Abstract
N-acetylglucosamine (GlcNAc), a glucosamine derivative, has a wide range of applications in pharmaceutical fields, and there is an increasing interest in the efficient production of GlcNAc genetic engineered bacteria. In this work, Escherichia coli ATCC 25947 (DE3) strain was engineered by a model-based dynamic regulation strategy achieving GlcNAc overproduction. First, the GlcNAc synthetic pathway was introduced into E. coli, and through flux balance analysis of the genome-scale metabolic network model, metabolic engineering strategies were generated to further increase GlcNAc yield. Knock-out of genes poxB and ldhA, encoding pyruvate oxidase and lactate dehydrogenase, increased GlcNAc titer by 5.1%. Furthermore, knocking out N-acetylmuramic acid 6-phosphate etherase encoded by murQ and enhancing glutamine synthetase encoded by glnA gene further increased GlcNAc titer to 130.8 g/L. Analysis of metabolic flux balance showed that GlcNAc production maximization requires the strict dynamic restriction of the reactions catalyzed by pfkA and zwf to balance cell growth and product synthesis. Hence, a dynamic regulatory system was constructed by combining the CRISPRi (clustered regularly interspaced short palindromic repeats interference) system with the lactose operon lacI and the transcription factor pdhR, allowing the cell to respond to the concentration of pyruvate and IPTG to dynamically repress pfkA and zwf transcription. Finally, the engineered bacteria with the dynamic regulatory system produced 143.8 g/L GlcNAc in a 30-L bioreactor in 55 h with a yield reaching 0.539 g/g glucose. Taken together, this work significantly enhanced the GlcNAc production of E. coli. Moreover, it provides a systematic, effective, and universal way to improve the synthetic ability of other engineered strains.