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Tricistronic overexpression of cytochrome P450 cam , putidaredoxin, and putidaredoxin reductase provides a useful cell-based catalytic system

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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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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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Correspondence to Donghak Kim.

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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

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