# Patent

## Title
Yeast mutant of kluyveromyces and method for ethanol production using the same

## Abstract

A yeast of the genus Kluyveromyces is modified to improve the ethanol yield from xylose by attenuation of at least one gene selected from the group consisting of the ADH1 gene derived from Kluyveromyces marxianus, a gene functionally equivalent to the ADH1 gene, the ADH4 gene derived from Kluyveromyces marxianus, and a gene functionally equivalent to the ADH4 gene.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/JP2012/057704 filed Mar. 26, 2013, claiming priority based on Japanese Patent Application No. 2011-076715, filed Mar. 30, 2011, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a mutant yeast of the genus Kluyveromyces and a method for ethanol production using the same.
BACKGROUND ART
Lignocellulose-containing biomass is an effective starting material for a useful alcohol, such as ethanol, or an organic acid. Lignocellulose-containing biomass includes woody biomass and herbaceous biomass. Lignocellulose-containing biomass, such as woody biomass, is mainly composed of cellulose, hemicellulose, and lignin. In order to produce a liquid fuel such as ethanol from lignocellulose-containing biomass, cellulose or hemicellulose is hydrolyzed (saccharified) into a constitutive monosaccharide, and the monosaccharide is converted into ethanol via fermentation. Cellulose is composed of glucose, and hemicellulose is mainly composed of arabinose and xylose. When producing ethanol with the use of lignocellulose-containing biomass, accordingly, it is preferable that xylose be effectively used as a fermentation substrate, in addition to glucose.
When producing ethanol from lignocellulose-containing biomass, production costs can be reduced if the saccharification process can be simultaneously carried out with the fermentation process without separating these processes from each other. Such technique is referred to as “the simultaneous saccharification and fermentation process.” The simultaneous saccharification and fermentation process requires the use of thermotolerant microorganisms capable of fermentation in the reaction temperature range for a carbohydrase (i.e., about 40 degrees C. or higher) and capable of using the xylose (pentose) as a substrate, in addition to glucose.
Examples of known thermotolerant yeasts include yeasts of the genus Kluyveromyces, such as Kluyveromyces marxianus. While a yeast of the genus Kluyveromyces is capable of ethanol fermentation with the utilization of xylose, the yield thereof has not been sufficient. For example, the results of functional analyses of the alcohol dehydrogenase gene of Saccharomyces cerevisiae (including a plurality of isomers) are reported in Non-Patent Documents 1 and 2. Patent Document 1 discloses a recombinant yeast strain capable of isomerizing xylose into xylulose, in which alcohol dehydrogenase activity has been reduced. Based on such finding, however, the influence of the deficiency or destruction of the alcohol dehydrogenase gene on the capacity for ethanol fermentation cannot be determined. In addition, functions of the alcohol dehydrogenase gene of a taxonomically different species (i.e., a yeast of the genus Kluyveromyces such as Kluyveromyces marxianus) cannot be evaluated.
PRIOR ART DOCUMENTS
Patent Document


    Patent Document 1: JP 2005-514951 A


Non-Patent Documents


    Non-Patent Document 1: FEMS Yeast Research 2, 2002, pp. 481-494
    Non-Patent Document 2: FEMS Yeast Research 8, 2008, pp. 967-978


