CN103289908A

CN103289908A - Metabolic engineering of arabinose- fermenting yeast cells

Info

Publication number: CN103289908A
Application number: CN2012103163389A
Authority: CN
Inventors: 安东尼斯·杰若恩·阿迪瑞安·马里斯·范; 雅各布斯·托马斯·普若克; 汉迪瑞克·乌特尔·威塞林克; 强汉斯·皮埃特·范迪肯; 亚伦·阿迪瑞安·文科勒; 强汉斯·汉迪瑞克·温迪·德
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2006-10-02
Filing date: 2007-10-01
Publication date: 2013-09-11
Anticipated expiration: 2027-10-01
Also published as: CN103289908B; PL2069476T3; DK2069476T3; CN101522886A; ES2601052T3; BRPI0719262A2; BRPI0719262B1; CN101522886B

Abstract

The invention relates to an eukaryotic cell expressing nucleotide sequences encoding the ara A, ara B and ara D enzymes whereby the expression of these nucleotide sequences confers on the cell the ability to use L-arabinose and/or convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product such as ethanol. Optionally, the eukaryotic cell is also able to convert xylose into ethanol.

Description

The eukaryotic metabolic engineering of fermentation pectinose

The application is dividing an application of Chinese invention patent application 200780036866.6 (PCT/NL2007/000246), and female case applying date is on October 1st, 2007, and exercise question is " the eukaryotic metabolic engineering of fermentation pectinose ".

Invention field

The present invention relates to following eukaryotic cell and use the technology of this cells produce tunning, described cell has and uses L-arabinose and/or L-arabinose is converted into L-ribulose and/or X 5P and/or is converted into the ability of desired fermentation product.

Background of invention

The ethanol dyestuff is considered to the valuable alternatives of fossil oil.From the hemicellulose level of plant biomass assign to the feasible production of the economics of ethanol need with suitable speed and high yield ferment simultaneously transform pentose and hexose these two.Yeast (especially Saccharomyces spp.) is the optimal candidate of this technology, because they all can be grown and ferment fast on hexose when aerobic and anaerobism.In addition, they to the toxicity environment of ligno-cellulose hydrolysate using than (modifying through general) bacterium have resistance many.

EP 1499708 has described the technology of making following S.cerevisiae bacterial strain, and described bacterial strain can be produced ethanol from L-arabinose.AraA by introducing Bacillus subtilis (the L-arabinose isomerase, modifies these bacterial strains from araB (L-ribulokinase) and araD (L-ribulose-5-P4-epimerase) gene of Escherichia coli.In addition, have extra sudden change in these strain gene groups, perhaps cross expression TAL1 (transaldolase) gene.Yet these genes have some shortcomings.They are at the limited condition bottom fermentation pectinose of oxygen.In addition, they have the low alcohol production speed (Becker and Boles, 2003) of 0.05g.g-1.h-1.In addition, these bacterial strains can not under anaerobic use L-arabinose.At last, these S.cerevisiae bacterial strains have the wild-type background, so they can not be used to some kinds of C5 sugar of co-fermentation.

WO 03/062430 and WO 06/009434 disclose the yeast strain that wood sugar can be converted into ethanol.These yeast strains can be worth directly xylose isomerase is turned to xylulose.

Still have the needs to the following alternative bacterial strain of producing ethanol, described bacterial strain operation is better, and vigor is more arranged and relative harsh working condition is had more resistance.

Description of drawings

The plasmid map of Fig. 1: pRW231 and pRW243.

Fig. 2: in the synthetic medium that contains 0.5% semi-lactosi (A) and 0.1% semi-lactosi+2% L-arabinose (B), the growth pattern of bacterial strain RWB219 (zero) and IMS0001 (●) shake-flask culture.Culture was cultivated 72 hours in the synthetic medium that contains semi-lactosi (A), was transferred to the synthetic medium that contains semi-lactosi and pectinose (B) then.By measuring OD ₆₆₀Measure growth.

Fig. 3 .S.cerevisiae IMS0001 is the growth velocity during the series transfer in the shake-flask culture thing, and described culture contains the synthetic medium of 2% (w/v) L-arabinose.The OD that each data point representative is measured from (index) growing period ₆₆₀The growth velocity of estimating.Closed and open circle represents bipartite serial shift experiment.

Fig. 4 .S.cerevisiae IMS0001 is the growth velocity between anaerobism SBR yeast phase in the synthetic medium that contains 2% (w/v) L-arabinose.The CO of each data point representative during the exponential growth ₂The growth velocity that spectrum (solid line) is estimated.

Fig. 5. the sugar consumption during the bacterial strain IMS0002 anaerobism batch fermentation and product form.Fermentation is carried out in 1 liter of synthetic medium, and described culture medium supplemented has 20g l ^-1Pectinose (A); 20g l ^-1Glucose and 20g l ^-1Pectinose (B); 30g l ^-1Glucose, 15g l ^-1Wood sugar and 15g l ^-1Pectinose (C).Sugar consumption and product during the anaerobism batch fermentation that carries out with the mixture of bacterial strain IMS0002 and RWB218 form.Fermentation is carried out in 1 liter of synthetic medium, and described culture medium supplemented has 30g l ^-1Glucose, 15g l ^-1Wood sugar and 15g l ^-1Pectinose (D).Symbol: glucose (●); Wood sugar (zero); Pectinose (■); CO from accumulative total ₂Produce the ethanol () that calculates; Ethanol (▲) by the HPLC measurement; The CO of accumulative total ₂Produce (△); Xylitol

Fig. 6. sugar consumption and the product selecting to be used for during the bacterial strain IMS0002 of anaerobic growth on wood sugar cell anaerobism batch fermentation form.Fermentation is carried out in 1 liter of synthetic medium, and described culture medium supplemented has 20g l ^-1Wood sugar and 20g l ^-1Pectinose (B).Symbol: wood sugar (zero); Pectinose (■); Ethanol (▲) by the HPLC measurement; The CO of accumulative total ₂Produce (△); Xylitol

Fig. 7. the sugar consumption during the bacterial strain IMS0003 anaerobism batch fermentation and product form.Fermentation is carried out in 1 liter of synthetic medium, and described culture medium supplemented has: 30g l ^-1Glucose, 15g l ^-1Wood sugar and 15g l ^-1Pectinose.Symbol: glucose (●); Wood sugar (zero); Pectinose (■); CO from accumulative total ₂Produce the ethanol () that calculates; Ethanol (▲) by the HPLC measurement; The CO of accumulative total ₂Produce (△).

Detailed Description Of The Invention

Eukaryotic cell

First aspect present invention relates to the eukaryotic cell that can express following nucleotide sequence, wherein, the expression of these nucleotide sequences is given cell use L-arabinose and/or L-arabinose is converted into L-ribulose and/or X 5P and/or is converted into desired fermentation product such as the ability of ethanol:

(a) nucleotide sequence of coding Arabinose isomerase (araA), wherein said nucleotide sequence is selected from the group of being made up of following:

(i) nucleotide sequence of coding araA, described araA comprises the aminoacid sequence that the aminoacid sequence with SEQ ID NO:1 has at least 55% sequence identity,

(ii) nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO:2 has at least 60% sequence identity,

(iii) nucleotide sequence, its complementary strand with (i) or the making nucleic acid molecular hybridization of sequence (ii),

(iv) nucleotide sequence, its sequence is because the degeneracy of genetic codon and different with the sequence of (iii) nucleic acid molecule;

(b) nucleotide sequence of coding L-ribulokinase (araB), wherein said nucleotide sequence is selected from the group of being made up of following:

(i) nucleotide sequence of coding araB, described araB comprises the aminoacid sequence that the aminoacid sequence with SEQ ID NO:3 has at least 20% sequence identity,

(ii) nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO:4 has at least 50% sequence identity,

(c) nucleotide sequence of coding L-ribulose-5-P-4-epimerase (araD), wherein said nucleotide sequence is selected from the group of being made up of following:

(i) nucleotide sequence of coding araD, described araD comprises the aminoacid sequence that the aminoacid sequence with SEQ ID NO:5 has at least 60% sequence identity,

(ii) nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO:6 has at least 60% sequence identity,

(iv) nucleotide sequence, its sequence is because the degeneracy of genetic codon and different with the sequence of (iii) nucleic acid molecule.

An embodiment preferred relates to the eukaryotic cell that can express following nucleotide sequence, thereby the expression of these nucleotide sequences is given cell use L-arabinose and/or L-arabinose is converted into L-ribulose and/or X 5P and/or is converted into desired fermentation product such as the ability of ethanol:

(i). nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO:2 has at least 60% sequence identity,

(ii). nucleotide sequence, the making nucleic acid molecular hybridization of its complementary strand and sequence (i),

(iii). nucleotide sequence, its sequence is because the degeneracy of genetic codon and different with the sequence of (ii) nucleic acid molecule;

(i). the nucleotide sequence of coding araB, described araB comprises the aminoacid sequence that the aminoacid sequence with SEQ ID NO:3 has at least 20% sequence identity,

(ii). nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO:4 has at least 50% sequence identity,

(iii). nucleotide sequence, its complementary strand with (i) or the making nucleic acid molecular hybridization of sequence (ii),

(iv). nucleotide sequence, its sequence is because the degeneracy of genetic codon and different with the sequence of (iii) nucleic acid molecule;

(i). the nucleotide sequence of coding araD, described araD comprises the aminoacid sequence that the aminoacid sequence with SEQ ID NO:5 has at least 60% sequence identity,

(ii). nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO:6 has at least 60% sequence identity,

(iv). nucleotide sequence, its sequence is because the degeneracy of genetic codon and different with the sequence of (iii) nucleic acid molecule.

Sequence identity and similarity

Sequence identity is defined as in this article: measure the relation between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequence by comparative sequences.Usually, identity or the similarity of comparative sequences on the whole length of the sequence that is compared.In this area, " identity " also represents serial correlation (relatedness) degree between amino acid or the nucleotide sequence, and described degree of relevancy is according to circumstances measured by the coupling between the character string of this class sequence.By an amino acid sequence of polypeptide and conservative aminoacid replacement and another polypeptide thereof are compared, measure " similarity " between two aminoacid sequences." identity " and " similarity " can easily be calculated by several different methods well known by persons skilled in the art.

The preferred method of measuring identity is designed to provide maximum match between tested sequence.The method of measuring identity and similarity is encoded in the obtainable computer program of the public.The preferred computer program means of identity and similarity comprises for example BestFit, BLASTP, BLASTN and FASTA (Altschul between the mensuration two sequences, S.F.et al., J.Mol.Biol.215:403-410 (1990), the public can obtain (BLAST Manual from NCBI and other source, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894).A kind of most preferred algorithm that uses is EMBOSS (http://www.ebi.ac.uk/emboss/align).Use EMBOSS to carry out aminoacid sequence preferred parameter relatively and open 10.0 as breach, breach extends 0.5, Blosum, 62 matrixes.Use EMBOSS to carry out nucleotide sequence preferred parameter relatively and open 10.0 as breach, breach extends the complete matrix of 0.5, DNA (DNA identity matrix).

Alternatively, when measuring the amino acid similarity degree, the technician also can consider so-called " guarding " aminoacid replacement, and this is that the technician is understood that.Conserved amino acid replaces the interchangeability of the residue refer to have similar side chain.For example, the amino acid group with aliphatic lateral chain is glycine, L-Ala, Xie Ansuan, leucine and Isoleucine; Amino acid group with aliphatics-hydroxyl measurement is Serine and Threonine; Amino acid group with amide containing side chain is l-asparagine and glutamine; Amino acid group with aromatic series side chain is phenylalanine, tyrosine and tryptophane; Amino acid group with basic side chain is Methionin, arginine and Histidine; Amino acid group with sulfur-containing side chain is halfcystine and methionine(Met).Preferred conserved amino acid substituting group is: Val-Leu-Isoleucine, phenylalanine-tyrosine, Methionin-arginine, L-Ala-Xie Ansuan and l-asparagine-glutamine.The replacement variant of aminoacid sequence disclosed herein is following variant, and wherein at least one residue is removed in the disclosed sequence, and inserts different residues in its position.Preferably, amino acid change is guarded.As follows to each naturally occurring amino acid whose preferred conservative replacement: Ala is to ser; Arg is to lys; Asn is to gln or his; Asp is to glu; Cys is to ser or ala; Gln is to asn; Glu is to asp; Gly is to pro; His is to asn or gln; Ile is to leu or val; Leu is to ile or val; Lys is to arg; Gln or glu; Met is to leu or ile; Phe is to met, leu or tyr; Ser is to thr; Thr is to ser; Trp is to tyr; Tyr is to trp or phe; Arrive ile or leu with Val.

