel-5'Ptdh3 and 3'Tadh1Spel oligos. The resulting PTDH3-AraA-T/\DH1 cassette was gel purified, cut at the 5'and 3' Spel sites and then ligated into pAKX002 cut with Nhel, resulting in plasmid pRW230.

The AraD construct was made by first amplifying a truncated version of the HXT7 promoter (SEQ ID NO:52) with oligos Sall5'Phxt7 and 5'AraDPhxt, the AraD gene with Phxt5' AraD and Tpgi3'AraD and the GPU terminator (SEQ ID NO:53) region with the 3'AraDTpgi and 3'TpgiSall oligos. The resulting fragments were extracted from gel and mixed in roughly equimolar amounts, after which a PCR was performed using the Sall5'Phxt7 and 3Tpgi1 Sail oligos. The resulting Pnxr7-AraD-Tpeg cassette was gel purified, cut at the 5'and 3' Sail sites and then ligated into pRW230 cut with Xhol, resulting in plasmid pRW231 (Figure 1).

[0084] Since too high an expression of the L-ribulokinase is detrimental to growth (Becker and Boles, 2003), the AraB gene was combined with the XKS1 gene, coding for xylulokinase, on an integration plasmid. For this, p415ADHXKS (Kuyper et al., 2005a) was first changed into pRW229, by cutting both p415ADFIXKS and pRS305 with Pvul and ligating the ADFIXKS-containing Pvul fragment from p415ADHXKS to the vector backbone from pRS305, resulting in pRW229. A cassette, containing the L. piantarum AraB gene between the PGM promoter (SEQ ID NO:51) and ADH1 terminator (SEQ ID NO:50) was made by amplifying the PGM promoter with the Sacl5'Ppgi1 and 5'AraBPpgi1 oligos, the AraB gene with the Ppgi5'AraB and Tadh3'AraB oligos and the ADH1 terminator with 3'AraBTadhl and 3'Tadh1Sacl oligos. The three fragments were extracted from gel and mixed in roughly equimolar amounts. On this mixture a PCR was performed using the Sacl-5'Ppgi1 and 3'Tadh1Sacl oligos. The resulting PpG|-|-AraB-T/\DH1 cassette was gel purified, cut at the 5'and 3' Sacl sites and then ligated into pRW229 cut with Sacl, resulting in plasmid pRW243 (Figure 1).

Strain RWB220 was transformed with pRW231 and pRW243 (table 2), resulting in strain IMS0001.

[0085] Restriction endonucleases (New England Biolabs, Beverly, MA, USA and Roche, Basel, Switzerland) and DNA ligase (Roche) were used according to the manufacturers' specifications. Plasmid isolation from E. coli was performed with the Qiaprep spin miniprep kit (Qiagen, Filden, Germany). DNA fragments were separated on a 1% agarose (Sigma, St. Louis, MO, USA) gel in 1*TBE (Sambrook et al, 1989). Isolation of fragments from gel was carried out with the Qiaquick gel extraction kit (Quiagen). Amplification of the (elements of the) AraA, AraB and AraD cassettes was done with Vent r DNA polymerase (New England Biolabs) according to the manufacturer's specification. The template was chromosomal DNA of S. cerevisiae CEN.PK113-7D for the promoters and terminators, or Lactobacillus piantarum DSM20205 for the Ara genes. The polymerase chain reaction (PCR) was performed in a Biometra TGradient Thermocycler (Biometra, Gottingen, Germany) with the following settings: 30 cycles of 1 min annealing at 55°C, 60°C or 65°C, 1 to 3 min extension at 75°C, depending on expected fragment size, and 1 min denaturing at 94°C.

Cultivation and media [0086] Shake-flask cultivations were performed at 30°C in a synthetic medium (Verduyn et al., 1992). The pH of the medium was adjusted to 6.0 with 2 Μ KOH prior to sterilisation. For solid synthetic medium, 1.5% of agar was added.

Pre-cultures were prepared by inoculating 100 ml medium containing the appropriate sugar in a 500-ml shake flask with a frozen stock culture. After incubation at 30°C in an orbital shaker (200 rpm), this culture was used to inoculate either shake-flask cultures or fermenter cultures. The synthetic medium for anaerobic cultivation was supplemented with 0.01 g I"1 ergosterol and 0.42 g I"1 Tween 80 dissolved in ethanol (Andreasen and Stier, 1953; Andreasen and Stier, 1954). Anaerobic (sequencing) batch cultivation was carried out at 30 °C in 2-1 laboratory fermenters (Applikon, Schiedam, The Netherlands) with a working volume of 1 I. The culture pH was maintained at pH 5.0 by automatic addition of 2 M KOH. Cultures were stirred at 800 rpm and sparged with 0.5 1 min'1 nitrogen gas (<10 ppm oxygen). To minimise diffusion of oxygen, fermenters were equipped with Norprene tubing (Cole Palmer Instrument company, Vernon Hills, USA). Dissolved oxygen was monitored with an oxygen electrode (Applisens, Schiedam, The Netherlands). Oxygen-limited conditions were achieved in the same experimental set-up by headspace aeration at approximately 0.05 I min"1.

Determination of dry weight [0087] Culture samples (10.0 ml) were filtered over preweighed nitrocellulose filters (pore size 0.45 Im; Gelman laboratory, Ann Arbor, USA). After removal of medium, the filters were washed with demineralised water and dried in a microwave oven (Bosch, Stuttgart, Germany) for 20 min at 360 W and weighed. Duplicate determinations varied by less than 1%.

Gas analysis [0088] Exhaust gas was cooled in a condensor (2 °C) and dried with a Permapure dryer type MD-110-48P-4 (Permapure, Toms River, USA). 02 and C02 concentrations were determined with a NGA 2000 analyser (Rosemount Analytical, Orrville, USA). Exhaust gasflow rate and specific oxygen-consumption and carbondioxide production rates were determined as described previously (Van Urk et al., 1988;Weusthuis et al., 1994). In calculating these biomass-specific rates, volume changes caused by withdrawing culture samples were taken account for.

Metabolite analysis [0089] Glucose, xylose, arabinose, xylitol, organic acids, glycerol and ethanol were analysed by HPLC using a Waters Alliance 2690 HPLC (Waters, Milford, USA) supplied with a BioRad HPX87H column (BioRad, Hercules, USA), a Waters 2410 refractive-index detector and aWaters 2487 UVdetector. The column was eluted at 60 °C with 0.5 g I"1 sulphuric acid at a flowrate of 0.6 ml min"1.

Assay for xylulose 5-phosphate (Zaldivar J., et al, Appl. Microbiol. Biotechnol., (2002), 59:436-442) [0090] For the analysis of intracellular metabolites such as xylulose 5-phosphate, 5 ml broth was harvested in duplicate from the reactors, before glucose exhaustion (at 22 and 26 h of cultivation) and after glucose exhaustion (42, 79 and 131 h of cultivation). Procedures for metabolic arrest, solid-phase extraction of metabolites and analysis have been described in detail by Smits H.P. et al. (Anal. Biochem., 261:36-42, (1998)). However, the analysis by high-pressure ion exchange chromatography coupled to pulsed amperometric detection used to analyze cell extracts, was slightly modified. Solutions used were eluent A, 75 mM NaOH, and eluent B, 500 mM NaAc. To prevent contamination of carbonate in the eluent solutions, a 50% NaOH solution with low carbonate concentration (Baker Analysed, Deventer, The Netherlands) was used instead of NaOH pellets. The eluents were degassed with Helium (He) for 30 min and then kept under a He atmosphere. The gradient pump was programmed to generate the following gradients: 100% A and 0% B (0 min), a linear decrease of A to 70% and a linear increase of B to 30% (0-30 min), a linear decrease of A to 30% and a linear increase of B to 70% (30-70 min), a linear decrease of A to 0% and a linear increase of B to 100% (70-75 min), 0% Aand 100% B (75-85 min), a linear increase of Ato 100% and a linear decrease of B to 0% (85-95 min). The mobile phase was run at a flowrate of 1 ml/min. Other conditions were according to Smits etal. (1998).

Carbon recovery [0091] Carbon recoveries were calculated as carbon in products formed, divided by the total amount of sugar carbon consumed, and were based on a carbon content of biomass of 48%. To correct for ethanol evaporation during the fermentations, the amount of ethanol produced was assumed to be equal to the measured cumulative production of CO2 minus the CO2 production that occurred due to biomass synthesis (5.85 mmol CO2 per gram biomass (Verduyn et al., 1990)) and the CO2 associated with acetate formation.

Selection for growth on L-arabinose [0092] Strain IMS0001 (CBS 120327 deposited at the CBS on 27/09/06), containing the genes encoding the pathways for both xylose (XylAand XKS1) and arabinose (AraA, AraB, AraD) metabolization, was constructed according the procedure described above. Although capable of growing on xylose (data not shown), strain IMS0001 did not seem to be capable of growing on solid synthetic medium supplemented with 2% L-arabinose. Mutants of IMS0001 capable of utilizing L-arabinose as carbon source for growth were selected by serial transfer in shake flasks and by sequencing-batch cultivation in fermenters (SBR).

For the serial transfer experiments, a 500-ml shake flask containing 100 ml synthetic medium containing 0.5% galactose were inoculated with either strain IMS0001, or the reference strain RWB219. After 72 hours, at an optical density at 660 nm of 3.0, the cultures were used to inoculate a new shake flask containing 0.1% galactose and 2% arabinose. Based on HPLC determination with D-ribulose as calibration standard, it was determined that already in the first cultivations of strain IMS0001, on medium containing a galactose/arabinose mixture, part of the arabinose was converted into ribulose and subsequently excreted to the supernatant. These HPLC analyses were performed using a Waters Alliance 2690 HPLC (Waters, Milford, USA) supplied 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 I"1 sulphuric acid at a flow rate of 0.6 ml min'1. In contrast to the reference strain RWB219, the OD660 °f the culture of strain IMS0001 increased after depletion of the galactose. When after approximately 850 hours growth on arabinose by strain IMS0001 was observed (figure 2), this culture was transferred at an ODfS60 of 1.7 to a shake flask containing 2% arabinose. Cultures were then sequentially transferred to fresh medium containing 2% arabinose at an Οϋββο of 2- 3. Utilization of arabinose was confirmed by occasionally measuring arabinose concentrations by HPLC (data not shown). The growth rate of these cultures increased from 0 to 0.15 h"1 in approximately 3600 hours (figure 3).

[0093] A batch fermentation under oxygen limited conditions was started by inoculating 1 I of synthetic medium supplemented with 2% of arabinose with a 100 ml shake flask culture of arabinose-grown IMS0001 cells with a maximum growth rate on 2% of L- arabinose of approximately 0.12 h"1. When growth on arabinose was observed, the culture was subjected to anaerobic conditions by sparging with nitrogen gas. The sequential cycles of anaerobic batch cultivation were started by either manual or automated replacement of 90% of the culture with synthetic medium with 20 g I"1 arabinose. For each cycle during the SBR fermentation, the exponential growth rate was estimated from the CO2 profile (figure 4). In 13 cycles, the exponential growth rate increased from 0.025 to 0.08 h"1. After 20 cycles a sample was taken, and plated on solid synthetic medium supplemented with 2% of L-arabinose and incubated at 30°C for several days. Separate colonies were re-streaked twice on solid synthetic medium with L-arabinose. Finally, a shake flask containing synthetic medium with 2% of L-L-arabinose was inoculated with a single colony, and incubated for 5 days at 30°C. This culture was designated as strain IMS0002 (CBS 120328 deposited at the Centraal Bureau voor Schimmelculturen (CBS) on 27/09/06). Culture samples were taken, 30% of glycerol was added and samples were stored at -80°C.

Mixed culture fermentation [0094] Biomass hydrolysates, a desired feedstock for industrial biotechnology, contain complex mixtures consisting of various sugars amongst which glucose, xylose and arabinose are commonly present in significant fractions. To accomplish ethanolic fermentation of not only glucose and arabinose, but also xylose, an anaerobic batch fermentation was performed with a mixed culture of the arabinose-fermenting strain IMS0002, and the xylose-fermenting strain RWB218. An anaerobic batch fermenter containing 800 ml of synthetic medium supplied with 30 g Γ1 D-glucose, 15 g Γ1 D-xylose, and 15 g I"1 L-arabinose was inoculated with 100 ml of pre-culture of strain IMS0002. After 10 hours, a 100 ml inoculum of RWB218 was added. In contrast to the mixed sugar fermentation with only strain IMS0002, both xylose and arabinose were consumed after glucose depletion (Fig. 5D). The mixed culture completely consumed all sugars, and within 80 hours 564.0 + 6.3 mmol I"1 ethanol (calculated from the CO2 production) was produced with a high overall yield of 0.42 g g"1 sugar. Xylitol was produced only in small amounts, to a concentration of 4.7 mmol I'”'.

Characterization of strain IMS0002 [0095] Growth and product formation of strain IMS0002 was determined during anaerobic batch fermentations on synthetic medium with either L-arabinose as the sole carbon source, or a mixture of glucose, xylose and L-arabinose. The pre-cultures for these anaerobic batch fermentations were prepared in shake flasks containing 100 ml of synthetic medium with 2% L-arabinose, by inoculating with -80°C frozen stocks of strain IMS0002, and incubating for 48 hours at 30°C.

[0096] Figure 5A shows that strain IMS0002 is capable of fermenting 20 g I"1 L-arabinose to ethanol during an anaerobic batch fermentation of approximately 70 hours. The specific growth rate under anaerobic conditions with L-arabinose as sole carbon source was 0.05 ± 0.001 h"1. Taking into account the ethanol evaporation during the batch fermentation, the ethanol yield from 20 g I'1 arabinose was 0.43 ± 0.003 g g'1. Without evaporation correction the ethanol yield was 0.35 ± 0.01 g g'1 of arabinose. No formation of arabinitol was observed during anaerobic growth on arabinose.

In Figure 5B, the ethanolic fermentation of a mixture of 20 g Γ"* glucose and 20 g I"1 L-arabinose by strain IMS0002 is shown. L-arabinose consumption started after glucose depletion. Within 70 hours, both the glucose and L-arabinose were completely consumed. The ethanol yield from the total of sugars was 0.42 ± 0.003 g g"1.

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

Table 1 shows the arabinose consumption rates and the ethanol production rates observed for the anaerobic batch fermentation of strain IMS0002. Arabinose was consumed with a rate of 0.23 - 0.75 g h""* g'1 biomass dry weight. The rate of ethanol produced from arabinose varied from 0.08 - 0.31 g h"1 g"1 biomass dry weight.

[0097] Initially, the constructed strain IMS0001 was able to ferment xylose (data not shown). In contrast to our expectations, the selected strain IMS0002 was not capable of fermenting xylose to ethanol (Fig 5C). To regain the capability of fermenting xylose, a colony of strain IMS0002 was transferred to solid synthetic medium with 2% of D-xylose, and incubated in an anaerobic jar at 30°C for 25 days. Subsequently, a colony was again transferred to solid synthetic medium with 2% of arabinose. After 4 days of incubation at 30°C, a colony was transferred to a shake flask containing synthetic medium with 2% arabinose. After incubation at 30°C for 6 days, 30% of glycerol was added, samples were taken and stored at -80°C. A shake flask containing 100 ml of synthetic medium with 2% arabinose was inoculated with such a frozen stock, and was used as preculture for an anaerobic batch fermentation on synthetic medium with 20 g I"1 xylose and 20 g I'1 arabinose. In figure 6, the fermentation profile of this batch fermentation is shown. Xylose and arabinose were consumed simultaneously. The arabinose was completed within 70 hours, whereas the xylose was completely consumed in 120 hours. At least 250 mM of ethanol was produced from the total of sugars, not taking into account the evaporation of the ethanol. Assuming an end biomass dry weight of 3.2 g I"1 (assuming a biomass yield of 0.08 g g'1 sugar), the end ethanol concentration estimated from the cumulative CO2 production (355 mmol I'1) was approximately 330 mmol I"1, corresponding to a 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).

