RESUMEN
cis, cis-muconic acid (MA) is an important platform chemical. Now, majority of reported engineered strains are genetically instable, the exogenous genes are expressed under the control of expensive inducer and the components of their fermentation medium are complex, thus large-scale microbial production of MA is limited due to the lack of suitable strains. Hence, it is still necessary to construct novel high-performance strain that is genetically stable, no induction and grows in simple inorganic fermentation medium. In this study, after 3 exogenous genes (aroZ, aroY, catA) for biosynthesis of MA were integrated into previously constructed 3-hydroshikimate producing Escherichia coli WJ060 strain and combinatorially regulated with 3 constitutive promoters with different strengths, 27 engineered strains were constructed. The best engineered strain, E. coli MA30 could produce 1.7 g/L MA in the simple inorganic fermentation medium without induction. To further enhance the production capacity of MA, the mutant library of E. coli MA30 was constructed by genome replication engineering and screened via high-throughput assay. After two-round screening, the new strain, E. coli MA30-G2 with improved production of MA was obtained, and the titer of MA increased more than 8%. Under the condition of 5 L fed-batch fermentation, E. coli MA30-G2 could produce about 11.5 g/L MA. Combinatorial regulation and high-throughput screening provide important reference to microbial production of other bio-based chemicals.