酵母蛋白酶A活性中心突变对蛋白酶A酶活力及菌株生长情况的影响

Effect of Site-directed Mutagenesis of Proteinase A Active Site Residues on Proteinase A Activities and the Growth Curves of Yeast Strains

蛋白酶A由PEP4基因编码,对酿酒酵母的生理生化功能起到重要的作用,蛋白酶A缺失或低表达对于理论研究和实际应用都具有重要价值。利用重叠延伸法构建了蛋白酶A第294位活性中心突变的PEP4基因表达载体,同时构建了野生型PEP4基因表达载体,并分别将两种表达载体转化到基因背景为PEP4缺失的菌株中。研究发现,蛋白酶A活性中心点突变的重组菌株与蛋白酶A缺失的重组菌株在细胞内外均不能检测到蛋白酶A活力,而转化野生型表达载体的菌株蛋白酶A活力与出发菌株基本一致。结果表明,活性中心点突变与基因完全缺失对菌株的影响一致;通过生长曲线的观察发现蛋白酶A活性中心点突变会导致生长速率的减慢。本研究对于工业菌株无痕敲除、提高应用安全性方面具有理论指导意义。

Proteinase A is encoded by PEP4 gene ,which plays an important role in the physiological and biochemical functions of Saccharomyces cerevisioe. The disruption or low expression of proteinase A is of great value for the theoretical research and practical application. In this paper, overlap extension PCR was used to construct PEP4 gene expression vector containing active site residues mutagenesis located in the 294th amino acid residues of PEP4 gene. Simultaneously, wild type PEP4 gene expression of vector was constructed. The two expression vectors were trans- formed into the genetic background PEP4 disruption strain. The results showed that proteinase A activities could not be detected both inside and outside the cells of the recombinant strain with proteinase A active site residues mutation and the genetic background PEP4 disruption strain. In contrast, the recombinant strain with wild type PEP4 gene expression vector displayed almost the same proteinase A activities as that of the host strain. As a conclusion, site-directed mutagenesis of proteinase A active site residues has the same effects as PEP4 disruption on the proteinase A activities of yeast strains. The growth curve showed that proteinase A active site residues mutation will lead to the slowing of yeast growth rate. This study had theoretical significance for industrial strains seamless knockout and in improving the application security.