Engineering Rhodosporidium toruloides for the production of polyhydroxybutyrate

English. 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.

English. Bioplastics from micro-factories A commonly used material in today’s society is plastics, which is a very cheap material with a lot of applications due to its many desirable characteristics. Some of these however, have downsides, such as the slow degradation which is a problem when the plastics end up in nature. Another issue is that most of the plastics produced today originate from fossil resources, which make them non-renewable. An alternative material that has gained a lot of attention in recent years due to its potential to solve both of these problems, is bioplastics: a type of plastics that can be produced from renewable sources and degraded e.g., in composts. One biopolymer that is of high interest for production of bioplastics is called polyhydroxybutyrate (PHB). PHB has characteristics that are similar to petroleum-based plastics and can be produced by different microorganisms. To produce PHB in microorganisms three genes are needed. The goal of this project was to insert these genes in a yeast that normally does not produce PHB and thereby turn the yeast cells into small micro-factories for biopolymers. The yeast that was chosen for this project is believed to have many desirable characteristics that will allow it to make higher amounts of PHB than other microbes. To make this possible, the genes were optimized so that they would be understood by the yeast and then bought from a company that make synthetic genes. The genes were delivered as small circular fragments of DNA, called plasmids. The three genes required for PHB production, together with one antibiotic resistance gene were then assembled together into one large piece of circular DNA (plasmid), by cutting-and-pasting the different pieces together using enzymes. To insert the genes in the yeast, the outer layer of the yeast cells (their “skin”) was weakened using a chemical treatment and an electric shock so that the new DNA could easier be transferred to the inside of the cell. Once inside, the DNA has to be integrated into the genome of the cell to be functional. The yeast has a DNA-repairing system that helps it survive damages to the DNA, that can take the new DNA and integrate it in the chromosome during the repairing. To find which cells have properly put the new DNA in its genome (three PHB genes and one antibiotic resistance gene), the cells are grown in presence of the antibiotic, and only the cells with the new DNA will survive. After these genetically modified yeast cells had been constructed, it was time to test if they worked as small micro-factories that produce PHB. The cells were cultivated for 3 days and samples to analyze if PHB was produced were taken. The cells were also evaluated for how fast they grew and consumed glucose. In the end, a yeast that could produce PHB was found. Future work can now try to make these micro-factories better and more efficient.