A novel tightly regulated gene expression system was developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-isopropylbenzoate (cumate) as an inducer. This novel expression system, referred to as the cumate gene switch, includes a specific expression vector, pNEW, that contains a partial T5 phage promoter combined with the Pseudomonas-based synthetic operator and the cymR repressor protein-encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer (cumate), which is nontoxic to the culture, water soluble, and inexpensive. The characteristics and potential of the expression system were determined. Using flow cytometry and fed-batch fermentations, we have shown that, with the newly developed cumate-regulated system, (i) higher recombinant product yields can be obtained than with the pET (isopropyl-␤-D-thiogalactopyranoside [IPTG])-induced expression system, (ii) expression is tightly regulated, (iii) addition of cumate quickly results in a fully induced and homogenous protein-expressing population in contrast to the bimodal expression profile of an IPTGinduced population, (iv) expression can be modulated by varying the cumate concentration, and (v) the cumate-induced population remains induced and fully expressing even at 8 h following induction, resulting in high yields of the target protein Furthermore, the cumate gene switch described in this article is applicable to a wide range of E. coli strains.A variety of gene expression systems exist for the production of recombinant proteins and as a tool in metabolic engineering. These systems differ in the hosts, plasmids, and promoters being utilized. The variety of existing expression systems reflects the diversity, complexity, and toxicity of the proteins being produced, requiring in certain instances that the product be expressed at various concentrations (14) or at different phases of cell growth (14) and be soluble (4), secreted (13) or compartmentalized (40,45). In an attempt to meet these challenges, the search for or the development of new hosts and expression vectors is ongoing.Gene transcription can be enhanced by replacing a promoter sequence naturally associated with that gene with a sequence of a stronger promoter (17, 42). The nature of the bioprocess will dictate the promoter of choice. A process that requires high-level expression will necessitate a strong constitutive or regulated promoter. A process requiring the expression of a product with toxicity issues for the cell will benefit from a regulated expression system that does not possess basal expression under repressed conditions (26, 37). The promoters P lac , P trp , P tac , P L , P T7 , P BAD , and P lacUV5 are commonly utilized for the construction of expression vectors (1, 18). Among these, P lacUV5 , P tac , and the combined system of P T7 with P lacUV5 are widely used, be...

The green fluorescent protein (GFP) was used as a model protein to study the recombinant protein production by the strain Methylobacterium extorquens ATCC 55366. Scale-up from shake flasks to 20 l fed-batch fermentation was achieved using methanol as a sole carbon and energy source and a completely minimal culture medium. Two different expression vectors were used to express GFP. Clone PCM-GFP containing the vector pCM110 with native promoter of the methanol dehydrogenase P mxaF produced approximately 100-fold more GFP than the clone PRK-GFP containing the vector pRK310 with the heterogeneous promoter P lac . Several fed-batch fermentations with and without selective pressure (tetracycline) were run in a 20 l stirred tank fermenter using the two different clones of M. extorquens. The methanol concentration was monitored with an on-line semiconductor gas sensor in the culture broth. It was maintained at a non-toxic level of 1.4 g l 31 with an adaptative control which regulates the methanol feed rate. The same growth profile was achieved in all fermentations. The maximum growth rate (W max ) was 0.18 h 31 with an overall yield (Y X =S ) of 0.3 g g 31 methanol. With this high cell density fermentation process, we obtained high levels (up to 4 g l 31 ) of GFP with the clone PCM-GFP. The maximum specific GFP production (Y GFP=X ) with this clone was 80 mg g 31 representing approximately 16% of the total cell protein. Additional feeding of pure oxygen to the fermenter permitted a longer phase of exponential growth but had no effect on the total yields of biomass and GFP. The specific GFP production of clone PCM-GFP remained unaffected in the presence or absence of selective pressure (tetracycline), within the initial 50 h of the fermentation culture. These results suggest that M. extorquens ATCC 55366 could be an interesting candidate for overexpression of recombinant proteins.

High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (P mxaF ) using the mini-Tn7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (gfp) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 site was identified in an intergenic region between glmS, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the attTn7 site is a noncoding region of the chromosome make the mini-Tn7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (bgl [␤-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens.Methylotrophic bacteria are a diverse group of microorganisms that are able to grow on single-carbon substrates, such as methane or methanol, as sole sources of organic carbon and energy. Methylotrophs are ubiquitous and inhabit different aquatic and terrestrial habitats, including the phyllosphere (21,23,32,33,37,39). One of the best-characterized methylotrophs, both genetically and physiologically, is the methanolutilizing organism Methylobacterium extorquens AM1 (9). This Methylobacterium species has received special attention as a potential source of industrially pertinent natural and recombinant proteins (5, 16). The industrial potential of M. extorquens as a producer of recombinant proteins is due to (i) the simplicity of its growth requirements and its utilization of a comparatively inexpensive substrate (methanol), (ii) the development and optimization of protocols for high-cell-density cultivation, (iii) the fact that the M. extorquens AM1 genome has been sequenced and annotated, and (iv) the development of genetic tools specifically for M. extorquens comprising novel cloning and expression vectors, efficient transposon mutagenesis, and simple and efficient electroporation protocols (5-7, 15, 28-30). The potential of M. extorquens and other pinkpigmented facultative methylotrophs as "cell factories" is further enhanced by their inherent abilities to produce natural products of great importance, including pyrroloquinoline q...

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...