酿酒酵母PDC5基因的缺失对2-苯乙醇合成的影响及相关代谢改造  

Improvement of 2-phenylethanol production by deleting gene PDC5 and related metabolic strategies in Saccharomyces cerevisiae

作　　者：朱灵桓 徐沙[1,2] 李由然 张梁[1,2] 石贵阳 ZHU Linghuan;XU Sha;LI Youran;ZHANG Liang;SHI Guiyang(School of Biotechnology,Jiangnan University,Wuxi 214122,China;National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;College of Food Science and Biology,Hebei University of Science and Technology,Shijiazhuang 050018,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]粮食发酵工艺与技术国家工程实验室(江南大学),江苏无锡214122 [3]河北科技大学食品与生物学院,河北石家庄050018

出　　处：《食品与发酵工业》2021年第16期22-30,共9页Food and Fermentation Industries

基　　金：国家自然科学基金项目(31571817)。

摘　　要：该文研究了酿酒酵母中编码丙酮酸脱羧酶的基因PDC5的缺失对2-苯乙醇合成的影响,并将其应用于2-苯乙醇高产菌株的代谢改造策略。首先利用CRISPR/Cas9双质粒敲除体系构建pdc5△突变株,通过优化过的培养条件进行摇瓶发酵,发现基因PDC5的缺失能够促进酵母合成2-苯乙醇。分别表达芳香族转氨酶Aro8p和Aro9p,发现在缺失Aro9p的突变株中过表达Aro8p能够促进2-苯乙醇的合成。由此构建了重组菌株RM59-810(pdc5△aro9△P_(TPI)-ARO8-T_(TT) P_(TPI)-ARO10-T_(TT)),在6.7 g/L L-苯丙氨酸培养基中培养120 h后2-苯乙醇的产量为3.85 g/L,摩尔转化率为0.67 mol/mol,是对照菌株的1.33倍,得率为0.5 g/g L-苯丙氨酸。该文为加强酿酒酵母合成2-苯乙醇的能力提供了一种新的代谢工程改造策略,为研究丙酮酸脱羧酶Pdc5p可能存在的调控作用提供了依据。As an important aromatic alcohols with favorable flavor and superior property,2-phenylethanol has been widely used in food,medicine,and chemical industry.In this study,the deletion of phenylpyruvate decarboxylase gene PDC5 was found to improve the synthesis of 2-phenylethanol,and related metabolic strategy of the Ehrlich pathway was rational reconstructed in Saccharomyces cerevisiae for further product-enhancement.Strain RM22(pdc5△)was obtained using CRISPR/Cas9,and improved 2-phenylethanol production was observed in the optimized culture conditions.Additionally,by expressing aromatic transaminase Aro8p and Aro9p respectively,we found that overexpressing of Aro8p in aro9-deleted strain was an efficient strategy to increase 2-phenylethanol production.To further im-prove the production of 2-phenylethanol,metabolic engineered strain RM-59810(pdc5△aro9△P_(TPI)-ARO8-T_(TT) P_(TPI)-ARO10-T_(TT))was con-structed,resulting in 3.85 g/L of 2-phenylethanol from 6.7 g/L phenylalanine after 120 h,and the molar conversion rate was 0.67 mol/mol,which was 1.33-fold than that of the control strain,with a yield of 0.5 g/g L-phenylalanine.In this study,a new efficient strategy was provided to improve 2-phenylethanol production in S.cerevisiae,and also provided a primary foundation for studying the possible regulato-ry effects of pyruvate decarboxylase Pdc5p.

关 键 词：酿酒酵母 PDC5 ARO8 2-苯乙醇 代谢改造 

分 类 号：TQ923[轻工技术与工程—发酵工程]