Whole‐Cell P450 Biocatalysis Using Engineered Escherichia coli with Fine‐Tuned Heme Biosynthesis

Abstract

By exploiting versatile P450 enzymes, whole-cell biocatalysis can be performed to synthesize valuable compounds in Escherichia coli. However, the insufficient supply of heme limits the whole-cell P450 biocatalytic activity. Here a strategy for improving intracellular heme biosynthesis to enhance the catalytic efficiencies of P450s is reported. After comparing the effects of improving heme transport and biosynthesis on P450 activities, intracellular heme biosynthesis is optimized through the integrated expression of necessary synthetic genes at proper ratios and the assembly of rate-limiting enzymes using DNA-guided scaffolds. The intracellular heme level is fine-tuned by the combined use of mutated heme-sensitive biosensors and small regulatory RNA systems. The catalytic efficiencies of three different P450s, BM3, sca-2, and CYP105D7, are enhanced through fine-tuning heme biosynthesis for the synthesis of hydroquinone, pravastatin, and 7,3′,4′-trihydroxyisoflavone as example products of chemical intermediate, drug, and natural product, respectively. This strategy of fine-tuned heme biosynthesis will be generally useful for developing whole-cell biocatalysts involving hemoproteins.

Efficient hydroxylation of flavonoids by using whole-cell P450 sca-2 biocatalyst in Escherichia coli

“…Since the CYP105 family is a three-component P450 enzyme, the most widely studied redox partners CamA (putidaredoxin reductase) and CamB (putidaredoxin) were employed in the whole-cell biocatalysis to transfer electrons from NAD(P)H to the heme-iron reactive center for O 2 activation. Thus, the genes encoding CamA and CamB were co-expressed with these five P450s genes using a pRSFDuet-1 plasmid in the C41(DE3) strain ( Hu et al, 2022a ), respectively, resulting in HFLA-1 to HFLA-5 strains. Subsequently, the hydroxylation of flavonoids by whole-cell as biocatalysts was compared using naringenin as a model substrate ( Figures 2A, B ).…”

“…P450s are thought to be reliable, effective, and ecofriendly biocatalysts for the synthesis of valuable compounds in recombinant hosts. In addition, compared to the utilization of purified or extracted P450s, whole-cell biotransformation has shown a clear advantage by providing the necessary precursors, the expensive cofactors NAD(P)H, and suitable environments for catalytic reactions ( Hu et al, 2022a ). Moreover, to exploit the versatile P450s for industrial applications, Escherichia coli is a widely applied and efficient system for whole-cell biotransformation ( Park et al, 2020 ).…”

Production of Natural Pigments Using Microorganisms

“…53 Hu et al effectively increased the heme content in E. coli by expressing the heme synthesis gene in appropriate proportions and assembling rate-limiting enzymes using DNA scaffolds. 101 Organic integration of various strategies may further enhance the production of porphyrins synthesized by microorganisms.…”

Expression and Characterization of a New Self‐Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

“…It has been reported that increased ALA concentration leads to decreased P450 content, due to the toxic effect of heme on cells. [28] Furthermore, the heme group contains iron in its center, and thus iron supplementation also affects the efficient production of heme and correctly folded P450 protein. Therefore, different concentrations of FeCl 3 were added to the expression media.…”