The hybrid nucleic acid sequence

The nucleotide sequence that is coded in the enzyme of expressing in the cell of the present invention also can define with the ability that SEQ ID NO.2,4,6,8,16,18,20,22,24,26,28,30 nucleotides sequence are listed in (or preferably under stringent hybridization condition) hybridization under the moderate hybridization conditions respectively by them.Stringent hybridization condition is defined as following condition in this article, it allows at least about 25, preferably about 50 Nucleotide, 75 or 100 and most preferably about 200 or the nucleotide sequence of more Nucleotide, under about 65 ℃ temperature, contain about 1M salt, preferably 6xSSC or have in any other solution of suitable ionic strength is hybridized, and is containing about 0.1M salt or still less, preferably 0.2xSSC or have in any other solution of suitable ionic strength washs under 65 ℃.Preferably, hybridization is spent the night, and namely carried out at least 10 hours, and preferably, washing is carried out at least one hour, changes washing soln at least twice.These conditions can allow to have about 90% or the sequence specific hybridization of higher sequence identity usually.

The moderate condition is defined as following condition in this article, at least 50 Nucleotide of described conditions permit, the nucleotide sequence of about 200 or more Nucleotide preferably, under about 45 ℃ temperature, contain about 1M salt, preferably 6xSSC or have in any other solution of suitable ionic strength is hybridized, and at room temperature contain about 0.1M salt, preferably 6xSSC or have in any other solution of suitable ionic strength washs.Preferably, hybridization is spent the night and is carried out, and namely carries out at least 10 hours, and preferably at least one hour is carried out in washing, and at least twice changes washing soln.These conditions can allow to have the sequence specific hybridization of 50% sequence identity at the most usually.Those skilled in the art can change these hybridization conditions, thereby identify the sequence that identity changes between 50% and 90% specifically.

AraA

A preferred nucleotide sequence that is coded in the Arabinose isomerase (araA) of expressing in the cell of the present invention is selected from the group of being made up of following:

(a) nucleotide sequence of coding araA polypeptide, described araA comprise the aminoacid sequence that the aminoacid sequence with SEQ ID NO.1 has 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity at least;

(b) nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO.2 has 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity at least;

(c) nucleotide sequence, its complementary strand with (a) or sequence of nucleic acid molecules (b) hybridization;

(d) nucleotide sequence, its sequence is because the degeneracy of genetic codon and different with the sequence of the nucleic acid molecule of (c).

Nucleotide sequence codified protokaryon or the eucaryon araA of coding araA namely have the araA of following aminoacid sequence, and described aminoacid sequence is identical with the aminoacid sequence of naturally occurring araA in prokaryotic organism or the eukaryote.When inventor's discovery and araB and araD coexpression, the following ability of concrete araA depends on that not too this araA is protokaryon or eucaryon source, and described ability is to give eukaryotic host cell to use pectinose and/or pectinose is converted into L-ribulose and/or X 5P and/or is converted into desired fermentation product such as the ability of ethanol.Or rather, this depends on the aminoacid sequence of araA and the dependency of SEQ ID NO.1 sequence.

AraB

A preferred nucleotide sequence that is coded in the L-ribulokinase (AraB) of expressing in the cell of the present invention is selected from the group of being made up of following:

(a) nucleotide sequence of the following polypeptide of coding, described polypeptide comprises the aminoacid sequence that the aminoacid sequence with SEQ ID NO.3 has 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity at least;

(b) nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO.5 has 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity at least;

Nucleotide sequence codified protokaryon or the eucaryon araB of coding araB namely have the araB of following aminoacid sequence, and described aminoacid sequence is relevant with the aminoacid sequence of naturally occurring araB in prokaryotic organism or the eukaryote.When inventor's discovery and araA and araD coexpression, the following ability of concrete araB depends on that not too this araB is protokaryon or eucaryon source, and described ability is given eukaryotic host cell use pectinose and/or pectinose is converted into L-ribulose and/or X 5P and/or is converted into desired fermentation product such as the ability of ethanol.Or rather, this depends on the aminoacid sequence of araB and the dependency of SEQ ID NO.3 sequence.

AraD

A preferred nucleotide sequence that is coded in the L-ribulose-5-P-4-epimerase (araD) of expressing in the cell of the present invention is selected from the group of being made up of following:

(e) nucleotide sequence of the following polypeptide of coding, described polypeptide comprises the aminoacid sequence that the aminoacid sequence with SEQ ID NO.5 has 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity at least;

(f) nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO.6 has 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity at least;

(g) nucleotide sequence, its complementary strand with (a) or sequence of nucleic acid molecules (b) hybridization;

(h) nucleotide sequence, its sequence is because the degeneracy of genetic codon and different with the sequence of the nucleic acid molecule of (c).

Nucleotide sequence codified protokaryon or the eucaryon araD of coding araD namely have the araD of following aminoacid sequence, and described aminoacid sequence is identical with the aminoacid sequence of naturally occurring araD in prokaryotic organism or the eukaryote.When inventor's discovery and araA and araB coexpression, the following ability of concrete araD depends on that not too this araD is protokaryon or eucaryon source, and described ability is given eukaryotic host cell use pectinose and/or pectinose is converted into L-ribulose and/or X 5P and/or is converted into desired fermentation product such as the ability of ethanol.Or rather, this depends on the aminoacid sequence of araD and the dependency of SEQ ID NO.5 sequence.

Shockingly, codon preference index (codon bias index) shows: compared with protokaryon araA, the araB described in the EP 1 499 708 and araD gene, Lactobacillus plantarum araA, araB and araD gene are more suitable for expressing in yeast.

It should be noted that L.plantarum is the biology that is considered to safety (GRAS) usually, it is safe by food registration body (food registration authorities) approval.Therefore, preferred nucleotide sequence is encoded respectively and is had respectively and above-mentioned SEQ ID NO:1,3 or 5 sequences araA, araB or the araD of relevant aminoacid sequence respectively.Preferred nucleotide sequence encode respectively fungi araA, araB or araD (for example from Basidiomycete) more preferably encode respectively from araA, araB or the araD of anaerobic fungi (anaerobic fungi that for example belongs to Neocallimastix, Caecomyces, Piromyces, Orpinomyces or Ruminomyces section).Perhaps, preferred nucleotide sequence encode respectively bacterium araA, araB or araD, it more preferably belongs to from Lactobacillus, most preferably from Lactobacillus plantarum kind preferably from gram positive bacterium.Preferably, one, two or three in araA, araB and the araD nucleotide sequence derive from the Lactobacillus genus, more preferably derive from Lactobacillus plantarum kind.The bacterium araA that expresses in cell of the present invention is not disclosed Bacillus subtilis araA (providing as SEQ ID NO:9) among the EP 1 499 708.The nucleotide sequence of SEQ ID NO:10 presentation code SEQ ID NO:9.The bacterium araB that expresses in cell of the present invention and araD are not araB and the araD (providing as SEQ ID NO:11 and SEQ ID NO:13) of disclosed Escherichia coli (E.coli) among the EP 1 499 708.The nucleotide sequence of SEQ ID NO:12 representative coding SEQ ID NO:11.The nucleotide sequence of SEQ ID NO:14 representative coding SEQ ID NO:13.

In order to improve the possibility that (bacterium) araA, araB and araD enzyme are expressed in eukaryotic host cell of the present invention such as yeast with activity form respectively, can make corresponding coding nucleotide sequence adapt to selected eukaryotic host cell and use to optimize its codon.The nucleotide sequence of coding araA, araB and araD enzyme (or other enzyme of the present invention, see below) can be expressed as codon adaptation indexI (CAI) to the adaptability that selected host cell codon uses.Codon adaptation indexI is defined as the codon use of gene in this article towards the adaptability of the codon use of the gene of highly expressing.The relative adaptability of every kind of codon (w) is the ratio for the use of every kind of codon of same amino acid and the use of maximum codon.The CAI index is defined as the geometrical mean of these relative adaptability values.Non-synonym and terminator codon (depending on genetic codon) foreclose.CAI value scope from 0 to 1, higher value representation maximum codon (see Sharp and Li, 1987, Nucleic Acids Research 15:1281-1295 more at high proportion; Also see Jansen et al., 2003, Nucleic Acids Res.31 (8): 2242-51).The nucleotide sequence that has adapted to preferably has at least 0.2,0.3,0.4,0.5,0.6 or 0.7 CAI.

In a preferred embodiment, the ability that cell uses L-arabinose and/or is translated into L-ribulose and/or X 5P is given in the expression of the nucleotide sequence of Nucleotide araA, the araB of definition and araD before coding this paper.Under the situation of not expecting to be bound by any theory, estimate that L-arabinose at first is converted into the L-ribulose, described L-ribulose is converted into X 5P subsequently, and described X 5P is the main molecules that enters pentose-phosphate pathway.In the context of the present invention, during " use L-arabinose " preferably was illustrated in 20 days at least, when having at least the 0.5%L-pectinose, under aerobic or anaerobic condition, cultivate through the optical density(OD) (OD of cell transformed in the 660nm place ₆₆₀) be enhanced about 0.5 to 1.0 or more.More preferably, OD ₆₆₀Be enhanced 0.5 to 1.5 or more.More preferably, have at least 1%, at least 1.5% or culturing cell during the 2%L-pectinose at least.Most preferably, culturing cell when having about 2%L-pectinose.

In the context of the present invention, during at least 20 days, have L-arabinose (with preferred concentration identical in the last period) but aerobic or anaerobic condition under when using suitable assay method to detect the L-ribulose of detection limit in the cultured cells, this cell can " be converted into the L-ribulose with L-arabinose ".Preferably, this assay method is the HPLC at the L-ribulose.

In the context of the present invention, during at least 20 days, when using suitable assay method to detect at least 2% X 5P increase under the aerobic or anaerobic condition that has L-arabinose (with preferred concentration identical in the last period) in the cultured cells, cell " can be converted into X 5P with L-arabinose ".Preferably, be described in Zaldivar J. at the assay method based on HPLC of X 5P, et al ((2002), Appl.Microbiol.Biotechnol., 59:436-442) in.This assay method is briefly described at experimental section.More preferably, describedly increase at least 5%, 10%, 15%, 20%, 25% or more.

In another preferred embodiment, during at least one month was by 1 year, when cultivating under the needs that have L-arabinose (with preferred concentration identical in the last period) or anaerobic condition, the ability that cell is converted into L-arabinose desired fermentation product is given in the expression of coding araA, the araB of this paper definition early and the nucleotide sequence of araD.More preferably, when cultivating under the condition that cell provides in last sentence, but use suitable assay method to detect the desired fermentation product of detection limit, then cell can be converted into desired fermentation product with L-arabinose.Further more preferably, this assay method is HPLC.Further more preferably, this tunning is ethanol.

Be used for the cell of encode respectively araA, araB and araD enzymatic conversion mentioned above following host cell preferably, it can enter wood sugar active or passive transport in the cell and in cell carry out xylose isomeraseization.Described cell preferably can carry out active glycolysis-.Described cell also can contain endogenous pentose-phosphate pathway, and can contain endogenous xylulokinase activity, thereby the xylulose that comes from xylose isomeraseization can be metabolised to pyruvic acid.Cell also preferably contains the enzyme that pyruvic acid is converted into desired fermentation product, described desired fermentation product such as ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1,3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics or cynnematin.Can make cell can produce butanols by one or more genes of introducing disclosed butanols approach among the WO2007/041269.

A kind of preferred cell is natural can to carry out the alcohol fermentation, preferably carries out the alcohol fermentation of anaerobism.Host cell also preferably has to the tolerance of the height of ethanol, to the height tolerance of low pH (can be lower than under 5,4,3 or 2.5 the pH grow) with to the height tolerance of organic acid such as lactic acid, acetic acid or formic acid and sugared degraded product such as furfural and hydroxymethylfurfural and to the height tolerance of the temperature that improves.Any of these feature of host cell or activity can naturally be present in the host cell, maybe can select or be introduced into by genetic modification by heredity.Proper host cell is for example fungi of eukaryotic microorganisms, yet optimum is yeast or filamentous fungus as host cell.

Yeast is defined as following eukaryotic microorganisms in this article, and it comprises all species (Alexopoulos, C.J., 1962, In:Introductory Mycology, the John Wiley of Eumycotina; Sons, Inc., New York), it is mainly with unicellular form growth.Yeast can maybe can pass through biological merisis by the unicellular thalloid growth of sprouting.The genus that belongs to one of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia as the preferred yeast of host cell.Preferably, yeast can carry out anaerobically fermenting, more preferably can carry out the alcohol fermentation of anaerobism.

Filamentous fungus is defined as comprising the eukaryotic microorganisms of all thread forms of Eumycotina in this article.The vegetative mycelium that the feature of these fungies is made up of chitin, Mierocrystalline cellulose and other complicated polysaccharide.Filamentous fungus of the present invention goes up different with yeast with heredity on form, on the physiology.The vegetalitas growth of filamentous fungus is by the mycelia elongation, and the carbon metabolism of most of filamentous fungus is obligate aerobic.The genus that belongs to one of Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium or Penicillium as the preferred filamentous fungus of host cell.

Over several years, having proposed to introduce multiple biology is used for from the suggestion of farm crop sugar production bio-ethanol.Yet in practice, all main bio-ethanol production technique all continue to use the yeast of Saccharomyces genus as ethanol producer.This is owing to the many attracting feature of Saccharomyces species for industrial technology, and namely to the tolerance of peracid, ethanol and infiltration, the ability of anaerobic growth also has its high pure fermentation capacity certainly.Preferred yeast species as host cell comprises S.cerevisiae, S.bulderi, S.barnetti, S.exiguus, S.uvarum, S.diastaticus, K.lactis, K.marxianus, K.fragilis.