SUMMARY OF THE INVENTION
Object to be Attained by the Invention
As described above, a yeast of the genus Kluyveromyces is capable of assimilating xylose and is thermotolerant, and there are high expectations that such microorganisms will be useful for the simultaneous saccharification and fermentation process. Concerning yeasts of the genus Kluyveromyces, however, the ethanol yield from xylose is very poor, and no means for improving such poor yield is known. Under the circumstances as described above, it is an object of the present invention to provide a mutant yeast of the genus Kluyveromyces modified so as to improve the ethanol yield from xylose and a method for ethanol production using such mutant yeast.
Means for Attaining the Object
The present inventors have conducted concentrated studies in order to attain the above object. As a result, they discovered that the ethanol yield from xylose of a yeast of the genus Kluyveromyces would be remarkably improved by attenuation of a particular gene(s) among a plurality of the alcohol dehydrogenase genes of the yeast, thereby completing the present invention. Specifically, the present invention includes the following features.
(1) A mutant yeast of the genus Kluyveromyces obtained by attenuation of at least one gene selected from the group consisting of the ADH1 gene derived from Kluyveromyces marxianus, a gene functionally equivalent to the ADH1 gene, the ADH4 gene derived from Kluyveromyces marxianus, and a gene functionally equivalent to the ADH4 gene.
(2) The mutant yeast according to (1), wherein the yeast of the genus Kluyveromyces is Kluyveromyces marxianus. 
(3) The mutant yeast according to (1), wherein the gene functionally equivalent to the ADH1 gene is derived from a yeast of the genus Kluyveromyces other than Kluyveromyces marxianus and encodes any of the proteins (a) to (c) below:
(a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2;
(b) a protein comprising an amino acid sequence having 90% or higher similarity to the amino acid sequence as shown in SEQ ID NO: 2 and having alcohol dehydrogenase activity; and
(c) a protein comprising an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by deletion, substitution, addition, or insertion of 1 or a plurality of amino acids and having alcohol dehydrogenase activity.
(4) The mutant yeast according to (1), wherein the gene functionally equivalent to the ADH4 gene is derived from a yeast of the genus Kluyveromyces other than Kluyveromyces marxianus and encodes any of the proteins (a) to (c) below:
(a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 4;
(b) a protein comprising an amino acid sequence having 90% or higher similarity to the amino acid sequence as shown in SEQ ID NO: 4 and having alcohol dehydrogenase activity; and
(c) a protein comprising an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 4 by deletion, substitution, addition, or insertion of 1 or a plurality of amino acids and having alcohol dehydrogenase activity.
(5) A method for ethanol production comprising: a step of culturing of the mutant yeast according to any of claims 1 to 4 in a xylose-containing medium; and a subsequent step of recovering of ethanol from the medium.
(6) The method for ethanol production according to (5), wherein the step of culturing is carried out in a reaction system containing the mutant yeast, lignocellulose-containing biomass, and a carbohydrase.
This description includes part or all of the content as disclosed in the description and/or drawings of Japanese Patent Application No. 2011-076715, which is a priority document of the present application.
Effects of the Invention
While the ethanol yield from glucose with the use of the mutant yeast according to the present invention is not substantially changed, the ethanol yield from xylose is significantly improved. Accordingly, the mutant yeast according to the present invention is capable of high-yield ethanol production in, for example, a medium containing xylose derived from lignocellulose-containing biomass such as woody biomass.
The method for ethanol production according to the present invention involves the use of a mutant yeast with significantly improved ethanol yield from xylose. Thus, the efficiency of ethanol production can be improved at significant levels.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a characteristic diagram showing changes in xylose concentration in a xylose-containing medium when four types of ADF-deficient strains are cultured therein.
FIG. 2 is a characteristic diagram showing changes in ethanol concentration in a xylose-containing medium when four types of ADF-deficient strains are cultured therein.
FIG. 3 is a characteristic diagram showing changes in concentration of yeast when four types of ADF-deficient strains are cultured in a xylose-containing medium.
FIG. 4 is a characteristic diagram showing changes in xylitol concentration in a glucose-containing medium when four types of ADF-deficient strains are cultured therein.
FIG. 5 is a characteristic diagram showing changes in ethanol concentration in a glucose-containing medium when four types of ADF-deficient strains are cultured therein.
FIG. 6 is a characteristic diagram showing changes in concentration of yeast when four types of ADF-deficient strains are cultured in a glucose-containing medium.
FIG. 7 shows the alignment of ADH1 derived from S. cerevisiae with ADH1 derived from K. marxianus. 
FIG. 8 shows the alignment of ADH1 derived from S. cerevisiae with ADH2 derived from K. marxianus. 
FIG. 9 shows the alignment of ADH1 derived from S. cerevisiae with ADH3 derived from K. marxianus. 
FIG. 10 shows the alignment of ADH1 derived from S. cerevisiae with ADH4 derived from K. marxianus. 
FIG. 11 shows the alignment of ADH2 derived from S. cerevisiae with ADH1 derived from K. marxianus. 
FIG. 12 shows the alignment of ADH2 derived from S. cerevisiae with ADH2 derived from K. marxianus. 
FIG. 13 shows the alignment of ADH2 derived from S. cerevisiae with ADH3 derived from K. marxianus. 
FIG. 14 shows the alignment of ADH2 derived from S. cerevisiae with ADH4 derived from K. marxianus. 
FIG. 15 shows the alignment of ADH1 derived from S. cerevisiae with ADH2 derived from S. cerevisiae. 



