Production of functionalized polyhydroxyalkanoates by genetically modified Methylobacterium extorquens strains
1 Microbial and Enzymatic Technology Group, Bioprocess Centre, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montréal, Québec, H4P 2R2, Canada
2 Laboratoire de Bioingénierie et de Biophysique de l'Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, 2500 Boulevard de l'Université, Sherbrooke, Québec, J1K 2R1, Canada
3 Biophysics and Nuclear Magnetic Resonance, Institute for Research in Immunology and Cancer, Université de Montréal, 2900 Boulevard Édouard-Montpetit, Montréal, Québec, H3T 1J4, Canada
4 Current Address: Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos, Viral Vaccine Program, Avenida Brasil 4365, 21045-900 Rio de Janeiro/RJ, Brazil
Microbial Cell Factories 2010, 9:70 doi:10.1186/1475-2859-9-70Published: 16 September 2010
Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications.
Our proprietary M. extorquens ATCC 55366 was found unable to yield functionalized PHAs when fed methanol and selected unsaturated carboxylic acids as secondary substrates. However, cloning of either the phaC1 or the phaC2 gene from P. fluorescens GK13, using an inducible and regulated expression system based on cumate as inducer (the cumate switch), yielded recombinant M. extorquens strains capable of incorporating modest quantities of C-C double bonds into PHA, starting from either C6= and/or C8=. The two recombinant strains gave poor results with C11=. The strain containing the phaC2 gene was better at using C8= and at incorporating C-C double bonds into PHA. Solvent fractioning indicated that the produced polymers were PHA blends that consequently originated from independent actions of the native and the recombinant PHA synthases.
This work constitutes an example of metabolic engineering applied to the construction of a methanol-utilizing bacterium capable of producing functionalized PHAs containing C-C double bonds. In this regard, the PhaC2 synthase appeared superior to the PhaC1 synthase at utilizing C8= as source of C-C double bonds and at incorporating C-C double bonds into PHA from either C6= or C8=. The M. ex-phaC2 strain is, therefore, a promising biocatalyst for generating advanced (functionalized) PHAs for future high value applications in various fields.