In a preferred embodiment, host cell of the present invention is to have used the nucleic acid construct transformed host cells, and described nucleic acid construct comprises araA, araB that coding defines as mentioned and the nucleotide sequence of araD enzyme.In a preferred embodiment, with three kinds of nucleic acid construct cotransformation host cells, every kind of nucleic acid construct comprises the nucleotide sequence of coding araA, araB or araD.The nucleic acid construct that comprises araA, araB and/or araD encoding sequence can be expressed araA, araB and/or araD enzyme in host cell.For this reason, can described in for example WO 03/0624430, make up nucleic acid construct.Host cell can comprise every kind of nucleic acid construct of single copy, but preferably comprises multiple copied.Nucleic acid construct can be maintained unbound state, and therefore comprises the sequence for self-replicating, for example the ARS sequence.Suitable free nucleic acid construct can be for example based on yeast 2 μ or pKD1 (Fleer et al., 1991, Biotechnology 9:968-975) plasmid.Yet preferably, every kind of nucleic acid construct is integrated in the genome of host cell with one or more copies.The genome that is integrated into host cell can take place at random by the reorganization that does not meet convention, but preferably nucleic acid construct is integrated in the genome of host cell by homologous recombination, as fungal molecule genetics field known (see for example WO 90/14423, EP-A-0 481 008, EP-A-0 635 574 and US 6,265,186).Therefore, in a preferred embodiment, cell of the present invention comprises the nucleic acid construct that contains araA, araB and/or araD encoding sequence, and can express araA, araB and/or araD enzyme.In a further preferred embodiment, araA, araB and/or araD encoding sequence are operably connected with promotor separately, described promotor causes that corresponding nucleotides sequence is listed in giving full expression in the cell, to give the ability that cell uses L-arabinose and/or L-arabinose is converted into L-ribulose and/or X 5P.Preferably, this cell is yeast cell.Therefore in aspect another, the present invention also comprises the nucleic acid construct of early setting forth as preamble.Preferably, this nucleic acid construct comprises the nucleotide sequence of coding araA, araB and/or araD.The nucleotide sequence of coding araA, araB or araD is definition early in this article all.

Further more preferably, the ability that cell is converted into L-arabinose the desired fermentation product that this paper defines is after a while given in the expression of corresponding nucleotide sequence in cell.In a further preferred embodiment, this tunning is ethanol.Further more preferably, this cell is yeast cell.

This paper uses term " to be operably connected " and refer to that polynucleotide element (or encoding sequence or nucleotide sequence) connects in functional mutual relationship.When nucleotide sequence was placed in the functional mutual relationship with another nucleotide sequence, it was " being operably connected ".For example, if promotor or enhanser can influence transcribing of encoding sequence, then it is operably connected with encoding sequence.What be operably connected means that the nucleotide sequence that is connected is normally continuous, and is continuous when needs engage two protein coding regions and meets reading frame.

This paper uses term " promotor " to refer to following nucleic acid fragment, it can bring into play function to control one or more gene transcription, for transcriptional orientation, be positioned at the upstream of genetic transcription starting point, and structurally by there being the binding site of DNA-dependent form RNA polymerase, transcriptional start point and any other dna sequence dna identify that described any other dna sequence dna includes but not limited to the transcription factor binding site point, prevent with activator binding site and the known direct or indirect work of those skilled in the art in order to regulate any other sequence from the amount of transcribing of this promotor." composing type " promotor is promoters active under most of environment and developmental condition." induction type " promotor is at environment or grows promoters active under the adjusting.

Can be used in the nucleotide sequence expression promoter of reaching coding araA, araB and/or araD can not be natural to the nucleotide sequence of the codase that will be expressed, and namely the promotor pair nucleotide sequence that is operably connected with it (encoding sequence) is allos.Although promotor is allos to the encoding sequence that is operably connected with it preferably, also promotor be homology also be preferred, be endogenous for host cell namely.Preferably, with be that intrinsic promotor is compared to encoding sequence, preferably can obtain pectinose, or pectinose and glucose, or wood sugar and pectinose, or wood sugar and pectinose and glucose are as carbon source, more preferably as main carbon source (namely more than 50% obtainable carbon source by pectinose, or pectinose and glucose, or wood sugar and pectinose, or wood sugar and pectinose and glucose composition), most preferably under the condition as sole carbon source, the promotor of (to nucleotide sequence) allos can be produced the transcript that comprises encoding sequence (or time per unit can be produced more transcript molecules, i.e. mRNA molecule) of high steady-state level.Suitable promotor comprises composing type and induction type natural promoter and the promotor through transforming in this context.A preferred promotor that is used for the present invention except meeting to katabolic product (glucose) prevent insensitive, and/or can preferably not need pectinose and/or wood sugar to be used for inducing.

Promotor with these features can extensively obtain, and is known to the skilled.The suitable example of this class promotor for example comprises the promotor from the glycolysis-gene, as phosphofructokinase (PPK), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK) promotor from yeast or filamentous fungus; About the more details visible (WO93/03159) from this class promotor of yeast.Other useful promotor is ribosomal protein encoding gene promotor, lactase gene promotor (LAC4), alcoholdehydrogenase promotor (ADH1, ADH4 etc.), Hydratase, phosphoenolpyruvate promotor (ENO), G-6-P isomerase promotor (PGI1, Hauf et al, 2000) or hexose (glucose) transport sub-promotor (HXT7) or glyceraldehyde-3-phosphate dehydrogenase (TDH3).The sequence of PGI1 promotor provides in SEQ ID NO:51.The sequence of HXT7 promotor provides in SEQ ID NO:52.The sequence of TDH3 promotor provides in SEQ ID NO:49.Other composing type and inducible promoter and enhanser or upstream activating sequence can be well known by persons skilled in the art.Can modify the promotor of using in the host cell of the present invention when needing, to influence its control feature.

A kind of preferred cell of the present invention is with araA, the araB of L.plantarum and the eukaryotic cell of araD gene transformation.More preferably, this eukaryotic cell is yeast cell, further more preferably is with araA, the araB of L.plantarum and the S.cerevisiae bacterial strain of araD gene transformation.Most preferably, this cell was CBS 120327 or CBS 120328, and it all is preserved in CBS Institute (Holland) on September 27th, 2006.

When being used for showing between given (reorganization) nucleic acid or peptide molecule and given host living beings or the host cell mutual relationship, term " homology " is understood that to represent that this nucleic acid or peptide molecule natively by host cell or biological generation of same species, are preferably produced by identical mutation or bacterial strain.Typically, if with the host cell homology, then the nucleic acid encoding sequence can be not that another promoter sequence in its natural surroundings or another secretory signal sequence of where applicable and/or terminator sequence are operably connected.When being used for showing the dependency of two nucleotide sequences, single-chain nucleic acid sequence of term " homology " expression can with the single-chain nucleic acid sequence hybridization of complementation.The hybridization degree can be depending on a large amount of factors, comprises the identity amount between the sequence and hybridization conditions such as temperature and salt concn as previously described.Preferably, the identity zone is greater than about 5bp, and more preferably the identity zone is greater than about 10bp.

During about nucleic acid (DNA or RNA) or protein use, term " allos " the following nucleic acid of expression or protein, they be not naturally occurring part in its biology in its existence, cell, genome or DNA or the RNA sequence, or the position that exists in genome or DNA or RNA sequence is different with its naturally occurring position.The nucleic acid of allos or protein are not endogenous to its cell that is introduced into, but derive from another cell synthetic or that reorganization produces.Usually, although not necessarily, this class nucleic acid encoding following proteins is wherein introduced or the cell of expressible dna is not produced described protein usually.Similarly, exogenous RNA is coded in the cell that has this exogenous RNA usually not expressed protein.Heterologous nucleic acids and protein also can be represented external nucleic acid or protein.Term heterologous nucleic acids or protein comprise following any nucleic acid or protein, and it can be identified as the cell of expressing this nucleic acid or protein by those skilled in the art is allos or external.The non-natural combination that also is applicable to nucleic acid or aminoacid sequence of term allos, namely in the combination sequence of at least two combinations to being external each other.

Can use and/or transform the preferred eukaryotic cell of L-arabinose and wood sugar

In a preferred embodiment, the cell of the present invention of expressing araA, araB and araD can use L-arabinose and/or be translated into L-ribulose and/or X 5P and/or this paper desired fermentation product of definition early, and demonstrates the ability of using wood sugar and/or wood sugar being converted into xylulose.Wood sugar is preferably a step isomerization steps (directly xylose isomerase being turned to xylulose) to the conversion of xylulose.Therefore this class cell can use L-arabinose can use wood sugar again." use " wood sugar preferably has the implication identical with " use " L-arabinose of this paper definition early.

The enzyme definition is according to the definition of using at xylose isomerase (EC 5.3.1.5), xylulokinase (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 (EC1.1.1.21) among the WO 06/009434.

In a preferred embodiment, as preamble early expression araA, the araB of definition and the eukaryotic cell of the present invention of araD have the ability that xylose isomerase is turned to xylulose, described in for example WO03/0624430 or WO 06/009434.By the nucleic acid construct transformed host cell with the nucleotide sequence that comprises the xylose isomerase of encoding, give the ability that this host cell turns to xylose isomerase xylulose.Be that wood sugar is to the direct isomerization of xylulose by transformed host cells with the ability that xylose isomerase turns to xylulose.This is understood that to represent that wood sugar is turned to xylulose by isomery in the single reaction by the xylose isomerase enzyme catalysis, with opposite to the two steps conversion of xylulose by the Xylitol intermediate product from wood sugar, described two steps transform respectively by Xylose reductase and xylitol dehydrogenase catalysis.

Nucleotide sequence coded xylose isomerase, its preferably of the present invention in transformed host cells with activity form expression.Therefore, the expression that nucleotides sequence is listed in the host cell produces xylose isomerase, this enzyme has the specific activity of every mg protein 10U xylose isomerase enzymic activity at least under 30 ℃, preferably have every mg 20U, 25U, 30U, 50U, 100U, 200U, 300 or the specific activity of 500U under 30 ℃ at least.Specific activity at the xylose isomerase of expressing in transformed host cells is defined as in this article: the amount of the xylose isomerase unit of enzyme activity of the protein of every mg host cell cell free lysate (for example free lysate of yeast cell).Being determined in the preamble of xylose isomerase enzymic activity described.

Preferably, the nucleotides sequence of coding xylose isomerase is listed in expression in the host cell and produces wood sugar is had following K _mXylose isomerase, described K _mBe less than 50,40,30 or 25mM, more preferably, to the K of wood sugar _mFor about 20mM or still less.

The following group of forming of an optional freedom of preferred nucleotide sequence of coding xylose isomerase:

(e) nucleotide sequence of the following polypeptide of coding, described polypeptide comprises the aminoacid sequence that the aminoacid sequence with SEQ ID NO.15 has 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity at least;

(f) nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO.16 has 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity at least;

Nucleotide sequence codified protokaryon or the eucaryon xylose isomerase of coding xylose isomerase, the xylose isomerase that namely has following aminoacid sequence, described aminoacid sequence is identical with the aminoacid sequence of naturally occurring xylose isomerase in prokaryotic organism or the eukaryote.The inventor finds that the following ability of concrete xylose isomerase depends on that not too this isomerase is protokaryon or eucaryon source, and described ability is given the ability that eukaryotic host cell turns to xylose isomerase xylulose.Or rather, this depends on the aminoacid sequence of isomerase and the dependency of Piromyces sequence (SEQ ID NO.7).Shockingly, compare with other known eucaryon isomerase, eucaryon Piromyces isomerase is more relevant with the protokaryon isomerase.Therefore, preferred nucleotide sequence coded xylose isomerase with following aminoacid sequence, described aminoacid sequence is relevant with Piromyces sequence defined above.A kind of preferred nucleotide sequence coded fungi xylose isomerase (for example from Basidiomycete), more preferably encode from the xylose isomerase of another anaerobic fungi, for example from the xylose isomerase of the anaerobic fungi that belongs to Neocallimastix, Caecomyces, Piromyces, Orpinomyces or Ruminomyces section.Perhaps, a kind of preferred nucleotide sequence coded bacterium xylose isomerase, preferably from gram negative bacterium, more preferably encode from Bacteroides guiding principle or the isomerase that belongs to from Bacteroides, most preferably from B.thetaiotaomicron (SEQ ID NO.15).

In order to improve the possibility that xylose isomerase is expressed in eukaryotic host cell such as yeast with activity form, can make the nucleotide sequence of coding xylose isomerase adapt to the eukaryotic host cell that preamble defines, use to optimize its codon.

