Bestowing Inducibility on the Cloned Methanol Dehydrogenase Promoter (P mxaF ) of Methylobacterium extorquens by Applying Regulatory Elements of Pseudomonas putida F1

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Abstract
P mxaF 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 P mxaF promoter in expression vectors, we adopted combinational methods (regulatory elements of the Pseudomonas putida F1 cym and cmt operons and Tn 7 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 P mxaF 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-Tn 7 transposon system. Cultures containing the chromosomally integrated cymR gene were subsequently transformed with pCHOI3 containing modified P mxaF (i.e., P mxaF 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 β-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 .