Engineering Rhodosporidium toruloides for the production of polyhydroxybutyrate

Abstract: The increased use of plastics has become an environmental problem where plastics, due to the slow degradation, accumulate in the oceans and landfills. Although plastics are a very cheap and useful material with desirable properties and a wide variety of applications, they are primarily made from petroleum-based resources which are non-renewable and of fossil origin. An alternative to these non-renewable plastics are bioplastics, which can be both bio-based and bio-degradable. Poly-3-D-hydroxybutyrate (PHB) is a promising precursor for bioplastics and is produced by various microorganisms. PHB is bio-based and biodegradable, possesses the characteristics of thermoplastics, and its mechanical properties are comparable to the chemical properties of polypropylene. PHB can be produced from acetyl-CoA in a three-step procedure involving the enzymes acetyl-CoA acetyltransferase, acetoacetyl-CoA reductase and polyhydroxyalkanoate synthase (PHB synthase). The enzymes are encoded by the genes PhaA, PhaB and PhaC respectively. A non-conventional oleaginous yeast is believed to potentially be a good producer of PHB as oleaginous yeasts are known to possess efficient routes for acetyl-CoA biosynthesis. In the present study, the codon-optimized genes PhaA1, PhaB1 and PhaC1 from Cupriavidus necator were successfully integrated into the genome of the oleaginous yeast Rhodosporidium toruloides using a recently developed electroporation protocol through random integration. Two clones were proven to produce PHB and further characterized. PHB production from glucose was demonstrated in shake-flasks experiments, where the best strain resulted in a PHB titer of 77 mg PHB/L, PHB yield of 3.7 mg PHB/g glucose and PHB yield on biomass of 0.0082 g PHB/g CDW. This study demonstrates that the codon-optimized genes are functional in the non-conventional oleaginous yeast used and that the production of PHB in the recombinant strains is possible.