酿酒酵母孢子表面展示系统的构建及应用

摘要

旨在建立1种酿酒酵母孢子表面展示系统,并将其应用于稀有糖合成.表面展示系统是运用微生物自身功能把外源蛋白或多肽展示在细胞表面,文中用磷酸甘油氧化酶(GPO)来验证酿酒酵母孢子表面展示系统的可行性.将编码肺炎链球菌来源的GPO的基因glpo与信号肽序列(ss)融合后连接到载体pRS42 4-TE Fpr上,将重组质粒pR S42 4-TEFpr-ss-glpo转化至酿酒酵母野生型及缺陷型菌株中并进行产孢,通过荧光观察、western blot分析以及酶活测定,表明酿酒酵母缺陷型孢子可以实现蛋白的表面展示.通过对GPO孢子表征,表明GPO孢子在pH为7.0、温度为30℃时活性最高,该孢子作为催化剂可以重复使用,当使用第3次时,活性可达最大活性的40％.最后将GPO孢子应用于“一锅四酶法”进行稀有糖L-果糖的合成,转化率为12％.%In this paper,a novel surface display system on yeast spore and its application for rare sugar synthesis were studied.Surface display system was used by microorganisms to display the exogenous proteins or peptides on their cell surface.Glycerophosphate oxidase (GPO) was used to verify the feasibility of the novel surface display system.The gene glpo GPO from Streptococcus pneumoniae was fused with signal sequenceand inserted into pRS424-TE-Fpr vector.The recombinant plasmid pRS424-TEFpr-ss-glpo was transformed into wild type and auxotroph of S.cerevisiae to sporulate.The results from fluorescence analysis,western blot detection and activity assays showed that S.cerevisiae spore surface display system was successfully constructed.The GPO spores were characterized and their optimum pH and temperature were 7.0 and 30℃,respectively.Moreover,the spores could be reused with activity of about 40％ of the maximum activity at the third time.Finally,GPO spores were employed in the one-pot four-enzyme system for synthesis of rare sugar L-fructose with yield of about 12％.