Development and use of a vector system for Methylophilus methylotrophus.
2015-11-19T09:07:28Z (GMT) by
The obligate methylotroph, Methylophilus methylotrophus, used by ICI for its Single Cell Protein production, may represent a valuable alternative host organism to E. coli for the commercial production of heterologous gene products. The organism has the advantages of being safe, coupled with an ability to grow well, in large quantities, on a cheap carbon source. This thesis describes the construction, characterisation and analysis of a series of plasmid cloning vectors designed for use in M.methylotrophus. The vectors are based on the IncQ plasmid R300B and maintain a broad host range with an increased capacity for easy-to-use cloning sites mainly derived from the E.coli plasmids pBR322 and pBR328. All the vectors thus carry antibiotic- resistance genes containing restriction sites which could lead to insertional inactivation as a means of detecting recombinants. One plasmid in particular, pGSS33, has four antibiotic-resistance genes all of which contain at least one such restriction site. The first demonstration of expression of a eukaryotic coding sequence, murine dihydrofolate reductase (DHFR), in M.methylotrophus has been described. This has been followed by expression of E.coli B-galactosidase and synthetic human ?-1 interferon, all making use of pGSS vectors. The pGSS15-DHFR plasmid, pDHFR2.43, may turn out to be a valuable test plasmid for studying the stability of cloned eukaryotic coding sequences in both E.coli and M.methylotrophus; it is readily detected (trimethoprim-resistant) and can be grown with or without selection. A start has been made towards providing increased expression from vectors carrying strong E.coli promoters (lacUV5 and synthetic trp) which have been demonstrated to work well in M.methylotrophus. Broad host range cosmid vectors have been constructed which have the potential to be used for the production of gene-banks in Gram-negative organisms other than E.coli. Copy numbers of the vector plasmids have been determined in E.coli strains and several different methods of measurement reviewed in an attempt to find one suitable for use with M.methylotrophus. An encouraging lead has been identified in the search for a high copy number plasmid, which coupled with a strong promoter, could provide the basis for a very efficient batch production process. Thus with the availability of easy-to-use cloning vectors, convenient delivery systems and the accumulated evidence of strong E.coli promoters working efficiently in M.methylotrophus, this organism can seriously be considered as a safe alternative host to E.coli..