Construction And Application Of PGK Promoter Library In Saccharomyces Cerevisiae

Acetate ester is the main aroma component of light-aroma Chinese Baijiu.Acetate ester is one of the most important volatile flavor substances that determine the quality and aroma of Chinese Baijiu,too.The acetate esters contents are different among the different types or different brands of Baijiu.Therefore,it is necessary to construct a series of Saccharomyces cerevisiae recombinant strains,which producing different levels of acetate esters.It is important to improve the quality of Chinese Baijiu.In metabolic engineering,promoter library is an important regulatory approache to regulate the expression of target gene precisely.In this study,PGK promoter library was constructed to precise regulate alcohol acetyltransferases encoded gene ATFI.These recombinant strains could generate different contents of acetate esters in Chinese Baijiu.The main research contents are as follows:(1)PGK promoter was mutated through EP-PCR and these PGKp mutants were screened by EGFP reporter gene.Twenty-eight mutants were selected to constitute the PGK promoter library and activity range between 10%and 141%of wild-type PGKp.The(p-galactosidase encoded gene lacZ was used as second reporter gene to assay the activity of mutants 8,17,18,22,25,27,28.The accuracy of EGFP reporter gene was confirmed and activity of seven mutants was 8<17<18<22<wild-type<25<27<28.(2)Seven selected promoters(8,17,18,22,25,27,28)were sequenced to analyze the the mutation site and these promoters were inserted into plasmid pUC-19.The locations of upstream activation sequence and several important transcription factors(Gcr1p,Rap1p,Abf1p,and Reb1p)were summarized by bioinformatics analysis.The base mutation located on the promoter element may be one of the reasons for the change of promoter activity.(3)Seven selected promoters(8,17,18,22,25,27,28)and wild-type PGKp were used for the overexpression of ATF1 with BAT2 deletion in Saccharomyces cerevisiae a45,namely a45-A8,a45-A17,a45-A18,a45-A22,a45-A25,a45-A27,a45-A28 and a45-AWT.The productions of ethyl acetate by these strains were 18.64,34.67,118.88,1 79.57,254.64,146.73,270.24,305.68 mg/L,respectively.Furthermore,the mRNA level of ATF1 gene and the activity of AATase were assayed.The results showed that different activity PGK promoter muants could regulate ATF1 gene expression and acetate ester production precisely.The results show that the activity of PGK promoter library varies widely and it will have a good application prospect.The PGK promoter library could regulate ATF1 gene expression precisely and recombinant strains could product different levels of acetate esters.The ability of PGKp mutant to regulate ATF1 gene was verified at ATF1 gene transcriptional level and alcohol acetyltransferase enzyme activity level.These strains meet the requirements of different styles and brands of Chinese Baijiu for different acetate ester content.These recombinant strains could improve the quality and aroma flavor in Chinese Baijiu.