EMBODIMENTS FOR CARRYING OUT THE INVENTION
The mutant yeast according to the present invention is derived from yeast of the genus Kluyveromyces by attenuation of a particular alcohol dehydrogenase gene(s), and the ethanol yield thereof from xylose is improved.
Examples of yeasts of the genus Kluyveromyces include yeasts such as K. aestuarii, K. africanus, K. bacillisporus, K. blattae, K. dobzhanskii, K. hubeiensis, K. lactis, K. lodderae, K. marxianus, K. nonfermentans, K. piceae, K. sinensis, K. thermotolerans, K. waltii, K. wickerhamii, and K. yarrowii. Specifically, the mutant yeast according to the present invention can be prepared by attenuation of a particular alcohol dehydrogenase gene of such yeast of the genus Kluyveromyces or a mutant thereof. Use of Kluyveromyces marxianus, which is known to be a thermotolerant yeast, as a yeast of the genus Kluyveromyces is particularly preferable. Kluyveromyces marxianus is not particularly limited. Known strains conserved in a distributable form at depository institutions or mutant strains derived from known strains can be used. An example of a known Kluyveromyces marxianus strain is the Kluyveromyces marxianus DMKU3-1042 strain. Examples of mutant strains derived from known strains include those derived from the Kluyveromyces marxianus DMKU3-1042 strain via destruction of the ura3 or leu2 gene for the purpose of impartation of auxotrophy.
The mutant yeast according to the present invention is obtained by attenuation of a particular alcohol dehydrogenase gene(s). The term “attenuation of gene” refers to reduction of the expression level of such gene and reduction of activity of an enzyme encoded by such gene. The gene expression level can be reduced by, for example, a method comprising destruction or deletion of a particular alcohol dehydrogenase gene, a method comprising destruction or deletion of an expression control region (e.g., a promoter) of such gene, or a method comprising expression of antisense RNA of such gene. Alternatively, the gene expression level can be reduced by, for example, the transposon method, the transgene method, the post-transcriptional gene silencing method, the RNAi method, the nonsense mediated decay (NMD) method, the ribozyme method, the antisense method, the micro-RNA (miRNA) method, or the small interfering RNA (siRNA) method. In addition, an inhibitor of alcohol dehydrogenase may be allowed to react with the gene, so as to reduce activity of an enzyme encoded by such gene. A particular alcohol dehydrogenase gene may be attenuated by performing any such methods in combination.
The mutant yeast according to the present invention has an improved ethanol yield from xylose. In other words, the capacity of the mutant yeast according to the present invention for xylose metabolism is improved. The term “capacity for xylose metabolism” used herein refers to the efficiency for fermentation (i.e., metabolization of xylose contained in a medium) that converts the substance into an alcohol. Thus, an improvement in the capacity for xylose metabolism is synonymous with the improvement in the reaction efficiency of such fermentation. The capacity of a yeast for xylose metabolism can be evaluated by conducting culture in a xylose-containing medium and quantifying the alcohol produced. Also, the capacity of a yeast for xylose metabolism can be evaluated using the uptake rate of xylose contained in a medium (i.e., the consumption rate) as the indicator. The uptake rate of xylose can be determined by serial measurement of the amount of decrease of xylose, the concentration of which at the initiation of culture is known.
The alcohol dehydrogenase gene to be attenuated is a particular alcohol dehydrogenase gene(s) among a plurality of alcohol dehydrogenase genes existing in a yeast of the genus Kluyveromyces. Specifically, four types of alcohol dehydrogenase genes of Kluyveromyces marxianus (i.e., KmADH1 to KmADH4) are known. The alcohol dehydrogenase genes of Kluyveromyces marxianus to be attenuated are the Km ADH1 gene and/or the Km ADH4 gene. The alcohol dehydrogenase genes of yeasts of the genus Kluyveromyces other than Kluyveromyces marxianus to be attenuated are the ADH gene functionally equivalent to the Km ADH1 gene and/or the ADH gene functionally equivalent to the Km ADH4 gene.
In the yeast of the genus Kluyveromyces other than Kluyveromyces marxianus, the ADH gene functionally equivalent to the Km ADH1 gene and the ADH gene functionally equivalent to the Km ADH4 gene can be identified by a conventional technique. For example, a plurality of alcohol dehydrogenase genes are first identified in yeasts of the genus Kluyveromyces other than Kluyveromyces marxianus. From among alcohol dehydrogenases encoded by such genes, subsequently, alcohol dehydrogenase comprising an amino acid sequence exhibiting the highest sequence similarity to the amino acid sequence of the alcohol dehydrogenase encoded by the Km ADH1 gene is identified. The alcohol dehydrogenase gene thus identified can be determined to be a gene functionally equivalent to the Km ADH1 gene of Kluyveromyces marxianus. A gene functionally equivalent to the Km ADH4 gene can be identified in the same manner. Sequence similarity is determined using a computer equipped with the BLAST (Basic Local Alignment Search Tool) Program, using the default settings.