Selection of strain IMS0003 [0098] Initially, the constructed strain IMS0001 was able to ferment xylose (data not shown). In contrast to our expectations, the selected strain IMS0002 was not capable of fermenting xylose to ethanol (Fig 5C). To regain the capability of fermenting xylose, a colony of strain IMS0002 was transferred to solid synthetic medium with 2% of D-xylose, and incubated in an anaerobic jar at 30°C for 25 days. Subsequently, a colony was again transferred to solid synthetic medium with 2% of arabinose. After 4 days of incubation at 30°C, a colony was transferred to a shake flask containing synthetic medium with 2% arabinose. After incubation at 30°C for 6 days, 30% of glycerol was added, samples were taken and stored at -80°C.

From this frozen stock, samples were spread on solid synthetic medium with 2% of L-arabinose and incubated at 30°C for several days. Separate colonies were re-streaked twice on solid synthetic medium with L-arabinose. Finally, a shake flask containing synthetic medium with 2% of L-arabinose was inoculated with a single colony, and incubated for 4 days at 30°C. This culture was designated as strain IMS0003 (CBS 121879 deposited at the CBS on 20/09/07). Culture samples were taken, 30% of glycerol was added and samples ware stored at -80°C.

Characterization of strain IMS0003 [0099] Growth and product formation of strain IMS0003 was determined during an anaerobic batch fermentation on synthetic medium with a mixture of 30 g I'1 glucose, 15 g I'1 D-xylose and 15 g Γ1 L-arabinose. The pre-culture for this anaerobic batch fermentation was prepared in a shake flasks containing 100 ml of synthetic medium with 2% L-arabinose, by inoculating with a -80°C frozen stock of strain IMS0003, and incubated for 48 hours at 30°C.

In figure 7, the fermentation profile of a mixture of 30 g Γ1 glucose, 15 g Γ1 D-xylose, and 15 g Γ1 L-arabinose by strain IMS0003 is shown. Arabinose consumption started after glucose depletion. Within 70 hours, the glucose, xylose and arabinose were completely consumed. Xylose and arabinose were consumed simultaneously. At least 406 mM of ethanol was produced from the total of sugars, not taking into account the evaporation of the ethanol. The final ethanol concentration calculated from the cumulative CO2 production was 572 mmol I'1, corresponding to an ethanol yield of 0.46 g g'1 of total sugar. In contrast to the fermentation of a mixture of glucose, xylose and arabinose by strain IMS0002 (figure 5C) or a mixed culture of strains IMS0002 and RWB218 (figure 5D), strain IMS0003 did not produce detectable amounts of xylitol.

TABLES

[0100]

Table 1: S. cerevisiae strains used. "TοΚΙλ Q· r\linr\c 1 icc»H in thic \AinrL·

Reference List [0101]

Andreasen AA, Stier TJ (1954) Anaerobic nutrition of Saccharomyces cerevisiae. II. Unsaturated fatty acid requirement for growth in a defined medium. J Cell Physiol 43:271-281

Andreasen AA, Stier TJ (1953) Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J Cell Physiol 41:23-36

Becker J, Boles E (2003) A modified Saccharomyces cerevisiae strain that consumes L-Arabinose and produces ethanol. Appl Environ Microbiol 69:4144-4150

Gietz R.D., Sugino A. (1988). Newyeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527-534.

Gietz, R. D., and R. A. Woods. 2002. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350:87-96.

Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996 Jul 1;24(13):2519-24.

Hauf J, Zmmermann FK, Muller S. Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae. Enzyme Microb Technol. 2000 Jun 1;26(9-10):688-698.

Inoue Η., H. Nojima and H. Okayama, Ugh efficiency transformation of Escherichia coli with plasmids. Gene 96 (1990), pp. 23-28

Kuyper M, Hårtog MMP, Toirkens MJ, Almering MJH, Winkler AA, Van Dijken JP, Prank JT (2005a) Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. Ferns Yeast Research 5:399-409

Kuyper M, Toirkens MJ, Diderich JA, Winkler AA, Van Dijken JP, Prank JT (2005b) Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. Ferns Yeast Research 5:925-934

Sambrook, K., Fritsch, E.F. and Maniatis, I. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Van Urk H, Mak PR, Scheffers WA, Van Dijken JP (1988) Metabolic responses of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose limitation to glucose excess. Yeast 4:283-291

Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1990) Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J Gen Microbiol 136:395-403

Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501-517

Weusthuis RA, Visser W, Prank JT, Scheffers WA, Van Dijken JP (1994) Effects of oxygen limitation on sugar metabolism in yeasts - a continuous-culture study of the Kluyver effect. Microbiology 140:703-715

SEQUENCE LISTING

[0102] <110> TU Delft AJA van Maris JT Prank HW Wisselink JP van Dijk AA Winkler JH de Winde <120> Metabolic engineering of arabinose-fermenting eukaryotic cells <130> P6011342PCT <150> EP06121633.9 <151 >2006-10-02 <150> US 60/848,357 <151 >2006-10-02 <160> 53 <170> Patentln version 3.3

<210> 1 <211>474 <212> PRT <213> Lactobacillus plantarum <400> 1

Met Leu Ser Val Pro Asp Tyr Glu Phe Trp Phe Val Thr Gly Ser Gin 15 10 15

His Leu Tyr Gly Glu Glu Gin Leu Lys ser val Ala Lys Asp Ala Gin 20 25 30

Asp Ile Ala Asp Lys Leu Asn Ala Ser Gly Lys Leu Pro Tyr Lys Val 35 40 45

Val Phe Lys Asp Val Met Thr Thr Ala Glu Ser Ile Thr Asn Phe Met 50 55 60

Lys Glu Val Asn Tyr Asn Asp Lys Val Ala Gly Val ile Thr Trp Met 65 70 75 80

His Thr Phe ser pro Ala Lys Asn Trp Ile Arg Gly Thr Glu Leu Leu 85 90 95

Gin Lys Pro Leu Leu His Leu Ala Thr Gin Tyr Leu Asn Asn ile Pro 100 105 110

Tyr Ala Asp ile Asp Phe Asp Tyr Met Asn Leu Asn Gin Ser Ala His 115 120 125

Gly Asp Arg Glu Tyr Ala Tyr Ile Asn Ala Arg Leu Gin Lys His Asn 130 135 140

Lys Ile val Tyr Gly Tyr Trp Gly Asp Glu Asp Val Gin Glu Gin Ile 145 150 155 160

Ala Arg Trp Glu Asp Val Ala Val Ala Tyr Asn Glu Ser Phe Lys Val 165 170 175

Lys val Ala Arg Phe Gly Asp Thr Met Arg Asn Val Ala val Thr Glu 180 185 190

Gly Asp Lys Val Glu Ala Gin Ile Lys Met Gly Trp Thr val Asp Tyr 195 200 205

Tyr Gly ile Gly Asp Leu val Glu Glu ile Asn Lys val ser Asp Ala 210 215 220

Asp val Asp Lys Glu Tyr Ala Asp Leu Glu ser Arg Tyr Glu Met Val 225 230 235 240

Gin val Asp Asn Asp Ala Asp Thr Tyr Lys His Ser Val Arg Val Gin 245 250 255

Leu Ala Gin Tyr Leu Gly Ile Lys Arg Phe Leu Glu Arg Gly Gly Tyr 260 265 270

Thr Ala Phe Thr Thr Asn Phe Glu Asp Leu Trp Gly Met Glu Gin Leu 275 280 285

Pro Gly Leu Ala Ser Gin Leu Leu ile Arg Asp Gly Tyr Gly Phe Gly 290 295 300

Ala Glu Gly Asp Trp Lys Thr Ala Ala Leu Gly Arg Val Met Lys Ile 305 310 315 320

Met Ser His Asn Lys Gin Thr Ala Phe Met Glu Asp Tyr Thr Leu Asp 325 330 335

Leu Arg His Gly His Glu Ala Ile Leu Gly ser His Met Leu Glu Val 340 345 350

Asp Pro Ser Ile Ala Ser Asp Lys Pro Arg Val Glu Val His Pro Leu 355 360 365

Asp Ile Gly Gly Lys Asp Asp Pro Ala Arg Leu val Phe Thr Gly Ser 370 375 380

Glu Gly Glu Ala Ile Asp val Thr val Ala Asp Phe Arg Asp Gly Phe 385 390 395 400

Lys Met Ile Ser Tyr Ala val Asp Ala Asn Lys Pro Glu Ala Glu Thr 405 410 415

Pro Asn Leu Pro Val Ala Lys Gin Leu Trp Thr Pro Lys Met Gly Leu 420 425 430

Lys Lys Gly Ala Leu Glu Trp Met Gin Ala Gly Gly Gly His His Thr 435 440 445

Met Leu Ser Phe Ser Leu Thr Glu Glu Gin Met Glu Asp Tyr Ala Thr 450 455 460

Met Val Gly Met Thr Lys Ala Phe Leu Lys 465 470

<210> 2 <211>1425 <212> DNA <213> Lactobacillus plantarum <400>2 atgttatcag tacctgatta tgagttttgg tttgttaccg gttcacaaca cctttatggt 60 gaagaacaat tgaagtctgt tgctaaggat gcgcaagata ttgcggataa attgaatgca 120

agcggcaagt taccttataa agtagtcttt aaggatgtta tgacgacggc tgaaagtatc . ISO accaacttta tgaaagaagt taattacaat gataaggtag ccggtgttat tacttggatg 240 cacacattct caccagctaa gaactggatt cgtggaactg aactgttaca aaaaccatta 300 ttacacttag caacgcaata tttgaataat attccatatg cagacattga ctttgattac 360 atgaacctta accaaagtgc ccatggcgac cgcgagtatg cctacattaa cgcccggttg 420 cagaaacata ataagattgt ttacggctat tggggcgatg aagatgtgca agagcagatt 480 gcacgttggg aagacgtcgc cgtagcgtac aatgagagct ttaaagttaa ggttgctcgc 540 tttggcgaca caatgcgtaa tgtggccgtt actgaaggtg acaaggttga agctcaaatt 600 aagatgggct ggacagttga ctattatggt atcggtgact tagttgaaga gatcaataag 660 gtttcggatg ctgatgttga taaggaatac gctgacttgg agtctcggta tgaaatggtc 720 caagttgata acgatgcgga cacgtataaa cattcagttc gggttcaatt ggcacaatat 780 ctgggtatta agcggttctt agaaagaggc ggttacacag cctttaccac gaactttgaa 840 gatctttggg ggatggagca attacctggt ctagcttcac aattattaat tcgtgatggg 900 tatggttttg gtgctgaagg tgactggaag acggctgctt taggacgggt tatgaagatt 960 atgtctcaca acaagcaaac cgcctttatg gaagactaca cgttagactt gcgtcatggt 1020 catgaagcga tcttaggttc acacatgttg gaagttgatc cgtctatcgc aagtgataaa 1080 ccacgggtcg aagttcatcc attggatatt gggggtaaag atgatcctgc tcgcctagta 1140 tttactggtt cagaaggtga agcaattgat gtcaccgttg ccgatttccg tgatgggttc 1200 aagatgatta gctacgcggt agatgcgaat aagccagaag ccgaaacacc taatttacca 1260 gttgctaagc aattatggac cccaaagatg ggcttgaaga agggtgcact agaatggatg 1320 caagctggtg gtggtcacca cacgatgctg tccttctcgt taactgaaga acaaatggaa 1380 gactatgcaa ccatggttgg catgactaag gcattcttaa agtaa 1425

<210> 3 <211> 533 <212> PRT <213> Lactobacillus plantarum <400>3

Met Asn Leu val Glu Thr Ala Gin Ala ile Lys Thr Gly Lys val Ser 15 10 15

Leu Gly ile Glu Leu Gly Ser Thr Arg ile Lys Ala val Leu ile Thr 20 25 30

Asp Asp Phe Asn Thr ile Ala Ser Gly Ser Tyr val Trp Glu Asn Gin 35 40 45

Phe Val Asp Gly Thr Trp Thr Tyr Ala Leu Glu Asp Val Trp Thr Gly 50 55 60

Ile Gin Gin Ser Tyr Thr Gin Leu Ala Ala Asp Val Arg Ser Lys Tyr 65 70 75 80

His Met Ser Leu Lys His ile Asn Ala ile Gly Ile Ser Ala Met Met 85 90 95

His Gly Tyr Leu Ala Phe Asp Gin Gin Ala Lys Leu Leu val Pro Phe 100 105 110

Arg Thr Trp Arg Asn Asn ile Thr Gly Gin Ala Ala Asp Glu Leu Thr 115 120 125

Glu Leu Phe Asp Phe Asn ile Pro Gin Arg Trp Ser ile Ala His Leu 130 135 140

Tyr Gin Ala Ile Leu Asn Asn Glu Ala His val Lys Gin Val Asp Phe 145 150 155 160 ile Thr Thr Leu Ala Gly Tyr Val Thr Trp Lys Leu ser Gly Glu Lys 165 170 175 val Leu Gly ile Gly Asp Ala ser Gly val Phe Pro ile Asp Glu Thr 180 185 190

Thr Asp Thr Tyr Asn Gin Thr Met Leu Thr Lys Phe Ser Gin Leu Asp 195 200 205

Lys Val Lys Pro Tyr Ser Trp Asp Ile Arg His Ile Leu Pro Arg Val 210 215 220

Leu Pro Ala Gly Ala ile Ala Gly Lys Leu Thr Ala Ala Gly Ala Ser 225 230 235 240

Leu Leu Asp Gin ser Gly Thr Leu Asp Ala Gly ser Val lie Ala Pro 245 250 255

Pro Glu Gly Asp Ala Gly Thr Gly Met Val Gly Thr Asn Ser Val Arg 260 265 270

Lys Arg Thr Gly Asn lie Ser Val Gly Thr ser Ala Phe ser Met Asn 275 280 285

Val Leu Asp Lys Pro Leu ser Lys Val Tyr Arg Asp lie Asp lie Val 290 295 300

Met Thr Pro Asp Gly Ser Pro Val Ala Met Val His Val Asn Asn Cys 305 310 315 320 ser ser Asp lie Asn Ala Trp Ala Thr lie Phe Arg Glu Phe Ala Ala 325 330 335

Arg Leu Gly Met Glu Leu Lys pro Asp Arg Leu Tyr Glu Thr Leu Phe ; 340 345 350

Leu Glu ser Thr Arg Ala Asp Ala Asp Ala Gly Gly Leu Ala Asn Tyr 355 360 365

Ser Tyr Gin ser Gly Glu Asn lie Thr Lys lie Gin Ala Gly Arg pro 370 375 380

Leu Phe Val Arg Thr Pro Asn ser Lys Phe ser Leu Pro Asn Phe Met 385 390 395 400

Leu Thr Gin Leu Tyr Ala Ala Phe Ala Pro Leu Gin Leu Gly Met Asp 405 410 415 lie Leu val Asn Glu Glu His Val Gin Thr Asp val Met lie Ala Gin 420 425 430

Gly Gly Leu Phe Arg Thr Pro Val lie Gly Gin Gin Val Leu Ala Asn 435 440 445

Ala Leu Asn lie pro lie Thr val Met Ser Thr Ala Gly Glu Gly Gly 450 455 460 pro Trp Gly Met Ala Val Leu Ala Asn Phe Ala cys Arg Gin Thr Ala 465 470 475 480

Met Asn Leu Glu Asp Phe Leu Asp Gin Glu Val Phe Lys Glu Pro Glu 485 490 495 ser Met Thr Leu ser Pro Glu Pro Glu Arg val Ala Gly Tyr Arg Glu 500 505 510

