Characterization of Cytochrome P450 Enzymes and Their Applications in Synthetic Biology

Jeffreys, Laura N.; Girvan, Hazel M.; McLean, Kirsty J.; Munro, Andrew W.

doi:10.1016/bs.mie.2018.06.013

Cited by 21 publications

(14 citation statements)

References 164 publications

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“…Here, multiple CYP716s are involved in the biosynthetic pathway. , In Arabidopsis , there are two CYP88A subfamily genes that have the same function and are involved in the biosynthesis of gibberellin, which continuously catalyzes the formation of kaurenoic acid to the gibberellic acid phytohormone precursor GA12 . In addition, similar sequences from different species may also have partially identical catalytic functions, such as CYP76AH1 and CYP76AH4, which are derived from S. miltiorrhiza and Rosmarinus officinalis , respectively. , CYPs have been suspected of holding the key to bioengineering and synthetic biology. , Via engineering of CYP proteins, one can increase the efficiency of catalytic product production and reduce yields of wasteful byproducts, thereby improving the overall efficiency of producing target compounds.…”

Section: Discussionsupporting

confidence: 93%

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Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology

Mao

Chen

et al. 2020

ACS Synth. Biol.

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Cytochrome P450s (CYPs) are important enzymes in the secondary metabolism of plants and have been recognized as key players in bioengineering and synthetic biology. Previously reported CYP76AH1 and CYP76AH3, having greater than 80% sequence homology, played a continuous catalytic role in the biosynthesis of tanshinones in Salvia miltiorrhiza. Homology modeling indicates that four sites might be responsible for differences in catalytic activity between the two enzymes. A series of modeling-based mutational variants of CYP76AH1 were designed to integrate the functions of the two CYPs. The mutant CYP76AH1D301E,V479F, which integrated the functions of CYP76AH1 and CYP76AH3, was found to efficiently catalyze C11 and C12 hydroxylation and C7 oxidation of miltiradiene substrates. Integration and utilization of CYP76AH1D301E,V479F by synthetic biology methods allowed the robust production of 11-hydroxy ferruginol, sugiol, and 11-hydroxy sugiol in yeast. The functionally integrated CYP gene after active site modifications improves catalytic efficiency by reducing the transfer of intermediate metabolites between component proteins. This provides a synthetic biology reference for improving the catalytic efficiencies of systems that produce plant natural products in microorganisms.

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Section: Discussionsupporting

confidence: 93%

“…23,24 CYPs have been suspected of holding the key to bioengineering and synthetic biology. 4,25 Via engineering of CYP proteins, one can increase the efficiency of catalytic product production and reduce yields of wasteful byproducts, thereby improving the overall efficiency of producing target compounds.…”

Section: ■ Discussionmentioning

confidence: 99%

Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology

Mao

Chen

et al. 2020

ACS Synth. Biol.

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“…The ferrous carbon monoxide spectrum of T252E was analyzed by the standard method using sodium dithionite (Na 2 S 2 O 4 ) as the reducing agent. , The treatment of the T252E mutant with dithionite to generate the ferrous form did not result in a significant shift in the position of the Soret band (Figure a, red spectrum). This contrasts with the reduction of WT CYP199A4 by dithionite, which shifted the Soret band from ∼418.5 to ∼414.0 nm, as expected for a ferrous five-coordinate high-spin heme iron (Figure S2).…”

