Heterologous Biosynthesis Of Ambrein In Engineered Escherichia Coli

Ambrein,a tricyclic triterpenoid,is the major constituent of ambergris which is one of the most valuable animal perfumery materials and traditional medicine.Ambrein is the source of ambergris odor,but also possess antinociceptive,antiinflammatory and other activities.Ambrein is generally used in the high-end perfume industry,but its rare in nature and difficulties in chemical synthesis restrict its wide range of applications in perfume and medicine industry.Nowadays,the biosynthesis of ambrein has been achieved through two-step enzyme catalytic reaction in vitro by D377 C SHC and BmeTC,in which squalene acts as substrate.Escherichia coli is a model microorganism and a good foreign gene expression vector,its endogenous isoprenoid pathway(MEP pathway)also provides a molecular basis for the biosynthesis of terpenoids and makes it possible to heterologously synthesize ambrein in engineered E.coli.In this paper,the squalene synthase gene erg9 was firstly integrated into the genome of E.coli by CRISPR-Cas9-mediated genome editing technique to construct engineered E.coli with squalene synthesis ability.The yield of squalene was 18.43mg/L.By introducing the GPP synthase from Arabidopsis thaliana into Escherichia coli,the production of squalene was increased by 1.17-fold to 21.54mg/L.After the construction of squalene synthesis module in Escherichia coli,the D377 C SHC gene and BmeTC gene were co-expressed on the plasmid vector and introduced into engineered E.coli to construct the complete ambrein synthesis pathway.The yield of ambrein in engineered E.coli was 1.47 mg / L after shaking flask fermentation.Finally,to construct complete ambrein synthesis pathway in the genome of E.coli,the integrated expression of D377 C SHC in the genome was carried out by means of genome-integrated approach.Compared with the results of D377 C SHC expression on the plasmid,the same intermediate metabolite was produced.