The nucleotide sequence of the coding region of the Km ADH1 gene and the amino acid sequence of alcohol dehydrogenase encoded by the Km ADH1 gene are shown in SEQ ID NOs: 1 and 2, respectively. The nucleotide sequence of the coding region of the Km ADH4 gene and the amino acid sequence of alcohol dehydrogenase encoded by the Km ADH4 gene are shown in SEQ ID NOs: 3 and 4, respectively.
The Km ADH1 gene and the Km ADH4 gene existing in Kluyveromyces marxianus are not limited to those specifically described above. That is, the Km ADH1 gene and the Km ADH4 gene may be those encoding proteins comprising amino acid sequences exhibiting 80% or higher, preferably 85% or higher, more preferably 90% or higher, further preferably 95% or higher, and most preferably 98% or higher similarity to the amino acid sequences as shown in SEQ ID NOs: 2 and 4, respectively, provided that such genes encode proteins having alcohol dehydrogenase activity. Sequence similarity is determined using a computer equipped with the BLAST (Basic Local Alignment Search Tool) Program, using the default settings.
The Km ADH1 gene and the Km ADH4 gene may be those encoding proteins comprising amino acid sequences derived from the amino acid sequences as shown in SEQ ID NOs: 2 and 4, respectively, by deletion, substitution, addition, or insertion of one or a plurality of (e.g., 2 to 35, preferably 2 to 30, more preferably 2 to 20, and further preferably 2 to 10) amino acids, provided that such genes encode proteins having alcohol dehydrogenase activity.
The Km ADH1 gene and the Km ADH4 gene may be polynucleotides hybridizing under stringent conditions to partial or full-length polynucleotides comprising nucleotide sequences complementary to the nucleotide sequences as shown in SEQ ID NOs: 1 and 3, respectively, provided that such genes encode proteins having alcohol dehydrogenase activity. Under stringent temperature and salt concentration conditions, a pair of polynucleotides having about 90%, preferably 95%, and more preferably 98% identity with each other form specific hybrids.
<Ethanol Production>
With the use of the mutant yeasts described above, ethanol fermentation can be carried out using a sugar such as xylose as a substrate. Since the mutant yeasts according to the present invention described above are excellent in terms of their capacity for xylose metabolism (i.e., the ethanol yield from xylose), such strains are particularly preferable for ethanol fermentation involving the use of a xylose-containing medium. In a xylose-containing medium, a yeast of the genus Kluyveromyces can grow, and it at least contains xylose as a sugar component that functions as a substrate for ethanol synthesis. A xylose-containing medium may contain a sugar component other than xylose, such as glucose.
A xylose-containing medium is not particularly limited, and it can be prepared by adding xylose to various types of synthetic media, including SD, YPD, YPAD, YM, and Yeast Nitrogen Base media. Alternatively, a sugar component contained in such known medium may be replaced with xylose, so as to prepare a xylose-containing medium.
Alternatively, a xylose-containing medium may be prepared from lignocellulose-containing biomass such as woody biomass or herbaceous biomass. Specifically, cellulose or hemicellulose contained in lignocellulose-containing biomass may be saccharified, and the resultant may then be used as a xylose-containing medium. Saccharification may be carried out via any conventional means without particular limitation. For example, saccharification may be carried out by a sulfuric acid method involving the use of dilute or concentrated sulfuric acid or by an enzymatic method involving the use of cellulase or hemicellulose. Woody biomass or herbaceous biomass may be subjected to conventional pre-treatment prior to saccharification. Such pre-treatment is not particularly limited, and examples thereof include degradation of lignin with a microorganism, grinding of woody biomass or herbaceous biomass, relaxation of the lignin structure via soaking in an ionic or alkaline solution, hydrothermal treatment via steaming in hot water, and ammonia treatment.
In particular, a mutant yeast prepared from Kluyveromyces marxianus is excellent in terms of thermotolerance, and ethanol fermentation can thus be performed at a relatively high temperature, such as 40 degrees C. or higher, preferably 35 degrees C. to 48 degrees C., and more preferably 40 degrees C. to 42 degrees C. In such a temperature range, carbohydrases such as cellulase and hemicellulose exhibit activity. Therefore, such mutant yeast is preferable for the so-called simultaneous saccharification and fermentation process involving the use of a carbohydrase. The simultaneous saccharification and fermentation process comprises saccharification of woody biomass with carbohydrase and ethanol fermentation from xylose carried out in the same reaction system. More specifically, a solution containing woody biomass, carbohydrase, and a mutant yeast is incubated at, for example, 40 degrees C. Thus, saccharification of woody biomass and fermentation of ethanol from xylose or glucose resulting from saccharification proceed, and ethanol can then be produced. In this process, the solution may be subjected to agitation or shaking.
