According to the New State Strategy of May, 2006, gasoline consumption in Japan is projected to be 60,000,000 kL by year 2030, of which 10% will be supplied by ethanol. Ethanol qualifies as a renewable energy, and it is produced by conversion of plant-derived components to ethanol by fermentation methods. For example, the budding yeast Saccharomyces cerevisiae, generally having high fermentation ability and high ethanol resistance, is a microorganism that has long been used to generate ethanol for production of alcoholic beverages, and it is also utilized in fuel ethanol production. What is known as “first generation” bioethanol is fuel ethanol produced using budding yeast or other microbes, using as the starting materials glucose from sugarcane and the like, or starch from corn and the like (which is easily convertable to glucose using enzymolysis, for example). Both domestically and abroad, fuel ethanol production has initially used glucose that can be assimilated by budding yeast, and starch that can be easily converted to glucose. On the other hand, since these materials are plants that are also used as foods and livestock feeds, there have been concerns that new problems will arise due to competition with use for foods. Therefore, expectations are increasing for “second-generation” bioethanols, with ethanol production from cellulosic biomass that are not usable for foods. Since cellulose can be decomposed into glucose by enzymes, it can likewise serve as a starting material for production of ethanol by fermentation. However, various problems are known to be associated with ethanol production from cellulosic biomass, depending on the combination of type of starting material, pretreatment method, saccharification process and fermentation process, and solutions to those problems are desired. While various resources have been considered for cellulosic starting materials, ligneous materials are the most promising, in terms of ensuring consistent ethanol production, because they are most abundant as cellulose resources. However, for efficient production of ethanol from ligneous materials, it is important for other components in addition to the cellulose that is the starting material for glucose. Ligneous materials are generally composed mainly of cellulose, hemicellulose and lignin, with a cellulose proportion of about 40% and a hemicellulose proportion of about 20% to 30%. Consequently, when a ligneous material, which contains a large amount of hemicellulose, is used as the starting material, it is desirable to accomplish ethanol conversion of the sugars such as xylose which are obtained from decomposition of hemicellulose by enzymes and the like. However, since budding yeast with high fermentation ability do not have functioning genes for assimilation of xylose, there is a problem in that they cannot produce ethanol from xylose. Therefore, the approach has been adopted of introducing xylose metabolizing enzymes of xylose-assimilating organisms into budding yeast, or enhancing endogenous metabolic functions to impart xylose-assimilating properties to budding yeast. Such exogenous xylose metabolizing enzymes include xylose reductase, xylitol dehydrogenase, xylulokinase, and xylose isomerase. In “Technological Research and Development for New Energies/New energy venture technological innovative projects (biomass)/Development of technique for bioethanol conversion from bamboo, for Kyushu Village Technology Architecture (2007-2008)” and “Development and research on processes for production of fuel ethanol from soft biomass” (2008-2009)”, the present inventors have reported on our creative development of yeast suitable for production of ethanol from xylose. With yeast breeding techniques, there have been created yeast with enhanced xylose metabolism and yeast that are resistant to growth inhibition even in high-concentration xylose environments (PTL 1). However, the causative gene has not yet been identified.
In order to allow production of ethanol from xylose using budding yeast, it is essential to combine xylose reductase (XR), as the initial gene of xylose metabolism, and xylitol dehydrogenase (XDH), or to use xylose isomerase (XI). Still, while introduction of these genes allows production of ethanol from xylose, the production efficiency is very low. This is because production of ethanol from xylose requires endogenous enzymes of budding yeast in addition to the enzyme from the introduced genes, and they are functionally inadequate. Widely employed attempts to compensate for this inadequacy include forced expression of endogenous enzymes of budding yeast by gene recombination, and introduction of genes for analogous enzymes with greater affinity for xylose, from other organisms that are xylose-assimilating. The compensating enzyme can be easily selected by referring to a metabolic map, and examples include transporters (such as Hxt5) for incorporating xylose from outside the cell, a xylulokinase (such as Xks1) and enzyme groups of oxidative or nonoxidative pentose phosphorylation pathways (such as Zwf1, Sol3, Gnd1, Rpe1, Rki1, Tkl1 or Tal1), and examples of enhanced xylose metabolic capacity by forced expression of these enzymes, or deletion of genes, have been reported (NPL 1).
