Abstract
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 PT7 and Pcat 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.