When recovering ethanol produced via fermentation from a carbon source such as xylose contained in a medium, any conventional technique can be employed without particular limitation. After the completion of ethanol fermentation described above, for example, a liquid phase containing ethanol is separated from a solid phase containing a mutant yeast and a solid component via solid-liquid separation. Thereafter, ethanol contained in the liquid phase is separated and purified via distillation, and high-purity ethanol can be thus recovered. The degree of ethanol purification can adequately be adjusted in accordance with the application of ethanol.
EXAMPLES
Hereafter, the present invention is described in greater detail with reference to the examples below, although the technical scope of the present invention is not limited to these examples.
Example 1
In this example, Kluyveromyces marxianus was used as a yeast of the genus Kluyveromyces to prepare alcohol dehydrogenase gene-deficient strains, and the ethanol yield from xylose was examined.
<Preparation of Strains>
<Preparation of ura3-leu2-Mutant Via Conjugation and Sporulation>
The RAK3605 strain, a ura3-strain of the Kluyveromyces marxianus DMKU3-1042 strain (Nonklang, S. et al., Appl. Environ. Microbiol. 74, pp. 7514-7521, 2008), was used as the type strain. An auxotrophic mutant derived from the K. marxianus DMKU3-1042 strain was demonstrated to become a diploid at low frequency. While a multiple auxotrophic mutant can be obtained via mutation by ultraviolet radiation, the probability of introduction of a mutation into chromosome DNA is consequently increased. In order to prepare a more stable strain, the strains described below were prepared via conjugation and sporulation for easy production of a multiple auxotrophic mutant from a diploid.
At the outset, the RAK3605 was irradiated with ultraviolet rays to obtain the lys-strain (the RAK3896 strain: ura3-lys2-), the ade-strain (the RAK3919 strain: ura3-ade2-), and the leu-strain (the RAK3966 strain: ura3-leu2-). URA3 of Saccharomyces cerevisiae was transformed into the chromosomes of such strains at random to obtain the RAK4088 strain (ura3-leu2-ScURA3), the RAK4152 strain (ura3-ade2-ScURA3), and the RAK4153 strain (ura3-lys2-ScURA3). The RAK4152 strain and the RAK4153 strain were streaked onto YPD medium (1% w/v yeast extract, 2% w/v peptone, 2% glucose, and 2% w/v agar) to be mixed with each other thereon, and the resultants were replicated in MM medium (0.17% w/v yeast nitrogen base w/o amino acids and ammonium sulfate, 0.5% w/v ammonium sulfate, 2% w/v glucose, and 2% w/v agar). The RAK4154 strains grown in MM medium (ura3-/ura3-ade2-/ADE2 and lys2-/LYS2 ScURA3/ScURA3) were inoculated into SPO medium used for S. cerevisiae (1% w/v potassium acetate, 0.1% w/v yeast extract, and 0.05% w/v glucose) for sporulation.
The obtained spores were separated and replicated in −A medium (MM+ uracil, tryptophan, histidine HCl, methionine, leucine, lysine HCl), −K medium (MM+ uracil, tryptophan, histidine HCl, methionine, leucine, adenine hemisulfate), −U medium (MM+ tryptophan, histidine HCl, methionine, leucine, lysine HCl, adenine hemisulfate) in order to obtain triple autotrophic strains (i.e., ura-, lys-, ade-). Three strains that could not grow in three media were obtained from the spores of the RAK4154 strain and these strains were designated to be the RAK4155 strains (ura3-, lys2-, ade2-). The RAK4088 strain was conjugated to the RAK4155 strain in the same manner to prepare the RAK4156 strain (ura3-/ura3-lys2-/LYS2 ade2-/ADE2 leu2-/LEU2 ScURA3/ScURA3). The resulting strain was subjected to sporulation to prepare the RAK4174 strain (leu2-ura3-).
<Preparation of Ku70-Deficient Strain>
K. marxianus causes non-homologous end-joining repair with high frequency. Accordingly, gene destruction cannot be easily achieved via homologous recombination repair as in the case of S. cerevisiae (Nonklang, S. et al., Appl. Environ. Microbiol. 74, pp. 7514-7521, 2008). Accordingly, a strain causing homologous recombination with high frequency was prepared by destroying the KU70 gene necessary for non-homologous end-joining repair. The RAK4736 strain (leu2-ura3-Kmku70Δ::ScLEU2) was prepared from the RAK4174 strain via destruction of KU70 (Abdel-Banat, B. M. et al., Yeast 27, 29-39, 2010).
<Preparation of ADH1-Deficient Strain>
A putative open reading frame of the ADH1 gene of the K. marxianus DMKU3-1042 strain was identified with the aid of Genetyx ver.10 (Genetyx Corporation). The ADH1 gene of the K. marxianus DMKU3-1042 strain was found to encode a protein comprising 348 amino acids. The pair of primers shown below were designed based on the nucleotide sequence information.








