Heterologous Biosynthesis And Optimization Of Soyasapogenol B In Engineered Saccharomyces Cerevisiae

Soyasapogenol B,an oleanane-type pentacyclic triterpene,can be obtained from soybean saponins through acid,microorganism or enzymatic hydrolysis,and has higher biological activity than soybean saponins.It is widely used in the pharmaceutical industry.Due to the low content of saponins in defatted soybeans,it is difficult to extract and purify them.Using synthetic biology and metabolic engineering methods to construct genetically engineered bacteria,heterologous synthesis of soyasapogenol B with important biological activities provides new hope for the struggle between humans and diseases.In this study,we first constructed a synthetic pathway for the precursor substance?-amyrin,by regulating the expression of key genes in the MVA pathway,studying the effects of single-copy and multi-copy expression of genes on yield,and studying the effects of different types of promoters on yield.The yield of ?-amyrin increased to17.6 mg/L,which was 25.8 times that of the original strain L01(0.68 mg/L).Secondly,to construct an engineering Saccharomyces cerevisiae strain that produces soyasapogenol B,select two sources of P450 enzymes and two sources of CPR permutation and combination,and carry out a suitability study.The yield of soya sapogenol B reached 0.96 mg/L.However,the CPR transmembrane domain derived from Glycyrrhiza uralensis was predicted,and the amino acid sequence of the transmembrane region was truncated.It was found that the product could not be detected after truncation,and the growth amount of the strain decreased by 60%compared with that of the complete gene expression.Optimize the expression of GgCYP and GgCPR to maximize the activity of the two P450 enzymes.Three Linkers of different lengths were selected to perform fusion expression and co-expression experiments of two P450 enzymes and reductase respectively.Protein fusion promotes the transfer of electrons between redox systems and improves the production of soyasapogenol B.After the first P450 enzyme CYP93E3 was fused with three different Linkers and the second P450 was co-expressed with CPR,a negative effect was produced.After CYP93E3 was co-expressed with CPR and CYP72A566 was fused with Linker 3,strain L18-9 was obtained.the production of soyasapogenol B was increased to 2.9 mg/L.Finally,Through optimizing the fermentation conditions in the shake flask and supplementing glucose fermentation in the 5L fermentor,the production of soyasapogenol B reached 5.81 mg/L,which was 41.5 times higher than the initial strain L06.