Be used for nucleotide sequence transformed host cells with coding xylose isomerase defined above and be preferably the host cell that wood sugar active or passive transport can be entered cell.This host cell preferably contains active glycolysis-.This cell also can contain endogenous pentose-phosphate pathway, and can contain endogenous xylulokinase activity, thereby the xylulose that comes from xylose isomeraseization can be metabolised to pyruvic acid.This host is also preferably contained the enzyme that pyruvic acid is converted into desired fermentation product, described desired fermentation product such as ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1,3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics or cynnematin.A kind of preferred host cell is natural can to carry out the alcohol fermentation, preferably carries out the alcohol fermentation of anaerobism.Host cell also preferably has to the tolerance of the height of ethanol, to the height tolerance of low pH (namely can be lower than under 5,4,3 or 2.5 the pH grow) with to the height tolerance of organic acid such as lactic acid, acetic acid or formic acid and sugared degraded product such as furfural and hydroxymethylfurfural with to the height tolerance of the temperature that improves.Any of these feature of host cell or activity can naturally be present in the host cell, maybe can be introduced into or modify by genetic modification.Proper host cell is for example fungi of eukaryotic microorganisms, yet optimum is yeast or filamentous fungus as host cell.Preferred yeast and filamentous fungus this paper define.

Wording host cell used herein has the implication identical with cell.

Cell of the present invention preferably transforms with following nucleic acid construct, and described nucleic acid construct comprises the nucleotide sequence of the xylose isomerase of encoding.Preferably the nucleic acid construct of Shi Yonging is identical with the nucleic acid construct of the nucleotide sequence that comprises coding araA, araB or araD of use.

In another embodiment preferred of the present invention, (it expresses araA, araB and araD to cell of the present invention, and demonstration turns to the direct isomery of wood sugar the ability of xylulose, define as preamble) also comprise following genetic modification, it improves the flux of pentose-phosphate pathway, described in WO 06/009434.Particularly, this genetic modification causes the raising of non-oxide part pentose-phosphate pathway flux.Cause that the genetic modification that the non-oxidizable part flux of pentose-phosphate pathway improves should be understood that to represent following modification in this article, with be that flux in the same bacterial strain of heredity is compared except causing the genetic modification that flux improves, the described modification to major general's flux improved 1.1,1.2,1.5,2,5,10 or 20 times.Flux that can the non-oxide part of following measurement pentose-phosphate pathway: modified host is grown at the wood sugar as sole carbon source, mensuration wood sugar specific consumption rate (specific xylose consumption rate) is opened and deduct the Xylitol specific production rate from the wood sugar specific consumption rate, if produce any Xylitol.Yet, the non-oxide part flux of pentose-phosphate pathway with proportional as the growth velocity on the wood sugar of sole carbon source, preferably with proportional as the anaerobic growth speed on the wood sugar of sole carbon source.As the growth velocity (μ on the wood sugar of sole carbon source _Max) and the flux of the non-oxide part of pentose-phosphate pathway between have linear relationship.Wood sugar specific consumption rate (Q _s) equal growth velocity (μ) divided by the productive rate (Y of biomass with respect to sugar _Xs), because biomass are constant (under a given set condition: the genetic background of anaerobism, growth medium, pH, bacterial strain etc. with respect to the productive rate of sugar; Be Q _s=μ/Y _Xs).Therefore, can reason out the flux that the non-oxide part of pentose-phosphate pathway improves from the raising of the maximum growth rate under these conditions.In a preferred embodiment, cell comprises following genetic modification, and it improves the flux of pentose-phosphate pathway, and has the wood sugar specific consumption rate of 346mg wood sugar/g biomass/h at least.

The genetic modification that improves the pentose-phosphate pathway flux can number of ways be introduced in the host cell.These approach comprise the more high stable state activity level of one or more enzymes that for example reach xylulokinase and/or the non-oxide part of pentose-phosphate pathway, and/or the non-specific aldose reductase activity of the steady-state level that reduces.Can be by selecting mutant (spontaneous or by chemical or radiation-induced) and/or influencing these changes of steady state activity level by recombinant DNA technology (for example passed through expression or inactivation gene respectively, described genes encoding is regulated enzyme or the factor of these genes).

In a preferred host cell, genetic modification comprised at least a enzyme of expression pentose-phosphate pathway (non-oxide part).Preferably, this enzyme is selected from the group of being made up of the enzyme of coding ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase, described in WO06/009434.

Can cross the multiple combination of the enzyme of expression pentose-phosphate pathway (non-oxide part).For example, being crossed the enzyme of expressing can be enzyme ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase at least; Or be enzyme ribulose-5-phosphate isomerase and transketolase at least; Or be enzyme ribulose-5-phosphate isomerase and transaldolase at least; Or be enzyme ribulose-5-phosphate epimerase and transketolase at least; Or be enzyme ribulose-5-phosphate epimerase and transaldolase at least; Or be enzyme transketolase and transaldolase at least; Or be enzyme ribulose-5-phosphate epimerase, transketolase and transaldolase at least; Or be enzyme ribulose-5-phosphate isomerase, transketolase and transaldolase at least; Or be enzyme ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase and transaldolase at least; Or be enzyme ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase and transketolase at least.In one embodiment of the invention, in host cell, cross expression ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and the various enzymes of transaldolase.More preferably following host cell, wherein genetic modification comprised at least and to cross expression transketolase and two kinds of enzymes of transaldolase because such host cell can be on wood sugar anaerobic growth.In fact, under some conditions, we find that the host cell of only expressing transketolase and transaldolase has excessively had the anaerobic growth speed identical with the host cell of expressing all four kinds of enzymes excessively on wood sugar, and described four kinds of enzymes are ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase.In addition, cross the host cell of expressing ribulose-5-phosphate isomerase and two kinds of enzymes of ribulose-5-phosphate epimerase and be to surpass only to cross and express isomerase or only cross the host cell of expressing epimerase and by preferred, express one of these enzymes and can produce metabolic imbalance because only cross.

This area can obtain multiple means and be used for crossing the expression enzyme at cell of the present invention.Particularly, can come the expression enzyme by the copy number that improves the gene of codase in the host cell, for example by in the genome of host cell, integrating extra gene copy, by expressing gene from episome multiple copied expression vector, or realize by the episome expression vector that introducing comprises multi-copy gene.

Perhaps, can reach enzyme cross expressing in host cell of the present invention by using following promotor, it is intrinsic that described promotor is not that coding was wanted the sequence of enzyme of expression, namely is the promotor of allos to the encoding sequence that is operably connected with it.To this purpose and stark suitable promotor defines in this article.

The encoding sequence that was used for expressing enzyme is homology with host cell of the present invention preferably.Yet, also can use equally with the encoding sequence of host cell allos of the present invention, described in WO 06/009434.

Being used for crossing the nucleotide sequence of expressing the ribulose-5-phosphate isomerase at host cell of the present invention is the nucleotide sequence that coding has the polypeptide of ribulose-5-phosphate isomerase activity, wherein preferably this polypeptide has the aminoacid sequence that has 50%, 60%, 70%, 80%, 90% or 95% identity at least with SEQ ID NO.17, or wherein this nucleotide sequence can under the moderate condition, preferably under stringent condition with the nucleotide sequence hybridization of SEQ ID NO.18.

Being used for crossing the nucleotide sequence of expressing the ribulose-5-phosphate epimerase at host cell of the present invention is the nucleotide sequence that coding has the polypeptide of ribulose-5-phosphate epimerization enzymic activity, wherein preferably this polypeptide has the aminoacid sequence that has 50%, 60%, 70%, 80%, 90% or 95% identity at least with SEQ ID NO.19, or wherein this nucleotide sequence can under the moderate condition, preferably under stringent condition with the nucleotide sequence hybridization of SEQ ID NO.20.

Being used for crossing the nucleotide sequence of expressing transketolase at host cell of the present invention is the nucleotide sequence that coding has the polypeptide of TKA, wherein preferably this polypeptide has the aminoacid sequence that has 50%, 60%, 70%, 80%, 90% or 95% identity at least with SEQ ID NO.21, or wherein this nucleotide sequence can under the moderate condition, preferably under stringent condition with the nucleotide sequence hybridization of SEQ ID NO.22.

Being used for crossing the nucleotide sequence of expressing transaldolase at host cell of the present invention is the nucleotide sequence that coding has the polypeptide of transaldolase activity, wherein preferably this polypeptide has the aminoacid sequence that has 50%, 60%, 70%, 80%, 90% or 95% identity at least with SEQ ID NO.23, or wherein this nucleotide sequence can under the moderate condition, preferably under stringent condition with the nucleotide sequence hybridization of SEQ ID NO.24.

When relating in genetically modified host cell when producing enzyme, expression is expressed in crossing of enzyme: compare with host cell not modified under the same terms, enzyme is produced with higher levels of specific enzyme activity.Usually, this expression is compared with not modified host cell under the same terms, and the protein (or being a plurality of protein under the situation of many subunits enzyme) with enzymic activity is produced with bigger amount, is produced with higher steady-state level in other words.Similarly, host cell not modified under this ordinary representation and the same terms is compared, and the mRNA that coding has the protein of enzymic activity is produced with greater amount, is produced with higher steady-state level in other words.Therefore, preferably by using suitable enzyme assay as described herein, express by the crossing of specific activity level determination enzyme of measuring enzyme in the host cell.Perhaps, can for example use the ratio steady-state level of the special antibody of enzyme by quantitative zymoprotein, or by the quantitative ratio steady-state level of the mRNA of this enzyme of coding, crossing of indirect measurement enzyme expressed.The latter is particularly useful for the enzyme of pentose-phosphate pathway, is not feasible easily for this approach enzyme assay, because the substrate of enzyme can commercially not obtain.Preferably, in host cell of the present invention, with except caused that the heredity of the genetic modification of expressing went up same bacterial strain and compare, be crossed the enzyme of expressing with at least 1.1,1.2,1.5,2,5,10 or 20 multiple by express.Should be appreciated that these cross expression level applicable to the steady-state level of the protein of the steady-state level of enzymic activity, enzyme, and the steady-state level of the transcript of codase.

In still another preferred embodiment, (it expresses araA, araB and araD to host cell of the present invention, and demonstration turns to the direct isomery of wood sugar the ability of xylulose, and comprise following genetic modification alternatively, it improves the flux of pentose-phosphate pathway, also comprises as mentioned before) genetic modification that improves the xylulokinase specific activity.Preferably, this genetic modification for example passed through to express the nucleotide sequence of coding xylulokinase, caused the expression of crossing of xylulokinase.The gene pairs host cell of coding xylulokinase can be endogenous, maybe can be the xylulokinase to host cell allos.Being used for crossing the nucleotide sequence of expressing xylulokinase at host cell of the present invention is the nucleotide sequence that coding has the polypeptide of xylulokinase activity, wherein preferably this polypeptide has the aminoacid sequence that has 50%, 60%, 70%, 80%, 90% or 95% identity at least with SEQ ID NO.25, or wherein this nucleotide sequence can under the moderate condition, preferably under stringent condition with the nucleotide sequence hybridization of SEQ ID NO.26.

A kind of especially preferred xylulokinase be with from the relevant wood sugar kinases of the xylulokinase xylB of Piromyces, described in WO 03/0624430.Being used for crossing a kind of preferred nucleotide sequence of expressing xylulokinase at host cell of the present invention is the nucleotide sequence that coding has the polypeptide of xylulokinase activity, wherein preferably this polypeptide has the aminoacid sequence that has 50%, 60%, 70%, 80%, 90% or 95% identity at least with SEQ ID NO.27, or wherein this nucleotide sequence can under the moderate condition, preferably under stringent condition with the nucleotide sequence hybridization of SEQ ID NO.28.

In host cell of the present invention, the genetic modification that improves the xylulokinase specific activity can make up with any modification that improves the pentose-phosphate pathway flux as mentioned above, but should make up optional to the present invention.Therefore, the host cell of the present invention that also comprises the genetic modification that improves the xylulokinase specific activity except the araA, the araB that express this paper definition and araD enzyme specifically comprises in the present invention.Be used in this area reaching with the analysis xylulokinase and cross the multiple means of expression with above described identical at the enzyme of pentose-phosphate pathway at host cell of the present invention.Preferably, in host cell of the present invention, with except caused that the heredity of the genetic modification of expressing went up same bacterial strain and compare, be crossed the xylulokinase of expressing with at least 1.1,1.2,1.5,2,5,10 or 20 multiple by express.Should be appreciated that the steady-state level of the protein of these steady-state level of crossing expression level and can be applicable to enzymic activity, enzyme, and the steady-state level of the transcript of codase.

