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Applied and Environmental Microbiology, December 2006, p. 7723-7729, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.02002-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Bestowing Inducibility on the Cloned Methanol Dehydrogenase Promoter (PmxaF) of Methylobacterium extorquens by Applying Regulatory Elements of Pseudomonas putida F1{triangledown}

Young J. Choi,1 Lyne Morel,1 Denis Bourque,1 Alaka Mullick,2 Bernard Massie,2 and Carlos B. Míguez1*

Microbial and Enzymatic Technology Group,1 Genomics & Gene Therapy Vectors Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada2

Received 23 August 2006/ Accepted 4 October 2006

PmxaF is a strong methanol-inducible promoter in Methylobacterium extorquens. When this promoter is cloned in expression vectors and used to drive heterologous gene expression, methanol inducibility is either greatly reduced or entirely lost. In order to bestow inducibility upon the cloned PmxaF promoter in expression vectors, we adopted combinational methods (regulatory elements of the Pseudomonas putida F1 cym and cmt operons and Tn7 transposon system) to control reporter gene expression at the transcriptional level in M. extorquens. An operator fragment (26 nucleotides) of the cmt operon was inserted downstream of the cloned PmxaF promoter in the broad-host-range expression vector (pCHOI3). The repressor gene (cymR) located upstream of the cym operon in P. putida F1 was amplified by PCR. To avoid cellular toxicity for M. extorquens caused by the overexpression of CymR, single and/or double copies of cymR were integrated into the chromosome of M. extorquens using the mini-Tn7 transposon system. Cultures containing the chromosomally integrated cymR gene were subsequently transformed with pCHOI3 containing modified PmxaF (i.e., PmxaF plus operator). In this construct, inducibility is afforded by cumate (p-isopropylbenzoate). In this report, we describe the inducible and tightly regulated expression of heterologous genes (bgl [for ß-galactosidase], est [for esterase], and gfp [for green fluorescent protein]) in M. extorquens. This is the first documented example of an inducible/regulated heterologous gene expression system in M. extorquens.

* Corresponding author. Mailing address: Microbial and Enzymatic Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Phone: (514) 496-6280. Fax: (514) 496-7251. E-mail: carlos.miguez{at}nrc-cnrc.gc.ca.

{triangledown} Published ahead of print on 13 October 2006.

Applied and Environmental Microbiology, December 2006, p. 7723-7729, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.02002-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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