ABSTRACT
In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in β-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 β-amyrin production. Compared with the control strain, the production of β-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the β-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 β-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.
Subject(s)
Ethanol , Fermentation , Metabolic Engineering , Saccharomyces cerevisiae , GeneticsABSTRACT
Objective:To reveal the dynamic changes of flavonoids secondary metabolites and relevant genes expressions in the process of germination of tartary buckwheat seeds by investigating the content of catechins，epicatechins，rutin，and quercetin，and the expressions of their relevant genes in tartary buckwheat sprouts and seedlings，in order to provide scientific basis for the selection of high-quality, high-nutrition tartary buckwheat sprouts.Method:Contents of catechin，epicatechin，rutin，and quercetin in tartary buckwheat sprouts and seedlings were detected by UPLC-ESI-QQQ-MS，and the expression levels of genes relating to flavonoids synthesis in tartary buckwheat sprouts and seedlings were detected by real-time quantitative PCR.Result:There were differences between tartary buckwheat sprouts and seedlings in the relative contents of catechin，epicatechin，rutin and quercetin，as well as the expressions of relevant genes in the synthesis pathway, including FtPAL，FtC4H，Ft4CL，FtCHS，FtCHI，FtF3H，FtF3'H，FtFLS，FtDFR，FtLAR，FtANS，FtANR. The contents of flavonoids and the expressions of relevant genes in tartary buckwheat sprouts were higher than those in tartary buckwheat seedlings.Conclusion:The higher accumulation of secondary metabolites and flavonoids in tartary buckwheat sprouts may be related to tartary buckwheat seeds' resistance to the external environment in the initial growth stage of germination. From the perspective of application，there are more flavonoids in tartary buckwheat sprouts than in tartary buckwheat seedlings, indicating that tartary buckwheat sprouts have a higher nutritional value.
ABSTRACT
In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-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 β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-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 β-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 β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.