Phe lie Gin Arg Tyr Gin Ala Gly Leu Pro Val Glu Ala Ala Ala Gly 515 520 525

Gin Ala lie Lys Tyr 530 <210> 4 <211> 1602

<212> DNA <213> Lactobacillus plantarum <400>4 atgaatttag ttgaaacagc ccaagcgatt aaaactggca aagtttcttt aggaattgag 60 cttggctcaa ctcgaattaa agccgttttg atcacggacg attttaatac gattgcttcg 120 ggaagttacg tttgggaaaa ccaatttgtt gatggtactt ggacttacgc acttgaagat 180 gtctggaccg gaattcaaca aagttatacg caattagcag cagatgtccg cagtaaatat 240 cacatgagtt tgaagcatat caatgctatt ggcattagtg ccatgatgca cggataccta 300 gcatttgatc aacaagcgaa attattagtt ccgtttcgga cttggcgtaa taacattacg . 360 gggcaagcag cagatgaatt gaccgaatta tttgatttca acattccaca acggtggagt 420 atcgcgcact tataccaggc aatcttaaat aatgaagcgc acgttaaaca ggtggacttc 480 ataacaacgc tggctggcta tgtaacctgg aaattgtcgg gtgagaaagt tctaggaatc 540 ggtgatgcgt ctggcgtttt cccaattgat gaaacgactg acacatacaa tcagacgatg 600 ttaaccaagt ttagccaact tgacaaagtt aaaccgtatt catgggatat ccggcatatt 660 ttaccgcggg ttttaccagc gggagccatt gctggaaagt taacggctgc cggggcgagc 720 ttacttgatc agagcggcac gctcgacgct ggcagtgtta ttgcaccgcc agaaggggat 780 gctggaacag gaatggtcgg tacgaacagc gtccgtaaac gcacgggtaa catctcggtg 840 ggaacctcag cattttcgat gaacgttcta gataaaccat tgtctaaagt ctatcgcgat 900 attgatattg ttatgacgcc agatgggtca ccagttgcaa tggtgcatgt taataattgt 960 tcatcagata ttaatgcgtg ggcaacgatt tttcgtgagt ttgcagcccg gttgggaatg 1020 gaattgaaac cggatcgatt atatgaaacg ttattcttgg aatcaactcg cgctgatgcg 1080 gatgctggag ggttggctaa ttatagttat caatccggtg agaatattac taagattcaa 1140 gctggtcggc cgctatttgt acggacacca aacagtaaat ttagtttacc gaactttatg 1200 ttgacccaat tatatgcggc gttcgcaccc ctccaacttg gtatggatat tcttgttaac 1260 gaagaacatg ttcaaacgga cgttatgatt gcacagggtg gattgttccg aacgccggta 1320 attggccaac aagtattggc caacgcactg aacattccga ttactgtaat gagtactgct 1380 ggtgaaggcg gcccatgggg gatggcagtg ttagccaact ttgcttgtcg gcaaactgca 1440 atgaacctag aagatttctt agatcaagaa gtctttaaag agccagaaag tatgacgttg 1500 agtccagaac cggaacgggt ggccggatat cgtgaattta ttcaacgtta tcaagctggc 1560 ttaccagttg aagcagcggc tgggcaagca atcaaatatt ag 1602

<210>5 <211 > 242 <212> PRT <213> Lactobacillus plantarum <400>5

Met Leu Glu Ala Leu Lys Gin Glu val Tyr Glu Ala Asn Met Gin Leu 15 10 15

Pro Lys Leu Gly Leu val Thr Phe Thr Trp Gly Asn val Ser Gly lie 20 25 30

Asp Arg Glu Lys Gly Leu Phe Val lie Lys pro ser Gly val Asp Tyr 35 40 45

Gly Glu Leu Lys Pro Ser Asp Leu Val Val Val Asn Leu Gin Gly Glu 50 55 60

Val Val Glu Gly Lys Leu Asn Pro Ser Ser Asp Thr Pro Thr His Thr 65 70 75 80 val Leu Tyr Asn Ala Phe Pro Asn lie Gly Gly lie Val His Thr His 85 90 95

Ser Pro Trp Ala Val Ala Tyr Ala Ala Ala Gin Met Asp Val Pro Ala 100 105 110

Met Asn Thr Thr His Ala Asp Thr Phe Tyr Gly Asp Val Pro Ala Ala 115 120 125

Asp Ala Leu Thr Lys Glu Glu lie Glu Ala Asp Tyr Glu Gly Asn Thr 130 135 140

Gly Lys Thr lie Val Lys Thr Phe Gin Glu Arg Gly Leu Asp Tyr Glu 145 150 155 160

Ala val Pro Ala Ser Leu val Ser Gin His Gly Pro Phe Ala Trp Gly 165 170 175

Pro Thr Pro Ala Lys Ala val Tyr Asn Ala Lys Val Leu Glu val Val 180 185 190

Ala Glu Glu Asp Tyr His Thr Ala Gin Leu Thr Arg Ala Ser Ser Glu 195 200 205

Leu Pro Gin Tyr Leu Leu Asp Lys His Tyr Leu Arg Lys His Gly Ala 210 215 220 ser Ala Tyr Tyr Gly Gin Asn Asn Ala His ser Lys Asp His Ala Val 225 230 235 240

Arg Lys

<210> 6 <211> 729 <212> DNA <213> Lactobacillus plantarum <400>6 atgctagaag cattaaaaca agaagtttat gaggctaaca tgcagcttcc aaagctgggc 60 ctggttactt ttacctgggg caatgtctcg ggcattgacc gggaaaaagg cctattcgtg 120 atcaagccat ctggtgttga ttatggtgaa ttaaaaccaa gcgatttagt cgttgttaac 180 ttacagggtg aagtggttga aggtaaacta aatccgtcta gtgatacgcc gactcatacg 240 gtgttatata acgcttttcc taatattggc ggaattgtcc atactcattc gccatgggca 300 gttgcctatg cagctgctca aatggatgtg ccagctatga acacgaccca tgctgatacg 360 ttctatggtg acgtgccggc cgcggatgcg ctgactaagg aagaaattga agcagattat 420 gaaggcaaca cgggtaaaac cattgtgaag acgttccaag aacggggcct cgattatgaa , 480 gctgtaccag cctcattagt cagccagcac ggcccatttg cttggggacc aacgccagct 540 aaagccgttt acaatgctaa agtgttggaa gtggttgccg aagaagatta tcatactgcg 600 caattgaccc gtgcaagtag cgaattacca caatatttat tagataagca ttatttacgt 660 aagcatggtg caagtgccta ttatggtcaa aataatgcgc attctaagga tcatgcagtt 720 cgcaagtaa 729 <210>7 <211 >437 <213> Piromyces species <400>7

Met Ala Lys Glu Tyr phe Pro Gin Ile Gin Lys Ile Lys Phe Glu Gly 15 10 15

Lys Asp ser Lys Asn pro Leu Ala Phe His Tyr Tyr Asp Ala Glu Lys 20 25 30

Glu Val Met Gly Lys Lys Met Lys Asp Trp Leu Arg Phe Ala Met Ala 35 40 45

Trp Trp His Thr Leu cys Ala Glu Gly Ala Asp Gin Phe Gly Gly Gly 50 55 60

Thr Lys ser Phe Pro Trp Asn Glu Gly Thr Asp Ala Ile Glu Ile Ala 65 70 75 80

Lys Gin Lys Val Asp Ala Gly phe Glu Ile Met Gin Lys Leu Gly ile 85 90 95

Pro Tyr Tyr cys Phe His Asp Val Asp Leu val ser Glu Gly Asn ser 100 105 110

Ile Glu Glu Tyr Glu Ser Asn Leu Lys Ala val val Ala Tyr Leu Lys 115 120 125

Glu Lys Gin Lys Glu Thr Gly Ile Lys Leu Leu Trp Ser Thr Ala Asn 130 135 140 val Phe Gly His Lys Arg Tyr Met Asn Gly Ala ser Thr Asn pro Asp 145 150 155 160

Phe Asp val val Ala Arg Ala Ile Val Gin Ile Lys Asn Ala Ile Asp 165 170 175

Ala Gly Ile Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg 180 185 190

Glu Gly Tyr Met Ser Leu Leu Asn Thr Asp Gin Lys Arg Glu Lys Glu 195 200 205

His Met Ala Thr Met Leu Thr Met Ala Arg Asp Tyr Ala Arg Ser Lys 210 215 220

Gly Phe Lys Gly Thr Phe Leu Ile Glu Pro Lys Pro Met Glu Pro Thr 225 230 235 240

Lys His Gin Tyr Asp Val Asp Thr Glu Thr Ala ile Gly Phe Leu Lys 245 250 255

Ala His Asn Leu Asp Lys Asp Phe Lys Val Asn ile Glu Val Asn His 260 265 270

Ala Thr Leu Ala Gly His Thr Phe Glu His Glu Leu Ala cys Ala Val 275 280 285

Asp Ala Gly Met Leu Gly Ser ile Asp Ala Asn Arg Gly Asp Tyr Gin 290 295 300

Asn Gly Trp Asp Thr Asp Gin phe Pro Ile Asp Gin Tyr Glu Leu val 305 310 315 320

Gin Ala Trp Met Glu Ile Ile Arg Gly Gly Gly Phe val Thr Gly Gly 325 330 335

Thr Asn Phe Asp Ala Lys Thr Arg Arg Asn ser Thr Asp Leu Glu Asp 340 345 350

Ile Ile Ile Ala His val Ser Gly Met Asp Ala Met Ala Arg Ala Leu 355 360 365

Glu Asn Ala Ala Lys Leu Leu Gin Glu ser Pro Tyr Thr Lys Met Lys 370 375 380

Lys Glu Arg Tyr Ala ser phe Asp Ser Gly lie Gly Lys Asp Phe Glu 385 390 395 400

Asp Gly Lys Leu Thr Leu Glu Gin val Tyr Glu Tyr Gly Lys Lys Asn 405 410 415

Gly Glu Pro Lys Gin Thr Ser Gly Lys Gin Glu Leu Tyr Glu Ala lie 420 425 430 val Ala Met Tyr Gin 435

<210> 8 <211>1669 <212> DNA <213> Piromyces species <400>8 gtaaatggct aaggaatatt tcccacaaat tcaaaagatt aagttcgaag gtaaggattc 60 taagaatcca ttagccttcc actactacga tgctgaaaag gaagtcatgg gtaagaaaat 120 gaaggattgg ttacgtttcg ccatggcctg gtggcacact ctttgcgccg aaggtgctga 180 ccaattcggt ggaggtacaa agtctttccc atggaacgaa ggtactgatg ctattgaaat 240 tgccaagcaa aaggttgatg ctggtttcga aatcatgcaa aagcttggta ttccatacta 300 ctgtttccac gatgttgatc ttgtttccga aggtaactct attgaagaat acgaatccaa 360 ccttaaggct gtcgttgctt acctcaagga aaagcaaaag gaaaccggta ttaagcttct 420 ctggagtact gctaacgtct tcggtcacaa gcgttacatg aacggtgcct ccactaaccc 480 agactttgat gttgtcgccc gtgctattgt tcaaattaag aacgccatag acgccggtat 540 tgaacttggt gctgaaaact acgtcttctg gggtggtcgt gaaggttaca tgagtctcct 600 taacactgac caaaagcgtg aaaaggaaca catggccact atgcttacca tggctcgtga 660 ctacgctcgt tccaagggat tcaagggtac tttcctcatt gaaccaaagc caatggaacc 720 aaccaagcac caatacgatg ttgacactga aaccgctatt ggtttcctta aggcccacaa 780 cttagacaag gacttcaagg tcaacattga agttaaccac gctactcttg ctggtcacac 840 tttcgaacac gaacttgcct gtgctgttga tgctggtatg ctcggttcca ttgatgctaa 900 ccgtggtgac taccaaaacg gttgggatac tgatcaattc ccaattgatc aatacgaact 960 cgtccaagct tggatggaaa tcatccgtgg tggtggtttc gttactggtg gtaccaactt 1020 cgatgccaag actcgtcgta actctactga cctcgaagac atcatcattg cccacgtttc 1080 tggtatggat gctatggctc gtgctcttga aaacgctgcc aagctcctcc aagaatctcc 1140 atacaccaag atgaagaagg aacgttacgc ttccttcgac agtggtattg gtaaggactt 1200 tgaagatggt aagctcaccc tcgaacaagt ttacgaatac ggtaagaaga acggtgaacc 1260 aaagcaaact tctggtaagc aagaactcta cgaagctatt gttgccatgt accaataagt 1320 taatcgtagt taaattggta aaataattgt aaaatcaata aacttgtcaa tcctccaatc 1380 aagtttaaaa gatcctatct ctgtactaat taaatatagt acaaaaaaaa atgtataaac 1440 aaaaaaaagt ctaaaagacg gaagaattta atttagggaa aaaataaaaa taataataaa 1500 caatagataa atcctttata ttaggaaaat gtcccattgt attattttca tttctactaa 1560 aaaagaaagt aaataaaaca caagaggaaa ttttcccttt tttttttttt tgtaataaat 1620 tttatgcaaa tataaatata aataaaataa taaaaaaaaa aaaaaaaaa 1669 <210>9 <211 >496

<212> PRT <213> Bacillus subtilis <400>9

Met Leu Gin Thr Lys Asp Tyr Glu phe Trp Phe val Thr Gly ser Gin 15 10 15

His Leu Tyr Gly Glu Glu Thr Leu Glu Leu val Asp Gin His Ala Lys 20 25 50 ser lie Cys Glu Gly Leu Ser Gly Ile ser ser Arg Tyr Lys lie Thr 35 40 45

His Lys Pro val Val Thr Ser Pro Glu Thr lie Arg Glu Leu Leu Arg 50 55 60

Glu Ala Glu Tyr ser Glu Thr Cys Ala Gly lie lie Thr Trp Met His 65 70 75 80

Thr Phe ser Pro Ala Lys Met Trp lie Glu Gly Leu Spr Ser Tyr Gin 85 90 95

Lys Pro Leu Met His Leu His Thr Gin Tyr Asn Arg Asp lie Pro Trp 100 105 110

Gly Thr lie Asp Met Asp phe Met Asn Ser Asn Gin Ser Ala His Gly 115 120 125

Asp Arg Glu Tyr Gly Tyr lie Asn Ser Arg Met Gly Leu Ser Arg Lys 130 135 140

Val lie Ala Gly Tyr Trp Asp Asp Glu Glu val Lys Lys Glu Met ser 145 150 155 160

Gin Trp Met Asp Thr Ala Ala Ala Leu Asn Glu Ser Arg His lie Lys 165 170 175

Val Ala Arg Phe Gly Asp Asn Met Arg His Val Ala Val Thr Asp Gly 180 185 190

Asp Lys val Gly Ala His Ile Gin phe Gly Trp Gin val Asp Gly Tyr 195 200 205

Gly Ile Gly Asp Leu Val Glu Val Met Asp Arg Ile Thr Asp Asp Glu 210 215 220

Val Asp Thr Leu Tyr Ala Glu Tyr Asp Arg Leu Tyr val Ile ser Glu 225 230 235 240

Glu Thr Lys Arg Asp Glu Ala Lys val Ala ser Ile Lys Glu Gin Ala 245 250 255

Lys Ile Glu Leu Gly Leu Thr Ala Phe Leu Glu Gin Gly Gly Tyr Thr 260 265 270

Ala Phe Thr Thr ser Phe Glu val Leu His Gly Met Lys Gin Leu pro 275 280 285

Gly Leu Ala Val Gin Arg Leu Met Glu Lys Gly Tyr Gly Phe Ala Gly 290 295 300

Glu Gly Asp Trp Lys Thr Ala Ala Leu Val Arg Met Met Lys Ile Met 305 310 315 320

Ala Lys Gly Lys Arg Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Phe 325 330 335

Glu Pro Gly Asn Glu Met Ile Leu Gly ser His Met Leu Glu val cys 340 345 350

Pro Thr Val Ala Leu Asp Gin pro Lys Ile Glu Val His Ser Leu ser 355 360 365

Ile Gly Gly Lys Glu Asp Pro Ala Arg Leu Val Phe Asn Gly Ile Ser 370 375 380

Gly Ser Ala Ile Gin Ala ser Ile val Asp Ile Gly Gly Arg Phe Arg 385 390 395 400

Leu val Leu Asn Glu val Asn Gly Gin Glu Ile Glu Lys Asp Met Pro 405 410 415

Asn Leu Pro val Ala Arg Val Leu Trp Lys Pro Glu Pro ser Leu Lys 420 425 430

Thr Ala Ala Glu Ala Trp Ile Leu Ala Gly Gly Ala His His Thr Cys 435 440 445

Leu Ser Tyr Glu Leu Thr Ala Glu Gin Met Leu Asp Trp Ala Glu Met 450 455 460

Ala Gly Ile Glu Ser val Leu Ile Ser Arg Asp Thr Thr Ile His Lys 465 470 475 480

Leu Lys His Glu Leu Lys Trp Asn Glu Ala Leu Tyr Arg Leu Gin Lys 485 490 495 <210> 10 <211 > 1511

