基于酿酒酵母多拷贝系统策略提高三乙酸内酯的产量  

Efficient production of triacetic acid lactone using a multi-copy integration system in Saccharomyces cerevisiae

作　　者：韦欢 林苹鑫 刘秀霞 刘春立[1,2] 杨艳坤 李业 白仲虎[1,2] WEI Huan;LIN Pingxin;LIU Xiuxia;LIU Chunli;YANG Yankun;LI Ye;BAI Zhonghu(National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,China;The Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;Environmental and Molecular Microbiology Laboratory,Korea Advanced Institute of Science and Technology,Daejeon 34141,South Korea)

机构地区：[1]江南大学,粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122 [2]江南大学,工业生物技术教育部重点实验室,江苏无锡214122 [3]韩国科学技术院环境与分子微生物实验室,韩国大田广域,34141

出　　处：《食品与发酵工业》2025年第4期1-10,共10页Food and Fermentation Industries

基　　金：国家自然科学基金(21878124)。

摘　　要：三乙酸内酯(triacetic acid lactone,TAL)是一种具有广泛应用前景的聚酮,可用作各种有机化合物的前体。为了提高酿酒酵母(Saccharomyces cerevisiae)中TAL的产量,该研究利用基因组重复序列整合的方法构建了S.cerevisiae多拷贝整合系统,应用该系统提高TAL的生产。首先以绿色荧光蛋白表征多拷贝Delta1整合,通过截短遗传霉素和潮霉素B两种抗生素基因的启动子以及增加抗生素浓度的方法增加筛选压力,进而提高整合效率和拷贝数,最优参数为将抗生素基因启动子截短至15 bp的同时采用160μg/mL的抗生素。将该优化系统随后用于新的多拷贝整合位点Delta2的表征。最后应用该系统合成TAL,通过HPLC分析TAL产量。结果表明,利用Delta1和Delta2序列表征绿色荧光蛋白的最高拷贝数分别为10和7,Delta1略优于Delta2。应用该系统在S.cerevisiae中合成TAL,Delta1和Delta2的产量分别为1.50、1.17 mmol/L,比单拷贝菌株产量分别提高460%和337%。该多拷贝系统有效提高了TAL的产量,为S.cerevisiae异源途径的表达提供了一种高效、模块化的多拷贝整合方法。Triacetic acid lactone(TAL)is a promising platform polyketide with broad applications,especially it can be used as a precursor for the synthesis of various organic compounds.This study characterized the repeating sequences on the yeast genome to integrate the TAL biosynthesis pathway into these sites for enhanced gene expression and TAL production by Saccharomyces cerevisiae.Firstly,this study used the green fluorescent protein as the reporter to characterize multi-copy integration by the Delta1 site.It showed that truncating the promoter of a selection marker gene(for geneticin or hygromycin B)or increasing the antibiotic concentration,were effective in improving the integration efficiency and copy numbers.The highest copy number of multi-copy integration was obtained when truncating the antibiotic gene promoter to 15 bp,and using an antibiotic at 160μg/mL.The optimization system was subsequently used to characterize the second multi-copy integration site,Delta2,with similar results.Then,the optimized multi-copy integration system was applied to introduce the TAL biosynthesis pathway into S.cerevisiae.The highest pathway copies using Delta1 and Delta2 sequences were 10 and 7,respectively,with Delta1 slightly better than Delta2.After 48 h fermentation in YTD medium,TAL titers of the Delta1-integration strain and the Delta2-integration strain were 1.50 mmol/L and 1.17 mmol/L,respectively,460%and 337%higher than the single-copy integration strain.This study demonstrates that the multi-copy integration system is an efficient approach to introducing heterologous pathways into S.cerevisiae to improve biocatalytic efficiency.

关 键 词：酿酒酵母 多拷贝整合 Delta整合 三乙酸内酯 启动子 

分 类 号：TQ926[轻工技术与工程—发酵工程]