Cloning Key Xylose-metabolic Genes And Construction Of Xylose-fermenting Recombinant Saccharomyces Cerevisiae Strain

Bioethanol is taken into account as many states'strategy as a sustainable, portable and renewable energy source. Cheap raw material and high ethanol yield are needed for commercial fuel-ethanol production; therefore the utilization of abundant and renewable lignocellulosics could make bioethanol more competitive and attractive. Efficient xylose fermentation to ethanol is essential to such a process. Native microorganisms and recombinant strains have not been proven sufficient to ferment xylose to ethanol, which has been the bottleneck for the bioconversion of lignocelluosics to ethanol. Research has been focused on the selection of microorganisms, the study of xylose metabolism, and the construction of efficient xylose-fermenting recombinant strains. In this dissertation, some xylose-utilizing yeast strains and lignocellulosic hydrolysate-utilizing yeast strains were identified and classified, and the relationship between them was studied with Molecular-Marking Technique. Some expression vectors with xylose-utilizing genes and xylose-fermenting recombinant Sacchaormyces cerevisiae strains were constructed using recombinant DNA technology. Xylose-metabolic engineering was also studied with these recombinant strains. Except Saccharomycess cerevisiae OB20, characters on morphology and physiology of 20 yeast species from 6 genera were in accord with the description in J. Lodder's Yeast Standard Classification and Identification Methods (1970 edition). These 6 genera included Candida, Pachysolen, Pichia, Brettanomyces, Schizosaccharomycess and Sacchaormyces. Differences in morphology and physiology were obvious between yeast genera but illegible between species. Therefore it was difficult to identify yeast species only by morphology and physiology of yeasts. Compared with traditional methods, random amplified polymorphic DNA technique (RAPD) could to be more precise, simple and convenient in yeast classification and identification. This is because RAPD could detect genome DNA of yeast that has been inheriting steadily and relationship between yeast species could be detected well and truly. Of 21 yeast species from 6 genera, 498 RAPD electrophoresis bands were detected as genome DNA and random amplified with 33 screened primers. There were 15.1 bands for each primer with polymorphic loci above 90%. The genetic distance between yeast species was calculated according to Nei's Genetic Distance and the clustering was done with UPGMA from RAPD data. It was shown that Candida shehatae NL05 and Candida shehatae NL01 were the same yeast species, and the genetic distance among these two and Candida shehatae NL02 was only 0.010. The genetic distance among 4 species of Saccharomycess cerevisiae was also 0.010, while that between 2 species of Candida spp Berkh was 0.020. For 2 species of Pachysolen tanophlis and 2 species of Pichia stipitis, the genetic distance between species was 0.070 and 0.052, respectively. The genetic distance for various yeasts was calculated as follows: 0.114 between Candida shehatae and Candida trophicalis, 0.493 among them and Pichia stipitis, 0.322 between Sachizosaccharomycess pombe and Brettanomyces custersii, 0.383 between Pachsolen tanophlis and Candida Krusei, 0.433 among them and Saccharomycess cerevisiae, and 0.471 among the above yeast strains and Candida utilis. The longest genetic distance calculated was among Candida sp Berkh and all the other yeast species which reached 0.567. Gene XYL1 coding xylose reductase and gene XYL2 coding xylitol dehydrogenase were cloned from the genome DNA of Pichia stipitis. DNA sequencing indicated that the length of XYL1 was 1977 bp and that of XYL2 was 1910 bp. The gene XYL1 was coded and a 319 AAR polypeptide (36120 Daltons) was produced, while the coding of XYL2 resulted in a 363 AAR polypeptide (38380 Daltons). Both genes were accord with the published data. The homogenetic coefficient between XYL1 and the gene sequence of gi 3260 in GeneBank was 98%, and the homogenetic coefficient between XYL2 and the gene sequence of gi 3262 in GeneBank was 99%. Analysis of the two genes showed that there were promoters and terminators but no intron in XYL1 and XYL2. This is essential to the expression of XYL1 and XYL2 in Saccharomyces cerevisiae. From yeast episome plasmid YEp24, expression vector YEp-X1 (10 kbp±), YEp-X2 (10 kbp±) and YEP-X1X2 (12 kbp±) containing genes XYL1, XYL2 and XYL1-XYL2, respectively, were recombined. These three recombinants of Saccharomyces cerevisiae, namely NLR02 (with YEp-X1), NLR03 (with YEp-X2), and NLR04 (with YEp-X1X2), were constructed by transferring mutant Saccharomyces cerevisaie (ura-trp) with LiAc reagent. Recombinant Saccharomyces cerevisaie NLR04 could grow well in xylose medium under aerobic, oxygen-limited, and anaerobic conditions, indicating that gene XYL1 and gene XYL2 from Pichia stipitis had been expressed in Saccharomyces cerevisiae. Due to some bottlenecks in pentose phosphate pathway, low-affinity xylose transferring system in cell membrane and the redox imbalance in recombinant yeast strain, recombinant Saccharomycess cerevisiae NLR04 utilized xylose inefficiently and produced mainly xylitol. Notable changes in xylose-metabolic flux in Saccharomyces cerevisiae NLR04 were observed with varied conditions. Xylitol and glycerol raised with glucose addition, and little xylose could be fermented to ethanol in anaerobic culture. After 11 days, recombinant Saccharomyces cerevisiae NLR04 could consume 2.4 g/l xylose and produced 0.50 g/l xylitol and 0.40 g/l ethanol under anaerobic conditions. The ethanol yield was 0.17 g/g (ethanol/ consumed xylose), 37.0% of theoretical yield. Differing in inducing strictly by xylose in Pichia stipitis and Candida shehatae, genes XYL1 and XYL2 could express consistently in recombinant Saccharomyces cerevisiae NLR04. Carbon source effected the expression of XYL2. Xylitol dehydrogenase (XDH) from Candida shehatae was strictly NADPH-dependent, but XDH from Pichia stipitis and recombinant Saccharomyces cerevisiae NLR04 was mainly NADPH-dependent even some NADH-dependent data were collected. It is supposed that there were some inhibitors for geneXYL2 expression in recombinant Saccharomyces cerevisiae NLR04 compared with Pichia stipitis and Candida shehatae, which might be a bottleneck of xylose-fermenting in recombinant Saccharomyces cerevisiae NLR04. In this study recombinant xylose-fermenting yeast strains were constructed, which expands and substantiates the strains'source and the theory for xylose-metabolic engineering of microorganisms and the bioconversion of lignocellulosics to ethanol.