<212> DNA <213> Bacillus subtilis <400> 10 atgagaaagg ggcagtttac atgcttcaga caaaggatta tgaattctgg tttgtgacag 60 gaagccagca cctatacggg gaagagacgc tggaactcgt agatcagcat gctaaaagca 120 tttgtgaggg gctcagcggg atttcttcca gatataaaat cactcataag cccgtcgtca 180 cttcaccgga aaccattaga gagctgttaa gagaagcgga gtacagtgag acatgtgctg 240 gcatcattac atggatgcac acattttccc ctgcaaaaat gtggatagaa ggcctttcct , 300 cttatcaaaa accgcttatg catttgcata cccaatataa tcgcgatatc ccgtggggta 360 cgattgacat ggattttatg aacagcaacc aatccgcgca tggcgatcga gagtacggtt 420 acatcaactc gagaatgggg cttagccgaa aagtcattgc cggctattgg gatgatgaag 480 aagtgaaaaa agaaatgtcc cagtggatgg atacggcggc tgcattaaat gaaagcagac 540 atattaaggt tgccagattt ggagataaca tgcgtcatgt cgcggtaacg gacggagaca 600 aggtgggagc gcatattcaa tttggctggc aggttgacgg atatggcatc ggggatctcg 660 ttgaagtgat ggatcgcatt acggacgacg aggttgacac gctttatgcc gagtatgaca 720 gactatatgt gatcagtgag gaaacaaaac gtgacgaagc aaaggtagcg tccattaaag 780 aacaggcgaa aattgaactt ggattaaccg cttttcttga gcaaggcgga tacacagcgt 840 ttacgacatc gtttgaagtg ctgcacggaa tgaaacagct gccgggactt gccgttcagc 900 gcctgatgga gaaaggctat gggtttgccg gtgaaggaga ttggaagaca gcggcccttg 960 tacggatgat gaaaatcatg gctaaaggaa aaagaacttc cttcatggaa gattacacgt 1020 accattttga accgggaaat gaaatgattc tgggctctca catgcttgaa gtgtgtccga 1080 ctgtcgcttt ggatcagccg aaaatcgagg ttcattcgct ttcgattggc ggcaaagagg 1140 accctgcgcg tttggtattt aacggcatca gcggttctgc cattcaagct agcattgttg 1200 atattggcgg gcgtttccgc cttgtgctga atgaagtcaa cggccaggaa attgaaaaag 1260 acatgccgaa tttaccggtt gcccgtgttc tctggaagcc ggagccgtca ttgaaaacag 1320 cagcggaggc atggatttta gccggcggtg cacaccatac ctgcctgtct tatgaactga 1380 cagcggagca aatgcttgat tgggcggaaa tggcgggaat cgaaagtgtt ctcatttccc 1440 gtgatacgac aattcataaa ctgaaacacg agttaaaatg gaacgaggcg ctttaccggc 1500 ttcaaaagta g 1511 <210 11 <211> 566 <212> PRT <213> E. coli <400 11

Met Ala lie Ala lie Gly Leu Asp Phe Gly ser Asp ser val Arg Ala 15 10 15

Leu Ala Val Asp cys Ala Ser Gly Glu Glu Ile Ala Thr Ser val Glu 20 25 30

Trp Tyr pro Arg Trp Gin Lys Gly Gin Phe Cys Asp Ala Pro Asn Asn 35 40 45

Gin Phe Arg His His Pro Arg Asp Tyr ile Glu Ser Met Glu Ala Ala 50 55 60

Leu Lys Thr Val Leu Ala Glu Leu Ser Val Glu Gin Arg Ala Ala val 65 70 75 80

Val Gly lie Gly val Asp Ser Thr Gly Ser Thr Pro Ala Pro lie Asp 85 90 95

Ala Asp Gly Asn val Leu Ala Leu Arg Pro Glu Phe Ala Glu Asn Pro 100 105 110

Asn Ala Met Phe Val Leu Trp Lys Asp His Thr Ala Val Glu Arg ser 115 120 125

Glu Glu lie Thr Arg Leu cys His Ala pro Gly Asn Val Asp Tyr Ser 130 135 140

Arg Tyr lie Gly Gly lie Tyr Ser Ser Glu Trp Phe Trp Ala Lys lie 145 150 155 160

Leu His val Thr Arg Gin Asp ser Ala val Ala Gin ser Ala Ala Ser 165 170 175

Trp lie Glu Leu Cys Asp Trp val Pro Ala Leu Leu ser Gly Thr Thr 180 185 190

Arg Pro Gin Asp lie Arg Arg Gly Arg Cys Ser Ala Gly His Lys ser 195 200 205

Leu Trp His Glu Ser Trp Gly Gly Leu Pro Pro Ala Ser Phe Phe Asp 210 215 220

Glu Leu Asp pro lie Leu Asn Arg His Leu Pro Ser Pro Leu Phe Thr 225 230 235 240

Asp Thr Trp Thr Ala Asp lie Pro val Gly Thr Leu cys Pro Glu Trp 245 250 255

Ala Gin Arg Leu Gly Leu Pro Glu Ser val val Ile ser Gly Gly Ala 260 265 270

Phe Asp cys His Met Gly Ala Val Gly Ala Gly Ala Gin Pro Asn Ala 275 280 285

Leu Val Lys Val lie Gly Thr Ser Thr cys Asp lie Leu lie Ala Asp 290 295 300

Lys Gin Ser val Gly Glu Arg Ala Val Lys Gly lie Cys Gly Gin Val 305 310 315 320

Asp Gly Ser Val Val Pro Gly Phe lie Gly Leu Glu Ala Gly Gin Ser 325 330 335

Ala Phe Gly Asp Ile Tyr Ala Trp Phe Gly Arg Val Leu Ser Trp Pro 340 345 350

Leu Glu Gin Leu Ala Ala Gin His Pro Glu Leu Lys Ala Gin lie Asn 355 360 365

Ala ser Gin Lys Gin Leu Leu Pro Ala Leu Thr Glu Ala Trp Ala Lys 370 375 380

Asn Pro Ser Leu Asp His Leu Pro val val Leu Asp Trp Phe Asn Gly 385 390 395 400

Arg Arg Ser Pro Asn Ala Asn Gin Arg Leu Lys Gly Val lie Thr Asp 405 410 415

Leu Asn Leu Ala Thr Asp Ala Pro Leu Leu Phe Gly Gly Leu lie Ala 420 425 430

Ala Thr Ala Phe Gly Ala Arg Ala lie Met Glu cys Phe Thr Asp Gin 435 440 445

Gly lie Ala Val Asn Asn Val Met Ala Leu Gly Gly lie Ala Arg Lys 450 455 460

Asn Gin val lie Met Gin Ala Cys cys Asp val Leu Asn Arg pro Leu 465 470 475 480

Gin lie val Ala ser Asp Gin cys cys Ala Leu Gly Ala Ala lie Phe 485 490 495

Ala Ala val Ala Ala Lys Val His Ala Asp lie Pro ser Ala Gin Gin 500 505 510

Lys Met Ala Ser Ala Val Glu Lys Thr Leu Gin Pro Arg ser Glu Gin 515 520 525

Ala Gin Arg Phe Glu Gin Leu Tyr Arg Arg Tyr Gin Gin Trp Ala Met 530 535 540

Ser Ala Glu Gin His Tyr Leu Pro Thr ser Ala pro Ala Gin Ala Ala 545 550 555 560

Gin Ala val Ala Thr Leu 565 <210> 12 <211> 1453 <212> DNA <213> E. coli <400> 12 atggcgattg caattggcct cgattttggc agtgattctg tgcgagcttt ggcggtggac 60 tgcgccagcg gtgaagagat cgccaccagc gtagagtggt atccccgttg gcaaaaaggg „ 120 caattttgtg atgccccgaa taaccagttc cgtcatcatc cgcgtgacta cattgagtca 180 atggaagcgg cactgaaaac cgtgcttgca gagcttagcg tcgaacagcg cgcagctgtg 240 gtcgggattg gcgttgacag taccggctcg acgcccgcac cgattgatgc cgacggtaac 300 gtgctggcgc tgcgcccgga gtttgccgaa aacccgaacg cgatgttcgt attgtggaaa 360 gaccacactg cggttgaaag aagcgaagag attacccgtt tgtgccacgc gccgggcaat 420 gttgactact cccgctatat tggcggtatt tattccagcg aatggttctg ggcaaaaatc 480 ctgcatgtga ctcgccagga cagcgccgtg gcgcaatctg ccgcatcgtg gattgagctg 540 tgcgactggg tgccagctct gctttccggt accacccgcc cgcaggatat tcgtcgcgga 600 cgttgcagcg ccgggcataa atctctgtgg cacgaaagct ggggcggctt gccgccagcc 660 agtttctttg atgagctgga cccgatcctc aatcgccatt tgccttcccc gctgttcact 720 gacacctgga ctgccgatat tccggtgggc accttatgcc cggaatgggc gcagcgtctc 780 ggcctgcctg aaagcgtggt gatttccggc ggcgcgtttg actgccatat gggcgcagtt 840 ggcgcaggcg cacagcctaa cgcactggta aaagttatcg gtacttccac ctgcgacatt 900 ctgattgccg acaaacagag cgttggcgag cgggcagtta aaggtatttg cggtcaggtt 960 gatggcagcg tggtgcctgg atttatcggt ctggaagcag gccaatcggc gtttggtgat 1020 atctacgcct ggttcggtcg cgtactcagc tggccgctgg aacagcttgc cgcccagcat 1080 ccggaactga aagcgcaaat caacgccagc cagaaacaac tgcttccggc gctgaccgaa 1140 gcatgggcca aaaatccgtc tctggatcac ctgccggtgg tgctcgactg gtttaacggt 1200 cgtcgctcgc caaacgctaa ccaacgcctg aaaggggtga ttaccgatct taacctcgct 1260 accgacgctc cgctgctgtt cggcggtttg attgctgcca ccgcctttgg cgcacgcgca 1320 atcatggagt gctttaccga tcaggggatc gccgtcaata acgtgatggc gctgggcggc 1380 atcgcgcgga aaaaccaagt cattatgcag gcctgctgcg acgtgctgaa tcgcccgctg 1440 caaattgttg cct 1453 <210> 13 <211> 231 <212> PRT <213> E. coli <400> 13

Met Leu Glu Asp Leu Lys Arg Gin val Leu Glu Ala Asn Leu Ala Leu 15 10 15 pro Lys His Asn Leu val Thr Leu Thr Trp Gly Asn val ser Ala Val 20 25 30

Asp Arg Glu Arg Gly Val Phe Val lie Lys Pro ser Gly Val Asp Tyr 35 40 45

Ser lie Met Thr Ala Asp Asp Met Val Val Val Ser lie Glu Thr Gly 50 55 60

Glu Val Val Glu Gly Ala Lys Lys Pro Ser Ser Asp Thr pro Thr His 65 70 75 80

Arg Leu Leu Tyr Gin Ala Phe Pro Ser lie Gly Gly lie Val His Thr 85 90 95

His ser Arg His Ala Thr lie Trp Ala Gin Ala Gly Gin ser lie Pro 100 105 110

Ala Thr Gly Thr Thr His Ala Asp Tyr Phe Tyr Gly Thr lie Pro cys 115 120 125

Thr Arg Lys Met Thr Asp Ala Glu lie Asn Gly Glu Tyr Glu Trp Glu 130 135 140

Thr Gly Asn Val lie Val Glu Thr Phe Glu Lys Gin Gly lie Asp Ala 145 150 155 160

Ala Gin Met Pro Gly Val Leu Val His ser His Gly Pro phe Ala Trp 165 170 175

Gly Lys Asn Ala Glu Asp Ala Val His Asn Ala lie val Leu Glu Glu 180 185 190

Val Ala Tyr Met Gly lie Phe cys Arg Gin Leu Ala Pro Gin Leu Pro 195 200 205

Asp Met Gin Gin Thr Leu Leu Asn Lys His Tyr Leu Arg Lys His Gly 210 215 220

Ala Lys Ala Tyr Tyr Gly Gin 225 230 <210> 14 <211 > 696 <212> DNA <213> E coli <400> 14 atgttagaag atctcaaacg ccaggtatta gaggccaacc tggcgctgcc aaaacataac 60 ctggtcacgc tcacatgggg caacgtcagc gccgttgatc gcgagcgcgg cgtctttgtg 120 atcaaacctt ccggcgtcga ttacagcatc atgaccgctg acgatatggt cgtggttagc 180 atcgaaaccg gtgaagtggt tgaaggtgcg aaaaagccct cctccgatac gccaactcac 240 cgactgctct atcaggcatt cccgtccatt ggcggcattg tgcacacaca ctcgcgccac 300 gccactatct gggcgcaggc gggccagtcg attccagcaa ccggcaccac ccacgccgac 360 tatttctacg gcaccattcc ctgcacccgc aaaatgaccg acgcagaaat caacggtgaa 420 tatgagtggg aaaccggtaa cgtcatcgta gaaaccttcg aaaaacaggg tatcgatgca . 480 gcgcaaatgc ccggcgtcct ggtccattct cacggcccat ttgcatgggg caaaaatgcc 540 gaagatgcgg tgcataacgc catcgtgctg gaagaggtcg cttatatggg gatattctgc 600 cgtcagttag cgccgcagtt accggatatg cagcaaacgc tgctgaataa acactatctg 660 cgtaagcatg gcgcgaaggc atattacggg cagtaa 696

<210> 15 <211 > 438 <212> PRT <213> Bacteroides thetaiotaomicron <400> 15

Met Ala Thr Lys Glu Phe Phe Pro Gly ile Glu Lys ile Lys Phe Glu 15 10 15

Gly Lys Asp ser Lys Asn Pro Met Ala Phe Arg Tyr Tyr Asp Ala Glu 20 25 30

Lys val lie Asn Gly Lys Lys Met Lys Asp Trp Leu Arg Phe Ala Met 35 40 45

Ala Trp Trp His Thr Leu Cys Ala Glu Gly Gly Asp Gin Phe Gly Gly 50 55 60

Gly Thr Lys Gin Phe Pro Trp Asn Gly Asn Ala Asp Ala lie Gin Ala 65 70 75 80

Ala Lys Asp Lys Met Asp Ala Gly Phe Glu Phe Met Gin Lys Met Gly 85 90 95 lie Glu Tyr Tyr Cys Phe His Asp Val Asp Leu Val ser Glu Gly Ala 100 105 110

Ser Val Glu Glu Tyr Glu Ala Asn Leu Lys Glu lie Val Ala Tyr Ala 115 120 125

Lys Gin Lys Gin Ala Glu Thr Gly lie Lys Leu Leu Trp Gly Thr Ala 130 135 140

Asn Val Phe Gly His Ala Arg Tyr Met Asn Gly Ala Ala Thr Asn Pro 145 150 155 160

Asp Phe Asp Val val Ala Arg Ala Ala Val Gin lie Lys Asn Ala lie 165 170 175

Asp Ala Thr lie Glu Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly 180 185 190

Arg Glu Gly Tyr Met ser Leu Leu Asn Thr Asp Gin Lys Arg Glu Lys 195 200 205

Glu His Leu Ala Gin Met Leu Thr lie Ala Arg Asp Tyr Ala Arg Ala 210 215 220

Arg Gly Phe Lys Gly Thr Phe Leu lie Glu Pro Lys Pro Met Glu Pro 225 230 235 240

Thr Lys His Gin Tyr Asp Val Asp Thr Glu Thr val lie Gly Phe Leu 245 250 255

Lys Ala His Gly Leu Asp Lys Asp Phe Lys Val Asn lie Glu Val Asn 260 265 270

His Ala Thr Leu Ala Gly His Thr Phe Glu His Glu Leu Ala Val Ala 275 280 285

Val Asp Asn Gly Met Leu Gly ser lie Asp Ala Asn Arg Gly Asp Tyr 290 295 300

Gin Asn Gly Trp Asp Thr Asp Gin Phe Pro lie Asp Asn Tyr Glu Leu 305 310 315 320

Thr Gin Ala Met Met Gin He lie Arg Asn Gly Gly Leu Gly Thr Gly 325 330 335

Gly Thr Asn Phe Asp Ala Lys Thr Arg Arg Asn ser Thr Asp Leu Glu 340 345 350

Asp lie Phe lie Ala His lie Ala Gly Met Asp Ala Met Ala Arg Ala 355 360 365

Leu Glu ser Ala Ala Ala Leu Leu Asp Glu ser Pro Tyr Lys Lys Met 370 375 380

Leu Ala Asp Arg Tyr Ala Ser Phe Asp Gly Gly Lys Gly Lys Glu Phe 385 390 395 400

Glu Asp Gly Lys Leu Thr Leu Glu Asp Val Val Ala Tyr Ala Lys Thr 405 410 415

Lys Gly Glu Pro Lys Gin Thr Ser Gly Lys Gin Glu Leu Tyr Glu Ala 420 425 430 lie Leu Asn Met Tyr Cys 435

