A Combinational Optimization Method for Efficient Production of Indigo by the Recombinant Escherichia coli with Expression of Monooxygenase and Malate Dehydrogenase.

Indigo pigment is a widely used pigment, and the use of biosynthesis to ferment indigo has become a hot research topic. Based on previous research, the indigo could be biosynthesized via the styrene oxygenation pathway, which is regulated by intracellular redox-cofactor rebalancing. In this work, the malate dehydrogenase ( mdh ) gene was selected as an NADH regeneration element to improve the intracellular cofactor regeneration level, and it was co-expressed with the styrene monooxygenase ( styAB ) gene by pET-28a(+) vector in E. coli for enhancing indigo production. The P T7 and P cat promoter was constructed to change the styAB gene and mdh gene from inducible expression to constitutive expression, since the expressing vector pET-28a(+) needs to be induced by IPTG. After different strategies of genetic manipulations, the styAB gene and mdh gene were successfully constitutively co-expressed by different promoters in E. coli , which obviously enhanced the monooxygenase activity and indigo production, as expected. The maximum yield of indigo in recombinant strains was up to 787.25 mg/L after 24 h of fermentation using 2.0 g/L tryptophan as substrate, which was nearly the highest indigo-producing ability using tryptophan as substrate in recent studies. In summary, this work provided a theoretical basis for the subsequent study of indigo biosynthesis and probably revealed a new insight into the construction of indigo biosynthesis cell factory for application.