In still another preferred embodiment, (it expresses araA, araB and araD to host cell of the present invention, and demonstration turns to the direct isomery of wood sugar the ability of xylulose, and comprise the genetic modification that improves the pentose-phosphate pathway flux alternatively, and/or also comprise the genetic modification that improves the xylulokinase specific activity, and all as previously mentioned) also comprise the genetic modification that reduces non-specific aldose reductase activity in the host cell.Preferably, reduce non-specific aldose reductase activity in the host cell by one or more genetic modifications, described genetic modification reduces the expression of non-specific aldose reductase or makes the gene inactivation of this enzyme of coding, described in WO 06/009434.Preferably, the expression of non-specific each endogenous copy of aldose reductase in genetic modification reduction or the inactivation host cell.Host cell can comprise the gene of the non-specific aldose reductase of coding of multiple copied owing to diploidy, polyploidy or dysploidy, and/or host cell can contain some kinds of different (same worker) enzymes with aldose reductase activity, different and each the free different genes encoding of its aminoacid sequence.In addition, each expression of gene of the non-specific aldose reductase of coding preferably is lowered or inactivation under this class situation.Preferably, part by deleting gene at least or by destroying gene with this gene inactivation or deletion, wherein the term gene also comprises any non-coding sequence in encoding sequence upstream or downstream in this linguistic context, and its (part) deletion or inactivation cause the reduction that non-specific aldose reductase activity is expressed in the host cell.Coding will reduce its active aldose reductase in host cell of the present invention nucleotide sequence is the nucleotide sequence that coding has the polypeptide of aldose reductase activity, wherein preferably this polypeptide has the aminoacid sequence that has 50%, 60%, 70%, 80%, 90% or 95% identity at least with SEQ ID NO.29, or wherein this nucleotide sequence can under the moderate condition, preferably under stringent condition with the nucleotide sequence hybridization of SEQ ID NO.30.

In host cell of the present invention, araA, the araB of this paper definition and the expression of araD enzyme and the genetic modification combination that reduces non-specific aldose reductase activity.The genetic modification that causes non-specific aldose reductase activity to reduce can make up with any modification of any modification that improves the pentose-phosphate pathway flux as mentioned above in host cell and/or raising xylulokinase specific activity, but these combinations are optional to the present invention.Therefore, comprising the host cell other genetic modification, that express araA, araB and araD that reduces non-specific aldose reductase activity specifically comprises in the present invention.

In a preferred embodiment, host cell is the CBS 120327 that is preserved in CBS association (Holland) on September 27th, 2006.

In still another preferred embodiment, the modified host cell that the present invention relates to also to adapt to L-arabinose utilization (use L-arabinose and/or be translated into the L-ribulose and/or X 5P and/or desired fermentation product) and adapt to xylose utilization alternatively, described host cell is by selecting spontaneous or (for example by radiation or chemical) the mutant acquisition of inducing, described mutant is grown at L-arabinose and optional wood sugar, preferably grow more preferably growth under anaerobic at L-arabinose and optional wood sugar as sole carbon source.The selection of mutant can be by for example Kuyper et al. (2004, FEMS Yeast Res.4:655-664) described culture continuous passage is carried out, and/or is undertaken by for example cultivating under selective pressure in the perseverance cultivation described in WO 06/009434 embodiment 4.This chosen process can continue as required.This chosen process was preferably finished in 1 year in a week.Yet the time period that can more grow of this chosen process if necessary.In this chosen process, culturing cell when having about 20g/l L-arabinose and/or about 20g/l wood sugar preferably.Being expected at the cell that obtains when this chosen process finishes is modified about its following ability, described ability is the ability of use L-arabinose and/or wood sugar, and/or L-arabinose is converted into the ability of L-ribulose and/or X 5P and/or desired fermentation product such as ethanol.In this linguistic context, " improved cell " can represent that the cell that obtains compares with the cell in its source, can use L-arabinose and/or wood sugar in more effective mode.For example, be expected under the identical condition, the cell of acquisition is grown better than the cell in its source: specific growth rate improves at least 2%.Preferably, this raising is at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more.As known as technical staff, specific growth rate can be from OD ₆₆₀Calculate.Therefore, by monitoring OD ₆₆₀, can the inference specific growth rate.In this linguistic context, " improved cell " can represent that also the cell that obtains compares with the cell in its source, in more effective mode L-arabinose is converted into L-ribulose and/or X 5P and/or desired fermentation product such as ethanol.For example, more L-ribulose and/or X 5P and/or desired fermentation product such as the ethanol of a large amount of the cells produce that obtains of expection: compare with the cell in its source under the same terms, at least a these compounds improve 2% at least.Preferably, this raising is at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more.In this linguistic context, " improved cell " can represent that also the cell that obtains compares with the cell in its source, in more effective mode wood sugar is converted into xylulose and/or desired fermentation product such as ethanol.For example, more xylulose and/or desired fermentation product such as the ethanol of a large amount of the cells produce that obtains of expection: compare at least a these compounds with the cell in its source under the same terms and improve 2% at least.Preferably, this raising is at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more.

In a preferred host cell of the present invention, at least a above-mentioned genetic modification (comprising the modification by selecting mutant to obtain) is given host cell on as carbon source, L-arabinose and optional wood sugar preferably as sole carbon source, preferably under anaerobic the ability of growth is preferably, modified host cell is not produced Xylitol basically, for example the Xylitol of Chan Shenging is lower than limit of detection, or for example is 5%, 2%, 1%, 0.5% or 0.3% of the basis carbon that is less than consumption with the mole.

Preferably, modified host cell has on as the L-arabinose of sole carbon source and optional wood sugar under aerobic conditions with at least 0.001,0.005,0.01,0.03,0.05,0.1,0.2,0.25 or the ability of the speed growth of 0.3h-1, if perhaps applicable words are under anaerobic with 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 0.2h ^-1The ability of speed growth.Preferably, modified host cell has on as the mixture of the glucose of sole carbon source and L-arabinose and optional wood sugar (1: 1 weight ratio) under aerobic conditions with at least 0.001,0.005,0.01,0.03,0.05,0.1,0.2,0.25 or the ability of the speed growth of 0.3h-1, if perhaps applicable words are under anaerobic with at least 0.001,0.005,0.01,0.03,0.05,0.1,0.12,0.15 or the ability of the speed growth of 0.2h-1.

Preferably, modified host cell has at least 346,350,400,500,600,650,700,750,800,900 or the L-arabinose of 1000mg/g cell/h and the specific consumption rate of optional wood sugar.Preferably, modified host cell has following tunning (as ethanol) productive rate with respect to L-arabinose and optional wood sugar, described productive rate be at least host cell the tunning with respect to glucose (as ethanol) productive rate 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 85%, 90%, 95% or 98%.More preferably, tunning (as the ethanol) productive rate with respect to L-arabinose and optional wood sugar of modified host cell equals the tunning with respect to glucose (as the ethanol) productive rate of host cell.The biomass yield with respect to L-arabinose and optional wood sugar of equally, modified host cell preferably be at least host cell with respect to 55%, 60%, 70%, 80%, 85%, 90%, 95% or 98% of the biomass yield of glucose.More preferably, the biomass yield with respect to-pectinose and optional wood sugar of modified host cell equals the biomass yield with respect to glucose of host cell.Be to be understood that two kinds of productive rates all under aerobic conditions compare or all under anaerobic compare in the comparison with respect to glucose and L-arabinose and the productive rate of optional wood sugar.

In a preferred embodiment, host cell was the CBS 120328 that is preserved in CBS association (Holland) on September 27th, 2006, or was preserved in the CBS 121879 of CBS association (Holland) on September 20th, 2007.

In a preferred embodiment, one or more enzymes of cell expressing, described enzyme gives cells produce at least a ability that is selected from down the tunning of group, this group is by ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1, and 3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics and cynnematin are formed.In a preferred embodiment, host cell of the present invention is the host cell for the production of ethanol.In another preferred embodiment, the present invention relates to for the production of the tunning except ethanol through transformed host cells.Non-ethanol fermentation product like this comprises in principle can be by any rough chemical (bulk chemical) or the fine chemicals of eukaryotic microorganisms such as yeast or filamentous fungus production.This class tunning comprises for example lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1,3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics and cynnematin.A preferred host cell of the present invention for the production of non-ethanol fermentation product is the host cell that contains following genetic modification, and described modification causes the alcoholdehydrogenase activity that reduces.

Method

On the other hand, the present invention relates to following zymotechnique, wherein host cell of the present invention is used to fermenting carbon source, and described carbon source comprises L-arabinose source and optional wood sugar source.Preferably, this L-arabinose source and wood sugar source are L-arabinose and wood sugar.In addition, the carbon source in the fermention medium also can comprise the glucose source.L-arabinose, wood sugar or glucose source can be L-arabinose, wood sugar or glucose equally, maybe can be any carbohydrate oligomer or the polymer that comprises L-arabinose, wood sugar or glucose unit, for example lignocellulose, xylan, Mierocrystalline cellulose, starch, arabinan etc.In order from this class carbohydrate, to discharge wood sugar or glucose.Can produce suitable carbohydrase (as zytase, dextranase, amylase etc.) to interpolation in the fermention medium or by modified host cell.Under one situation of back, modified host cell can be by genetically engineered for producing and secreting this class carbohydrase.Another advantage of using oligomer or polymeric glucose source is that it makes and during fermentation can keep (more) low free glucose concn, and the carbohydrase of rate limiting amount is for example used in described fermentation.Therefore, this so that can prevent metabolism and the transhipment non-glucose sugar such as the required system of wood sugar prevent.In a kind of preferred technology, modified host cell fermentation L-arabinose (being wood sugar alternatively) and glucose the two, preferably fermentation simultaneously, preferably use modified host cell in this case, described host cell is prevented insensitive to the glucose that hinders diauxic growth (diauxic growth).Except the source as the L-arabinose of carbon source, optional wood sugar (and glucose), fermention medium also can comprise the modified required proper composition of host cell growth.The composition that is used for the fermention medium of microorganism such as yeast and filamentous fungus growth is well known in the art.

In a kind of preferred technology, technology for the production of the tunning that is selected from down group is provided, this group is by ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1,3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics and cynnematin are formed, and wherein this technology comprises step:

(a) the modified host cell fermentation that defines with this paper contains the substratum in pectinose and optional wood sugar source; Alternatively

(b) reclaim described tunning.

Zymotechnique is for the production of tunning such as ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1, the technology of 3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics (as penicillin G and penicillin v and fermentation derivative thereof) and cynnematin.This zymotechnique can be the zymotechnique of aerobic or anaerobism.Anaerobic ferment process is defined as the zymotechnique that carries out when not having oxygen in this article, or wherein substantially not oxygen consumed, preferably consume and to be less than 5,2.5 or 1mmol/L/h, more preferably consume the technology of 0mmol/L/h (being that oxygen consumption is undetectable) and wherein organic molecule performance electron donor and two kinds of effects of electron acceptor(EA).When not having oxygen, the NADH that produces in glycolysis-and biomass form can not oxidized phosphorylation oxidation.In order to address this problem, many microorganisms use one of pyruvic acid or derivatives thereof as electronics and hydrogen acceptor, thus regeneration of NAD+.Therefore, in a kind of preferred anaerobic ferment process, pyruvic acid is used as electronics (and hydrogen acceptor), and be reduced to tunning such as ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1,3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics and cynnematin.In a preferred embodiment, zymotechnique is anaerobism.The zymotechnique of anaerobism is favourable, because it is more cheap than aerobic technology: need specific equipment still less.In addition, the expection anaerobic process provide higher product productive rate than Aerobic processes.Under aerobic conditions, common biomass yield is than higher under the anaerobic condition.Therefore, usually under aerobic conditions, lower under the product productivity ratio anaerobic condition of expection.According to the inventor, technology of the present invention is the anaerobic ferment process that the first use of developing so far comprises the substratum in L-arabinose source.

In another preferred embodiment, zymotechnique carries out under the condition that is in the oxygen restriction.More preferably, zymotechnique is aerobic and is under the condition of oxygen restriction.The zymotechnique of oxygen restriction is following technology, and wherein oxygen consumption is subjected to be transferred to from gas the restriction of the oxygen of liquid.The degree of the actual mixing of the amount by entering air-flow and the fermentation equipment of composition and use/mass transfer characteristic measurement oxygen restriction.Preferably, in the technology under being in the oxygen restricted condition, the speed of oxygen consumption is at least 5.5mmol/L/h, more preferably is at least 6mmol/L/h, further more preferably is at least 7mmol/L/h.

Zymotechnique preferably carries out under to the suitableeest temperature of modified cell.Therefore, for most of yeast or fungal cell, be less than 42 ℃, preferably be less than under 38 ℃ the temperature and carry out zymotechnique.For yeast or filamentous fungal host cell, zymotechnique preferably carries out being lower than under 35,33,30 or 28 ℃ the temperature and being higher than under 20,22 or 25 ℃ the temperature.

A kind of preferred technology is the technology for the production of ethanol, and wherein this technology comprises step: the substratum that (a) contains pectinose and optional wood sugar source with the modified host cell fermentation of this paper definition; (b) reclaims ethanol alternatively.Fermention medium also can comprise also by the glucose source of fermentation for ethanol.In a preferred embodiment, the zymotechnique for the production of ethanol is anaerobism.In preamble, being defined of anaerobism.In another preferred embodiment, the zymotechnique for the production of ethanol is aerobic.In another preferred embodiment, be in for the production of the zymotechnique of ethanol under the condition of oxygen restriction, more preferably be under the condition of aerobic and oxygen restriction.The condition of oxygen restriction defines in preamble.