Section: Resultsmentioning

confidence: 99%

An Altered Heme Environment in an Engineered Cytochrome P450 Enzyme Enables the Switch from Monooxygenase to Peroxygenase Activity

et al. 2022

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Cytochrome P450 heme-thiolate monooxygenases are exceptionally versatile enzymes which insert an oxygen atom into the unreactive C–H bonds of organic molecules. They source O2 from the atmosphere and usually derive electrons from nicotinamide cofactors via electron transfer proteins. The requirement for an expensive nicotinamide adenine dinucleotide (phosphate) cofactor and the redox protein partners can be bypassed by driving the catalysis using hydrogen peroxide (H2O2). We demonstrate that the mutation of a highly conserved threonine residue, involved in dioxygen activation, to a glutamate shuts down monooxygenase activity in a P450 enzyme and converts it into a peroxygenase. The reason for this switch in the threonine to glutamate (T252E) mutant of CYP199A4 from Rhodopseudomonas palustris HaA2 was linked to the lack of a spin state change upon the addition of the substrate. The crystal structure of the substrate-bound form of this mutant highlighted a modified oxygen-binding groove in the I-helix and the retention of the iron-bound aqua ligand. This ligand interacts with the glutamate residue, which favors its retention. Electron paramagnetic resonance confirmed that the ferric heme aqua ligand of the mutant substrate-bound complex had altered characteristics compared to a standard ferric heme aqua complex. Significant improvements in peroxygenase activity were demonstrated for the oxidative demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and veratric acid to vanillic acid (up to 6-fold). The detailed characterization of this engineered heme peroxygenase will facilitate the development of new methods for driving the biocatalytic generation of oxygenated organic molecules via selective C–H bond activation using heme enzymes.

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“…3 Most P450 systems are composed of a P450 monooxygenase and one or two additional proteins to constitute an electron transfer chain. 4 Unlike other independent monooxygenase, the activities of P450 monooxygenases highly rely on interactions with their redox partners, 5,6 demonstrating the need for combinatorically engineering the multicomponents of a P450 system for tighter coupling. For instance, coupling P450 monooxygenase with exogenous redox partner(s) would gain higher oxidative activities than coexpression with the native redox partner(s).…”

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confidence: 99%

“…Steroid biosynthesis interlinks with numbers of oxidative reactions which has always been catalyzed by cytochrome P450 systems . Most P450 systems are composed of a P450 monooxygenase and one or two additional proteins to constitute an electron transfer chain . Unlike other independent monooxygenase, the activities of P450 monooxygenases highly rely on interactions with their redox partners, , demonstrating the need for combinatorically engineering the multicomponents of a P450 system for tighter coupling.…”

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confidence: 99%

Pregnenolone Overproduction in Yarrowia lipolytica by Integrative Components Pairing of the Cytochrome P450scc System

et al. 2019

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Microbial production of steroid drugs exhibits great potentials in much greener and more sustainable manners, in which engineering multiple cytochrome P450s is the prerequisite requirement. The pairing of multicomponents of P450 systems is a tremendous challenge. Herein, biosynthesis of pregnenolone (a common precursor of steroid drugs) in Yarrowia lipolytica was taken as a typical instance to explore the engineering strategy of the cytochrome P450 side-chain cleavage enzyme (P450scc) system. The mature forms of the components belonging to P450scc system, CYP11A1, adrenodoxin (Adx), and adrenodoxin reductase (AdR), were coexpressed in a former constructed campesterol producing strain. To maximize pregnenolone production, an integrative components pairing strategy was proposed for pairing the component sources and balancing the expression levels of CYP11A1, Adx, and AdR. Led by the above approaches, a 2344-fold improvement of pregnenolone titer was achieved at the shake flask level. Consequently, a highest reported pregnenolone titer of 78.0 mg/L in microbes was obtained in a 5 L bioreactor. Our study not only highlights the integrative components pairing of cytochrome P450scc as a general strategy for engineering other cytochrome P450s, but also provides a feasible and efficient platform of Y. lipolytica for other steroids production.

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Characterization of Cytochrome P450 Enzymes and Their Applications in Synthetic Biology

Cited by 21 publications

References 164 publications

Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology

Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology

An Altered Heme Environment in an Engineered Cytochrome P450 Enzyme Enables the Switch from Monooxygenase to Peroxygenase Activity

Pregnenolone Overproduction in Yarrowia lipolytica by Integrative Components Pairing of the Cytochrome P450scc System

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