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 Saccharo⁃myces cerevisiae and analysis of its ability in producing lactic acid

【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网站检索牛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p AUR123-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-螺旋和无规则卷曲。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,同时产生约70 g/L乙醇,乳酸产出率约为0.15 g/g葡萄糖。【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的发酵能力,且赋予了酿酒酵母以200 g/L葡萄糖为碳源约制备出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。

【Objective】The purpose of present study was to explore the possibility of production of food grade lactic acid by the fermentation of bovine lactate dehydrogenase A genetically modified(LDHA)Saccharomyces cerevisiae,and to provide technical support for the large-scale production of lactic acid by genetically modified commercial S.cerevisiae.【Method】The coding sequence of bovine LDHA gene was downloaded from NCBI website.Synonymous codons in the coding sequence were replaced by preferable codons of S.cerevisiae.Then,the sequence was artificially synthesized and introduced into S.cerevisiae pAUR123 expression vectors.Recombinant expression vector pAUR123-LDHA was intro‐duced into commercial EC1118 strain of S.cerevisiae,and Aureobasidin A was used to screen the genetically modified S.cerevisiae.Genetically modified and non-genetically modified S.cerevisiae EC1118 strains were separately inoculated in YPD liquid medium containing 200 g/L glucose for 5 d.During the period,the activity of LDHA and ability to produce lactic acid by fermentation in 2 groups of S.cerevisiae were tested to evaluate the feasibility of fermentation and produc‐tion of lactic acid by bovine genetically modified LDHA S.cerevisiae.【Result】The coding sequence of bovine LDHA gene was 1170 bp long and encoded 389 amino acid residues.Up to 58.35%of synonymous codons in the coding se‐quence of bovine LDHA gene were replaced by preferable codons of S.cerevisiae.The estimated half-life of bovine LDHA in S.cerevisiae was>20 h,and its secondary structure was rich inα-helix and irregular coil.After replacement,the co-ding sequence of bovine LDHA gene was carried by expression vector pAUR123 and introduced into S.cerevisiae.One strain of genetically modified S.cerevisiae,named EC1118-LDHA,was obtained after being screened by Aureobasidin A.The genetically modified strain of S.cerevisiae EC1118-LDHA used 200 g/L glucose as carbon source,and the activity of LDHA was 4.7±1.2 mU/mg after fermentation for 24 h,and about 30 g/L lactic acid and 70 g/L ethanol were produced after fermentation for 2-3 d.The production rate of lactic acid was approximately 0.15 g/g glucose.【Conclusion】Introdu-cing the coding sequence of the bovine LDHA gene into S.cerevisiae after replacing the preferable codons of S.cerevisiae successfully constructs a genetically modified bovine LDHA gene in S.cerevisiae.The introduction of bovine LDHA gene can not affect the fermentation ability of commercial S.cerevisiae,and endow S.cerevisiae the ability to produce about 30 g/L lactic acid with 200 g/L glucose as the carbon source.Therefore,it is completely feasible to produce food-grade lactic acid by using bovine LDHA genetically modified S.cerevisiae.