<210> 16 <211 > 1317 <212> DNA <213> Bacteroides thetaiotaomicron <400> 16 atggcaacaa aagaattttt tccgggaatt gaaaagatta aatttgaagg taaagatagt „ 60 aagaacccga tggcattccg ttattacgat gcagagaagg tgattaatgg taaaaagatg 120 aaggattggc tgagattcgc tatggcatgg tggcacacat tgtgcgctga aggtggtgat 180 cagttcggtg gcggaacaaa gcaattccca tggaatggta atgcagatgc tatacaggca 240 gcaaaagata agatggatgc aggatttgaa ttcatgcaga agatgggtat cgaatactat 300 tgcttccatg acgtagactt ggtttcggaa ggtgccagtg tagaagaata cgaagctaac 360 ctgaaagaaa tcgtagctta tgcaaaacag aaacaggcag aaaccggtat caaactactg 420 tggggtactg ctaatgtatt cggtcacgcc cgctatatga acggtgcagc taccaatcct 480 gacttcgatg tagtagctcg tgctgctgtt cagatcaaaa atgcgattga tgcaacgatt 540 gaacttggcg gagagaatta tgtgttttgg ggtggtcgtg aaggctatat gtctcttctg 600 aacacagatc agaaacgtga aaaagaacac cttgcacaga tgttgacgat tgctcgtgac 660 tatgcccgtg cccgtggttt caaaggtact ttcctgatcg aaccgaaacc gatggaaccg 720 actaaacatc aatatgacgt agatacggaa actgtaatcg gcttcctgaa agctcatggt 780 ctggataagg atttcaaagt aaatatcgag gtgaatcacg caactttggc aggtcacact 840 ttcgagcatg aattggctgt agctgtagac aatggtatgt tgggctcaat tgacgccaat 900 cgtggtgact atcagaatgg ctgggataca gaccaattcc cgatcgacaa ttatgaactg 960 actcaggcta tgatgcagat tatccgtaat ggtggtctcg gtaccggtgg tacgaacttt 1020 gatgctaaaa cccgtcgtaa ttctactgat ctggaagata tctttattgc tcacatcgca 1080 ggtatggacg ctatggcccg tgcactcgaa agtgcagcgg ctctgctcga cgaatctccc 1140 tataagaaga tgctggctga ccgttatgct tcatttgatg ggggcaaagg taaagaattt 1200 gaagacggca agctgactct ggaggatgtg gttgcttatg caaaaacaaa aggcgaaccg 1260 aaacagacta gcggcaagca agaactttat gaggcaattc tgaatatgta ttgctaa 1317

<210> 17 <211 > 258 <212> PRT <213> Saccharomyces cerevisiae <400> 17

Met Ala Ala Gly val Pro Lys lie Asp Ala Leu Glu Ser Leu Gly Asn 15 10 15

Pro Leu Glu Asp Ala Lys Arg Ala Ala Ala Tyr Arg Ala val Asp Glu 20 25 30

Asn Leu Lys Phe Asp Asp His Lys lie lie Gly lie Gly Ser Gly ser 35 40 45

Thr Val val Tyr Val Ala Glu Arg Ile Gly Gin Tyr Leu His Asp Pro 50 55 60

Lys Phe Tyr Glu val Ala Ser Lys Phe lie Cys lie pro Thr Gly Phe 65 70 75 80

Gin ser Arg Asn Leu lie Leu Asp Asn Lys Leu Gin Leu Gly ser lie 85 90 95

Glu Gin Tyr Pro Arg lie Asp lie Ala Phe Asp Gly Ala Asp Glu Val 100 105 110

Asp Glu Asn Leu Gin Leu lie Lys Gly Gly Gly Ala cys Leu Phe Gin 115 120 125

Glu Lys Leu val Ser Thr ser Ala Lys Thr Phe lie Val val Ala Asp 130 135 140 ser Arg Lys Lys ser Pro Lys His Leu Gly Lys Asn Trp Arg Gin Gly 145 150 155 160 val Pro lie Glu lie Val Pro ser ser Tyr Val Arg val Lys Asn Asp 165 170 175

Leu Leu Glu Gin Leu His Ala Glu Lys Val Asp lie Arg Gin Gly Gly 180 185 190

Ser Ala Lys Ala Gly Pro val val Thr Asp Asn Asn Asn Phe lie lie 195 200 205

Asp Ala Asp phe Gly Glu lie ser Asp Pro Arg Lys Leu His Arg Glu 210 215 220 lie Lys Leu Leu Val Gly Val Val Glu Thr Gly Leu Phe lie Asp Asn 225 230 235 240

Ala ser Lys Ala Tyr phe Gly Asn ser Asp Gly ser val Glu val Thr 245 250 255

Glu Lys

<210> 18 <211 >2467 <212> DNA <213> Saccharomyces cerevisiae <400> 18 ggatccaaga ccattattcc atcagaatgg aaaaaagttt aaaagatcac ggagattttg 60 ttcttctgag cttctgctgt ccttgaaaac aaattattcc gctggccgcc ccaaacaaaa 120 acaaccccga tttaataaca ttgtcacagt attagaaatt ttctttttac aaattaccat 180 ttccagctta ctacttccta taatcctcaa tcttcagcaa gcgacgcagg gaatagccgc 240 tgaggtgcat aactgtcact tttcaattcg gccaatgcaa tctcaggcgg acgaataagg 300 gggccctctc gagaaaaaca aaaggaggat gagattagta ctttaatgtt gtgttcagta , 360 attcagagac agacaagaga ggtttccaac acaatgtctt tagactcata ctatcttggg 420 tttgatcttt cgacccaaca actgaaatgt ctcgccatta accaggacct aaaaattgtc 480 cattcagaaa cagtggaatt tgaaaaggat cttccgcatt atcacacaaa gaagggtgtc 540 tatatacacg gcgacactat cgaatgtccc gtagccatgt ggttaggggc tctagatctg 600 gttctctcga aatatcgcga ggctaaattt ccattgaaca aagttatggc cgtctcaggg 660 tcctgccagc agcacgggtc tgtctactgg tcctcccaag ccgaatctct gttagagcaa 720 ttgaataaga aaccggaaaa agatttattg cactacgtga gctctgtagc atttgcaagg 780 caaaccgccc ccaattggca agaccacagt actgcaaagc aatgtcaaga gtttgaagag 840 tgcataggtg ggcctgaaaa aatggctcaa ttaacagggt ccagagccca ttttagattt 900 actggtcctc aaattctgaa aattgcacaa ttagaaccag aagcttacga aaaaacaaag 960 accatttctt tagtgtctaa ttttttgact tctatcttag tgggccatct tgttgaatta 1020 gaggaggcag atgcctgtgg tatgaacctt tatgatatac gtgaaagaaa attcatgtat 1080 gagctactac atctaattga tagttcttct aaggataaaa ctatcagaca aaaattaatg 1140 agagcaccca tgaaaaattt gatagcgggt accatctgta aatattttat tgagaagtac 1200 ggtttcaata caaactgcaa ggtctctccc atgactgggg ataatttagc cactatatgt 1260 tctttacccc tgcggaagaa tgacgttctc gtttccctag gaacaagtac tacagttctt 1320 ctggtcaccg ataagtatca cccctctccg aactatcatc ttttcattca tccaactctg 1380 ccaaaccatt atatgggtat gatttgttat tgtaatggtt ctttggcaag ggagaggata 1440 agagacgagt taaacaaaga acgggaaaat aattatgaga agactaacga ttggactctt 1500 tttaatcaag ctgtgctaga tgactcagaa agtagtgaaa atgaattagg tgtatatttt 1560 cctctggggg agatcgttcc tagcgtaaaa gccataaaca aaagggttat cttcaatcca 1620 aaaacgggta tgattgaaag agaggtggcc aagttcaaag acaagaggca cgatgccaaa 1680 aatattgtag aatcacaggc tttaagttgc agggtaagaa tatctcccct gctttcggat 1740 tcaaacgcaa gctcacaaca gagactgaac gaagatacaa tcgtgaagtt tgattacgat 1800 gaatctccgc tgcgggacta cctaaataaa aggccagaaa ggactttttt tgtaggtggg 1860 gcttctaaaa acgatgctat tgtgaagaag tttgctcaag tcattggtgc tacaaagggt 1920 aattttaggc tagaaacacc aaactcatgt gcccttggtg gttgttataa ggccatgtgg 1980 tcattgttat atgactctaa taaaattgca gttccttttg ataaatttct gaatgacaat 2040 tttccatggc atgtaatgga aagcatatcc gatgtggata atgaaaattg gatcgctata 2100 attccaagat tgtcccctta agcgaactgg aaaagactct catctaaaat atgtttgaat 2160 aatttatcat gccctgacaa gtacacacaa acacagacac ataatataca tacatatata 2220 tatatcaccg ttattatgcg tgcacatgac aatgcccttg tatgtttcgt atactgtagc 2280 aagtagtcat cattttgttc cccgttcgga aaatgacaaa aagtaaaatc aataaatgaa 2340 gagtaaaaaa caatttatga aagggtgagc gaccagcaac gagagagaca aatcaaatta „ 2400 gcgctttcca gtgagaatat aagagagcat tgaaagagct aggttattgt taaatcatct 2460 cgagctc 2467

<210> 19 <211 > 238 <212> PRT <213> Saccharomyces cerevisiae <400> 19

Met val Lys Pro ile ile Ala pro ser ile Leu Ala ser Asp Phe Ala 15 10 15

Asn Leu Gly cys Glu cys His Lys val ile Asn Ala Gly Ala Asp Trp 20 25 30

Leu His ile Asp val Met Asp Gly His Phe val Pro Asn ile Thr Leu 35 40 45

Gly Gin Pro Ile Val Thr ser Leu Arg Arg Ser Val Pro Arg Pro Gly 50 55 60

Asp Ala Ser Asn Thr Glu Lys Lys pro Thr Ala Phe Phe Asp cys His 65 70 75 80

Met Met Val Glu Asn Pro Glu Lys Trp val Asp Asp Phe Ala Lys Cys 85 90 95

Gly Ala Asp Gin Phe Thr Phe His Tyr Glu Ala Thr Gin Asp Pro Leu 100 105 110

His Leu Val Lys Leu ile Lys Ser Lys Gly Ile Lys Ala Ala Cys Ala 115 120 125 ile Lys pro Gly Thr ser val Asp val Leu phe Glu Leu Ala Pro His 130 135 140

Leu Asp Met Ala Leu val Met Thr Val Glu Pro Gly Phe Gly Gly Gin 145 150 155 160

Lys Phe Met Glu Asp Met Met pro Lys Val Glu Thr Leu Arg Ala Lys 165 170 175

Phe Pro His Leu Asn Ile Gin Val Asp Gly Gly Leu Gly Lys Glu Thr 180 185 190

Ile Pro Lys Ala Ala Lys Ala Gly Ala Asn Val Ile Val Ala Gly Thr 195 200 205 ser Val Phe Thr Ala Ala Asp Pro His Asp Val Ile ser Phe Met Lys 210 215 220

Glu Glu Val ser Lys Glu Leu Arg ser Arg Asp Leu Leu Asp 225 230 235

<210> 20 <211> 1328 <212> DNA <213> Saccharomyces cerevisiae <400> 20 gttaggcact tacgtatctt gtatagtagg aatggctcgg tttatgtata ttaggagatc 60 aaaacgagaa aaaaatacca tatcgtatag tatagagagt ataaatataa gaaatgccgc 120 atatgtacaa ctaatctagc aaatctctag aacgcaattc cttcgagact tcttctttca 180 tgaaggagat aacatcgtgc gggtcagctg cagtgaaaac actggtacca gcgacaataa 240 cgttggcacc ggctttggcg gctttcggga tggtctcctt gcccaaacca ccatcgactt 300 ggatattcaa atgggggaac ttggctctca aagtttccac ttttggcatc atgtcttcca 360 tgaatttttg gcctccaaac ccaggttcca cagtcataac aagagccata tccaaatgag 420 gagctagttc aaataaaacg tcaacagaag taccaggttt gatggcgcat gcagctttga 480 tgcccttaga cttaatcaac ttaactaaat gcaaagggtc ttgtgtggcc tcgtagtgga 540 acgtaaattg gtcagcacca catttagcaa aatcgtcgac ccatttttca ggattttcaa 600 ccatcatgtg acaatcgaag aacgcagtgg gcttcttttc tgtgttgcta gcatcgccag 660 ggcgtggcac agaacgacgt agggaggtaa caattggttg gcccagagta atgtttggaa 720 caaaatggcc gtccatgaca tcgatatgta accaatctgc gccggcgttg atgaccttat 780 gacattcgca acccaagttg gcgaagtcag aagcaaggat actgggagct ataattggtt 840 tgaccatttt ttcttgtgtg tttacctcgc tcttggaatt agcaaatggc cttcttgcat 900 gaaattgtat cgagtttgct ttatttttct ttttacgggc ggattctttc tattctggct 960 ttcctataac agagatcatg aaagaagttc cagcttacgg atcaagaaag tacctataca 1020 tatacaaaaa tctgattact ttcccagctc gacttggata gctgttcttg ttttctcttg 1080 gcgacacatt ttttgtttct gaagccacgt cctgctttat aagaggacat ttaaagttgc 1140 aggacttgaa tgcaattacc ggaagaagca accaaccggc atggttcagc atacaataca 1200 catttgatta gaaaagcaga gaataaatag acatgatacc tctcttttta tcctctgcag 1260 cgtattattg tttattccac gcaggcatcg gtcgttggct gttgttatgt ctcagataag 1320 cgcgtttg 1328

<210>21 <211 > 680 <212> PRT <213> Saccharomyces cerevisiae <400 21

Met Thr Gin Phe Thr Asp Ile Asp Lys Leu Ala Val Ser Thr Ile Arg 15 10 15

Ile Leu Ala Val Asp Thr Val Ser Lys Ala Asn Ser Gly His Pro Gly 20 25 30

Ala Pro Leu Gly Met Ala Pro Ala Ala His val Leu Trp Ser Gin Met 35 40 45

Arg Met Asn Pro Thr Asn Pro Asp Trp Ile Asn Arg Asp Arg Phe Val 50 55 60

Leu ser Asn Gly His Ala val Ala Leu Leu Tyr ser Met Leu His Leu 65 70 75 80

Thr Gly Tyr Asp Leu Ser Ile Glu Asp Leu Lys Gin Phe Arg Gin Leu 85 90 95

Gly Ser Arg Thr Pro Gly His Pro Glu Phe Glu Leu Pro Gly Val Glu 100 105 110

Val Thr Thr Gly Pro Leu Gly Gin Gly ile Ser Asn Ala Val Gly Met 115 120 125

Ala Met Ala Gin Ala Asn Leu Ala Ala Thr Tyr Asn Lys Pro Gly Phe 130 135 140

Thr Leu ser Asp Asn Tyr Thr Tyr Val Phe Leu Gly Asp Gly Cys Leu 145 150 155 160

Gin Glu Gly ile Ser Ser Glu Ala Ser ser Leu Ala Gly His Leu Lys 165 170 175

Leu Gly Asn Leu Ile Ala Ile Tyr Asp Asp Asn Lys ile Thr Ile Asp 180 185 190

Gly Ala Thr ser Ile ser Phe Asp Glu Asp val Ala Lys Arg Tyr Glu 195 200 205

Ala Tyr Gly Trp Glu Val Leu Tyr Val Glu Asn Gly Asn Glu Asp Leu 210 215 220

Ala Gly Ile Ala Lys Ala Ile Ala Gin Ala Lys Leu Ser Lys Asp Lys 225 230 235 240

Pro Thr Leu Ile Lys Met Thr Thr Thr Ile Gly Tyr Gly ser Leu His 245 250 255

Ala Gly ser His Ser Val His Gly Ala Pro Leu Lys Ala Asp Asp val 260 265 270

Lys Gin Leu Lys ser Lys Phe Gly Phe Asn Pro Asp Lys Ser phe val 275 280 285 val Pro Gin Glu val Tyr Asp His Tyr Gin Lys Thr Ile Leu Lys pro 290 295 300

