[Regulation of ß-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells].

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in ß-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the ß-amyrin production. Compared with the control strain, the production of ß-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the ß-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of ß-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

[Study of heterologous efficient synthesis of ß-amyrin and high-density fermentation].

In this study, the synthetic pathway of ß-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing ß-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced ß-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of ß-amyrin synthesis for further improving ß-amyrin production, resulting the strain Y2-C2-4 which produced ß-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of ß-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of ß-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of ß-amyrin-based triterpenoids.