A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration

Tsuyoshi Mouri, Junji Michizoe, Hirofumi Ichinose, Noriho Kamiya, Masahiro Goto

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.

Original languageEnglish
Pages (from-to)514-520
Number of pages7
JournalApplied Microbiology and Biotechnology
Volume72
Issue number3
DOIs
Publication statusPublished - Sep 1 2006

Fingerprint

glycerol dehydrogenase
Camphor 5-Monooxygenase
Biocatalysts
Cytochromes
Mixed Function Oxygenases
Glycerol
Escherichia coli
Regeneration
NAD
Proteins
Camphor
Enzymes
Hydroxylation
Electrons
Oxygenases
Pseudomonas putida
Oxygenation
Molecular oxygen
Substrates
Catalysis

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

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title = "A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration",
abstract = "A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.",
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T1 - A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration

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AU - Michizoe, Junji

AU - Ichinose, Hirofumi

AU - Kamiya, Noriho

AU - Goto, Masahiro

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AB - A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.

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