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Applied and Environmental Microbiology, January 2009, p. 337-344, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.01758-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6{triangledown} ,{dagger}

Daniel J. Koch,1 Mike M. Chen,1 Jan B. van Beilen,2 and Frances H. Arnold1*

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125,1 Département de Biologie Moléculaire Végétale, Université de Lausanne, Lausanne, Switzerland2

Received 30 July 2008/ Accepted 8 November 2008

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47{Delta}B) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.

* Corresponding author. Mailing address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125. Phone: (626) 395-4162. Fax: (626) 568-8743. E-mail: frances{at}cheme.caltech.edu

{triangledown} Published ahead of print on 14 November 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, January 2009, p. 337-344, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.01758-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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