In addition, while not considered to be involved in the metabolic pathway based on metabolic maps, genes that have been reported to influence the metabolic pathway include PET18, TEC1, ARR1 (NPL 2), MNI1, RPA49 (NPL 3), YLR042C (NPL 3, NPL 4), ALP1, ISC1, RPL20B, BUD21 (NPL 5), PHO13 (NPLs 6 and 7) and FPS1 (NPL 8). In addition, PTL 2 reports a yeast with enhanced expression of acetaldehyde dehydrogenase, PTL 3 reports some xylose-assimilating yeasts with enhanced expression of one or more genes including HXT10, HXT11, HXT14, GIT1, RGT2, ARO1, ARO7, PHA2, TRP5, PYC1, PYC2 and PDA1, PTL 4 reports yeast transformed so as to overexpress formate dehydrogenase, and PTL 5 reports a yeast with loss of glycine-synthesizing protein and/or methionine-synthesizing protein gene expression, each with improved production efficiency of ethanol from xylose. These reports suggest that enzymes that are not directly found in the conversion pathway from xylose to ethanol in metabolic maps also indirectly influence conversion efficiency.
As mentioned above, in order to achieve efficient conversion from xylose to ethanol, merely the information relating to enzymes involved in the metabolic pathway from xylose to ethanol in a metabolic map is insufficient, it being also necessary to study genes and proteins that indirectly contribute to enhancing their conversion efficiency. For this purpose, the most comprehensive and effective approach may be said to be to obtain variants imparted with enhanced metabolic capacity by introduction of mutations and the use of appropriate screening methods. In a preceding project, the present inventors have made use of natural mutations and breeding techniques to successfully create yeast variants with improved xylose-assimilating properties. At the current time, however, it has not been possible to analyze which genes and which mutations of those genes are contributing to those properties.
Even when it is possible to obtain distinctive variants by natural mutations and breeding techniques, it has been necessary to sequence the entire genome of the microorganism in order to directly identify the causative genes, but the conventional Sanger method has been time-consuming and impractical. Consequently, experimental methods have been carried out for acquiring certain regions of the entire genome in which such genes are found, taking advantage of molecular biological methods and genetic methods. For example, there is a method of fragmenting variant genomic DNA, obtaining transformants exhibiting similar properties from among the transformants obtained by transfer thereof into the parent strain, and analyzing the transferred DNA. Other methods include combining different strains to discover genes in the neighborhood of the causative gene. The trouble with such methods is the extremely long times required, and difficulties often arise when recessive mutations or multiple gene mutations are involved. On the other hand, several devices have been developed in recent years, known as “next-generation sequencers”, that are considerably more rapid than the conventional Sanger method. With next-generation sequencers it is possible to obtain sequence information matching entire microbial genomes, and by analyzing the entire genomes of obtained variants, it has become possible to identify causative genes. In actuality, however, with next-generation sequencing data, especially with types in which sequencing is performed in parallel on a large scale, the huge number of read nucleotide bases that are obtained as a final result are less precise on the individual nucleotide level compared to conventional Sanger sequencing, and numerous errors are included. Furthermore, since mutations are assumed to occur randomly, variant genomes presumably include very large numbers of “neutral” mutations that do not contribute to the phenotype. Consequently, in approaches where causative genes are identified by genomic analysis by a next-generation sequencer for variants, there is an essential need for 1) analysis methods that compensate for the low precision on the nucleotide base level that is characteristic of next-generation sequencers, and 2) removal of neutral mutations that are unrelated to the mutations of interest, but such methods have not yet been established.