Research On Salt-Tolerant Gene GPD1of Yeast During High-Salt Liquid Fermentation Of Soysauce

The quality of soy sauce, a key to the industry, depends on the flavor of soy sauce which is closely related to the yeast strains added in the fermentation process.During high-salt liquid fermentation of soy sauce, Z rouxii, is very important for the flavor formation. The concentration of salt is17%in the high salt liquid fermentation of soy sauce, which causes a high osmotic stress. In this environment, the metabolic activity of the yeast is low, where yeasts need to have the ability of the adjustment to the poor condition. Hence, a higher salt-tolerant mutant strain Z rouxii3-2(S3-2) was constructed from the wild type Z rouxii (S) using genome shuffling in our lab. The mechanism of salt-tolerant yeast S has not been yet fully, so the salt tolerance gene GPD1was studied, from comparative analysis of gene sequence, the molecular level of transcriptional protein structure and over-expression of gene to clarify the salt-tolerant mechanism of S yeast.To better understand the osmo-adaption mechanism, firstly the genes sequence of S3-2GPD1and SGPD1were identified after inserting the plasmid pUC19, respectively. It was found that three mutations occurred in the GPD1gene sequence of the strain S3-2compared with that of the strain S. In ORF, adenine (A) of S was replaced into guanine-quadruplexes (G) of S3-2at665bp; in promoter, adenine (A) was lacked in S3-2compared with S at66bp; in terminator, guanine-quadruplexes (G) was added in S3-2at77bp. After sequencing, the second structures of S3-2GPD1and SGPDI were constituted to analyze the quality of coding proteins for further research, and one mutation point (Lysine223Arginine) was changed. Secondly the transcription levels of S3-2GPD1and SGPDI at different salt content were estimated using RT-PCR analysis, and the results showed that the transcription levels and salt tolerance of S3-2GPD1were higher than those of SGPDI. Finally the plasmids YEplac195, Y-S and Y-S3-2were transformed into strain S. cerevisiae (W303-A), forming the engineered strains W-Y, W-S and W-S3-2. respectively. The results of index determination indicated that the salt-tolerance of W-S and W-S3-2were distinctly improved compared to the control W-YIn conclusion, the results suggested that the increase in salt tolerance of yeast strains was not due to changes in protein spatial structure of GPD1but because of raising the level of transcription. In addition, the increase of the level of transcription would benefit the safety of S3-2and the security of the fermentation of soy sauce. The enzyme of Gpdlp which can induct the production of glycerol can improve the ability of response to the external environment.