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Applied and Environmental Microbiology, September 2007, p. 5711-5715, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.00791-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters{triangledown} ,{dagger}

Sung Kuk Lee,1,{ddagger} Howard H. Chou,2,{ddagger} Brian F. Pfleger,1,§ Jack D. Newman,1,¶ Yasuo Yoshikuni,2 and Jay D. Keasling1,2,3*

Department of Chemical Engineering,1 Department of Bioengineering, University of California, Berkeley, California 94720,2 Synthetic Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California3

Received 9 April 2007/ Accepted 11 July 2007

Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (PBAD) system that is more compatible with IPTG (isopropyl-ß-D-1-thiogalactopyranoside) induction of a lactose-inducible (Plac) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.

* Corresponding author. Mailing address: Berkeley Center for Synthetic Biology, 717 Potter Street, Building 977, Mail Code 3224, University of California, Berkeley, CA 94720-3224. Phone: (510) 495-2620. Fax: (510) 495-2630. E-mail: keasling{at}berkeley.edu

{triangledown} Published ahead of print on 20 July 2007.

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

{ddagger} These authors contributed equally to this work.

§ Present address: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109.

Present address: Amyris Biotechnologies, 5980 Horton Street, Ste. 450, Emeryville, CA 94608.

Applied and Environmental Microbiology, September 2007, p. 5711-5715, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.00791-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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