Gly val Glu Ala Asn Asn Lys Trp Asn Lys Leu Phe ser Glu Tyr Gin 305 310 315 320

Lys Lys Phe Pro Glu Leu Gly Ala Glu Leu Ala Arg Arg Leu Ser Gly 325 330 335

Gin Leu Pro Ala Asn Trp Glu ser Lys Leu pro Thr Tyr Thr Ala Lys 340 345 350

Asp ser Ala val Ala Thr Arg Lys Leu Ser Glu Thr val Leu Glu Asp 355 360 365

Val Tyr Asn Gin Leu Pro Glu Leu Ile Gly Gly Ser Ala Asp Leu Thr 370 375 380

Pro Ser Asn Leu Thr Arg Trp Lys Glu Ala Leu Asp Phe Gin Pro Pro 385 390 395 400

Ser ser Gly Ser Gly Asn Tyr Ser Gly Arg Tyr Ile Arg Tyr Gly Ile 405 410 415

Arg Glu His Ala Met Gly Ala Ile Met Asn Gly Ile ser Ala Phe Gly 420 425 430

Ala Asn Tyr Lys Pro Tyr Gly Gly Thr Phe Leu Asn Phe val Ser Tyr 435 440 445

Ala Ala Gly Ala Val Arg Leu Ser Ala Leu ser Gly His Pro val Ile 450 455 460

Trp Val Ala Thr His Asp Ser Ile Gly Val Gly Glu Asp Gly pro Thr 465 470 475 480

His Gin Pro Ile Glu Thr Leu Ala His Phe Arg ser Leu Pro Asn ile 485 490 495

Gin val Trp Arg Pro Ala Asp Gly Asn Glu val Ser Ala Ala Tyr Lys 500 505 510

Asn Ser Leu Glu ser Lys His Thr Pro Ser Ile Ile Ala Leu Ser Arg 515 520 525

Gin Asn Leu Pro Gin Leu Glu Gly ser Ser Ile Glu Ser Ala Ser Lys 530 535 540

Gly Gly Tyr val Leu Gin Asp Val Ala Asn pro Asp Ile Ile Leu Val 545 550 555 560

Ala Thr Gly ser Glu val Ser Leu Ser val Glu Ala Ala Lys Thr Leu 565 570 575

Ala Ala Lys Asn Ile Lys Ala Arg val Val ser Leu pro Asp phe Phe 580 585 590

Thr Phe Asp Lys Gin Pro Leu Glu Tyr Arg Leu Ser Val Leu Pro Asp 595 600 605

Asn val Pro ile Met Ser Val Glu val Leu Ala Thr Thr cys Trp Gly 610 615 620

Lys Tyr Ala His Gin Ser phe Gly Ile Asp Arg Phe Gly Ala Ser Gly 625 630 635 640

Lys Ala Pro Glu Val Phe Lys Phe phe Gly phe Thr pro Glu Gly Val 645 650 655

Ala Glu Arg Ala Gin Lys Thr Ile Ala Phe Tyr Lys Gly Asp Lys Leu 660 665 670

Ile Ser Pro Leu Lys Lys Ala Phe 675 680

<210> 22 <211 >2046 <212> DNA <213> Saccharomyces cerevisiae <400> 22 atggcacagt tctccgacat tgataaactt gcggtttcca ctttaagatt actttccgtt 60 gaccaggtgg aaagcgcaca atctggccac ccaggtgcac cactaggatt ggcaccagtt 120 gcccatgtaa ttttcaagca actgcgctgt aaccctaaca atgaacattg gatcaataga 180 gacaggtttg ttctgtcgaa cggtcactca tgcgctcttc tgtactcaat gctccatcta 240 ttaggatacg attactctat cgaggacttg agacaattta gacaagtaaa ctcaaggaca 300 ccgggtcatc cagaattcca ctcagcggga gtggaaatca cttccggtcc gctaggccag 360 ggtatctcaa atgctgttgg tatggcaata gcgcaggcca actttgccgc cacttataac 420 gaggatggct ttcccatttc cgactcatat acgtttgcta ttgtagggga tggttgctta 480 caagagggtg tttcttcgga gacctcttcc ttagcgggac atctgcaatt gggtaacttg 540 attacgtttt atgacagtaa tagcatttcc attgacggta aaacctcgta ctcgttcgac 600 gaagatgttt tgaagcgata cgaggcatat ggttgggaag tcatggaagt cgataaagga 660 gacgacgata tggaatccat ttctagcgct ttggaaaagg caaaactatc gaaggacaag 720 ccaaccataa tcaaggtaac tactacaatt ggatttgggt ccctacaaca gggtactgct 780 ggtgttcatg ggtccgcttt gaaggcagat gatgttaaac agttgaagaa gaggtggggg 840 tttgacccaa ataaatcatt tgtagtacct caagaggtgt acgattatta taagaagact „ 900 gttgtggaac ccggtcaaaa acttaatgag gaatgggata ggatgtttga agaatacaaa 960 accaaatttc ccgagaaggg taaagaattg caaagaagat tgaatggtga gttaccggaa 1020 ggttgggaaa agcatttacc gaagtttact ccggacgacg atgctctggc aacaagaaag 1080 acatcccagc aggtgctgac gaacatggtc caagttttgc ctgaattgat cggtggttct 1140 gccgatttga caccttcgaa tctgacaagg tgggaaggcg cggtagattt ccaacctccc 1200 attacccaac taggtaacta tgcaggaagg tacattagat acggtgtgag ggaacacgga 1260 atgggtgcca ttatgaacgg tatctctgcc tttggtgcaa actacaagcc ttacggtggt 1320 acctttttga acttcgtctc ttatgctgca ggagccgtta ggttagccgc cttgtctggt 1380 aatccagtca tttgggttgc aacacatgac tctatcgggc ttggtgagga tggtccaacg 1440 caccaaccta ttgaaactct ggctcacttg agggctattc caaacatgca tgtatggaga 1500 cctgctgatg gtaacgaaac ttctgctgcg tattattctg ctatcaaatc tggtcgaaca 1560 ccatctgttg tggctttatc acgacagaat cttcctcaat tggagcattc ctcttttgaa 1620 aaagccttga agggtggcta tgtgatccat gacgtggaga atcctgatat tatcctggtg 1680 tcaacaggat cagaagtctc catttctata gatgcagcca aaaaattgta cgatactaaa 1740 aaaatcaaag caagagttgt ttccctgcca gacttttata cttttgacag gcaaagtgaa 1800 gaatacagat tctctgttct accagacggt gttccgatca tgtcctttga agtattggct 1860 acttcaagct ggggtaagta tgctcatcaa tcgttcggac tcgacgaatt tggtcgttca 1920 ggcaaggggc ctgaaattta caaattgttc gatttcacag cggacggtgt tgcgtcaagg 1980 gctgaaaaga caatcaatta ctacaaagga aagcagttgc tttctcctat gggaagagct 2040 ttctaa 2046

<210> 23 <211 > 335 <212> PRT <213> Saccharomyces cerevisiae <400> 23

Met ser Glu Pro Ala Gin Lys Lys Gin Lys val Ala Asn Asn ser Leu 15 10 15

Glu Gin Leu Lys Ala Ser Gly Thr val val val Ala Asp Thr Gly Asp 20 25 30

Phe Gly ser Ile Ala Lys Phe Gin Pro Gin Asp Ser Thr Thr Asn Pro 35 40 45

Ser Leu Ile Leu Ala Ala Ala Lys Gin Pro Thr Tyr Ala Lys Leu ile 50 55 60

Asp Val Ala Val Glu Tyr Gly Lys Lys His Gly Lys Thr Thr Glu Glu 65 70 75 80

Gin Val Glu Asn Ala Val Asp Arg Leu Leu Val Glu Phe Gly Lys Glu 85 90 95

Ile Leu Lys Ile Val Pro Gly Arg Val Ser Thr Glu Val Asp Ala Arg 100 105 110

Leu Ser Phe Asp Thr Gin Ala Thr Ile Glu Lys Ala Arg His Ile Ile 115 120 125 .

Lys Leu Phe Glu Gin Glu Gly Val Ser Lys Glu Arg Val Leu Ile Lys 130 135 140

Ile Ala ser Thr Trp Glu Gly ile Gin Ala Ala Lys Glu Leu Glu Glu 145 150 155 160

Lys Asp Gly Ile His Cys Asn Leu Thr Leu Leu Phe ser Phe val Gin 165 170 175

Ala Val Ala cys Ala Glu Ala Gin Val Thr Leu Ile Ser pro Phe val 180 185 190

Gly Arg Ile Leu Asp Trp Tyr Lys Ser ser Thr Gly Lys Asp Tyr Lys 195 200 205

Gly Glu Ala Asp Pro Gly Val Ile Ser Val Lys Lys Ile Tyr Asn Tyr 210 215 220

Tyr Lys Lys Tyr Gly Tyr Lys Thr Ile Val Met Gly Ala ser Phe Arg 225 230 235 240

Ser Thr Asp Glu Ile Lys Asn Leu Ala Gly Val Asp Tyr Leu Thr ile 245 250 255

Ser pro Ala Leu Leu Asp Lys Leu Met Asn ser Thr Glu Pro Phe Pro 260 265 270

Arg Val Leu Asp Pro val Ser Ala Lys Lys Glu Ala Gly Asp Lys ile 275 280 285 ser Tyr ile Ser Asp Glu Ser Lys phe Arg Phe Asp Leu Asn Glu Asp 290 295 300

Ala Met Ala Thr Glu Lys Leu Ser Glu Gly Ile Arg Lys Phe Ser Ala 305 310 315 320

Asp ile Val Thr Leu Phe Asp Leu ile Glu Lys Lys val Thr Ala 325 330 335

<210> 24 <211 >2046 <212> DNA <213> Saccharomyces cerevisiae <400> 24 atggcacagt tctccgacat tgataaactt gcggtttcca ctttaagatt actttccgtt 60 gaccaggtgg aaagcgcaca atctggccac ccaggtgcac cactaggatt ggcaccagtt 120 gcccatgtaa ttttcaagca actgcgctgt aaccctaaca atgaacattg gatcaataga 180 gacaggtttg ttctgtcgaa cggtcactca tgcgctcttc tgtactcaat gctccatcta 240 ttaggatacg attactctat cgaggacttg agacaattta gacaagtaaa ctcaaggaca 300 ccgggtcatc cagaattcca ctcagcggga gtggaaatca cttccggtcc gctaggccag 360 ggtatctcaa atgctgttgg tatggcaata gcgcaggcca actttgccgc cacttataac 420 gaggatggct ttcccatttc cgactcatat acgtttgcta ttgtagggga tggttgctta 480 caagagggtg tttcttcgga gacctcttcc ttagcgggac atctgcaatt gggtaacttg 540 attacgtttt atgacagtaa tagcatttcc attgacggta aaacctcgta ctcgttcgac 600 gaagatgttt tgaagcgata cgaggcatat ggttgggaag tcatggaagt cgataaagga 660 gacgacgata tggaatccat ttctagcgct ttggaaaagg caaaactatc gaaggacaag 720 ccaaccataa tcaaggtaac tactacaatt ggatttgggt ccctacaaca gggtactgct 780 ggtgttcatg ggtccgcttt gaaggcagat gatgttaaac agttgaagaa gaggtggggg 840 tttgacccaa ataaatcatt tgtagtacct caagaggtgt acgattatta taagaagact 900 gttgtggaac ccggtcaaaa acttaatgag gaatgggata ggatgtttga agaatacaaa 960 accaaatttc ccgagaaggg taaagaattg caaagaagat tgaatggtga gttaccggaa 1020 ggttgggaaa agcatttacc gaagtttact ccggacgacg atgctctggc aacaagaaag 1080 acatcccagc aggtgctgac gaacatggtc caagttttgc ctgaattgat cggtggttct 1140 gccgatttga caccttcgaa tctgacaagg tgggaaggcg cggtagattt ccaacctccc 1200 attacccaac taggtaacta tgcaggaagg tacattagat acggtgtgag ggaacacgga 1260 atgggtgcca ttatgaacgg tatctctgcc tttggtgcaa actacaagcc ttacggtggt 1320 acctttttga acttcgtctc ttatgctgca ggagccgtta ggttagccgc cttgtctggt 1380 aatccagtca tttgggttgc aacacatgac tctatcgggc ttggtgagga tggtccaacg 1440 caccaaccta ttgaaactct ggctcacttg agggctattc caaacatgca tgtatggaga 1500 cctgctgatg gtaacgaaac ttctgctgcg tattattctg ctatcaaatc tggtcgaaca 1560 ccatctgttg tggctttatc acgacagaat cttcctcaat tggagcattc ctcttttgaa 1620 aaagccttga agggtggcta tgtgatccat gacgtggaga atcctgatat tatcctggtg 1680 tcaacaggat cagaagtctc catttctata gatgcagcca aaaaattgta cgatactaaa 1740 aaaatcaaag caagagttgt ttccctgcca gacttttata cttttgacag gcaaagtgaa 1800 gaatacagat tctctgttct accagacggt gttccgatca tgtcctttga agtattggct 1860 acttcaagct ggggtaagta tgctcatcaa tcgttcggac tcgacgaatt tggtcgttca 1920 ggcaaggggc ctgaaattta caaattgttc gatttcacag cggacggtgt tgcgtcaagg . 1980 gctgaaaaga caatcaatta ctacaaagga aagcagttgc tttctcctåt gggaagagct 2040 ttctaa 2046

<210> 25 <211 > 600 <212> PRT <213> Saccharomyces cerevisiae <400> 25

Met Leu Cys Ser Val ile Gin Arg Gin Thr Arg Glu Val Ser Asn Thr 15 10 15

Met Ser Leu Asp Ser Tyr Tyr Leu Gly Phe Asp Leu Ser Thr Gin Gin 20 25 30

Leu Lys cys Leu Ala lie Asn Gin Asp Leu Lys ile val His Ser Glu 35 40 45

Thr Val Glu Phe Glu Lys Asp Leu Pro His Tyr His Thr Lys Lys Gly 50 55 60

Val Tyr lie His Gly Asp Thr lie Glu cys Pro val Ala Met Trp Leu 65 70 75 80

Glu Ala Leu Asp Leu Val Leu Ser Lys Tyr Arg Glu Ala Lys Phe Pro 85 90 95

Leu Asn Lys Val Met Ala Val ser Gly Ser Cys Gin Gin His Gly ser 100 105 110

Val Tyr Trp Ser Ser Gin Ala Glu ser Leu Leu Glu Gin Leu Asn Lys 115 120 “' " " " 125

Lys Pro Glu Lys Asp Leu Leu His Tyr val ser ser val Ala Phe Ala 130 135 140

Arg Gin Thr Ala Pro Asn Trp Gin Asp His Ser Thr Ala Lys Gin Cys 145 150 155 160

Gin Glu Phe Glu Glu Cys lie Gly Gly Pro Glu Lys Met Ala Gin Leu 165 170 175

Thr Gly Ser Arg Ala His Phe Arg Phe Thr Gly Pro Gin lie Leu Lys 180 185 190 lie Ala Gin Leu Glu Pro Glu Ala Tyr Glu Lys Thr Lys Thr lie ser 195 200 205

Leu val Ser Asn Phe Leu Thr ser lie Leu Val Gly His Leu Val Glu 210 215 220

Leu Glu Glu Ala Asp Ala Cys Gly Met Asn Leu Tyr Asp lie Arg Glu 225 230 235 240

Arg Lys Phe ser Asp Glu Leu Leu His Leu lie Asp ser ser ser Lys 245 250 255

Asp Lys Thr lie Arg Gin Lys Leu Met Arg Ala Pro Met Lys Asn Leu 260 265 270 lie Ala Gly Thr lie Cys Lys Tyr Phe lie Glu Lys Tyr Gly Phe Asn 275 280 285

