Study On Optimization Of Rare Ginsenoside CK Produced By Saccharomyces Cerevisiae

Rare ginsenoside CK has attracted much attention because of its various pharmacological properties such as anti-inflammatory,anti-allergic,anti-aging,anti-diabetic and protecting liver and nerves.At present,microbial and enzymatic transformation to prepare CK have high substrate cost and long transformation cycle,which is difficult to meet application requirements.De novo synthesis of microbial cell factories is one of the effective strategies to solve the shortage of CK.In this paper,on the basis of CK-producing Saccharomyces cerevisiae chassis cells,in order to increase the production of CK,the upstream pathway of CK synthesis is optimized:(1)Added flexible linker between ERG20 and ERG9 genes from Saccharomyces cerevisiae BY4742 for gene fusion,and used overlap technology to construct ERG20-ERG9 fusion box;(2)Used overlap technology to construct HMGR small box derived from Staphylococcus aureus;(3)Used Gibson assembly technology to efficiently recombine the four fragments of p AUR112 vector,URA3,HMGR and ERG20-ERG9 in yeast to construct a complete expression box;(4)Transformed the linearized expression box into the ginsenoside CK-producing chassis yeast CK9 by codon optimization of Pg DDS,CYP716A47,46 t ATR1 and UGTPg1 in the early stage of our research group;(5)Did fermentation test and detected CK and squalene production.Main research results:(1)Constructed an expression box with a size of 6963 bp that optimizes the three key rate-limiting enzyme genes of ERG20,ERG9 and HMGR,which has a Saccharomyces cerevisiae genome ? sequence at both ends;(2)Among the CK9-?UMEs strains optimized in the upstream pathway,the highest production of CK was measured to be 64.40 mg/L,which was about 3.3 times of the 19.18 mg/L yield of the initial chassis yeast CK9;(3)Fed-batch fermentation is more conducive to the growth of CK-producing yeast strains and increases CK production;(4)Optimized the upstream pathway can better increase the supply of CK precursor squalene.Conclusion:(1)Adding a flexible linker between ERG20 and ERG9 genes for gene fusion can promote the synthesis of CK by the recombinant strain;(2)Obtaining a stable strain CK9-?UME-46# with high CK production after optimizing the upstream pathway.Innovation and significance:(1)Innovation points:(1)Selected the natural truncated HMGR gene derived from Staphylococcus aureus,plus the ERG20 and ERG9 genes derived from Saccharomyces cerevisiae BY4742 to optimize the upstream synthesis pathway;(2)Added flexible linker:Gly-Ser-Thr-Ser-Ser-Gly-Ser-Gly between ERG20 and ERG9 genes for gene fusion.(2)Significance: This experiment is aimed at the optimization of the three genes of ERG20,ERG9 and HMGR,which can provide more squalene to synthesize CK,so subsequent optimization studies on CK can focus on the cell attenuation in the downstream pathway.