Abstract
BackgroundMethylotrophic (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.ResultsOur 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.ConclusionsThis 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.
Highlights
Methylotrophic bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol
As can be seen, feeding of : 4-pentenoic acid (C5)-fatty acids led to accumulation of the copolymer PHBV solely and, no trace of C-C double bounds could be detected in the PHAs produced upon feeding C5-fatty acids containing a C-C double bond
Given the extent of our screening efforts, it may be reasonably concluded that the wildtype M. extorquens ATCC 55366 strain was unable to accumulate functionalized PHAs containing C-C double bonds
Summary
Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Carbon double bonds are comparatively inert but can be transformed into reactive functional groups under mild reaction conditions. Following this approach, polyhydroxyalkanoates with carboxyl, hydroxyl, epoxy and halogenic groups have been produced ([14] and references therein). Most functionalized PHAs that have been produced to date belong to the family of medium-chain-length (MCL, C ≥ 6) polyhydroxyalkanoates, in which all monomers have six carbon atoms or more. These PHAs are characterized by rubber-like mechanical properties with low melting temperatures (
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