In this technology, the volume production power (productivity) of ethanol is preferably and per hour whenever rises to few 0.5,1.0,1.5,2.0,2.5,3.0,5.0 or 10.0g ethanol.Alcohol yied with respect to L-arabinose and optional wood sugar and/or glucose in the technology is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or 98%.Alcohol yied is defined as the per-cent of theoretical maximum yield in this article, and described theoretical maximum yield is every g glucose or wood sugar 0.51g ethanol for glucose and L-arabinose with optional wood sugar.In another preferred embodiment, the present invention relates to the technology for the production of the tunning that is selected from down group, this group is by lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1, and 3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics and cynnematin are formed.This optimal process ground comprises step: the substratum that (a) contains L-arabinose and optional wood sugar source with modified host cell fermentation defined above; (b) reclaims described tunning alternatively.In a kind of preferred technology, substratum also contains the glucose source.

In causing the zymotechnique of the present invention of alcohol production, can be by relatively quoting some advantages as proof with known ethanol fermentation technology:

-anaerobic technique is possible.

The condition of-oxygen restriction also is possible.

-can obtain higher alcohol yied and alcohol production speed.

The bacterial strain of-use may be able to use L-arabinose and optional wood sugar.

Except above-mentioned zymotechnique, provide another kind of zymotechnique as another aspect of the present invention, wherein use two kinds of different cell fermentations to comprise at least two kinds of carbon sources that are selected from down the carbon source of group at least, this group is made up of source, wood sugar source and the glucose source of L-arabinose, but is not limited only to this.In this zymotechnique, " at least two kinds of different cells " represents that this optimal process ground is co-fermentation technology.In a preferred embodiment, use two kinds of different cells: a kind of is the cell of the present invention that defines as preamble, it can use L-arabinose and/or be translated into the L-ribulose and/or X 5P and/or desired fermentation product such as ethanol, and optionally can use wood sugar; Another kind is the bacterial strain of definition among WO 03/062430 and/or the WO 06/009434 for example, and it can use wood sugar and/or be translated into desired fermentation product such as ethanol.The cell that can use wood sugar preferably directly turns to xylose isomerase the bacterial strain of the ability of xylulose (in a step) as the demonstration of preamble definition.These two kinds of different bacterial strains are preferably cultivated when having L-arabinose source, wood sugar source and optional glucose source.But three kinds of co-cultivation or how different cells, and/or can use three kinds or more carbon sources, as long as at least a cell can use at least a carbon source of existence and/or be translated into desired fermentation product such as ethanol.Statement " using at least a carbon source " has the implication identical with statement " use L-arabinose ".Statement " its (being carbon source) is converted into desired fermentation product " has the implication identical with statement " L-arabinose is converted into desired fermentation product ".

In a preferred embodiment, the present invention relates to produce the technology of the tunning that is selected from down group, this group is by ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1,3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics and cynnematin are formed, and wherein this technology comprises the steps:

(a) with the cell of the present invention of preamble definition with can use wood sugar and/or show the cell fermentation substratum that directly wood sugar is converted into the ability of xylulose, described substratum contains L-arabinose source and wood sugar source at least, wherein each cell is tunning and alternatively with L-arabinose and/or wood-sugar fermentation

(b) reclaim described tunning.

All embodiment preferred of above-mentioned zymotechnique also are the embodiment preferred of this further zymotechnique: identify tunning, identify L-arabinose source and wood sugar source, fermentation condition (condition, the temperature of carrying out this technology, alcohol production power, the alcohol yied of aerobic or anaerobic condition, oxygen restriction).

Genetic modification

In order in host cell of the present invention as mentioned above, to cross the expression enzyme, and for other genetic modification of host cell (being preferably yeast), use multiple nucleic acid construct transformed host cell of the present invention by means commonly known in the art.These class methods are for example known from manual of standards, Sambrook and Russel (2001) for example " 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).The method that is used for fungal host cells conversion and genetic modification can be known from for example EP-A-0635574, WO 98/46772, WO 99/60102 and WO 00/37671.

Above described for crossing at host cell of the present invention and expressed the promotor of using in the nucleic acid construct of enzyme.At the nucleic acid construct that was used for expressing, 3 of the nucleotide sequence of codase '-hold preferably to be operably connected with the Transcription Termination subsequence.Preferably, this Transcription Termination subsequence can be operated in the yeast cell that the host cell of selecting is for example selected.Under any circumstance, the selection of terminator is not critical; It can be for example from any yeast genes, although if terminator also can be worked sometimes during from the eukaryotic gene of non-yeast.Transcription termination sequence also preferably comprises polyadenylation signal.Preferred terminator sequence is alcoholdehydrogenase (ADH1) and PGI1 terminator.More preferably, ADH1 and PGI1 terminator are all from S.cerevisiae (being respectively SEQ ID NO:50 and SEQ ID NO:53).

Alternatively, selectable marker can be present in the nucleic acid construct.This paper uses the gene of term " marker " presentation code proterties or phenotype, and described proterties or phenotype allow to select or screen the host cell that contains this marker.Marker gene can be antibiotics resistance gene, and wherein suitable microbiotic can be used to never be selected through cell transformed in the cell transformed.Yet, preferably use the non-antibiotic resistance markers, as the auxotrophy marker (URA3, TRP1, LEU2).In a preferred embodiment, be marker-free with the nucleic acid construct transformed host cells.The method of be used for to make up not having a reorganization marker gene host cell is open and based on the use of two-way mark thing at EP-A-0635574.Perhaps, the marker that can screen such as green fluorescent protein, lacZ, luciferase, E.C. 2.3.1.28, β-Pu Taotanggansuanmei can be impregnated in the nucleic acid construct of the present invention, allow screening through cell transformed.

Optional other element that can be present in the nucleic acid construct of the present invention includes but not limited to that one or more leader sequence, enhanser, conformity gene and/or acceptor gene, intron sequences, kinetochore, telomer and/or matrix are adhered to (MAR) sequence.Nucleic acid construct of the present invention also can comprise the sequence for self-replicating, as the ARS sequence.Suitable episome nucleic acid construct can be for example based on 2 μ or pKD1 (Fleer et al., 1991, the Biotechnology 9:968-975) plasmid of yeast.Perhaps, nucleic acid construct can comprise for the sequence of integrating, and preferably integrates by homologous recombination.This class sequence therefore can be with the host cell gene group in be used for the sequence of the target site homology integrated.Nucleic acid construct of the present invention can provide with known mode itself, described mode is usually directed to for example restriction enzyme digestion (restricting) and the technology that is connected nucleic acid/nucleotide sequence, at this Technical Reference manual of standards Sambrook and Russel (2001) " Molecular Cloning:A Laboratory Manual (3rd edition); Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press. for example

The method that is used for yeast or fungi inactivation and gene disruption is (seeing for example Fincham, 1989, Microbiol Rev.53 (1): 148-70 or EP-A-0 635 574) well known in the art.

In presents and claim thereof, verb " comprises " and version uses with its nonrestrictive implication, in the project of representing this word back is included in, but do not get rid of the project of clearly not mentioning.In addition, indefinite article "/kind " (" a " or " an ") is not got rid of existence more than the possibility of an element when relating to element, unless context requires to exist and only exist an element clearly.Therefore, indefinite article "/kind " (" a " or " an ") ordinary representation " at least one/kind ".

The present invention also describes by following embodiment, and described embodiment should not be interpreted as limiting the scope of the invention.

Embodiment

Plasmid and strain construction

Bacterial strain

The Sachharomyces cerevisiae bacterial strain of the consumption L-arabinose of describing in this work himself is the derivative of RWB217 based on bacterial strain RWB220.RWB217 is the CEN.PK bacterial strain, in the pentose-phosphate pathway of wherein encoding gene TAL1, the TKL1 of expression of enzymes, RPE1, RKI1 cross to be expressed (Kuyper et al., 2005a).In addition, the gene of coding aldose reductase (GRE3) is lacked.Bacterial strain RWB217 also contains two kinds of plasmids: single copy plasmid, and it has for LEU2 marker and the episome of crossing expression xylulokinase (XKS1); The multiple copied plasmid, it has URA3 as the marker of expressing xylulose isomerase XylA.At the growth that improves at wood sugar RWB217 is selected step, this describes in Kuyper et al. (2005b).This step obtained two kinds of pure bacterial strain RWB218 (Kuyper et al., 2005b) and RWB219.Difference between RWB218 and the RWB219 is after selecting step, by containing glucose as the mineral substance substratum coating of carbon source and again line obtain RWB218, and use wood sugar as carbon source to RWB219.

Bacterial strain RWB219 is contained non-selectively cultivation on the YP (YPD) of glucose as carbon source, thereby promoting losing of two kinds of plasmids.After being coated on the YPD, by observe the single bacterium colony of test plasmid loss at uridylic and leucine auxotrophy.Transform two kinds of bacterial strains that plasmid is all lost with the pSH47 that contains the cre recombinase, cross the KanMX box (Guldener et al., 1996) that still exists after the expression construct thereby remove integration RKI1.The bacterium colony that will have plasmid is resuspended in the Yeast protein peptone substratum (YP) (10g/l yeast extract and 20g/l peptone are all from BD Difco Belgium) that contains 1% semi-lactosi, hatches 1 hour at 30 ℃.About 200 cells are coated on the YPD.The bacterium colony that check obtains of losing at KanMX marker (G418 resistance) and pSH47 (URA3).The bacterial strain called after RWB220 that KanMX marker and pSH47 plasmid are all lost then.For the bacterial strain that obtains in this patent, to test, transform RWB220 with pRW231 and pRW243, obtain bacterial strain IMS0001.

During making up, bacterial strain is at compound YP:10g l ^-1Yeast extract (BD Difco), 20g l ^-1Peptone (BD Difco) is gone up or is kept on synthetic medium (MY) (Verduyn et al., 1992), and described culture medium supplemented has glucose (2%) as carbon source (YPD or MYD) and be supplemented with 1.5% agar under the situation of flat board.After the plasmid conversion, bacterial strain is coated on the MYD.The conversion of yeast is carried out according to Gietz and Woods (2002).Plasmid is gone up amplification at Escherichia coli strain X L-1 blue (Stratagene, La Jolla, CA, the U.S.).Conversion is carried out according to Inoue et al. (1990).E.coli cultivates on LB (Luria-Bertani) flat board or in liquid TB (Terrific Broth) substratum, for separating of plasmid (Sambrook et al, 1989).

Plasmid

In order to grow at L-arabinose, yeast need be expressed three kinds of different genes: L-arabinose isomerase (AraA), L-ribulokinase (AraB) and L-ribulose-5-P-4-epimerase (AraD) (Becker and Boles, 2003).In this work, we are chosen in AraA, AraB and the AraD that expresses among the S.cerevisiae from lactic-acid-bacterium Lactobacillus plantarum.Because final purpose is that L-arabinose and other sugar are made up consumption as the D-wood sugar, so the assortment of genes that the gene of the bacterium L-arabinose approach of will encoding and encoding D-wood sugar consumes is on same plasmid.

In order to obtain high-caliber expression, L.plantarum AraA is connected forward (FWD) with the AraD gene to be had among the plasmid pAKX002 of 2 μ XylA.

TDH3 promotor (SEQ ID NO:49) by using SpeI5 ' Ptdh3 and 5 ' AraAPtdh3 amplification brachymemma version, use Ptdh5 ' AraA and Tadh3 ' AraA amplification AraA gene and with 3 ' AraATadh1 and 3 ' Tadh1-SpeI amplifying ADH, 1 terminator (SEQ ID NO:50), structure AraA box.From gel, extract three kinds of fragments and it is mixed with equimolar amount roughly.Use SpeI-5 ' Ptdh3 and 3 ' Tadh1SpeI oligomer that this mixture is carried out PCR.The P that gel-purified obtains _TDH3-AraA-T _ADH1Box, 5 ' and the cutting of 3 ' SpeI site, connect to advance then among the pAKX002 with the NheI cutting, obtain plasmid pRW230.

Following manufacturing AraD construct: the HXT7 promotor (SEQ ID NO:52) of at first using oligomer SalI5 ' Phxt7 and 5 ' AraDPhxt amplification brachymemma version, use Phxt5 ' AraD and Tpgi3 ' AraD amplification AraD gene and distinguish with 3 ' AraDTpgi and 3 ' TpgiSalI oligomer amplification GPI1 terminator (SEQ ID NO:53).From gel, extract the fragment obtain and it is mixed with equimolar amount roughly, use SalI5 ' Phxt7 and 3 ' Tpgi1SalI oligomer to carry out PCR then.The P that gel-purified obtains _HXT7-AraD-T _PGI1Box, 5 ' and the cutting of 3 ' SalI site, connect to advance then among the pRW230 with the XhoI cutting, obtain plasmid pRW231 (Fig. 1).

Because the high expression level of crossing of L-ribulokinase is (Becker and Boles, 2003) that are harmful to growth, thus with the XKS1 assortment of genes of AraB gene and coding xylulokinase on integrated plasmid.For this reason, at first following with p415ADHXKS (Kuyper et al., 2005a) change into pRW229: with PvuI cutting p415ADHXKS and pRS305 the two, and will be connected with carrier main chain from pRS305 from the PvuI fragment that contains ADHXKS of p415ADHXKS, obtain pRW229.

