Metabolic Engineering of the 5-Aminolevulinate Biosynthetic Pathway in E. coli Improves Efficiency of...

Biocatalysis using heme-dependent enzymes provides a powerful synthetic platform to facilitate a variety of chemical transformations required for organic synthesis. Despite recent advances in biocatalysis, recombinant expression systems for hemoproteins leave much room for improvement due to the strict regulation of heme biosynthesis in the host organism. To develop an efficient cofactor supplementation system for the expression of active holohemoproteins, we describe metabolic engineering of the heme biosynthetic pathway in E. coli. Through incorporation of a heterogeneous C4 pathway involving 5-aminolevulinic acid synthase of Paracoccus denitrificans, it was found that the concentrations of 5-aminolevulinic acid and heme in the engineered cells are increased during cultivation, and the expression level of the holohemoproteins is significantly improved. Notably, the heme content in the engineered cells is even higher than that produced by conventional cultivation methods, which add 5-aminolevulinic acid into the culture medium. Furthermore, we also demonstrate the application of this engineered E. coli cells in whole-cell and lysate-based biocatalysis using various types of heme-dependent enzymes. Considering the recent demand for biocatalysis, the system developed in this study will serve as a new practical and versatile platform for hemoprotein-based biocatalysis.