KmADH1-167-ASC:
(SEQ ID NO: 5)







5′-GGGGGCACTTCGAACGCTGAAGTATCTTCATCTGGAGTATACCTTTT


 


TTTCGCCACTGGAggcgcgcccggg-3′


 








KmADH1 + 1070c-TDHu:
(SEQ ID NO: 6)







5′-TACCATATCAAAAGGGTCCTTGCTTATTTGGAAGTGTCAACGACAAT


 


TCTACCAATGATTtggcagtattgataatgag-3′






In the nucleotide sequences of the primers shown above, uppercase letters represent regions homologous to ADH1. ScURA3 was synthesized from the pST106 vector (Ano, A. et al., Biosci. Biotechnol. Biochem., 73, pp. 633-640, 2009) with the use of the above pair of primers and transformed into the RAK4736 strain. As a result, the RAK6148 strain (ura3-leu2-ku70Δ::ScLEU2 adh1Δ::ScURA3) was obtained.
<Preparation of ADH4-Deficient Strain>
A putative open reading frame of the ADH4 gene of the K. marxianus DMKU3-1042 strain was identified with the aid of Genetyx ver.10 (Genetyx Corporation). The ADH4 gene of the K. marxianus DMKU3-1042 strain was found to encode a protein comprising 379 amino acids. The pair of primers shown below were designed based on the nucleotide sequence information.








KmADH4-60-ASC:
(SEQ ID NO: 7)







5′-CGTACACCCTCAAGCTCATCGCCCGTACACCCACATTATACTATTAA


 


TAAACCACAAACAggcgcgcccggg-3′


 








KmADH4 + 1206c:
(SEQ ID NO: 8)







5′-GAAGGATCATCCAAATGAAAAGAAAGGGACGTTAAGTTAGCATAGCT


 


TAGTTGGACTGAGtggcagtattgataatgag-3′






In the nucleotide sequences of the primers shown above, uppercase letters represent regions homologous to ADH4. ScURA3 was synthesized from the pST106 vector with the use of the above pair of primers and transformed into the RAK4736 strain. As a result, the RAK6150 strain (ura3-leu2-ku70Δ::ScLEU2 adh4Δ::ScURA3) was prepared.
<Preparation of ADH2-Deficient Strain>
A putative open reading frame of the ADH2 gene of the K. marxianus DMKU3-1042 strain was identified with the aid of Genetyx ver.10 (Genetyx Corporation). The ADH2 gene of the K. marxianus DMKU3-1042 strain was found to encode a protein comprising 348 amino acids, as with the case of the ADH1 gene. The pair of primers shown below were designed based on the nucleotide sequence information.










KmADH2-764:
(SEQ ID NO: 9)









5′-CCCACCCACCCACTGCTACA-3′



 










KmADH2-1c:
(SEQ ID NO: 10)









5′-catttctagttgttggttgttgttt-3′






The upstream region of the ADH2 open reading frame was synthesized using the pair of primers and the DMKU3-1042 genome of K. marxianus as a template.
Also, the pair of primers shown below were designed based on the nucleotide sequence of the ADH2 gene.










KmADH2 + 1045:
(SEQ ID NO: 11)









5′-GCGGACTAACTAGCCCATTAGT-3′



 










KmADH2-2141c:
(SEQ ID NO: 12)









5′-CCCCACGCACAACGTAAACCTT-3′






The downstream region of the ADH2 open reading frame was synthesized using the pair of primers and the DMKU3-1042 genome of K. marxianus as a template.
Separately, the ScURA3 gene was synthesized from S. cerevisiae BY4704 (MATa ade2Δ::hisG his3Δ200 leu2Δ0 met15Δ0 trp1Δ63) in accordance with a conventional technique.
The thus-obtained ScURA3 gene was fused to a position between the ADH2 upstream region and the ADH2 downstream region for transformation into the RAK3605 strain. As a result, the RAK6396 strain (ura3-adh2Δ::ScURA3) was obtained.
<Preparation of ADH3-Deficient Strain>
A putative open reading frame of the ADH3 gene of the K. marxianus DMKU3-1042 strain was identified with the aid of Genetyx ver.10 (Genetyx Corporation). The ADH3 gene of the K. marxianus DMKU3-1042 strain was found to encode a protein comprising 375 amino acids. The pair of primers shown below were designed based on the nucleotide sequence thereof.










KmADH3-842:
(SEQ ID NO: 13)









5′-GGCCTGGGTTACCACTGGTCCCCTG-3′



 










KmADH3-1c:
(SEQ ID NO: 14)









5′-tgttgcgtgatattttctgtgcctg-3′






The upstream region of the ADH3 open reading frame was synthesized using the pair of primers and the DMKU3-1042 genome of K. marxianus as a template.
Also, the pair of primers shown below were designed based on the nucleotide sequence of the ADH3 gene.










