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.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Mouri
authors.forename: Junji
authors.initials: J
authors.lastname: Michizoe
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Ichinose
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
mesh_terms: D002164:Camphor / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D006900:Hydroxylation; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D009243:NAD; D002164:Camphor; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; D019475:Camphor 5-Monooxygenase