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

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 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 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-Tn7 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 ␤-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.Methylotrophic bacteria are a diverse group of microorganisms with the ability to utilize single-carbon (C 1 ) substrates more reduced than carbon dioxide as their sole source of carbon and energy. Among the methylotrophs, members of the genus Methylobacterium have been described as being ubiquitous, participating in a myriad of favorable interactions with nature (24,26,27). Furthermore, Methylobacterium spp. naturally produce several substances of commercial importance, including poly-␤-hydroxybutyrate (3, 4), vitamin B 12 (28), pyrroloquinoline quinone (1, 10), and carotenoids (29). Over the past decade, Methylobacterium extorquens AM1 has been extensively studied and characterized both genetically and physiologically (6,18,20). The wealth and depth of understanding of M. extorquens and closely related strains suggest the potential of M. extorquens as a source of industrially pertinent natural products and recombinant proteins. The salient aspects for this potential have been described elsewhere (2, 8, 9), and they include (i) simple and inexpensive cultivation requirements, (ii) optimized high-cell-density fermentation protocols, (iii) available genome sequence for M. extorquens AM1, and (iv) availability of suitable genetic tools for M. extorquens (8,13,19,21,22). Application of these tools has made it possible to overexpress a variety of recombinant proteins in the range of 3 to 6 g/liter under high-cell-density growth conditions (8, 9, 13). However, inducible/regula...

New Vectors for Chromosomal Integration Enable High-Level Constitutive or Inducible Magnetosome Expression of Fusion Proteins in Magnetospirillum gryphiswaldense

“…In similar approaches, Choi and coworkers integrated double copies of the cym repressor into Methylobacterium extorquens, thereby achieving tight repression of an inducible promoter (45). In the same organism, expression of five chromosomal copies of gfp resulted in 20-fold-higher expression than single-copy expression (46).…”

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

“…Obviously, although it has been reported several times that heterologous expression in M. extorquens strains is much stronger when it is controlled by P mxaF than when it is controlled by P lac (26,(29)(30)(31), the transcript levels of the RCM genes in all recombinant strains were sufficiently high to generate substantial RCM activities in our case. More importantly, transformation with pBBR1MCS-3 resulted in slightly higher 2-HIBA titers than transformation with the pCM80 expression system.…”

“…In addition, a toxicity of the mutase proteins has not been previously observed when the RCM Ktus genes are expressed in E. coli BL21 via the pBBR1MCS-3 vector (12). Likewise, similar inhibitory effects of heterologous proteins have not been reported in most studies on recombinant AM1 (26,32,(39)(40)(41) or other M. extorquens strains (29)(30)(31). In contrast, Chou and coworkers (42) found that rates of expression of a nonorthologous formaldehyde oxidation pathway in strain AM1 that were too high led to a growth rate that was one-third that of the wild type.…”

Production and characterization of polyhydroxyalkanoates by recombinant Methylobacterium extorquens: Combining desirable thermal properties with functionality

“…This new technology for inducible and regulated gene expression was d e v e l o p e db yo u rg r o u pf o rM. extorquens [28,29]. It may be described as follows: (A) In the absence of the chemical inducer (p-isopropylbenzoate = cumate), the repressor protein cymR is bound to the operator site upstream of the gene of interest and transcription is blocked; (B) addition of cumate as inducer leads to formation of the cymR-cumate complex, followed by detachment of cymR from the operator and activation of the transcription of the downstream gene.…”