By using SacI5 ' Ppgi1 and 5 ' AraBPpgi1 oligomer amplification PGI1 promotor, use Ppgi5 ' AraB and Tadh3 ' AraB oligomer amplification AraB gene, with with 3 ' AraBTadh1 and 3 ' Tadh1SacI oligomer amplifying ADH, 1 terminator, make the box that contains L.plantarum AraB gene between PGI1 promotor (SEQ ID NO:51) and the ADH1 terminator (SEQ ID NO:50).From gel, extract three kinds of fragments and it is mixed with equimolar amount roughly.Use SacI-5 ' Ppgi1 and 3 ' Tadh1SacI oligomer that this mixture is carried out PCR.The P that gel-purified obtains _PGI1-AraB-T _ADH1Box, 5 ' and the cutting of 3 ' SacI site, connect to advance then among the pRW22 with the SacI cutting, obtain plasmid pRW243 (Fig. 1).

Transform bacterial strain RWB220 with pRW231 and pRW243 (table 2), obtain bacterial strain IMS0001.

Restriction endonuclease (New England Biolabs, Beverly, MA, the U.S. and Roche, Basel, Switzerland) and dna ligase (Roche) use according to the explanation of manufacturers.(Qiagen, Hilden is Germany) from the E.coli separation quality grain to prepare test kit with the centrifugal a small amount of of Qiaprep.Among 1 * TBE (Sambrook et al, 1989) on 1% agarose (Sigma, St.Louis, MO, the U.S.) gel the DNA isolation fragment.With Qiaquick gel extraction kit (Quiagen) isolated fragment from the gel.The amplification of AraA, AraB and AraD box (element) is finished with VentR archaeal dna polymerase (New England Biolabs) according to the explanation of manufacturers.The template of promotor and terminator is the chromosomal DNA of S.cerevisiae CEN.PK113-7D, and the template of Ara gene is the chromosomal DNA of Lactobacillus plantarum DSM20205.Polymerase chain reaction (PCR) Biometra TGradient Thermocycler (Biometra,

Germany) carry out in, use setting: annealed 1 minute at 55 ℃, 60 ℃ or 65 ℃, extend 1 to 3 minute (fragment length that depends on expectation) and 94 ℃ of sex change 1 minute, carry out 30 circulations at 75 ℃.

Cultivate and substratum

In synthetic medium (Verduyn et al., 1992), carry out shake-flask culture at 30 ℃.Use 2M KOH with the pH regulator to 6.0 of substratum before the sterilization.For the solid synthetic medium, add 1.5% agar.

By shaking the 100ml substratum that contains suitable sugar in the bottle with freezing microbial strain culture inoculation 500-ml, prepare pre-culture.300 ℃ in orbital shaker (200rpm), hatch after, use this culture inoculation shake-flask culture thing or fermentor cultivation thing.The synthetic medium that is used for the anaerobism cultivation is supplemented with the 0.42g l that is dissolved in ethanol ^-1Tween 80 and 0.01g l ^-1Ergosterol (Andreasen and Stier, 1953; Andreasen and Stier, 1954).Anaerobism (sequencing) batch culture is carried out in working volume is 2 liters of laboratory ferment jars (Applikon, Schiedam, Switzerland) of 1 liter under 30 ℃.By automatic interpolation 2M KOH culture pH is maintained pH 5.0.With 800rpm stir culture thing, and with 0.5 l min ^-1Nitrogen bubble (＜10ppm oxygen).For the diffusion of minimum oxygen, to fermentor tank assembling Norprene transfer lime (Cole Palmer Instrument company, Vernon Hills, USA).With oxygen electrode (Applisens, Schiedam, Holland) monitoring dissolved oxygen.By with about 0.05l min ^-1The head space inflation reaches the condition of oxygen restriction in identical experimental installation.

The mensuration of dry weight

Nitrocellulose filter paper (the aperture 0.45lm that is weighing in advance; Gelman laboratory, Ann Arbor USA) goes up filtration culture samples (10.0ml).After removing substratum, with deionized water wash filter paper and in microwave oven (Bosch, Stuttgart, Germany) in dry 20 minutes of 360W and weigh.Bipartite mensuration changes less than 1%.

Gasometry

Gas is discharged in cooling in condenser (2 ℃), and with MD-110-48P-4 type Permapure moisture eliminator (Permapure, Toms River, USA) drying.(Rosemount Analytical, Orrville USA) measure O with NGA 2000 analysers ₂And CO ₂Concentration.(Van Urk et al., 1988 as discussed previously; Weusthuis et al., 1994) measure and discharge gas flow rate and the specific consumption rate of oxygen and the specific production rate of carbonic acid gas.When calculating these biomass-specific speed, consider by taking out the stereomutation that culture samples causes.

Metabolite analysis

Use Waters Alliance 2690 HPLC (Waters, Milford, USA) analyze glucose, wood sugar, pectinose, Xylitol, organic acid, glycerine and ethanol by HPLC, described equipment is equipped with BioRad HPX 87H post (BioRad, Hercules, USA), Waters 2410 become optical index detector and Waters 2487UV detector.Use 0.5g l down at 60 ℃ ^-1Sulfuric acid with the flow velocity wash-out post of 0.6ml min-1.

To the mensuration of X 5P (Zaldivar J., et al, Appl.Microbiol.Biotechnol., (2002), 59:436-442)

For analysis of cells intracellular metabolite product such as X 5P, (cultivation 42,79 and 131 hours) duplicate mobile phone 5ml nutrient solution from reactor after (cultivation the 22nd and 26 hour) and glucose exhaust before glucose exhausts.Described in detail by Smits H.P.et al. (Anal.Biochem., 261:36-42, (1998)) for the step that stops metabolism, solid phase extractions meta-bolites and analysis.Yet, slightly modified described high pressure ion exchange chromatogram and the pulse Amperometric Detection Coupled coupling that is used for the analysis of cells extract by the analysis of high pressure ion exchange chromatogram.The solution that uses is elutriant A, 75mM NaOH and elutriant B, 500mM NaAc.In order to prevent the pollution of carbonate in the elutriant, use the NaOH solution (Baker Analysed, Deventer, Holland) with low-carbonate concentration to replace the NaOH piece.With helium (He) elutriant was outgased 30 minutes,, remain under the helium pressure then.Gradient pump is programmed for the following gradient of generation: 100% A and 0% B (0 minute), the linearity of A to 70% reduces and the linearity of B to 30% improves (0-30 minute), the linearity of A to 30% reduces and the linearity of B to 70% improves (30-70 minute), the linearity of A to 0 reduces and the linearity of B to 100% improves (70-75 minute), 0% A and 100% B (75-85 minute), the linearity of A to 100% improves and the linearity of B to 0% reduces (85-95 minute).Moving phase moves with 1ml/ minute flow velocity.Other condition is according to Smits et al. (1998).

Carbon reclaims

Carbon reclaims carbon in the product be calculated as formation divided by the sugar charcoal total amount that consumes, and based on 48% biomass carbon content.In order to proofread and correct the accumulative total CO that the amount of alcohol that supposition produces equals to measure at fermentation ethanol evaporation therebetween ₂Production deducts owing to the synthetic CO that takes place of biomass ₂Produce (every gram biomass 5.85mmol CO ₂(Verduyn et al., 1990)) with form relevant CO with acetate ₂Produce.

Select at the growth on L-arabinose

Make up bacterial strain IMS0001 (CBS 120327, are preserved in CBS 27/09/06) according to above-mentioned steps, it contains the gene of coding wood sugar (XylA and XKS1) and the two pathways metabolism of pectinose (AraA, AraB, AraD).As if although can be in wood sugar growth (data not shown), bacterial strain IMS0001 can not grow at the solid synthetic medium that is supplemented with the 2%L-pectinose.Select to utilize L-arabinose as the IMS0001 mutant of carbon source for growth by the transfer of the series in shaking bottle with by the sequencing batch culture (SBR) in fermentor tank.

For serial shift experiment, the 500-ml that contains the 100ml synthetic medium with bacterial strain IMS0001 or reference strain RWB219 inoculation shakes bottle, and described substratum contains 0.5% semi-lactosi.After 72 hours, under 3.0 660nm absorbancy, use culture to inoculate the new bottle that shakes, it contains 0.1% semi-lactosi and 2% pectinose.Based on measuring with the HPLC of D-ribulose as calibration criterion, measured in cultivating the first time of bacterial strain IMS0001, on the substratum that contains semi-lactosi/pectinose mixture, the part pectinose has been converted into ribulose and has secreted to supernatant liquor subsequently.Use Waters Alliance 2690 HPLC (Waters, Milford USA) carries out these HPLC and analyzes, and described equipment is equipped with BioRad HPX 87H post (BioRad, Hercules, USA), Waters2410 become optical index detector and Waters 2487UV detector.Use 0.5g l down at 60 ℃ ^-1Sulfuric acid with 0.6ml min ^-1Flow velocity wash-out post.RWB219 is opposite with reference strain, the OD of bacterial strain IMS0001 culture ₆₆₀After exhausting semi-lactosi, improve.When observing bacterial strain IMS0001 in pectinose growth after about 850 hours (Fig. 2), with this culture with 1.7 OD ₆₆₀Be transferred to and contain the shaking in the bottle of 2% pectinose.Then with the OD of culture with 2-3 ₆₆₀Sequencing is to the mailbox substratum that contains 2% pectinose, by measuring the utilization (data not shown) that arabinose concentrations confirms pectinose with HPLC discontinuously.The growth velocity of these cultures is increased to 0.15h from 0 in about 3600 hours ^-1(Fig. 3).

By with 1 liter of synthetic medium that is supplemented with 2% pectinose of 100ml shake-flask culture thing inoculation, begin the batch fermentation under the oxygen restricted condition, described shake-flask culture thing is the shake-flask culture thing of the IMS0001 cell of pectinose cultivation, it is with about 0.12h ^-1Maximum growth rate grow at the 2%of L-arabinose.When observing when pectinose is grown, by with nitrogen bubble to culture anaerobic condition in addition.Contain 20g l by manually or automatically 90% culture being replaced with ^-1The synthetic medium of pectinose, the sequential loop of beginning anaerobism batch culture.For each circulation between the SBR yeast phase, from CO ₂Spectrum estimation index growth velocity (Fig. 4).In 13 circulations, exponential growth speed is from 0.025h ^-1Be increased to 0.08h ^-1Take a sample after 20 circulations, be coated on the solid synthetic medium that is supplemented with 2% L-arabinose, and hatch a couple of days at 30 ℃.Rule again twice at the bacterium colony that contains on the solid synthetic medium of L-arabinose separating.At last, contain with single colony inoculation the 2%L-pectinose synthetic medium shake bottle, and hatched 5 days at 30 ℃.This culture is named as bacterial strain IMS0002 (CBS 120328, are preserved in Centraal Bureau voor Schimmelculturen (CBS) 27/09/06).Take culture samples, add 30% glycerine and sample is stored under-80 ℃.

The mixed culture fermentation

The biomass by hydrolyzation product is the raw material of industrial biotechnology expectation, and it contains the complex mixture of being made up of multiple sugar, and glucose, wood sugar and pectinose are present in the significant fraction usually in the described sugar.In order to finish the ethanol fermentation of glucose and pectinose and wood sugar, carry out the anaerobism batch fermentation with the mixed culture of pectinose fermentation strain IMS0002 and wood-sugar fermentation bacterial strain RWB218.Pre-culture with 100ml bacterial strain IMS0002 is inoculated the anaerobism batch fermentation jar that contains the 800ml synthetic medium, and described synthetic medium contains 30g l ^-1D-glucose, 15g l ^-1D-wood sugar and 15g l ^-1L-arabinose.Add 100ml RWB218 inoculum after 10 hours.With only to carry out mixing sugar fermentation with bacterial strain IMS0002 opposite, exhausting afterwards at glucose, wood sugar and pectinose all are consumed (Fig. 5 D).The mixed culture completely consumed all sugar, and in 80 hours, produced 564.0 ± 6.3mmol l with the high overall yield of 0.42g g-1 sugar ^-1Ethanol is (from CO ₂Produce and calculate).Xylitol only produces in a small amount, and concentration is 4.7mmol l ^-1

Sign to bacterial strain IMS0002

Growth and the product of measuring bacterial strain IMS0002 during anaerobism batch fermentation on the synthetic medium form, and described substratum contains L-arabinose as unique carbon source, or contains the mixture of glucose, wood sugar and L-arabinose.Containing the shaking in the bottle of synthetic medium that 100ml contains the 2%L-pectinose, by with-80 ℃ of freezing bacterial classification inoculations of bacterial strain IMS0002 and under 30 ℃, hatched 48 hours, for the preparation of the pre-culture of this anaerobism batch fermentation.

