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
左梦娜 1何佳宁 1尹千禧 1王凤梅 2马利兵1
作者信息
- 1. 内蒙古科技大学生命科学与技术学院,内蒙古包头 014010
- 2. 包头轻工职业技术学院食品药品学院,内蒙古包头 014035
- 折叠
摘要
[目的]探究转牛乳酸脱氢酶A基因(LDHA)酿酒酵母发酵制备食品级乳酸的可行性,为采用转基因商业酿酒酵母规模化制备乳酸提供技术支撑.[方法]从NCBI网站下载牛LDHA基因编码序列,并将编码序列中的同义密码子替换为酿酒酵母偏好密码子,经人工合成后导入酿酒酵母表达载体pAUR123,再将重组表达载体pAUR123-LDHA导入商业化酿酒酵母EC1118株,采用Aureobasidin A筛选转基因酿酒酵母.将转基因及非转基因酿酒酵母EC1118株分别接种于含200 g/L葡萄糖的YPD液体培养基中连续培养5 d,期间检测2组酿酒酵母的LDHA活性及其发酵产乳酸能力,以评估转牛LDHA基因酿酒酵母发酵制备乳酸的可行性.[结果]牛LDHA基因编码序列长1170 bp,共编码389个氨基酸残基;以酿酒酵母偏好密码子替换牛LDHA基因编码序列中的同义密码子,其替换率高达58.35%.牛LDHA在酿酒酵母中的预计半衰期>20 h,其二级结构富含α-螺旋及无规则卷曲.替换后的牛LDHA基因编码序列由表达载体pAUR123携带导入酿酒酵母中,经Aureobasidin A筛选获得1株转基因酿酒酵母菌株,命名为EC1118-LDHA.转基因酿酒酵母EC1118-LDHA株以200 g/L葡萄糖为碳源,发酵24 h后的LDHA活性达4.7±1.2 mU/mg,发酵2～3 d后约制备出30 g/L乳酸,同时约产生70 g/L乙醇,乳酸产出率约为0.15 g/g葡糖糖.[结论]将替换酿酒酵母偏好密码子后的牛LDHA基因编码序列导入酿酒酵母中能成功构建转牛LDHA基因酿酒酵母,牛LDHA基因的导入不会影响商业化酿酒酵母发酵能力,且赋予了酿酒酵母以200 g/L葡萄糖为碳源制备约30 g/L乳酸的能力.因此,采用转牛LDHA基因酿酒酵母制备食品级乳酸完全可行.
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引用本文复制引用
基金项目
国家自然科学基金项目(31660340)
内蒙古自然科学基金项目(2022MS03017)
内蒙古科技大学基本科研业务费专项(2023RCTD024)
出版年
2023
国家科技期刊平台
NSTL
万方数据