Bioretrosynthesis of Functionalized N‐Heterocycles from Glucose via One‐Pot Tandem Collaborations of Designed Microbes

Abstract The design of multistrain systems has markedly expanded the prospects of using long biosynthetic pathways to produce natural compounds. However, the cooperative use of artificially engineered microbes to synthesize xenobiotic chemicals from renewable carbohydrates is still in its infancy. Here, a microbial system is developed for the production of high‐added‐value N‐heterocycles directly from glucose. Based on a retrosynthetic analysis, eleven genes are selected, systematically modulated, and overexpressed in three Escherichia coli strains to construct an artificial pathway to produce 5‐methyl‐2‐pyrazinecarboxylic acid, a key intermediate in the production of the important pharmaceuticals Glipizide and Acipimox. Via one‐pot tandem collaborations, the designed microbes remarkably realize high‐level production of 5‐methyl‐2‐pyrazinecarboxylic acid (6.2 ± 0.1 g L⁻¹) and its precursor 2,5‐dimethylpyrazine (7.9 ± 0.7 g L⁻¹). This study is the first application of cooperative microbes for the total biosynthesis of functionalized N‐heterocycles and provides new insight into integrating bioretrosynthetic principles with synthetic biology to perform complex syntheses.