This thesis aimed to investigate how plant monoterpenes can be used to produce biobased plastics. Monoterpenes are volatile compounds, produced by plants to defend themselves against insects and pathogens or to attract pollinators. Many monoterpenes have a characteristic odour, and are used by humans in all kinds of products for their nice smell or taste. For example the monoterpene (+)-limonene has a fresh citrus odour, and is used in cosmetics and sodas. Recently, however, it was demonstrated that the chemical structure of some monoterpenes may also be suitable to serve as a feed stock for the synthesis of commodity chemicals and biomaterials.
Plants produce monoterpenes in specialized structures, such as glandular trichomes. Trichomes are gland-like structures on the leaf surface that serve as small biochemical factories. Plants produce and store monoterpenes and other volatile compounds in these trichomes. However, the amount of monoterpenes in plants is often not large enough for bulk applications. Therefore, I set out to investigate which genes plants use to produce monoterpenes, and if I can express these genes in a better production platform, in order to produce larger amounts of monoterpenes.
Monoterpenes consist of 10 carbon atoms and are synthesized from the precursor geranyl diphosphate (GPP) in the plastids of the plant cell. After synthesis of the monoterpene backbone, usually several structural modifications, for example oxidation, take place, by other enzymes in the cell. Chapter 1 of this thesis introduces what monoterpenes are, how they are synthesized in plants and how they can be produced by metabolic engineering in heterologous hosts like micro-organisms for human applications.
One of the best studied monoterpenes is limonene. Chapter 2 reviews the existing and potential applications of limonene as well as the state of the art in its microbial production. The chapter describes which genes have been used for the biosynthesis of limonene, as well as the strategies that have been employed to enhance the production in micro-organisms.
Chapter 3 describes our production of limonene using the micro-organism Saccharomyces cerevisieae (yeast). For this purpose, a mutated yeast strain was used, which produces a small amount of GPP as precursor for limonene biosynthesis. Limonene has a chiral centre, which means it can exist in two enantiomers, (+) or (-), which are mirror images. I showed that it is possible to produce both forms in yeast by introducing limonene synthase genes from different plant species. It turned out that it is not straightforward to harvest limonene from yeast cultures, as it is very volatile and does not mix well with the culture broth. Therefore, a system was developed to trap limonene from the yeast culture headspace during production. Compared to other limonene harvesting systems, this resulted in a better yield.
Chapter 4 describes how a natural derivative of limonene, methylperillate, can be converted to plastic. Methylperillate has a suitable structure to be converted into a polymer building block. To demonstrate this, methylperillate was converted to the bulk chemical terephthalic acid, which is the building block of polyethylene terephthalate (PET). Due to the high structural similarity between methylperillate and terephthalic acid, a short chemical synthesis route consisting of two steps could be developed.
For the large scale application of methylperillate for biobased commodity chemicals, it would be useful to produce methylperillate in micro-organisms. Methylperillate has the same backbone as limonene, but with a methylated carboxyl group at the C7-position. At the onset of this thesis not much was known about the enzymes involved in the biosynthesis of such methylated carboxyl groups. In Chapter 5, I characterise a biosynthetic pathway to methylperillate. After screening several plant species we found that Salvia dorisiana, a sage species, can produce methylperillate in the glandular trichomes on its leaves. Trichomes were isolated from the leaves and used as the source, using genomics techniques, for the isolation of four genes, which I showed are involved in the biosynthesis of methylperillate. Production of methylperillate was established in the tobacco-like model plant, Nicotiana benthamiana, using these four Salvia genes. In the future these genes could also be used in yeast or other microbes to produce methylperillate in fermenters.
In Chapter 6 the research results of this thesis are discussed. A perspective is provided on producing bioplastics from compounds like methylperillate. Questions addressed in this chapter include how much monoterpenes should be produced to realistically use them for the production of biomaterials, and which possible solutions can be foreseen to produce monoterpenes on a larger scale. One future scenario is to focus on the use of monoterpenes for more specific, high-value applications by taking advantage of their natural chirality.
All in all, this research is an important first step to use specific molecules from plants as an alternative source for biomaterials. Potentially, this will decrease dependence on fossil oil, and improve sustainability of production processes.