Abstract
The catalytic turnover of cytochrome P450 cam from Pseudomonas putida requires two auxiliary reduction partners, putidaredoxin (Pd) and putidaredoxin reductase (PdR). We report the functional expression in Escherichia coli of tricistronic constructs consisting of P450 cam encoded by the first cistron and the auxiliary proteins, Pd and PdR by the second and the third. Transformed bacterial whole cells efficiently oxidized (1R)-(+)-camphor to 5-exo-hydroxycamphor and, interestingly, limonene to (−)-perillyl alcohol. These bioengineered E. coli cells possess a heterologous self-sufficient P450 catalytic system that may have advantages in terms of low cost and high yield for the production of fine chemicals.
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References
Guengerich FP (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem Res Toxicol 14:611–650
Katagiri M, Ganguli BN, Gunsalus IC (1968) A soluble cytochrome P-450 functional in methylene hydroxylation. J Biol Chem 243:3543–3546
Kim D, Guengerich FP (2004) Selection of human cytochrome P450 1A2 mutants with selectivity enhanced catalytic activity for heterocyclic amine N-hydroxylation. Biochemistry 43:981–988
Lamb DC, Guengerich FP, Kelly SL, Waterman MR (2006) Exploiting Streptomyces coelicolor A3(2) P450s as a model for application in drug discovery. Expert Opin Drug Metab Toxicol 2:27–40
Miyazawa M, Shindo M, Shimada T (2002) Metabolism of (+)- and (−)-limonenes to respective carveols and perillyl alcohols by CYP2C9 and CYP2C19 in human liver microsomes. Drug Metab Dispos 30:602–607
Murphy TM, Vu H, Nguyen T (1998) The superoxide synthases of rose cells. Plant Physiol 117:1301–1305
Ortiz de Montellano PR (2005) Cytochrome P450: structure, mechanism, and biochemistry, 3rd edn. Plenum Press, New York
Paine MJI, Scrutton NS, Munro AW, Gutierrez A, Roberts GCK, Wolf CR (2004) Electron transfer partners of cytochrome P450, 3rd edn. Plenum Press, New York
Parikh A, Gillam EM, Guengerich FP (1997) Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat Biotechnol 15:784–788
Poulos TL, Perez M, Wagner GC (1982) Preliminary crystallographic data on cytochrome P-450CAM. J Biol Chem 257:10427–10429
Reddy BS, Wang CX, Samaha H, Lubet R, Steele VE, Kelloff GJ, Rao CV (1997) Chemoprevention of colon carcinogenesis by dietary perillyl alcohol. Cancer Res 57:420–425
Sibbesen O, De Voss JJ, Montellano PR (1996) Putidaredoxin reductase-putidaredoxin-cytochrome p450cam triple fusion protein. Construction of a self-sufficient Escherichia coli catalytic system. J Biol Chem 271:22462–22469
Sibbesen O, Zhang Z, Ortiz de Montellano PR (1998) Cytochrome P450cam substrate specificity: relationship between structure and catalytic oxidation of alkylbenzenes. Arch Biochem Biophys 353:285–296
Trudgill PW, DuBus R, Gunsalus IC (1966) Mixed function oxidation V. Flavin interaction with a reduced diphosphopyridine nucleotide dehydrogenase, one of the enzymes participating in camphor lactonization. J Biol Chem 241:1194–1205
van Beilen JB, Holtackers R, Luscher D, Bauer U, Witholt B, Duetz WA (2005) Biocatalytic production of perillyl alcohol from limonene by using a novel Mycobacterium sp. cytochrome P450 alkane hydroxylase expressed in Pseudomonas putida. Appl Environ Microbiol 71:1737–1744
Acknowledgments
This work was supported by grants from the U. S. Public Health Service (R01 GM25515) and by a Korea Research Foundation Grant funded by the Korean Government(MOEHRD, Basic Research Promotion Fund) (KRF-2007-331-E00025)
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Fig. 1
GC-MS analysis of camphor hydroxylation by CYP101:Pd:PdR tricistronic system. Conversion of camphor to hydroxycamphor by CYP101:Pd:PdR tricistronic whole cell system for 1 h is 49%
Fig. 2
GC-MS analysis of limonene hydroxylation by CYP101:Pd:PdR tricistronic system. Conversion of limonene to perillyl alcohol by CYP101:Pd:PdR tricistronic whole cell system for 1 h is 55%
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Kim, D., Ortiz de Montellano, P.R. Tricistronic overexpression of cytochrome P450 cam , putidaredoxin, and putidaredoxin reductase provides a useful cell-based catalytic system. Biotechnol Lett 31, 1427–1431 (2009). https://doi.org/10.1007/s10529-009-0016-3
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DOI: https://doi.org/10.1007/s10529-009-0016-3