Regulation of heterologous recombinant protein expression

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Methylotrophic or methanotrophic bacteria such as Methylobacterium are transformed with a gene of interest, and expression of the gene is regulated by means of a cumate repressor protein and an operator sequence which is operatively linked to the gene of interest, and the addition of an external agent. Specifically, the cymR repressor and cmt operator from Pseudomonas putida may serve to regulate gene expression in methylotrophic or methanotrophic bacteria with the addition of cumate.

Methylotrophic bacteria are ubiquitous, inhabiting different aquatic and terrestrial habitats including the phyllosphere. In addition to their important role in the cycling of single carbon compounds in the environment, methanol-utilizing methylotrophs have been shown to enter into plant-growth-promoting phytosymbiotic relationships (1, 2). Amongst the methylotrophs, several Methylobacterium species have been well described in the literature. One of the best characterized strains within the Methylobacterium genus, both genetically and physiologically, is the methanol utilizing Methylobacterium extorquens AM1 (3). Another well characterized M

extorquens strain, ATCC 55366, was isolated for its capacity to produce high quantities of poly-8-hydroxybutyric acid. A fed-batch fermentation system was developed specifically for this strain to further enhance its growth potential and PHB

productivity. See also U.S. Patent no. 5,302,525, which issued Nov. 23, 1992.

Methylobacterium extorquens has received special attention as a potential source of industrially pertinent natural and recombinant proteins (4, 5). The industrial potential of M extorquens as a producer of recombinant proteins is due to: (i) the simplicity of its growth requirements and a comparably inexpensive substrate (methanol); (ii) the development and optimization of protocols for high cell density cultivation;

(iii) the sequencing and annotation of the M extorquens AM1 genome; and (iv) the development of genetic tools specifically for M extorquens comprising novel cloning and expression vectors, efficient transposon mutagenesis, integrative expression vectors and protocols, and simple and efficient electroporation protocols (6-8). The potential of M.

extorquens and other pink pigmented facultative methylotrophs (PPFMs) as "cell factories"

is f L' CA 02619989 2008-05-22 = PCTICA rats" of MARCH 2007 2 2 .63 ..07 further enhanced by their inherent capabilities to produce natural products of great import including pyrroloquinoline quinone (9, 10), vitamin B12 (11), poly-B-hydroxybutyric acid (12, 13), caratenoids (14), and phytohormones (15, 16). By applying the molecular tools and high cell-density fermentation technologies mentioned above, it has been possible to overexpress a variety of recombinant proteins in M.

extorquens strains ATCC 55366 and AM1. Representative proteins overexpressed in these two M. extorquens strains include the green fluorescent protein, esterase from Lactobacillus casei, enterocin P from Enterococcus faeciwn, and haloalkane dehalogenase from Xanthobacter autotrophicus (4, 17, 18, 19). Several of these activities are the subject of our pending U.S. Patent Publication No. 2006 0234336, filed November 29, 2002. Miguez et al.

However, as it concerns Methylobacterium extorquens strains AM1 and ATCC

55366, practical and tight inducible expression of recombinant genes has not been attained (20).

Inducible heterologous promoters have been tested in M extorquens ATCC 55366, in particular Piac and XPL and PR however expression is both leaky and weak. M. extorquens possesses native methanol inducible promoters, notably promoters which are located upstream of genes that encode for the methanol dehydrogenase and other proteins required for its activity, and for enzymes required for the synthesis of the methanol dehydrogenase prosthetic group, the pyrroloquinoline quinone. Of these, promoter PinxilF has been thoroughly scrutinized both biochemically and in expression studies. Although it is a very strong promoter (4, 17, 20), in practical terms it is essentially constitutive. Expression of the green fluorescent gene (gfp) under the control of Pllaap occurred even when the culture was grown repeatedly on succinate.