KmADH3 + 1076:
(SEQ ID NO: 15)









5′-TGGAACAAGGTAAGATCTTGGG-3′



 










KmADH3 + 2069c:
(SEQ ID NO: 16)









5′-TTGCAGGATCCAGAATGGGTCAGTG-3′






The downstream region of the ADH3 open reading frame was synthesized using the pair of primers and the DMKU3-1042 genome of K. marxianus as a template.
As with the case of the ADH2-deficient strain, ScURA3 was fused to a position between the ADH3 upstream region and the ADH3 downstream region for transformation into the RAK3605 strain. As a result, the RAK6398 strain (ura3-adh3Δ::ScURA3) was obtained.
<Ethanol Fermentation Test>
The ADH1-deficient strain, the ADH2-deficient strain, the ADH3-deficient strain, and the ADH4-deficient strain prepared in the manner described above were subjected to an ethanol fermentation test using glucose and an ethanol fermentation test using xylose.
<Pre-Culture>
A platinum loopful of the deficient strains described above was introduced into a 50-ml assist tube supplemented with 20 ml of YPX (20 g/l xylose) medium, and shake culture was carried out at 30 degrees C. and 140 rpm for 6 to 8 hours. Subsequently, a 2.5% yeast solution was subjected to shake culture in a 500-ml triangular flask supplemented with 200 ml of YPX (20 g/l xylose) medium at 30 degrees C. and 140 rpm overnight. The cultured yeast strains were centrifuged and washed three times with sterile water to prepare a yeast suspension (OD 600 of 30).
<Main Culture>
Culture was conducted under the conditions shown in Table 1, and sugar consumption, growth of yeast strains, and ethanol production were examined. Culture was conducted with an initial glucose concentration of 50 g/l and with an initial xylose concentration of 20 g/l, respectively.







