Restructuring Of Biosynthetic Pathway Of Physcion In Saccharomyces Cerevisiae

Anthraquinone physcion and emodin have biological activities such as antiviral,anti-inflammatory,antibacterial,immunosuppressive and antitumor,so they have been widely concerned by scholars at home and abroad.The predecessors have produced emodin on the scale of preparation of wild-type Aspergillus ochrae,and the yield is higher than that isolated from medicinal materials.Therefore,exploring the biosynthesis of fungi-derived physcion is of great significance for its efficient production.At present,the biosynthesis from emodin to physcion is not yet clear.In vitro studies have shown that emodin is catalyzed into physcion by methyltransferase.Therefore,we propose that the precursor of physcion is likely to be emodin,which is catalyzed by a specific oxygen-methyltransferase to hydroxymethylate the C₆ position of emodin to produce physcion.In order to reconstruct the biosynthetic pathway of physcion in Saccharomyces cerevisiae,this study first excavated 16 candidate oxygen-methyltransferases based on bioinformatics,and preferentially selected 5 enzymes in E.coli for functional identification through biotransformation experiments,they are AurJ,Fsr2,Bik3,PtaI,and PgmB.The experimental results show that in addition to AurJ,Fsr2,Bik3,PtaI and PgmB can catalyze emodin to produce physcion,of which the concentration of physcion in PtaI and PgmB is approximately 19.72μg/mL and 18.87μg/mL,respectively,the catalytic effect is higher than the other two enzymes,and successfully screened emodin oxygen-methyltransferase.Secondly,in this experiment,an engineering strain of Saccharomyces cerevisiae that produced physcion precursor,emodin,was constructed.Using the delta site,NpgA was successfully integrated into the genome of yeast W303 to construct W303-NpgA engineering strain.Expressing polyketide synthase and thioesterase SlACAS-HyTE in W303-NpgA and BJ5464-NpgA,and a small amount of by-product emodin was detected.The experimental results show that W303-NpgA and BJ5464-NpgA can successfully express polyketide synthase,which is an ideal host strain for heterologous synthesis of polyketide products.To increase emodin production,the point mutations ACC1^{ser659ala,ser1157ala}(ACC1^**)and wild-type ACC1 were integrated into the r DNA multicopy sites of the W303-NpgA and BJ5464-NpgA genomes,respectively,to construct W303-NpgA-ACC1,W303-NpgA-ACC1^**,BJ5464-NpgA-ACC1 and BJ5464-NpgA-ACC1^**engineering strains with over-expressed malonyl-Co A precursors.Then expressed SlACAS-HyTE,the test results showed that the yield of emodin increased by 1.5,2.7,1.7 and 3.4 times respectively,the highest was 0.64μg/mL.Finally,in order to explore whether the heterologous synthesized emodin and physcion have an inhibitory effect on Saccharomyces cerevisiae cells,an antibacterial experiments with different concentration gradients were conducted by K-B paper diffusion method,and the results showed that their minimum inhibitory concentration was 8 mg/mL,has little effect on subsequent engineering strains that efficiently synthesize emodin and physcion.In this paper,genetic engineering was used to construct an engineering strain of Saccharomyces cerevisiae that produced polyketides,and realized the heterologous synthesis of emodin in Saccharomyces cerevisiae strains.Four fungus-derived emodin oxygen-methyltransferase were screened by in vitro functional identification,laying the foundation for the future construction of the biosynthetic pathway of physcion in Saccharomyces cerevisiae.