利用人工锌指文库选育高乙醇耐受性工业酵母菌株

Breeding of robust industrial ethanol-tolerant Saccharomyces cerevisiae strain by artificial zinc finger protein library

摘要:

选育高乙醇耐性的酿酒酵母菌株对提高燃料乙醇的发酵效率具有重要意义。锌指蛋白广泛存在于多种生物中，对基因的转录和翻译起重要的调节作用。利用人工设计的锌指蛋白可定向设计锌指序列及其排列顺序，实现对细胞内多个基因的全局调控。由于与环境胁迫反应相关的基因很多，因此可利用人工锌指蛋白技术获得耐受性提高的微生物重组菌。文中将人工锌指文库转入到酿酒酵母模式菌株S288c，选育了具有高乙醇耐受性的重组菌株M01，并分离了与乙醇耐受性提高相关的人工锌指蛋白表达载体pRS316ZFP-M01，转入工业酿酒酵母Sc4126，在含有不同浓度乙醇的平板上，工业酵母Sc4126的重组菌株表现出显著的耐受性提高。在高糖培养基 (250 g/L) 条件下进行乙醇发酵，发现重组菌的乙醇发酵效率明显快于野生型，发酵时间提前24 h，且发酵终点乙醇浓度提高6.3％。结果表明人工锌指文库能够提高酵母的乙醇耐受性，为构建发酵性能优良的酵母菌种奠定了基础。

Abstract:

Breeding of robust industrial Saccharomyces cerevisiae strains with high ethanol tolerance is of great significance for efficient fuel ethanol production. Zinc finger proteins play important roles in gene transcription and translation, and exerting control on the regulation of multiple genes. The sequence and localization of the zinc finger motif can be designed and engineered, and the artificial zinc finger protein can be used to regulate celluar metabolism. Stress tolerance of microbial strains is related to multiple genes. Therefore, it is possible to use artificially-designed zinc finger proteins to breed stress tolerant strains. In this study, a library containing artificial zinc finger protein encoding genes was transformed into the model yeast strain S288c. A recombinant strain named M01 with improved ethanol tolerance was obtained. The plasmid in M01 was isolated, and then transformed into the industrial yeast strain Sc4126. Ethanol tolerance of the recombinant strain of Sc4126 were significantly improved. When high gravity ethanol fermentation using 250 g/L glucose was performed, comparing with the wild-type strain, fermentation time of the recombinant strain was decreased by 24 h and the final ethanol concentration was enhanced by 6.3%. The results of this study demonstrate that artificial zinc finger proteins are able to exert control on stress tolerance of yeast strains, and these results provide basis to construct robust industrial yeast strains for efficient ethanol fermentation.