Fig. 5 A is presented at during about 70 hours anaerobism batch fermentation, and bacterial strain IMS0002 can be with 20g l ^-1The L-arabinose fermentation is ethanol.When using L-arabinose as sole carbon source, the specific growth rate under the anaerobic condition is 0.05 ± 0.001h ^-1Consider the ethanol evaporation during the batch fermentation, from 20g l ^-1The alcohol yied of pectinose is 0.43 ± 0.003g g ^-1Do not evaporate timing, alcohol yied is 0.35 ± 0.01g g ^-1Pectinose.During the anaerobic growth on the pectinose, do not observe the formation of arabitol.

In Fig. 5 B, showed the 20g l of bacterial strain IMS0002 ^-1Glucose and 20g l ^-1The ethanol fermentation of the mixture of L-arabinose.After exhausting, glucose begins to consume L-arabinose.In 70 hours, glucose and L-arabinose are all by completely consumed.Alcohol yied from total reducing sugar is 0.42 ± 0.003g g ^-1

In Fig. 5 C, showed the 30g l of bacterial strain IMS0002 ^-1Glucose, 15g l ^-1D-wood sugar and 15g ^-1The fermentation pattern of the mixture of L-arabinose.In 80 hours, glucose and pectinose are all by completely consumed.In the 100mM wood sugar only 20mM consumed by bacterial strain IMS0002.In addition, observe the formation of 20mM Xylitol.Significantly, wood sugar is converted into Xylitol by bacterial strain IMS0002.Therefore, the alcohol yied from total reducing sugar is lower than above-mentioned fermentation: 0.38 ± 0.001g g ^-1Alcohol yied from total glucose and pectinose is similar to other fermentation: 0.43 ± 0.001g g ^-1

Table 1 has shown pectinose wear rate and the alcohol production speed of observing at the anaerobism batch fermentation of bacterial strain IMS0002.Pectinose is with 0.23-0.75g h ^-1g ^-1The speed of biomass dry weight is consumed.Produce the speed of ethanol from 0.08 to 0.31g h from pectinose ^-1g ^-1The biomass dry weight changes.

At first, the bacterial strain IMS0001 of structure can xylose-fermenting (data not shown).Opposite with our expection, selected bacterial strain IMS0002 can not be ethanol (Fig. 5 C) with wood-sugar fermentation.In order to obtain the ability of xylose-fermenting again, the colony lift of bacterial strain IMS0002 to the solid synthetic medium that contains the 2%D-wood sugar, and was hatched under 30 ℃ 25 days in anaerobic jar.Subsequently again with colony lift to the solid synthetic medium that contains 2% pectinose.30 ℃ hatch 4 days after, to containing the shaking in the bottle of synthetic medium, described synthetic medium contains 2% pectinose with colony lift.30 ℃ hatch 6 days after, add 30% glycerine, take a sample and be stored under-80 ℃.Shake bottle also as containing 20g l with so freezing bacterial classification inoculation ^-1Wood sugar and 20g l ^-1The pre-culture of anaerobism batch fermentation on the synthetic medium of pectinose, the described bottle that shakes contains the synthetic medium that 100ml contains 2% pectinose.In Fig. 6, shown the fermentation pattern of this batch fermentation.Wood sugar and pectinose are consumed simultaneously.Pectinose is depleted in 70 hours, and wood sugar is depleted in 120 hours.From total reducing sugar, produce 250mM ethanol at least, do not counted the evaporation of ethanol.Suppose 3.2g l ^-1Final biomass dry weight (suppose 0.08g g ^-1The biomass yield of sugar), then by the CO that adds up ₂Produce (355mmol l ^-1) the final alcohol concn estimated is about 330mmol l ^-1, corresponding to 0.41g g ^-1The alcohol yied of pentose.Except ethanol, glycerine and organic acid, produced Xylitol (about 5mM) in a small amount.

The selection of bacterial strain IMS0003

At first, the bacterial strain IMS0001 of structure can xylose-fermenting (data not shown).Opposite with our expection, selected bacterial strain IMS0002 can not be ethanol (Fig. 5 C) with wood-sugar fermentation.In order to obtain the ability of xylose-fermenting again, the colony lift of bacterial strain IMS0002 to the solid synthetic medium that contains the 2%D-wood sugar, and was hatched under 30 ℃ 25 days in anaerobic jar.Subsequently again with colony lift to the solid synthetic medium that contains 2% pectinose.30 ℃ hatch 4 days after, to containing the shaking in the bottle of synthetic medium, described synthetic medium contains 2% pectinose with colony lift.30 ℃ hatch 6 days after, add 30% glycerine, take a sample and be stored under-80 ℃.

The sample that will come from this freezing bacterial classification is coated with at the solid synthetic medium that contains the 2%L-pectinose, and hatches a couple of days at 30 ℃.Rule again twice at the bacterium colony that contains on the solid synthetic medium of L-arabinose separating.At last, contain with single colony inoculation the 2%L-pectinose synthetic medium shake bottle, and hatched 4 days at 30 ℃.This culture is named as bacterial strain IMS0003 (CBS 121879, are preserved in CBS 20/09/07).Take culture samples, add 30% glycerine and sample is stored under-80 ℃.

Sign to bacterial strain IMS0003

Growth and the product of measuring bacterial strain IMS0003 during anaerobism batch fermentation on the synthetic medium form, and described substratum contains 30g l ^-1Glucose, 15g l ^-1D-wood sugar and 15g l ^-1The mixture of L-arabinose.Containing the shaking in the bottle of synthetic medium that 100ml contains the 2%L-pectinose, by with-80 ℃ of freezing bacterial classification inoculations of bacterial strain IMS0003 and under 30 ℃, hatched 48 hours, for the preparation of the pre-culture of this anaerobism batch fermentation.

In Fig. 7, shown the 30g l of bacterial strain IMS0003 ^-1Glucose, 15g l ^-1D-wood sugar and 15g l ^-1The fermentation pattern of L-arabinose mixture.After exhausting, glucose begins to consume pectinose.In 70 hours, glucose, wood sugar and pectinose are all by completely consumed.Wood sugar and pectinose are consumed simultaneously.From total reducing sugar, produce 406mM ethanol at least, do not count the evaporation of ethanol.CO from accumulative total ₂Producing the final alcohol concn that calculates is 572mmol l ^-1, corresponding to 0.46g g ^-1The alcohol yied of total reducing sugar.Opposite to the fermentation of glucose, wood sugar and pectinose mixture with the mixed culture (Fig. 5 D) of bacterial strain IMS0002 (Fig. 5 C) or bacterial strain IMS0002 and RWB218, but bacterial strain IMS0003 does not produce the Xylitol of detection limit.

Form

Table 1: the S.cerevisiae bacterial strain of use.

Table 2: the plasmid of use

Table 3: the oligomer that uses in this work

Table 4:

Observed maximum glucose and pectinose specific consumption rate and ethanol specific production rate during the anaerobism batch fermentation of S.cerevisiae IMS0002.

q _Glu: the glucose specific consumption rate

q _Ara: the pectinose specific consumption rate

q _{Eth, glu}: the ethanol specific production rate of growing period on glucose

q _{Eth, ara}: the ethanol specific production rate of growing period on pectinose

The reference tabulation

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).New yeast-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，Zimmermann 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 H.，H.Nojima and H.Okayama，High efficiency transformation of Escherichia coli with plasmids.Gene 96(1990)，pp.23-28

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

Kuyper M，Toirkens MJ，Diderich JA，Winkler AA，Van Dijken JP，Pronk JT(2005b) Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain.Fems 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，Pronk 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

Claims

1. can express the eukaryotic cell of following nucleotide sequence, wherein the expression of these nucleotide sequences is given that cell uses L-arabinose and/or L-arabinose is converted into L-ribulose and/or X 5P and/or is converted into the ability of desired fermentation product:

I. the encode nucleotide sequence of araA, described araA comprise the aminoacid sequence that the aminoacid sequence with SEQ ID NO:1 has at least 55% sequence identity,

Ii. nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO:2 has at least 60% sequence identity,

Iii. nucleotide sequence, its complementary strand with (i) or the making nucleic acid molecular hybridization of sequence (ii),

Iv. nucleotide sequence, its sequence is because the degeneracy of genetic codon and different with the sequence of (iii) nucleic acid molecule;

I. the encode nucleotide sequence of araB, described araB comprise the aminoacid sequence that the aminoacid sequence with SEQ ID NO:3 has at least 20% sequence identity,

Ii. nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO:4 has at least 50% sequence identity,

I. the encode nucleotide sequence of araD, described araD comprise the aminoacid sequence that the aminoacid sequence with SEQ ID NO:5 has at least 60% sequence identity,

Ii. nucleotide sequence, it comprises the nucleotide sequence that the nucleotide sequence with SEQ ID NO:6 has at least 60% sequence identity,

Iv. nucleotide sequence, its sequence is because the degeneracy of genetic codon and different with the sequence of (iii) nucleic acid molecule.

2. according to the cell of claim 1, in wherein said araA, araB and the araD nucleotide sequence one, two or three derive from the genus of Lactobacillus, preferably derive from the kind of Lactobacillus plantarum.

3. according to the cell of claim 1 or 2, wherein said cell is yeast cell, preferably belongs to one of subordinate: Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia.

4. according to the cell of claim 3, wherein said yeast cell belongs to one of to sow: S.cerevisiae, S.bulderi, S.barnetti, S.exiguus, S.uvarum, S.diastaticus, K.lactis, K.marxianus or K.fragilis.

5. according to each cell in the claim 1 to 4, the nucleotide sequence of araA, araB and/or araD of wherein encoding is operably connected with following promotor, described promotor causes that corresponding nucleotides sequence is listed in giving full expression in the described cell, thereby gives the ability that cell uses L-arabinose and/or L-arabinose is converted into L-ribulose and/or X 5P and/or desired fermentation product.

6. according to each cell in the claim 1 to 5, wherein said cell shows the ability that directly xylose isomerase is turned to xylulose.

7. according to the cell of claim 6, wherein said cell comprises genetic modification, and described genetic modification can improve the flux of pentose-phosphate pathway.

8. according to the cell of claim 6 or 7, wherein said genetic modification comprised at least one gene of expressing the non-oxide part of pentose-phosphate pathway.

9. cell according to Claim 8, wherein said gene is selected from the group of being made up of following: the gene of coding ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase.

10. cell according to Claim 8, wherein said genetic modification comprised crosses the gene of expressing coding transketolase and transaldolase at least.

11. each cell in 10 according to Claim 8, wherein this cell also comprises the genetic modification that improves the xylulokinase specific activity.

12. according to the cell of claim 11, wherein said genetic modification comprised the gene of expressing the coding xylulokinase.

13. each cell in 12 according to Claim 8, it is endogenous wherein being crossed the described cell of described gene pairs of expressing.

14. according to each cell in the claim 5 to 13, wherein said cell comprises the genetic modification that reduces non-specific aldose reductase activity in the described cell.

15. according to the cell of claim 14, wherein said genetic modification reduces the expression of the non-specific aldose reductase gene of coding, or makes described gene inactivation.

16. according to the cell of claim 15, wherein by at least part of of the described gene of disappearance or by destroying described gene, make this gene inactivation.

17. according to the cell of claim 14 or 15, the expression of each gene in described cell of the non-specific aldose reductase of wherein encoding is lowered or inactivation.

18. according to each cell in the aforementioned claim, wherein said tunning is selected from the group of being made up of following: ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1,3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics and cynnematin.

19. comprise the nucleic acid construct of following nucleotide sequence: the coding araA nucleotide sequence, the coding araB nucleotide sequence and/or the coding araD nucleotide sequence, all described nucleotide sequences all such as in claim 1 or 2 definition.

20. the technology for the production of the tunning that is selected from down group, described group by ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1,3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics and cynnematin are formed, and described technology comprises:

(a) come fermention medium with the modified cell that defines in each in the claim 1 to 18, described substratum contains pectinose source and optional wood sugar source, thereby described cell is tunning with pectinose and optional wood-sugar fermentation; Alternatively

(b) reclaim described tunning.

21. the technology for the production of the tunning that is selected from down group, described group by ethanol, lactic acid, 3-hydroxyl-propionic acid, vinylformic acid, acetic acid, succsinic acid, citric acid, oxysuccinic acid, fumaric acid, amino acid, 1,3-propane-glycol, ethene, glycerine, butanols, beta-lactam antibiotics and cynnematin are formed, and described technology comprises:

(a) with in the claim 1 to 18 in each definition cell and can use wood sugar and/or show that the cell that the direct isomery of wood sugar is turned to the ability of xylulose comes fermention medium, described substratum contains pectinose source and wood sugar source at least, thereby every kind of cell can both be tunning with pectinose and/or wood-sugar fermentation; Alternatively

(b) reclaim described tunning.

22. according to the technology of claim 20 or 21, wherein said substratum also contains the glucose source.

23. according to each technology in the claim 20 to 22, wherein said tunning is ethanol.

24. according to the technology of claim 23, wherein said ethanol volume production power is every liter of 0.5g ethanol at least per hour.

25. according to the technology of claim 23 or 24, wherein said alcohol yied is at least 30%.

26. according to each technology in the claim 20 to 25, wherein said technology is anaerobism.

27. according to each technology in the claim 20 to 25, wherein said technology is aerobic, preferably carries out under the limited condition of oxygen.