De Novo Biosynthesis of 1,5-Pentanediol by Metabolically Engineered Escherichia coli.

1,5-Pentanediol (1,5-PDO) is a five-carbon aliphatic diol widely used as a raw material for synthesis of polyurethanes, polyesters, plastics, or fibers. Recently, the de novo synthesis of 1,5-PDO has been established, but the accumulation of intermediates and low yield of product limit its further application. In this study, based on the l-lysine high-producing Escherichia coli, an efficient microbial cell factory containing a 5-hydroxyvalerate synthesis module (5-HV) and a 1,5-PDO synthesis module was designed. By screening the enzymes of different modules, a 1,5-PDO-synthesizing recombinant strain with the best combination of MmCAR, Yahk, and GabT was obtained. The amino acid residues in the adenosine domain of CAR were rationally mutated to glutamic acid to obtain the variant MmCARQ302E, which had enhanced activity against 5-HV and reduced its accumulation. Subsequently, the accumulation of 5-HV was further reduced by enhancing the expression of CAR through RBS engineering and fixing CAR with the help of EutM protein scaffold. In addition, the endogenous gene ycjQ of E. coli was deleted to reduce the reoxidation of 1,5-PDO. Finally, the 1,5-PDO yield reached 12.9 g/L under the optimized fermentation conditions, achieving efficient biosynthesis of 1,5-PDO and lower accumulation of 5-HV, which is the highest yield reported in E. coli so far.