Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia. Here, we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase (bFMO) for the efficient production of indirubin in Escherichia coli. A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity, resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5- and 2.0-fold, respectively. A combined modification at the two regions (K223R/D317S) achieved a 6.6-fold improvement in catalytic efficiency (kcat/Km) due to enhancing π–π stacking interactions stabilization. Finally, an engineered E. coli strain was constructed by metabolic engineering, which could produce 860.7 mg/L (18 mg/L/h) indirubin, the highest yield ever reported. This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin. Graphical Abstract

Background: The production of bioactive compounds using microbial hosts is considered a safe, cost competitive and scalable approach. However, the efficient engineering of cell factories with well stability, such as for the production of L-aspartate family amino acids and derivatives, remains an outstanding challenge.Results: In the work, the toxin/antitoxin system and genome modification strategy were used to construct a stable Escherichia coli strain for L-homoserine production. The metabolic engineering strategies were focused on the enhancement of precursors for L-homoserine synthesis, reinforcement of the NADPH generation and efflux transporters using CRISPR-Cas9 system at the genome level. To improve the plasmid stability, two strategies were explored, including construction of the aspartate-auxotrophic and hok/sok systems. Constructing the auxotrophic complementation system to maintain plasmid stability was failed herein. The plasmid stability was improved by introducing the hok/sok system, resulting in 6.1 g/L (shake flask) and 44.4 g/L (5 L fermenter) L-homoserine production of the final engineered strain SHL19 without antibiotics addition. Moreover, the hok/sok system was also used to improve the plasmid stability for ectoine production, resulting in 36.7% and 46.5% higher titer of ectoine at shake flask and 5L fermenter without antibiotics addition, respectively. Conclusion: This work provides valuable strategies to improve plasmid stability for producing L-aspartate family amino acids and derivatives and eliminate environmental concerns associated with the application of antibiotics.