Thr Asn cys Lys val ser Pro Met Thr Gly Asp Asn Leu Ala Thr lie 290 295 300 cys Ser Leu Pro Leu Arg Lys Asn Asp val Leu val ser Leu Gly Thr 305 310 315 320

Ser Thr Thr val Leu Leu Val Thr Asp Lys Tyr His Pro Ser Pro Asn 325 330 335

Tyr His Leu phe lie His Pro Thr Leu Pro Asn His Tyr Met Gly Met 340 345 350 lie cys Tyr cys Asn Gly ser Leu Ala Arg Glu Arg lie Arg Asp Glu 355 360 365

Leu Asn Lys Glu Arg Glu Asn Asn Tyr Glu Lys Thr Asn Asp Trp Thr 370 375 380

Leu Phe Asn Gin Ala Val Leu Asp Asp Ser Glu Ser Ser Glu Asn Glu 385 390 ’""395 ’ 400

Leu Gly val Tyr Phe Pro Leu Gly Glu lie Val Pro Ser Val Lys Ala 405 410 415

Ile Asn Lys Arg Val lie Phe Asn Pro Lys Thr Gly Met ile Glu Arg 420 425 430

Glu Val Ala Lys Phe Lys Asp Lys Arg His Asp Ala Lys Asn lie Val 435 440 445

Glu Ser Gin Ala Leu Ser Cys Arg Val Arg Ile Ser Pro Leu Leu Ser 450 455 460

Asp Ser Asn Ala ser Ser Gin Gin Arg Leu Asn Glu Asp Thr lie Val 465 470 475 480

Lys Phe Asp Tyr Asp Glu Ser Pro Leu Arg Asp Tyr Leu Asn Lys Arg 485 490 495

Pro Glu Arg Thr phe Phe val Gly Gly Ala Ser Lys Asn Asp Ala lie 500 505 510

Val Lys Lys phe Ala Gin Val lie Gly Ala Thr Lys Gly Asn phe Arg 515 520 525

Leu Glu Thr Pro Asn Ser cys Ala Leu Gly Gly Cys Tyr Lys Ala Met 530 535 540

Trp Ser Leu Leu Tyr Asp Ser Asn Lys lie Ala Val Pro Phe Asp Lys 545 550 555 560

Phe Leu Asn Asp Asn Phe Pro Trp His Val Met Glu Ser lie Ser Asp 565 570 575

Val Asp Asn Glu Asn Trp Asp Arg Tyr Asn ser Lys lie Val Pro Leu 580 585 590

Ser Glu Leu Glu Lys Thr Leu lie 595 600

<210> 26 <211 >2467 <212> DNA <213> Saccharomyces cerevisiae <400> 26 ggatccaaga ccattattcc atcagaatgg aaaaaagttt aaaagatcac ggagattttg 60 ttcttctgag cttctgctgt ccttgaaaac aaattattcc gctggccgcc ccaaacaaaa 120 acaaccccga tttaataaca ttgtcacagt attagaaatt ttctttttac aaattaccat 180 ttccagctta ctacttccta taatcctcaa tcttcagcaa gcgacgcagg gaatagccgc 240 tgaggtgcat aactgtcact tttcaattcg gccaatgcaa tctcaggcgg acgaataagg 300 gggccctctc gagaaaaaca aaaggaggat gagattagta ctttaatgtt gtgttcagta 360 attcagagac agacaagaga ggtttccaac acaatgtctt tagactcata ctatcttggg 420 tttgatcttt cgacccaaca actgaaatgt ctcgccatta accaggacct aaaaattgtc 480 cattcagaaa cagtggaatt tgaaaaggat cttccgcatt atcacacaaa gaagggtgtc 540 tatatacacg gcgacactat cgaatgtccc gtagccatgt ggttaggggc tctagatctg 600 gttctctcga aatatcgcga ggctaaattt ccattgaaca aagttatggc cgtctcaggg 660 tcctgccagc agcacgggtc tgtctactgg tcctcccaag ccgaatctct gttagagcaa 720 ttgaataaga aaccggaaaa agatttattg cactacgtga gctctgtagc atttgcaagg 780 caaaccgccc ccaattggca agaccacagt actgcaaagc aatgtcaaga gtttgaagag 840 tgcataggtg ggcctgaaaa aatggctcaa ttaacagggt ccagagccca ttttagattt 900 actggtcctc aaattctgaa aattgcacaa ttagaaccag aagcttacga aaaaacaaag 960 accatttctt tagtgtctaa ttttttgact tctatcttag tgggccatct tgttgaatta „ 1020 gaggaggcag atgcctgtgg tatgaacctt tatgatatac gtgaaagaaa attcatgtat 1080 gagctactac atctaattga tagttcttct aaggataaaa ctatcagaca aaaattaatg 1140 agagcaccca tgaaaaattt gatagcgggt accatctgta aatattttat tgagaagtac 1200 ggtttcaata caaactgcaa ggtctctccc atgactgggg ataatttagc cactatatgt 1260 tctttacccc tgcggaagaa tgacgttctc gtttccctag gaacaagtac tacagttctt 1320 ctggtcaccg ataagtatca cccctctccg aactatcatc ttttcattca tccaactctg 1380 ccaaaccatt atatgggtat gatttgttat tgtaatggtt ctttggcaag ggagaggata 1440 agagacgagt taaacaaaga acgggaaaat aattatgaga agactaacga ttggactctt 1500 tttaatcaag ctgtgctaga tgactcagaa agtagtgaaa atgaattagg tgtatatttt 1560 cctctggggg agatcgttcc tagcgtaaaa gccataaaca aaagggttat cttcaatcca 1620 aaaacgggta tgattgaaag agaggtggcc aagttcaaag acaagaggca cgatgccaaa 1680 aatattgtag aatcacaggc tttaagttgc agggtaagaa tatctcccct gctttcggat 1740 tcaaacgcaa gctcacaaca gagactgaac gaagatacaa tcgtgaagtt tgattacgat 1800 gaatctccgc tgcgggacta cctaaataaa aggccagaaa ggactttttt tgtaggtggg 1860 gcttctaaaa acgatgctat tgtgaagaag tttgctcaag tcattggtgc tacaaagggt 1920 aattttaggc tagaaacacc aaactcatgt gcccttggtg gttgttataa ggccatgtgg 1980 tcattgttat atgactctaa taaaattgca gttccttttg ataaatttct gaatgacaat 2040 tttccatggc atgtaatgga aagcatatcc gatgtggata atgaaaattg gatcgctata 2100 attccaagat tgtcccctta agcgaactgg aaaagactct catctaaaat atgtttgaat 2160 aatttatcat gccctgacaa gtacacacaa acacagacac ataatataca tacatatata 2220 tatatcaccg ttattatgcg tgcacatgac aatgcccttg tatgtttcgt atactgtagc 2280 aagtagtcat cattttgttc cccgttcgga aaatgacaaa aagtaaaatc aataaatgaa 2340 gagtaaaaaa caatttatga aagggtgagc gaccagcaac gagagagaca aatcaaatta 2400 gcgctttcca gtgagaatat aagagagcat tgaaagagct aggttattgt taaatcatct 2460 cgagctc 2467

<210> 27 <211 >494 <212> PRT <213> Piromyces species <400> 27

Met Lys Thr Val Ala Gly Ile Asp Leu Gly Thr Gin Ser Met Lys Val 15 10 15

Val Ile Tyr Asp Tyr Glu Lys Lys Glu lie lie Glu Ser Ala Ser Cys 20 25 SO

Pro Met Glu Leu Ile ser Glu Ser Asp Gly Thr Arg Glu Gin Thr Thr 35 40 45

Glu Trp Phe Asp Lys Gly Leu Glu Val Cys phe Gly Lys Leu Ser Ala 50 55 60

Asp Asn Lys Lys Thr lie Glu Ala lie Gly lie ser Gly Gin Leu His 65 70 75 80

Gly Phe Val Pro Leu Asp Ala Asn Gly Lys Ala Leu Tyr Asn lie Lys 85 90 95

Leu Trp Cys Asp Thr Ala Thr Val Glu Glu Cys Lys lie lie Thr Asp 100 105 110

Ala Ala Gly Gly Asp Lys Ala Val lie Asp Ala Leu Gly Asn Leu Met 115 120 125

Leu Thr Gly Phe Thr Ala Pro Lys lie Leu Trp Leu Lys Arg Asn Lys 130 135 140 pro Glu Ala phe Ala Asn Leu Lys Tyr lie Met Leu Pro His Asp Tyr 145 150 155 160

Leu Asn Trp Lys Leu Thr Gly Asp Tyr Val Met Glu Tyr Gly Asp Ala 165 170 175 ser Gly Thr Ala Leu Phe Asp Ser Lys Asn Arg cys Trp Ser Lys Lys 180 185 190 lie Cys Asp lie lie Asp Pro Lys Leu Leu Asp Leu Leu Pro Lys Leu 195 200 205 lie Glu Pro ser Ala Pro Ala Gly Lys Val Asn Asp Glu Ala Ala Lys 210 215 220

Ala Tyr Gly lie Pro Ala Gly lie Pro Val ser Ala Gly Gly Gly Asp 225 230 235 240

Asn Met Met Gly Ala Val Gly Thr Gly Thr Val Ala Asp Gly Phe Leu 245 250 255

Thr Met Ser Met Gly Thr Ser Gly Thr Leu Tyr Gly Tyr Ser Asp Lys 260 265 270

Pro lie ser Asp Pro Ala Asn Gly Leu Ser Gly Phe Cys ser Ser Thr 275 280 285

Gly Gly Trp Leu Pro Leu Leu Cys Thr Met Asn Cys Thr Val Ala Thr 290 295 300

Glu Phe val Arg Asn Leu Phe Gin Met Asp lie Lys Glu Leu Asn Val 305 310 315 320

Glu Ala Ala Lys Ser Pro Cys Gly Ser Glu Gly Val Leu val lie Pro 325 330 335

Phe Phe Asn Gly Glu Arg Thr Pro Asn Leu Pro Asn Gly Arg Ala Ser 340 345 350 lie Thr Gly Leu Thr Ser Ala Asn Thr Ser Arg Ala Asn lie Ala Arg 355 360 365

Ala ser Phe Glu Ser Ala Val Phe Ala Met Arg Gly Gly Leu Asp Ala 370 375 380

Phe Arg Lys Leu Gly Phe Gin Pro Lys Glu lie Arg Leu lie Gly Gly 385 390 395 400

Gly Ser Lys ser Asp Leu Trp Arg Gin lie Ala Ala Asp lie Met Asn 405 410 415

Leu Pro lie Arg val Pro Leu Leu Glu Glu Ala Ala Ala Leu Gly Gly 420 425 430

Ala val Gin Ala Leu Trp cys Leu Lys Asn Gin ser Gly Lys cys Asp 435 440 445 lie Val Glu Leu Cys Lys Glu His lie Lys lie Asp Glu Ser Lys Asn 450 455 460

Ala Asn Pro lie Ala Glu Asn val Ala Val Tyr Asp Lys Ala Tyr Asp 465 470 475 480

Glu Tyr cys Lys Val val Asn Thr Leu Ser pro Leu Tyr Ala 485 490

<210> 28 <211> 2041 <212> DNA <213> Piromyces species <400> 28 attatataaa ataactttaa ataaaacaat ttttatttgt ttatttaatt attcaaaaaa 60 aattaaagta aaagaaaaat aatacagtag aacaatagta ataatatcaa aatgaagact 120 gttgctggta ttgatcttgg aactcaaagt atgaaagtcg ttatttacga ctatgaaaag 180 aaagaaatta ttgaaagtgc tagctgtcca atggaattga tttccgaaag tgacggtacc 240 cgtgaacaaa ccactgaatg gtttgacaag ggtcttgaag tttgttttgg taagcttagt 300 gctgataaca aaaagactat tgaagctatt ggtatttctg gtcaattaca cggttttgtt 360 cctcttgatg ctaacggtaa ggctttatac aacatcaaac tttggtgtga tactgctacc 420 gttgaagaat gtaagattat cactgatgct gccggtggtg acaaggctgt tattgatgcc * 480 cttggtaacc ttatgctcac cggtttcacc gctccaaaga tcctctggct caagcgcaac 540 aagccagaag ctttcgctaa cttaaagtac attatgcttc cacacgatta cttaaactgg 600 aagcttactg gtgattacgt tatggaatac ggtgatgcct ctggtaccgc tctcttcgat 660 tctaagaacc gttgctggtc taagaagatt tgcgatatca ttgacccaaa acttttagat 720 ttacttccaa agttaattga accaagcgct ccagctggta aggttaatga tgaagccgct 780 aaggcttacg gtattccagc cggtattcca gtttccgctg gtggtggtga taacatgatg 840 ggtgctgttg gtactggtac tgttgctgat ggtttcctta ccatgtctat gggtacttct 900 ggtactcttt acggttacag tgacaagcca attagtgacc cagctaatgg tttaagtggt 960 ttctgttctt ctactggtgg atggcttcca ttactttgta ctatgaactg tactgttgcc 1020 actgaattcg ttcgtaacct cttccaaatg gatattaagg aacttaatgt tgaagctgcc 1080 aagtctccat gtggtagtga aggtgtttta gttattccat tcttcaatgg tgaaagaact 1140 ccaaacttac caaacggtcg tgctagtatt actggtctta cttctgctaa caccagccgt 1200 gctaacattg ctcgtgctag tttcgaatcc gccgttttcg ctatgcgtgg tggtttagat 1260 gctttccgta agttaggttt ccaaccaaag gaaattcgtc ttattggtgg tggttctaag 1320 tctgatctct ggagacaaat tgccgctgat atcatgaacc ttccaatcag agttccactt 1380 ttagaagaag ctgctgctct tggtggtgct gttcaagctt tatggtgtct taagaaccaa 1440 tctggtaagt gtgatattgt tgaactttgc aaagaacaca ttaagattga tgaatctaag 1500 aatgctaacc caattgccga aaatgttgct gtttacgaca aggcttacga tgaatactgc 1560 aaggttgtaa atactcttte tccattatat gcttaaattg ccaatgtaaa aaaaaatata 1620 atgccatata attgccttgt caatacactg ttcatgttca tataatcata ggacattgaa 1680 tttacaaggt ttatacaatt aatatctatt atcatattat tatacagcat ttcattttct 1740 aagattagac gaaacaattc ttggttcctt gcaatataca aaatttacat gaatttttag 1800 aatagtctcg tatttatgcc caataatcag gaaaattacc taatgctgga ttcttgttaa 1860 taaaaacaaa ataaataaat taaataaaca aataaaaatt ataagtaaat ataaatatat 1920 aagtaatata aaaaaaaagt aaataaataa ataaataaat aaaaattttt tgcaaatata 1980 taaataaata aataaaatat aaaaataatt tagcaaataa attaaaaaaa aaaaaaaaaa 2040 a 2041

<210> 29 <211 > 327 <212> PRT <213> Saccharomyces cerevisiae <400> 29

Met ser ser Leu Val Thr Leu Asn Asn Gly Leu Lys Met Pro Leu val 15 10 15

Gly Leu Gly cys Trp Lys ile Asp Lys Lys val cys Ala Asn Gin ile » 20 25 30

Tyr Glu Ala Ile Lys Leu Gly Tyr Arg Leu Phe Asp Gly Ala cys Asp 35 40 45

Tyr Gly Asn Glu Lys Glu val Gly Glu Gly Ile Arg Lys Ala ile ser 50 55 60

Glu Gly Leu Val ser Arg Lys Asp Ile Phe val Val Ser Lys Leu Trp 65 70 75 80

Asn Asn Phe His His pro Asp His val Lys Leu Ala Leu Lys Lys Thr 85 90 95

Leu Ser Asp Met Gly Leu Asp Tyr Leu Asp Leu Tyr Tyr Ile His Phe 100 105 110

Pro Ile Ala Phe Lys Tyr val Pro Phe Glu Glu Lys Tyr Pro Pro Gly 115 120 125

Phe Tyr Thr Gly Ala Asp Asp Glu Lys Lys Gly His Ile Thr Glu Ala 130 135 140

His Val Pro Ile Ile Asp Thr Tyr Arg Ala Leu Glu Glu Cys Val Asp 145 150 155 160

Glu Gly Leu ile Lys Ser Ile Gly Val Ser Asn Phe Gin Gly ser Leu 165 170 175

Ile Gin Asp Leu Leu Arg Gly cys Arg Ile Lys Pro val Ala Leu Gin 180 185 190 ile Glu His His Pro Tyr Leu Thr Gin Glu His Leu val Glu Phe Cys 195 200 205

