| Lignocellulose represents the largest source of renewable carbohydrate on Earth, and its commercial conversion into chemical products has attracted people’s attention.The comprehensive utilization of biomass has been becoming more and more serious to alleviate the outstanding social problems, such as resource shortage. But the lack of a robust organism to ferment xylose is also one of the current bottleneck problems, and to solve this problem, lots of works need to be done, among which screening of new better strain to metabolic xylose is the key, while genetic engineering is an important adjunct for the microbial strain improvement.For a long period, the application of xylose-utilizing yeasts has been limited by their relatively low xylose conversion efficiency, strict oxygen supply conditions and glucose effect on xylose fermentation. The xylose-utilizing yeasts of Spathaspora passalidarum(S. passalidarum), Candida amazonensis(C. amazonensis) and Candida jeffriesii(C. jeffriesii) were isolated recently, and Scheffersomyces stipitis(S. stipitis) and Candida tenuis(C. tenuis) have been studied for decades. Their stress tolerance(temperature tolerance, ethanol tolerance, osmotic tolerance), utilization ability of carbon and nitrogen sources, and xylose fermentation performance under different environmental conditions were studied in this study at first. Meanwhile, the antibiotics sensitivity and genome ploidy of the tested yeasts were also detected. The results revealed that S. passalidarum is a potentially valuable candidate for its higher temperature tolerance up to 44?C, broader utilization of carbon and nitrogen sources, higher consumption rate of xylose and a maximum ethanol yield of 0.44 g×g-1. Although S. passalidarum is a diploid strain, the sensitivity to common antibiotics made it easy for further genetic manipulation. In addition, C. amazonensis may be promising yeast for the advantage in cellobiose metabolism and for its higher native capacity of xylose fermentation with a higher xylitol yield of 0.83 g×g-1. C. amazonensis showed a higher acid tolerance which was capable to ferment at p H2.5, and its optimum fermentation temperature was 35?C. However, the resistance to tested antibiotics may increase the economic costs of its genetic manipulation.According to the genetic coding characteristics of CTG clade yeasts, the genetic expression system with a series of genetic tools applicable in xylose yeasts was constructed. The PA series plasmids based on autonomously replicating sequence Ss ARS2 transformed S. stipitis successfully and the gfp was functional expressed by site-directed mutagenesis of the sole CTG codon to TTG. The PR series plasmids based on 18 S r DNA were shuttle vectors with independent intellectual property right and were capable of stable integration into genome in a wide range of heterologous hosts. The plasmid PRACTH-gfpm was studied as the object to transform S. stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and Saccharomyces cerevisiae(S. cerevisiae), and the stability of plasmid in S. stipitis transformants was up to 99.48%. In addition, the gfp was functional expressed in all the tested yeasts under the control of Sp ADH1 promoter and Sp CYC1 terminator.Finally, the expression system was further proved to be useful in multiple yeast hosts by construction of the plasmid PRACTH-ldh which contains the L-lactate dehydrogenase gene(ldh L) derived from Lactobacillus plantarum. The lactate acid metabolism pathway of S. stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and S. cerevisiae was put up by transforming the recombinant plasmid PRACTH-ldh, and resulted in the distribution of L-lactate acid flow in engineered strain cells when metabolism xylose and glucose. Among them, C. amazonensis exhibited the highest lactate acid fermentation capacity which accumulated 44 g×L-1 of lactate acid with51.54 g×L-1 xylose and the highest production yield was 0.85 g×g-1. |
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