Analysis of genetic bases for efficient expression of heterologous proteins in Saccharomyces cerevisiae

为了构建高效表达外源蛋白的酿酒酵母超级宿主，突变体命名为bel(基础表达水平)，表明报告蛋白产量增加。对bel突变体的分析表明，bel突变通过影响染色质或更高的染色体组织顺序，导致各种基因转录增加。通过对酿酒酵母交配型调控突变株的筛选，筛选出一个新的突变株，命名为HMLα2-102。利用HMLα2-102突变和SIR3基因的温度敏感突变抑制HML和HMR位点交配型信息沉默拷贝的表达，构建了外源基因温控表达的双模式表达系统。对PH084的转录分析表明，在PI饥饿条件下，PHO84被强烈下调。将PHO84启动子与PHO81的温度敏感突变相结合，建立了另一种温度可调控的表达系统。针对利用酿酒酵母作为外源蛋白生产宿主的问题，建立了利用显性选择标记与蓝菌素相结合的原养酿酒菌株转化体系，为酿酒酵母超级宿主细胞高效生产外源蛋白提供了有用的信息。

Toward to construction of Saccharomyces cerevisiae super host for efficient expression of heterologous proteins, mutants designated bel (basal expression level) showing increased production of reporter protein. Analysis of bel mutants revealed that bel mutations caused increased transcription of various genes through affecting chromatin or higher order of chromosomal organization. It was suggested that the use of bel mutation could lead to further increased promoter activity of some set of genes.From the screening of mutants displaying disregulation in mating-type control in S. cerevisiae, a novel mutations designated hml alpha 2-102 was isolated. In combination of hml alpha 2-102 mutation with a temperature sensitive mutation in the SIR3 gene which acts to repress the expression of silent copy of mating-type informations residing at HML and HMR locus, a system for temperature-controlled expression of a foreign gene with dual mode was developed.The PH084 gene was cloned and found to encode an inorganic phosphate (Pi) transporter in S. cerevisiae. Analysis of transcriptin of PH084 revealed that PHO84 is strongly derepressed under Pi starved condition. Combining of promoter of PHO84 with a temperature sensitive mutation in PHO81 which is necessary for expression of the PHO84 gene led to the establishment of another temperature regulatable expression system. Using this system, human lysozyme and rice alpha -amylase was efficiently produced.Aiming at the use of brewing strains as host for production of heterologous proteins, transformation system of prototrophic brewing strains using a dominant selective marker in combination with cerulenin was developed.All of these studies provided useful informations for breeding of super host cell of S. cerevisiae for efficient production of heterologous proteins.