Semi-rational Modification Of Cytochrome P450 CYP72A154 And Application In The Synthesis Of Glycyrrhetinic Acid By Yeast

Glycyrrhetinic acid is a kind of ingredient used in medicinal herbs,and has various pharmacological activities such as antiviral,antibacterial,anti-AIDS,and prevention and treatment of tumors,and is widely used in various fields such as food,cosmetics,and medicine.In the early stage of the laboratory,a yeast engineering strain C-CPR12 with glycyrrhetinic acid was synthesized,but the yield of glycyrrhetinic acid was very low.Among them,11-oxo-β-amyrin catalyzed by P450 enzyme CYP72A154to glycyrrhetinic acid.The oxidation process is an important limiting step in the synthesis of glycyrrhetinic acid.Therefore,based on the previous studies,the design of the 11-oxo-β-amyrin pathway was carried out with the haploid Saccharomyces cerevisiae CENPK2-1C as host,and the semi-rational mutation of CYP72A154 was carried out.It is expected to improve the catalytic activity of the enzyme and the level of synthesis of glycyrrhetinic acid.The main findings are as follows:Theβ-amyrin synthetase gene bAS（derived from Glycyrrhiza glabra）,the monooxygenase gene CYP88D6（derived from Glycyrrhiza uralensis fisch）and the cytochrome reductase gene AtCPR1（derived from Arabidopsis thaliana）were amplified using a laboratory-preserved plasmid as templates.The gene expression cassette was constructed by OE-PCR method,and the 11-oxo-β-amyrin synthesis pathway was successfully integrated into the yeast strain CEN.PK2-1C to obtain the engineering yeast CO.The 11-oxo-β-amyrin was successfully synthesized by gas chromatography-mass spectrometry.The yield reached 8.37 mg/L,indicating the successful construction of 11-oxo-β-amyrin in yeast.The synthetic pathway provides a host basis for semi-rational transformation of CYP72A154.In order to increase the activity of CYP72A154,the functional sites of P450 enzyme substrate binding pockets（SRSs）were analyzed by CYP450 database,and six mutation sites were identified.Six mutants and yeast strains（V148T,G238F,W344Y,I382Y,R446A,L508V）of CYP72A154 were successfully obtained by site-directed mutagenesis.The levels of active oxygen produced by redox reaction and the synthesis level of glycyrrhetinic acid were determined.The favorable mutation V148T found that the engineered strain V148T had the lowest level of intracellular and extracellular reactive oxygen species,and had little cytotoxicity.The glycyrrhetinic acid synthesis reached the highest value of46.812μg/L,which was 1.59 times higher than that of the control strain GAO.In order to improve the supply of precursors of yeast engineering yeast in the synthesis of glycyrrhetinic acid,the engineering strain ScM（overexpressing acetoacetyl-CoA transferase gene ERG10,HMG-CoA synthase gene ERG13,HMG reductase gene tHMG and other genes）and Sgib（overexpression of prenyl diphosphate isomerase IDI,pyrophosphate synthase ERG20,squalene synthase ERG9 and other genes）were used as a host and introduced the synthetic pathway of 11-oxo-β-amyrin and the mutant pRS42K-V148T,the engineering yeast GA1 and GA2 was constructed and produced 96.5μg/L and 83.7μg/L of glycyrrhetinic acid,respectively,which were 106%and 78.8%higher than the optimized V148T strain.Further,from the redox system,the production of glycyrrhetinic acid was increased to 125.92μg/L by co-expression of Saccharomyces cerevisiae cytochrome oxidase gene CYB5and Arabidopsis thaliana cytochrome reductase AtCPR2.By overexpressing glucose-6-phosphate dehydrogenase ZWF1 and NADH kinase POS5,the intracellular NADPH supply level was enhanced,and the glycyrrhetinic acid yield was 182.37μg/L.The fermentation conditions were optimized from the basic fermentation conditions of carbon source,nitrogen source,pH,temperature and inoculum.The optimal fermentation conditions were determined:glucose 10 g/L,peptone 20 g/L,inoculum OD₆₀₀=0.1 The pH was 6,and the fermentation temperature was 28°C.Using ethanol feed fermentation,the yield of glycyrrhetinic acid reached 782.6μg/L,which was 42.43 times higher than the initial control yeast strainThe above results indicated that the glycyrrhetinic acid synthesis pathway was successfully constructed in Saccharomyces cerevisiae,and optimized by key enzymes,pathways and fermentation processes,significantly improved the synthesis level of glycyrrhetinic acid.