Construction And Optimization Of Glycyrrhetinic Acid Artificial Synthetic Pathway In S.cerevisiae

Glycyrrhetinic acid is a pentacyclic terpene compound and is widely used in medicine,cosmetics and food and other fields due to its wide range of pharmacological activity and special intermediates structure.Glycyrrhetinic acid has anti-inflammatory,antiviral,hypolipidemic,prevention and treatment of cancer and a variety of anti AIDS effect,is also a very important antidote.Glycyrrhetinic acid is mainly produced by hydrolysis of glycyrrhizic acid,whose production does not conform to the concept of green development,bacacse of complex production process,long cycle,high energy consumption,environmental pollution,high cost and low product yield.With the continuous development of metabolic engineering and synthetic biology,artificial construction of microorganisms in the natural products synthesis has shown a unique advantage,and has become the focus of international industrial biotechnology research.In this study,by means of molecular biology and metabolic engineering,the introduction of heterologous glycyrrhetinic acid synthesis pathway in Saccharomyces cerevisiae,optimization of controlling pathway and fermentation,and changing the source of carbon source,provide Saccharomyces cerevisiae in the high yield of glycyrrhetinic acid.The results of the study the following:Through the literature retrieval and analysis of the whole genome sequence of Saccharomyces cerevisiae,we determined the promoter and terminator to control the functional gene.According to the codon preference of Saccharomyces cerevisiae,we optimized the codon of ?-amyrin synthase b AS,P450 monooxygenase CYP88D6 and CYP72A154 from Glycyrrhiza glabra,then cloned the P450 reductase At CPR1 and At CPR2 in Arabidopsis thaliana.According to promoter-functional gene-terminator pattern,we constructe gene expression cassette by PCR,including beta amyrin synthase,P450 monooxygenase and P450 reductase,besides we integrated by homologous recombination and overlap extension into haploid Saccharomyces cerevisiae CEN.PK2-1C genome,realizing construction of synthetic pathway of glycyrrhetinic acid.HPLC and mass spectrometry was used to demonstrate the successful synthesis of glycyrrhetinic acid,the yield reached 25.5?g/L.Then,the suitability of glycyrrhetinic acid synthesis pathway and the chassis host were analyzed that we designed the glycyrrhetinic acid synthesis pathway to different host chassis,by haploid Saccharomyces cerevisiae CEN.PK2-1C,CEN.PK2-1D and diploid INVSc1 respectively.The fermentation results showed that the haploid Saccharomyces cerevisiae was more favorable for the synthesis of glycyrrhetinic acid,which was 100% higher than that of the diploid Saccharomyces cerevisiae INVSc1.We use linear precursor 2,3-oxidosqualene of triterpene synthesis as control node,overexpress of endogenous genes squalene monooxygenase(ERG1)in Saccharomyces cerevisiae responsible for squalene into 2,3-oxidation of squalene,and coexpress of squalene synthase(ERG9)in order to improve the squalene supply,increasing the yield of glycyrrhetinic acid to 36.1 ?g/L.From the point of view of fermentation engineering,the engineering yeast Saccharomyces cerevisiae was cultured in fed batch fermentation.The fermentation results showed that the yield of glycyrrhetinic acid increased by 71.8% when the feed batch fermentation used glucose as carbon source,and the yield reached 48.6?g/L when the feed batch fermentation adds ethanol,increasing by 73% than glucose fed batch fermentation.The results showed that ethanol as carbon source was more favorable for the synthesis of glycyrrhetinic acid.