Construction of bovine LDHA genetically modified Saccharomyces cerevisiae and analysis of its ability in producing lactic acid

【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 introduced 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 production 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 sequence 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 in α-helix and irregular coil. After replacement, the coding 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】Introducing 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 30g/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.