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 expressi… Show more
“…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 ).…”
Section: Resultsmentioning
confidence: 99%
“…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 ).…”
The hydroxylation is an important way to generate the functionalized derivatives of flavonoids. However, the efficient hydroxylation of flavonoids by bacterial P450 enzymes is rarely reported. Here, a bacterial P450 sca-2mut whole-cell biocatalyst with an outstanding 3′-hydroxylation activity for the efficient hydroxylation of a variety of flavonoids was first reported. The whole-cell activity of sca-2mut was enhanced using a novel combination of flavodoxin Fld and flavodoxin reductase Fpr from Escherichia coli. In addition, the double mutant of sca-2mut (R88A/S96A) exhibited an improved hydroxylation performance for flavonoids through the enzymatic engineering. Moreover, the whole-cell activity of sca-2mut (R88A/S96A) was further enhanced by the optimization of whole-cell biocatalytic conditions. Finally, eriodictyol, dihydroquercetin, luteolin, and 7,3′,4′-trihydroxyisoflavone, as examples of flavanone, flavanonol, flavone, and isoflavone, were produced by whole-cell biocatalysis using naringenin, dihydrokaempferol, apigenin, and daidzein as the substrates, with the conversion yield of 77%, 66%, 32%, and 75%, respectively. The strategy used in this study provided an effective method for the further hydroxylation of other high value-added compounds.
“…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 ).…”
Section: Resultsmentioning
confidence: 99%
“…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 ).…”
The hydroxylation is an important way to generate the functionalized derivatives of flavonoids. However, the efficient hydroxylation of flavonoids by bacterial P450 enzymes is rarely reported. Here, a bacterial P450 sca-2mut whole-cell biocatalyst with an outstanding 3′-hydroxylation activity for the efficient hydroxylation of a variety of flavonoids was first reported. The whole-cell activity of sca-2mut was enhanced using a novel combination of flavodoxin Fld and flavodoxin reductase Fpr from Escherichia coli. In addition, the double mutant of sca-2mut (R88A/S96A) exhibited an improved hydroxylation performance for flavonoids through the enzymatic engineering. Moreover, the whole-cell activity of sca-2mut (R88A/S96A) was further enhanced by the optimization of whole-cell biocatalytic conditions. Finally, eriodictyol, dihydroquercetin, luteolin, and 7,3′,4′-trihydroxyisoflavone, as examples of flavanone, flavanonol, flavone, and isoflavone, were produced by whole-cell biocatalysis using naringenin, dihydrokaempferol, apigenin, and daidzein as the substrates, with the conversion yield of 77%, 66%, 32%, and 75%, respectively. The strategy used in this study provided an effective method for the further hydroxylation of other high value-added compounds.
“…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.…”
Section: Metabolic Pathways Of Natural Porphyrin Pigmentsmentioning
Pigments are involved in many aspects of human life, including food, cosmetics, and textiles. At present, the pigment market is mainly occupied by synthetic pigments. However, synthetic pigments have gradually presented safety and environmental problems. Therefore, humans have begun to focus on the use of natural pigments. In contrast to the extraction of pigments from plants and animals, the production of natural pigments by microbial fermentation is not affected by season and region. This review highlights recent advances in microbial synthesis of natural pigments, classifying them into various groups, including flavonoids, isoprenoids, porphyrins, N-heterocyclics, polyketides, and others. The biosynthetic pathways for each group are elucidated along with the latest progress made in enhancing production efficiency for both natural and non-natural microorganisms. Additionally, challenges associated with economically producing natural pigments using microorganisms are also discussed. This review provides a reference for researchers to replace synthetic pigments with natural pigments.
“…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.…”
Section: Enhanced Expression Of the P450 Azc1mentioning
Oxyfunctionalization of non-activated carbon bonds by P450 monooxygenases has drawn great industrial attraction. Selfsufficient P450s containing catalytic heme and reductase domains in a single polypeptide chain offer many advantages since they do not require external electron transfer partners.Here, we report the first P450 enzyme identified and expressed from Azorhizobium caulinodans. Firstly, expression conditions of P450 AZC1 were optimized for enhanced expression in E.coli. The highest P450 content was obtained in E.coli Rosetta DE3 plysS when it was incubated in TB media supplemented with 0.75 mM IPTG, 0.5 mM ALA, and 0.75 mM FeCl 3 at 25 °C for 24 hours. Subsequently, the purified enzyme showed a broad substrate spectrum including fatty acids, linear and cyclic alkanes, aromatics, and pharmaceuticals. Finally, P450 AZC1 showed optimal activity at pH 6.0 and 40 °C and a broad pH and temperature profile, making it a promising candidate for industrial applications.
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