Lys Leu His Asp ile Gin Val val Ala Tyr Ser Ser Phe Gly Pro Gin 210 215 220 ser Phe Ile Glu Met Asp Leu Gin Leu Ala Lys Thr Thr Pro Thr Leu 225 230 235 240

Phe Glu Asn Asp Val Ile Lys Lys val ser Gin Asn His Pro Gly ser 245 250 255

Thr Thr ser Gin Val Leu Leu Arg Trp Ala Thr Gin Arg Gly Ile Ala 260 265 270 val Ile Pro Lys ser Ser Lys Lys Glu Arg Leu Leu Gly Asn Leu Glu 275 280 285

Ile Glu Lys Lys Phe Thr Leu Thr Glu Gin Glu Leu Lys Asp Ile Ser 290 295 300

Ala Leu Asn Ala Asn Ile Arg Phe Asn Asp Pro Trp Thr Trp Leu Asp 305 310 315 320

Gly Lys phe Pro Thr Phe Ala 325

<210> 30 <211> 984 <212> DNA <213> Saccharomyces cerevisiae <400> 30 atgtcttcac tggttactct taataacggt ctgaaaatgc ccctagtcgg cttagggtgc 60 tggaaaattg acaaaaaagt ctgtgcgaat caaatttatg aagctatcaa attaggctac 120 cgtttattcg atggtgcttg cgactacggc aacgaaaagg aagttggtga aggtatcagg 180 aaagccatct ccgaaggtct tgtttctaga aaggatatat ttgttgtttc aaagttatgg 240 aacaattttc accatcctga tcatgtaaaa ttagctttaa agaagacctt aagcgatatg 300 ggacttgatt atttagacct gtattatatt cacttcccaa tcgccttcaa atatgttcca 360 tttgaagaga aataccctcc aggattctat acgggcgcag atgacgagaa gaaaggtcac 420 atcaccgaag cacatgtacc aatcatagat acgtaccggg ctctggaaga atgtgttgat 480 gaaggcttga ttaagtctat tggtgtttcc aactttcagg gaagcttgat tcaagattta 540 ttacgtggtt gtagaatcaa gcccgtggct ttgcaaattg aacaccatcc ttatttgact 600 caagaacacc tagttgagtt ttgtaaatta cacgatatcc aagtagttgc ttactcctcc 660 ttcggtcctc aatcattcat tgagatggac ttacagttgg caaaaaccac gccaactctg 720 ttcgagaatg atgtaatcaa gaaggtctca caaaaccatc caggcagtac cacttcccaa 780 gtattgctta gatgggcaac tcagagaggc attgccgtca ttccaaaatc ttccaagaag 840 gaaaggttac ttggcaacct agaaatcgaa aaaaagttca ctttaacgga gcaagaattg 900 aaggatattt ctgcactaaa tgccaacatc agatttaatg atccatggac ctggttggat 960 ggtaaattcc ccacttttgc ctga 984

<210>31 <211 > 31 <212> DNA <213> ARTIFICIAL <220 <223> primer <400 31 gactagtcga gtttatcatt atcaatactg c 31 <210> 32 <211> 49 <212> DNA <213> Artificial <220> <223> primer <400> 32 ctcataatca ggtactgata acattttgtt tgtttatgtg tgtttattc 49 <210> 33 <211> 49 <212> DNA <213> artificial <220> <223> primer <400> 33 gaataaacac acataaacaa acaaaatgtt atcagtacct gattatgag 49 <210> 34 <211> 48 <212> DNA <213> artificial <220> <223> primer aatcataaat cataagaaat tcgcttactt taagaatgcc ttagtcat 48 <210> 35 <211> 48 <212> DNA <213> artificial <220> <223> primer <400> 35 atgactaagg cattcttaaa gtaagcgaat ttcttatgat ttatgatt 48 <210> 36 <211> 36 <212> DNA <213> artificial <220> <223> primer <400> 36 cactagtctc gagtgtggaa gaacgattac aacagg 36 <210> 37 <211 > 31 <212> DNA <213> artificial <220> <223> primer <400> 37 cgagctcgtg ggtgtattgg attataggaa g 31 <210> 38 <211> 48 <212> DNA <213> artificial <220> <223> primer <400> 38 ttgggctgtt tcaactaaat tcatttttag gctggtatct tgattcta 48 <210> 39 <211> 48 <212> DNA <213> artificial <220> <223> primer <400> 39 tagaatcaag ataccagcct aaaaatgaat ttagttgaaa cagcccaa 48 <210> 40 <211> 48 <212> DNA <213> artificial <220> <223> primer <400> 40 aatcataaat cataagaaat tcgctctaat atttgattgc ttgcccag 48 <210>41 <211> 48 <212> DNA <213> artificial <220> <223> primer <400> 41 ctgggcaagc aatcaaatat tagagcgaat ttcttatgat ttatgatt 48 <210> 42 <211 > 31 <212> DNA <213> artificial <220> <223> primer <400> 42 tgagctcgtg tggaagaacg attacaacag g 31 <210> 43 <211> 28 <212> DNA <213> artificial <220> <223> primer <400> 43 acgcgtcgac tcgtaggaac aatttcgg 28 <210> 44 <211> 50 <212> DNA <213> artificial <220> <223> primer <400> 44 cttcttgttt taatgcttct agcatttttt gattaaaatt aaaaaaactt 50 <210> 45 <211> 50 <212> DNA <213> artificial <220> <223> primer <400> 45 aagttttttt aattttaatc aaaaaatgct agaagcatta aaacaagaag 50

<210> 46 <211> 46 <212> DNA <213> artificial <220> <223> primer <400> 46 ggtatatatt taagagcgat ttgtttactt gcgaactgca tgatcc 46 <210> 47 <211> 46 <212> DNA <213> artificial <220> <223> primer <400> 47 ggatcatgca gttcgcaagt aaacaaatcg ctcttaaata tatacc 46 <210> 48 <211> 33 <212> DNA <213> artificial <220> <223> primer <400> 48 cgcagtcgac cttttaaaca gttgatgaga acc 33 <210> 49 <211 > 676 <212> DNA <213> artificial <220> <223> promoter <400> 49 tcgagtttat cattatcaat actgccattt caaagaatac gtaaataatt aatagtagtg 60 attttcctaa ctttatttag tcaaaaaatt agccttttaa ttctgctgta acccgtacat 120 gcccaaaata gggggcgggt tacacagaat atataacatc gtaggtgtct gggtgaacag 180 tttattcctg gcatccacta aatataatgg agcccgcttt ttaagctggc atccagaaaa 240 aaaaagaatc ccagcaccaa aatattgttt tcttcaccaa ccatcagttc ataggtccat 300 tctcttagcg caactacaga gaacaggggc acaaacaggc aaaaaacggg cacaacctca 360 atggagtgat gcaacctgcc tggagtaaat gatgacacaa ggcaattgac ccacgcatgt 420 atctatctca ttttcttaca ccttctatta ccttctgctc tctctgattt ggaaaaagct 480 gaaaaaaaag gttgaaacca gttccctgaa attattcccc tacttgacta ataagtatat 540 aaagacggta ggtattgatt gtaattctgt aaatctattt cttaaacttc ttaaattcta 600 cttttatagt tagtcttttt tttagtttta aaacaccaag aacttagttt cgaataaaca 660 cacataaaca aacaaa 676 <210> 50 <211 > 326 <212> DNA <213> artificial <220> <223> terminator <400> 50 gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 120 ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc 180 tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca attgtagata 240 tgctaactcc agcaatgagt tgatgaatct cggtgtgtat tttatgtcct cagaggacaa 300 cacctgttgt aatcgttctt ccacac 326 <210>51 <211 > 374 <212> DNA <213> artificial <220> <223> promoter <400> 51 gtgggtgtat tggattatag gaagccacgc gctcaacctg gaattacagg aagctggtaa 60 ttttttgggt ttgcaatcat caccatctgc acgttgttat aatgtcccgt gtctatatat 120 atccattgac ggtattctat ttttttgcta ttgaaatgag cgttttttgt tactacaatt 180 ggttttacag acggaatttt ccctatttgt ttcgtcccat ttttcctttt ctcattgttc * 240 tcatatctta aaaaggtcct ttcttcataa tcaatgcttt cttttactta atattttact 300 tgcattcagt gaattttaat acatattcct ctagtcttgc aaaatcgatt tagaatcaag 360 ataccagcct aaaa 374 <210> 52 <211 > 390 <212> DNA <213> artificial <220> <223> promoter <400> 52 ctcgtaggaa caatttcggg cccctgcgtg ttcttctgag gttcatcttt tacatttgct 60 tctgctggat aattttcaga ggcaacaagg aaaaattaga tggcaaaaag tcgtctttca 120 aggaaaaatc cccaccatct ttcgagatcc cctgtaactt attggcaact gaaagaatga 180 aaaggaggaa aatacaaaat atactagaac tgaaaaaaaa aaagtataaa tagagacgat 240 atatgccaat acttcacaat gttcgaatct attcttcatt tgcagctatt gtaaaataat 300 aaaacatcaa gaacaaacaa gctcaacttg tcttttctaa gaacaaagaa taaacacaaa 360 aacaaaaagt ttttttaatt ttaatcaaaa 390 <210> 53 <211 > 302 <212> DNA <213> artificial <220> <223> terminator <400> 53 acaaatcgct cttaaatata tacctaaaga acattaaagc tatattataa gcaaagatac 60 gtaaattttg cttatattat tatacacata tcatatttct atatttttaa gatttggtta 120 tataatgtac gtaatgcaaa ggaaataaat tttatacatt attgaacagc gtccaagtaa 180 ctacattatg tgcactaata gtttagcgtc gtgaagactt tattgtgtcg cgaaaagtaa 240 aaattttaaa aattagagca ccttgaactt gcgaaaaagg ttctcatcaa ctgtttaaaa 300 gg 302

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description • EP1499708A f0Q031 f00181 £00191 [0019] • WQ03062430A [0004] [00711 • W006009434A 830041 F0Q33F ΓΡ0341 [00431 fOOSSI Γ00601 f0071l • W02007041289A f00211 • W0030624430A Γ00261 F00341 Γ00561 • WQ9014423A Γ00261 • EP0481008AF0D2S1 • EP0835574A 100261 Γ00721 f0074f Γ00761 • US82651883 f0026f • WQ9303159A F00301 • WQ9848772A Γ00721 • W09960102A rø0721 • WQ0037871A 100721 • EPOcif 21833A [0102] . US60848357B F01021

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Claims (26)

A yeast cell capable of expressing the following nucleotide sequences, wherein the expression of these nucleotide sequences imparts the yeast cell the ability to utilize L-arabinose and / or convert L-arabinose to L-ribulose and / or xylulose-5-phosphate and / or to ethanol in which yeast cell is expressed: (a) a nucleotide sequence encoding an arabinose isomerase (araA), wherein the nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding an araA, which araA comprises an amino acid sequence, having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1; ii. nucleotide sequences comprising a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 2; iii. nucleotide sequences whose complementary strand hybridizes to a nucleic acid molecule with the sequence of (i) or (ii); (b) a nucleotide sequence encoding an L-ribulokinase (araB), wherein the nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding an araB, which araB comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 3; ii. nucleotide sequences comprising a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 4; iii. nucleotide sequences whose complementary strand hybridizes to a nucleic acid molecule with the sequence of (i) or (ii); and (c) a nucleotide sequence encoding an L-ribulose-5-P-4 epimerase (araD), wherein the nucleotide sequence is selected from the group consisting of: i. an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5; ii. nucleotide sequences comprising a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 6; iii. nucleotide sequences whose complementary strand hybridizes to a nucleic acid molecule with the sequence of (i) or (ii), wherein hybridization at points iii. is determined under hybridization conditions which allow a nucleic acid sequence of 200 nucleotides to hybridize at a temperature of 65 ° C in a solution comprising approx. 1 M salt and wash at 65 ° C in a solution of approx. 0.1 M, where the hybridization is performed for 10 hours and washing is performed for 1 hour with two shifts of the wash solution. The yeast cell of claim 1, wherein one, two or three of the araA, araB and araD nucleotide sequences are derived from a Lactobacillus genus, preferably a Lactobacillus plant species. The yeast cell of claim 1 or 2, wherein the cell is a yeast cell which preferably belongs to one of the genera: Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia. The yeast cell of claim 3, wherein the yeast cell belongs to one of the species: S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, 5. uvarum, S. diastaticus, K. lactis, K. marxianus or K. fragilis. A yeast cell according to any one of claims 1 to 4, wherein the nucleotide sequences encoding araA, araB and / or araD are operably linked to a promoter that catalyzes sufficient expression of the corresponding nucleotide sequences in the cell to confer cell capability. to utilize L-arabinose and / or convert L-arabinose to L-ribulose and / or xylulose-5-phosphate and / or to ethanol. The yeast cell of any one of claims 1 to 5, wherein the yeast cell exhibits the ability to directly isomerize xylose to xylulose. The yeast cell of claim 6, wherein the yeast cell comprises a gene modification which increases the flow in the pentose phosphate reaction pathway. The yeast cell of claim 6 or 7, wherein the gene modification comprises overexpression of at least one gene of the non-oxidative portion of the pentose phosphate pathway. The yeast cell of claim 8, wherein the gene is selected from the group consisting of the genes encoding ribulose-5-phosphate isomerase, ribulose-5-phosphate pimerase, transketolase, and transaldolase. The yeast cell of claim 8, wherein the gene modification comprises overexpression of at least the genes encoding a transketolase and a transaldolase. The yeast cell according to any one of claims 8 to 10, wherein the yeast cell further comprises a gene modification which increases the specific xylulose kinase activity. The yeast cell of claim 11, wherein the gene modification comprises the overexpression of a gene encoding a xylulose kinase. The yeast cell of any one of claims 8 to 12, wherein the overexpressed gene is endogenous to the cell. The yeast cell of any of claims 5 to 13, wherein the yeast cell comprises a gene modification that reduces nonspecific aldose reductase activity in the yeast cell. The yeast cell of claim 14, wherein the gene modification reduces the expression of or inactivates a gene encoding a nonspecific aldose reductase. The yeast cell of claim 15, wherein the gene is inactivated by deletion of at least a portion of the gene or by destruction of the gene. The yeast cell of claim 14 or 15, wherein the expression of each gene in the yeast cell encoding a nonspecific aldose reductase is reduced or inactivated. A yeast cell according to any one of the preceding claims, wherein the fermentation product is ethanol. A nucleic acid construct comprising a nucleic acid sequence encoding an araA, a nucleic acid sequence encoding an araB, and / or a nucleic acid sequence encoding an araD, all as defined in claims 1 or 2. A process for producing ethanol, comprising: a. Fermenting a medium containing a source of arabinose and optionally xylose, with a modified yeast cell according to any one of claims 1 to 18, wherein the yeast cell ferments arabinose and optionally xylose to ethanol; and optionally b. recovery of ethanol. A method of producing ethanol, comprising: a. Fermenting a medium containing at least one source of L-arabinose and one source of xylose, with a yeast cell according to any one of claims 1 to 18 and a yeast cell. capable of utilizing xylose and / or exhibiting the ability to directly isomerize xylose to xylulose, whereby each yeast cell ferments L-arabinose and / or xylose to ethanol; and optionally b. recovery of the fermentation product. The method of claim 20 or 21, wherein the medium also contains a source of glucose. A process according to any one of claims 20-22, wherein the volumetric ethanol productivity is at least 0.5 g ethanol per liter. liter per liter. hour. The process of claim 23, wherein the ethanol yield is at least 30%. The method of any one of claims 20 to 24, wherein the method is anaerobic. The method of any one of claims 20 to 24, wherein the process is aerobic and preferably performed under oxygen limited conditions.