表达人源HPD基因对酿酒酵母孢子壁二酪氨酸层正确组装的干扰  

Expression of human HPD gene interferes correct assembly of the dityrosine layer on the spore wall of Saccharomyces cerevisiae

作　　者：王晓文 殷政 高晓冬[1] 中西秀树[1] WANG Xiaowen;YIN Zheng;GAO Xiaodong;NAKANISHI Hideki(School of Bioengineering, Jiangnan University, Wuxi 214122, China)

机构地区：[1]江南大学生物工程学院

出　　处：《食品与发酵工业》2019年第15期17-23,共7页Food and Fermentation Industries

基　　金：国家自然科学基金(21576118);自主科研重点项目(JUSRP51629B);江苏高校品牌专业建设工程资助项目;国家轻工技术与工程一流学科自主课题(LITE2018-015)

摘　　要：研究人类基因4-羟苯丙酮酸二加氧酶(4-hydroxyphenylpyruvate dioxygenase,HPD)的表达对酿酒酵母( Saccharomyces cerevisiae )产孢过程及酵母孢子壁组装的影响。将 HPD 导入酿酒酵母中,比较人源化 HPD 酵母与野生型酵母的表型差异,分析 HPD 对酵母产孢及孢子壁的影响。与野生型酵母相比,人源化 HPD 酵母对产孢率无影响,但孢子自身荧光强度下降,乙醚敏感性增加,荧光增白剂(calcofluor white,CFW)染色荧光强度增强,该结果表明孢子壁出现缺陷,其二酪氨酸层组装过程中部分缺失。进一步研究发现,人源化 HPD 酵母产孢时,Hpd蛋白的催化产物尿黑酸(homogentisate,HGA)在产孢过程中会被氧化从而引起培养基颜色变化,且二酪氨酸复合物会大量泄漏至培养基中,最终导致二酪氨酸层不能正确组装。该研究结果为酿酒酵母二酪氨酸层的形成机制提供了新的认知。This study analyzed the effects of expressing human gene 4- hydroxyphenylpyruvate dioxygenase (HPD) on sporulation and spore wall assembly in Saccharomyces cerevisiae. HPD was introduced into S. cerevisiae , and the phenotype differences between humanized HPD yeast and wild-type yeast were compared. It was found that compared with wild-type yeast, humanized HPD yeast had no effect on the sporulation rate, while the fluorescence intensity of spores decreased, ether sensitivity increased, and the fluorescence intensity of calcofluor white increased. This indicated that HPD caused the defect of spore wall and part of the dityrosine layer missing during the assembly process. Moreover, expressed HPD gene changed the sporulation medium color and increased the fluorescence intensity, as homogentisate, the catalytic product of HPD , was oxidized during sporulation. Furthermore, a large number of dityrosine complex leaked into the sporulating medium and dityrosine layer could not assemble correctly in consequence. Overall, this study provides a new insight into the mechanisms of forming dityrosine layer in S. cerevisiae.

关 键 词：酿酒酵母 4-羟苯丙酮酸二加氧酶(HPD) 产孢子 二酪氨酸层 氧化性 组装 

分 类 号：TS261.11[轻工技术与工程—发酵工程]