稳定遗传的染色体组合整合酿酒酵母重组菌株的构建

Development of genetically stable recombinant Saccharomyces cerevisiae strains using combinational chromosomal integration

摘要:

染色体整合表达可获得稳定遗传的基因工程菌，是工业酿酒酵母育种的重要手段。pAUR135整合载体是抗生素标记基因可循环使用的载体，添加特定的同源臂后可构建在酵母染色体上稳定遗传的菌株。目前对酵母菌的分子育种常需要对多个基因进行过表达，利用pAUR135载体可将不同基因分别整合在不同染色体或相同染色体的不同位点，这种组合整合表达方法可对不同基因的表达强度比例进行调节，构建表型优化的工业酿酒酵母菌株。本研究以木糖代谢途径基因为例，构建了3个pAUR135整合载体，将3个木糖代谢基因依次整合到工业酿酒酵母染色体的不同位点，获得了染色体组合整合表达的代谢工程菌株。与将这3个基因整合在同一个位点的对照重组菌株相比，染色体组合整合的重组菌株木糖利用率提高了24.4%–35.5%。多基因染色体组合整合方法从新的角度对工业酵母进行代谢工程改造，所获得的工程菌株不带有任何外来基因和选择标记，可以保持性状的稳定，是工业酿酒酵母分子育种的新方法。

Abstract:

Chromosomal integration enables stable phenotype and therefore has become an important strategy for breeding of industrial Saccharomyces cerevisiae strains. pAUR135 is a plasmid that enables recycling use of antibiotic selection marker, and once attached with designated homologous sequences, integration vector for stable expression can be constructed. Development of S. cerevisiae strains by metabolic engineering normally demands overexpression of multiple genes, and employing pAUR135 plasmid, it is possible to construct S. cerevisiae strains by combinational integration of multiple genes in multiple sites, which results in different ratios of expressions of these genes. Xylose utilization pathway was taken as an example, with three pAUR135-based plasmids carrying three xylose assimilation genes constructed in this study. The three genes were sequentially integrated on the chromosome of S. cerevisiae by combinational integration. Xylose utilization rate was improved 24.4%–35.5% in the combinational integration strain comparing with that of the control strain with all the three genes integrated in one location. Strain improvement achieved by combinational integration is a novel method to manipulate multiple genes for genetic engineering of S. cerevisiae, and the recombinant strains are free of foreign sequences and selection markers. In addition, stable phenotype can be maintained, which is important for breeding of industrial strains. Therefore, combinational integration employing pAUR135 is a novel method for metabolic engineering of industrial S. cerevisiae strains.