南方农业学报2023，Vol.54Issue(12)：3719-3726,8.DOI:10.3969/j.issn.2095-1191.2023.12.026
转牛LDHA基因酿酒酵母构建及产乳酸能力分析
Construction of bovine LDHA genetically modified Saccharomy-ces cerevisiae and its ability in the production of lactic acid
摘要
Abstract
[Objective]The purpose of present study was to explore the possibility of production of food grade lactic acid by the fermentation of commercial transgenic Saccharomyces cerevisiae,and to provide technical support for the large-scale production of lactic acid by transgenic commercial Saccharomyces cerevisiae.[Method]The coding se-quence(CDS)of bovine(Bos taurus)lactate dehydrogenase A(LDHA)gene was downloaded from NCBI.Synonymous codons in the CDS of bovine LDHA gene were replaced by preferable codons used in Saccharomyces cerevisiae,then,the sequence was artificially synthesized and cloned into pAUR123 expression vectors.Recombinant pAUR123 vectors were introduced into competent commercial EC1118 strain of Saccharomyces cerevisiae,and antibiotic Aureobasidin A was used to screen the transgenic Saccharomyces cerevisiae..Transgenic and non-transgenic Saccharomyces cerevisiae were separately seeded and cultured in YPD liquid medium containing 200 g/L glucose for 5 days,the activity of LDHA and the production of lactic acid were tested to evaluate the feasibility of production of lactic acid by Saccharomyces cerevi-siae transfected with bovine LDHA gene.[Result]The CDS of bovine LDHA gene is 1170 bp,codes 389 amino acids.58.35%codons in the coding sequence of bovine LDHA gene were replaced by synonymous,preferable codons used in Saccharomyces cerevisiae.The estimated half-life of bovine LDHA enzyme in Saccharomyces cerevisiae was more than 20 h.α-helix and coil enrich in the secondary structure of bovine LDHA enzyme.After preferable codons replacement,the CDS of bovine LDHA gene was integrated into pAUR123 expression vectors,and then recombinant pAUR123 vectors were transformed into Saccharomyces cerevisiae.One strain of transgenic Saccharomyces cerevisiae,named EC1118-LDHA,was obtained after screened by antibiotic Aureobasidin A.The transgenic strain of Saccharomyces cerevisiae,EC1118-LDHA,could use 200 g/L glucose as carbon source,the activity of LDHA enzyme was 4.7±1.2 mU/mg total pro-tein after fermentation for 24 h,and 30 g/L lactic acid and 70 g/L ethanol were also produced after fermentation for 2-3 d.The production rate of lactic acid was approximately 0.15 g/g glucose.[Conclusion]Transgenic Saccharomyces cerevisiae with bovine LDHA gene can be constructed after the CDS of bovine LDHA gene,in which some codons are replaced by preferred codons of Saccharomyces cerevisiae,are introduced into Saccharomyces cerevisiae.The introduction of bovine LDHA gene can not affect the fermentation ability of commercial Saccharomyces cerevisiae,and endow Saccharomyces cerevisiae the ability to produce about 30 g/L lactic acid with 200 g/L glucose as the carbon source.Therefore,it is fea-sible to produce food-grade lactic acid by using Saccharomyces cerevisiae transferred with bovine LDHA gene.关键词
酿酒酵母/牛/乳糖脱氢酶A基因(LDHA)/乳酸/发酵Key words
Saccharomyces cerevisiae/bovine/lactate dehydrogenase A gene/lactic acid/fermentation分类
农业科技引用本文复制引用
左梦娜,何佳宁,尹千禧,王凤梅,马利兵..转牛LDHA基因酿酒酵母构建及产乳酸能力分析[J].南方农业学报,2023,54(12):3719-3726,8.基金项目
国家自然科学基金项目(31660340) （31660340）
内蒙古自然科学基金项目(2022MS03017) （2022MS03017）
内蒙古科技大学基本科研业务费专项(2023RCTD024) National Natural Science Foundation of China(31660340) （2023RCTD024）
Natural Science Foundation of Inner Mongolia Autonomous Region(2022MS03017) （2022MS03017）
Fundamental Research Funds for Inner Mongolia University of Science&Technology(2023RCTD024) （2023RCTD024）
