Combining Protein Engineering and Pathway Engineering to Achieve Green Biosynthesis of l-Hypaphorine in Escherichia coli from Glucose

l-Hypaphorine (l-HYP) is an l-tryptophan-derived alkaloid with significant medicinal value. The objective of this study tried to use Escherichia coli as a chassis cell to produce l-HYP from glucose by fermentation. To do this, the optimized methyltransferases were first mined based on the data of the phylogenetic tree of methyltransferase, indicating that the methyltransferase from Mycobacterium smegmatis with two mutation sites (i.e., EgtDM252 V,E282A and MsE) was designed as the best enzyme for producing l-HYP using l-tryptophan as a substrate. After this, a mutant MsET163G with high catalytic efficiency was constructed by semirational directed evolution focusing on the substrate binding cavity. To achieve the self-supply of l-tryptophan (l-Trp) and S-adenosyl methionine (SAM) in E. coli, the biosynthetic pathway of l-Trp and SAM was reconstructed. As a result, the final strain E. coli HYP-12 could produce 200.5 ± 2.6 mg/L of l-HYP with a glucose conversion efficiency (α) of 2.01% in shake-flask fermentation. This is the first report on producing l-HYP from glucose by microbial fermentation, which provides a real possibility for industrial l-HYP production using environmentally friendly methods rather than extraction from plant and provides a general concept for constructing other nonendogenous products' high-producing strains.