Reprogramming Escherichia coli metabolism for butyrolactam production by the temperature-powered multi-genes control circuit

Biomanufacturing offers a promising alternative for the production of the building block of nylons, of which butyrolactam is an important platform chemical. To date, there is few studies on butyrolactam biosynthesis, and static control characterized by orientation, irreversibility and ineffectiveness often impedes synthesis efficiency. Here, the chassis Escherichia coli was systematically engineered for butyrolactam synthesis by stepwise optimization strategies. In particular, inadequate α-ketoglutarate pool was identified as the main bottleneck, and then the CRISPRa and CRISPRi served as ON- and OFF-switch for establishing the temperature-powered bioswitch (thereafter renamed AITB circuit), achieving simultaneous transcriptional down- and up-regulation of the multi-genes. In the fed-batch culture, the highest titer of 59.87 g/L butyrolactam was produced by redirecting α-ketoglutarate catabolism. Further, with cassava starch as the substrate, a total of 14.96 g/L butyrolactam was generated, providing the possibility for bioproduct synthesis from renewable feedstock. This study provides efficient tool for developing high-performance biorefineries for bio-based compounds production of interest.