|dc.description.abstract||Omega-3 fatty acids (ω-3 FA) are essential to human health. However, the primary source of ω-3 FA are declining fish stocks. A more sustainable alternative is the cultivation of marine bacteria, however, the yields to date are less than favourable compared with those of fish. Oleaginous bacteria, when genetically engineered with the 16-27 kb pfaA-E gene cluster that encodes ω-3 FA synthesis, may potentially overcome this yield deficiency. One such species is Rhodococcus opacus PD630, which is able to accumulate up to 87% of its cell dry weight as fatty acids. This fatty acid accumulation makes R. opacus PD630 highly desirable as a biotechnological producer strain. However, before ω-3 FA may be produced by genetically engineered R. opacus PD630, work must be undertaken to develop expression vectors carrying the pfaA-E gene cluster. New transformation protocols, capable of handling an expression vector of 30-35 kb, twice the size of the largest reported to have been transformed into a Rhodococcus spp. to date, must also be developed.
This thesis describes the development of new protocols, based on electroporation and sonoporation, for the transformation of R. opacus PD630. An electroporation based protocol was successfully developed, with an efficiency of transformation of 5.317 x10^4 R. opacus PD630 transformants per µg of DNA using the E. coli -Rhodococcus spp. shuttle vector pBS305, compared with 81.15 for the most efficient method previously reported, and 7.158 x10^6 for Escherichia coli DH5α. A novel method for the generation and recovery of R. opacus PD630 protoplasts for transformation was successfully developed. Protoplast electroporation was found to be ineffective, though protoplasts were able to be used for the extraction of plasmid DNA in the identification of putative transformants. Sonoporation was found to be an ineffective means of transformation, both in E. coli DH5α and R. opacus PD630.
The development of rhodococcal vectors carrying pfa genes was attempted with LT-PCR from Shewanella baltica chromosomal DNA, and both restriction cloning and PCR of the transgenic expression vector pDHA4. LT-PCR was found to be ineffective at amplifying the 16-20 kb pfa gene cluster of S. baltica, even after optimisation to minimise non-target binding. Restriction cloning of pDHA4 was successful in obtaining the pfa gene cluster, though at a yield insufficient for further use in cloning. PCR amplification of the gene cluster proved to be successful, however, an absence of selective pressure for the gene cluster when inserted into transgenic vectors, and a failure to recover any transformants in E. coli DH5α, meant that this approach was abandoned. PCR amplification of the Rhodococcus spp. gene expression and plasmid maintenance genes from the E. coli -Rhodococcus spp. shuttle vector pTip QT1 was successful. These genes were able to be inserted into pDHA4, although no transformants of E. coli DH5α carrying the construct were able to be recovered. De novo DNA synthesis is a possibility for the construction of the desired transgenic vector.
To determine if pDHA4, a purportedly E. coli-only plasmid, could be maintained by R. opacus PD630, a derivative lacking the pfa gene cluster, pDHA4VO, was constructed. Expression of the chloramphenicol resistance gene of pDHA4VO in a confirmed R. opacus PD630 transformant, suggested that R. opacus PD630 was capable of the heterologous expression of DNA from pDHA4. This is the first reported incidence of heterologous expression in R. opacus PD630 without the use of an E. coli -Rhodococcus spp. shuttle vector. Transformation was attempted by electroporation with pDHA4, although no confirmed R. opacus PD630 transformants were recovered. Differences in the GC content between pDHA4 and pDHA4VO are thought to be a contributing factor, and may be indicative of the range of GC contents of DNA which R. opacus PD630 is capable of expressing heterologously.||