TABLE 1







Conditions of experiments













Initial

Number of


Liquid amount
Container
O.D.
Temperature
revolutions





25 ml
50-ml assist tube
1
30 degrees C.
140 rpm









Growth of yeast strains was evaluated by measuring the yeast density using a spectrophotometer (UV-1800, λ=600, Shimadzu Corporation). The amount of sugar and the concentration of ethanol in the medium were measured using the HPLC RI detector (Shimadzu Corporation).
<Results>
FIG. 1 to FIG. 3 show “changes in xylose concentration in a medium,” “changes in ethanol concentration in a medium,” “changes in xylitol concentration in a medium,” and “changes in concentration of yeasts” from when the deficient strains were cultured in a medium containing xylose as a sugar component. In comparison with a wild-type strain, all the ADH-deficient strains showed lower rates of xylose consumption, as shown in FIG. 1. The significantly lower rate of xylose consumption was observed particularly in the ADH2-deficient strain and the ADH3-deficient strain. As shown in FIG. 2, the ADH1-deficient strain and the ADH4-deficient strain exhibited the ethanol yield from xylose, which was significantly improved over that of a wild-type strain. However, no improvement was observed in the ethanol yield attained with the ADH2-deficient strain or the ADH3-deficient strain. As shown in FIG. 3, the capacity of the ADH1-deficient strain and the ADH4-deficient strain for growth of yeast strains was found to be equivalent to that of a wild-type strain. In comparison with a wild-type strain, however, the capacity of the ADH2-deficient strain and the ADH3-deficient strain for growth of yeast strains was found to be poorer.
FIG. 4 to FIG. 6 show “changes in glucose concentration in a medium,” “changes in ethanol concentration in a medium,” “changes in glycerol concentration in a medium,” and “changes in concentration of yeasts” when the deficient strains were cultured in a medium containing glucose as a sugar component. As shown in FIG. 4 to FIG. 6, the ethanol yield attained by culturing the deficient strains using glucose was equivalent to or lower than that attained with a wild-type strain. That is, destruction of the ADH genes was found to have no influence on glucose intake, ethanol fermentation from glucose, ethanol productivity in ethanol fermentation utilizing glucose, or other activity. In the case of yeasts of the genus Kluyveromyces, however, attenuation of the ADH1 gene and the ADH4 gene of was found to specifically improve the ethanol yield from xylose.
FIGS. 7 to 14 show the results of examination of homology at amino acid levels between ADH1 or ADH2 derived from S. cerevisiae and each of ADH1 to ADH4 derived from K. marxianus for reference purposes. FIG. 7 shows the alignment of ADH1 derived from S. cerevisiae with ADH1 derived from K. marxianus. FIG. 8 shows the alignment of ADH1 derived from S. cerevisiae with ADH2 derived from K. marxianus. FIG. 9 shows the alignment of ADH1 derived from S. cerevisiae with ADH3 derived from K. marxianus. FIG. 10 shows the alignment of ADH1 derived from S. cerevisiae with ADH4 derived from K. marxianus. FIG. 11 shows the alignment of ADH2 derived from S. cerevisiae with ADH1 derived from K. marxianus. FIG. 12 shows the alignment of ADH2 derived from S. cerevisiae with ADH2 derived from K. marxianus. FIG. 13 shows the alignment of ADH2 derived from S. cerevisiae with ADH3 derived from K. marxianus. FIG. 14 shows the alignment of ADH2 derived from S. cerevisiae with ADH4 derived from K. marxianus. 
As shown in FIG. 7, the identity and the similarity between ADH1 derived from S. cerevisiae and ADH1 derived from K. marxianus were 79% and 97%, respectively. As shown in FIG. 8, the identity and the similarity between ADH1 derived from S. cerevisiae and ADH2 derived from K. marxianus were 86% and 96%, respectively. As shown in FIG. 9, the identity and the similarity between ADH1 derived from S. cerevisiae and ADH3 derived from K. marxianus were 79% and 96%, respectively. As shown in FIG. 10, the identity and the similarity between ADH1 derived from S. cerevisiae and ADH4 derived from K. marxianus were 80% and 94%, respectively. As shown in FIG. 11, the identity and the similarity between ADH2 derived from S. cerevisiae and ADH1 derived from K. marxianus were 79% and 97%, respectively. As shown in FIG. 12, the identity and the similarity between ADH2 derived from S. cerevisiae and ADH2 derived from K. marxianus were 84% and 96%, respectively. As shown in FIG. 13, the identity and the similarity between ADH2 derived from S. cerevisiae and ADH3 derived from K. marxianus were 78% and 96%, respectively. As shown in FIG. 14, the identity and the similarity between ADH2 derived from S. cerevisiae and ADH4 derived from K. marxianus were 80% and 95%, respectively.
FIG. 15 shows the alignment of ADH1 derived from S. cerevisiae with ADH2 derived from S. cerevisiae. As shown in FIG. 15, the identity and the similarity between ADH1 derived from S. cerevisiae and ADH2 derived from S. cerevisiae were 93% and 99%, respectively.
As shown in FIGS. 7 to 14, the identity and the similarity between each of ADH1 to ADH4 derived from K. marxianus and ADH1 or ADH2 derived from S. cerevisiae are very high, and it is difficult to deduce the functions of ADH1 to ADH4 derived from K. marxianus from comparison with ADH1 and ADH2 derived from S. cerevisiae. Therefore, the outcome to the effect that the ethanol yield from xylose can be specifically improved by attenuation of the ADH1 or ADH4 gene of a yeast of the genus Kluyveromyces can be astonishing.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

## Claims

1. A mutant of Kluyveromyces marxianus obtained by attenuation of an ADH1 gene and/or an ADH4 gene, wherein the ADH1 gene encodes a protein selected from the group consisting of (a) and (b), and wherein the ADH4 gene encodes a protein selected from the group consisting of (c) and (d):
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2; and
(b) a protein comprising an amino acid sequence having 90% or higher similarity to the amino acid sequence of SEQ ID NO: 2 and having alcohol dehydrogenase activity,
(c) a protein comprising the amino acid sequence of SEQ ID NO: 4; and
(d) a protein comprising an amino acid sequence having 90% or higher similarity to the amino acid sequence of SEQ ID NO: 4 and having alcohol dehydrogenase activity.

2. A method for producing ethanol, comprising:
(1) culturing a mutant of Kluyveromyces marxianus in a xylose-containing medium, said mutant having been obtained by attenuation of an ADH1 gene and/or an ADH4 gene, wherein the ADH1 gene encodes a protein selected from the group consisting of (a) and (b), and wherein the ADH4 gene encodes a protein selected from the group consisting of (c) and (d):
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2; and
(b) a protein comprising an amino acid sequence having 90% or higher similarity to the amino acid sequence of SEQ ID NO: 2 and having alcohol dehydrogenase activity,
(c) a protein comprising the amino acid sequence of SEQ ID NO: 4; and
(d) a protein comprising an amino acid sequence having 90% or higher similarity to the amino acid sequence of SEQ ID NO: 4 and having alcohol dehydrogenase activity, and

(2) recovering ethanol from the medium.

3. The method according to claim 2, wherein said culturing is carried out in a reaction system containing the mutant of Kluyveromyces marxianus, lignocellulose-containing biomass, and a carbohydrase.