Whole‐Cell Biotransformation of Benzene to Phenol Catalysed by Intracellular Cytochrome P450BM3 Activated by External Additives

Karasawa, Masayuki; Stanfield, Joshua Kyle; Yanagisawa, Sota; Okada, Shoji; Watanabe, Yoshihito

doi:10.1002/anie.201804924

Cited by 47 publications

(42 citation statements)

References 36 publications

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“…Cofactor regeneration and cofactor-free P450 systems have also found applications in whole-cell biocatalysts. Watanabe and associates developed E. coli as a whole-cell biocatalyst vehicle to mediate the hydroxylation of benzene into phenol by WT P450 BM3 in the presence of decoy molecules (165). A novel whole-cell P450 photobiocatalysis system driven by the electrons from eosin Y instead of redox partners and cofactors was used for the bioconversion of pharmaceuticals with engineered bacterial P450s and human P450s (150).…”

Section: P450-related Metabolic Engineeringmentioning

confidence: 99%

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

et al. 2019

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Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C–H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.

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Section: P450-related Metabolic Engineeringmentioning

confidence: 99%

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

et al. 2019

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“…For instance, binding site redesign has been widely adopted for altering substrate specificity and further product outcome in terms of regio-and stereospecificity [130][131][132][133]. Decoy molecule strategy, in which a wisely designed molecule cheats the hosting enzyme [134], activating it and shifting substrate specificity, has been used to perform peroxygenation [135,136] alkanes [137] and benzene [138] also within vial cells [139]. Directed evolution has emerged as the method of election for reprogramming P450s and other heme-proteins toward abiological reactivities [106,107].…”

Section: Engineering Natural Scaffoldsmentioning

confidence: 99%

Mimochrome, a metalloporphyrin‐based catalytic Swiss knife†

Leone

Chino

Nastri

et al. 2020

Biotech and App Biochem

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Over the years, mimochromes, a class of miniaturized porphyrin‐based metalloproteins, have proven to be reliable but still versatile scaffolds. After two decades from their birth, we retrospectively review our work in mimochrome design and engineering, which allowed us developing functional models. They act as electron‐transfer miniproteins or more elaborate artificial metalloenzymes, endowed with peroxidase, peroxygenase, and hydrogenase activities. Mimochromes represent simple yet functional synthetic models that respond to metal ion replacement and noncovalent modulation of the environment, similarly to natural heme‐proteins. More recently, we have demonstrated that the most active analogue retains its functionality when immobilized on nanomaterials and surfaces, thus affording bioconjugates, useful in sensing and catalysis. This review also briefly summarizes the most important contributions to heme‐protein design from leading groups in the field.

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“…Crystallisation of the P450BM3 haem domain in complex with decoy molecules has proven challenging, with only a few successful examples having been reported by us . Recently, two powerful third‐generation decoy molecules, N ‐( S )‐ibuprofenoyl‐ l ‐phenylalanine (SIbuPhe) and N ‐enanthyl‐ l ‐prolyl‐ l ‐phenylalanine (C7ProPhe, Figure a,b), have been demonstrated to be particularly suited to the hydroxylation of benzene in vitro and in vivo in whole‐cell biotransformations . However, numerous attempts at crystallising P450BM3 with these two decoy molecules using vapour‐diffusion crystallisation were futile, yielding only crystals of the substrate‐free enzyme.…”

Section: Resultsmentioning

confidence: 99%

Crystals in Minutes: Instant On‐Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain

et al. 2020

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Despite CYP102A1 (P450BM3) representing one of the most extensively researchedmetalloenzymes,crystallisation of its haem domain upon modification can be ac hallenge. Crystal structures are indispensable for the efficient structurebased design of P450BM3 as ab iocatalyst. The abietane diterpenoid derivative N-abietoyl-l-tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild-type P450BM3 haem domain, with visible crystals forming within 2hours and diffracting to an ear-atomic resolution of 1.22 . Using these crystals as seeds in across-microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoym olecules and diverse artificial metalloporphyrins binding various ligand molecules,a sw ell as heavily tagged haem-domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.

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Whole‐Cell Biotransformation of Benzene to Phenol Catalysed by Intracellular Cytochrome P450BM3 Activated by External Additives

Cited by 47 publications

References 36 publications

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

Mimochrome, a metalloporphyrin‐based catalytic Swiss knife†

Crystals in Minutes: Instant On‐Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain

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