Whole‐cell‐based CYP153A6‐catalyzed (S)‐limonene hydroxylation efficiency depends on host background and profits from monoterpene uptake via AlkL

Biotechnology and Bioengineering - Tập 110 Số 5 - Trang 1282-1292 - 2013
Sjef Cornelissen1,2,3, Mattijs K. Julsing1,2,3, Jan Volmer1,2,3, Ole Riechert1,2,3, Andreas Schmid1,2,3, Bruno Bühler1,2,3
1Department of Biochemical and Chemical Engineering, Laboratory of Chemical Biotechnology, TU Dortmund University, Emil-Figge-Strasse 66, 44227, Dortmund, Germany
2fax: +49-231-755-7382
3telephone: +49-231-755-7384

Tóm tắt

AbstractLiving microbial cells are considered to be the catalyst of choice for selective terpene functionalization. However, such processes often suffer from side product formation and poor substrate mass transfer into cells. For the hydroxylation of (S)‐limonene to (S)‐perillyl alcohol by Pseudomonas putida KT2440 (pGEc47ΔB)(pCom8‐PFR1500), containing the cytochrome P450 monooxygenase CYP153A6, the side products perillyl aldehyde and perillic acid constituted up to 26% of the total amount of oxidized terpenes. In this study, it is shown that the reaction rate is substrate‐limited in the two‐liquid phase system used and that host intrinsic dehydrogenases and not CYP153A6 are responsible for the formation of the undesired side products. In contrast to P. putida KT2440, E. coli W3110 was found to catalyze perillyl aldehyde reduction to the alcohol and no oxidation to the acid. Furthermore, E. coli W3110 harboring CYP153A6 showed high limonene hydroxylation activities (7.1 U g). The outer membrane protein AlkL was found to enhance hydroxylation activities of E. coli twofold in aqueous single‐phase and fivefold in two‐liquid phase biotransformations. In the latter system, E. coli harboring CYP153A6 and AlkL produced up to 39.2 mmol (S)‐perillyl alcohol L within 26 h, whereas no perillic acid and minor amounts of perillyl aldehyde (8% of the total products) were formed. In conclusion, undesired perillyl alcohol oxidation was reduced by choosing E. coli's enzymatic background as a reaction environment and co‐expression of the alkL gene in E. coli represents a promising strategy to enhance terpene bioconversion rates. Biotechnol. Bioeng. 2013; 110: 1282–1292. © 2012 Wiley Periodicals, Inc.

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Biotechnology and Bioengineering

Tập 110 Số 5

1282-1292

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