Improved production of the non-native cofactor F420 in Escherichia coli

Abstract

The deazaflavin cofactor F420 is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F420 biosynthesis to optimize F420 production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F420 biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space–time yield of F420 compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F420-production system and will allow the recombinant in vivo use of F420-dependent enzymes for biocatalysis and protein engineering applications.

Introducing an Artificial Deazaflavin Cofactor in Escherichia coli and Saccharomyces cerevisiae

“…Recently, Shah et al showed that the F 420 yield in E. coli can be increased up to 2.33 μmol L –1 by varying carbon sources, which demonstrates the potential for improving non-natural cofactor production by using simple methods. 14 The FOP yield presented here closely resembles the F 420 yield from M. smegmatis , which is 1.43 μmol L –1 . The estimated FOP productivity per unit of time (0.078 μmol L –1 h –1 ), however, is much higher than that of F 420 by M. smegmatis .…”

“…coli can produce the cofactor as well, albeit with lower yields than that produced naturally in M. smegmatis (∼27 nmol L –1 of culture in E. coli, compared to 1.43 μmol L –1 in M. smegmatis ). ,, The heterologous production yield of the cofactor can be improved through optimizing growth conditions as it was demonstrated in a follow-up study …”

“… 10 , 12 , 13 The heterologous production yield of the cofactor can be improved through optimizing growth conditions as it was demonstrated in a follow-up study. 14 …”

Asymmetric ene-reduction of α,β-unsaturated compounds by F420-dependent oxidoreductases A (FDOR-A) enzymes fromMycobacterium smegmatis

“…Alongside recent work incorporating F420 biosynthesis into common microbial cell factories, such as E. coli, 13,[23][24][25] this work should facilitate the wider adoption of FDORs as biocatalysts.…”

“…Using induced-fit computational docking, plausible Michaelis structures were obtained from which we could rationalize the observed enantioselectivity and propose a mechanism for the reduction of the substrates. Alongside recent work incorporating F 420 biosynthesis into common microbial cell factories, such as E. coli , 13, 23–25 this work should facilitate the wider adoption of FDORs as biocatalysts.…”