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.

Automated summary

In this study, the malate dehydrogenase (mdh) gene was selected as an NADH regeneration element to enhance the intracellular cofactor regeneration level and improve indigo production in E. coli. The constitutive co-expression of the styrene monooxygenase (styAB) gene and mdh gene was successfully achieved by different promoters, resulting in increased monooxygenase activity and indigo production. The findings of this study provide a theoretical basis for further research on indigo biosynthesis and offer insights into the construction of indigo biosynthesis cell factories for practical applications.