Platform for the production of cyclic and aromatic compounds in Saccharomyces cerevisiae

Abstract

The switch from a petrol based economy to a biobased economy has brought researchers to investigate how biotechnology may be utilized to create cell factories producing already known compounds, while production of new-to-nature or designed compounds are also receiving attention. The understanding of natural product chemistry has long been improving, especially with the application of molecular biology to investigate the enzymatic machinery behind the biosynthesis of natural products. The understanding of polyketide synthases, which produces the natural products polyketides, has also brought about engineering attempts of these complex enzymes and it has to some degree been possible to design novel polyketide scaffolds. The goal of this PhD project was to investigate the possibility to create a programmable platform for creation of polyketides with a given chain length and folding pattern. Initially, the possibility of combining type III PKSs with cyclases from type II PKS systems with Saccharomyces cerevisiae as production organism was investigated (Chapter 3 - Manuscript I). This approach was an attempt to divide the polyketide chain synthesis and the folding of the polyketide chain, into different enzymes making combinatorial experiments possible. It was found that a variety type III PKSs were functionally expressed in S. cerevisiae producing polyketide chains of C6 to C16 depending on the expressed PKS. With the combination of the C16 producing octaketide synthase (OKS) and two different cyclases directing either the C7-C12 or C9-C14 folding patterns, it was shown that it was possible to direct the folding of the type III PKS polyketide product. This was a proof-of-concept step towards making a platform to design polyketides with a certain chain length and folding pattern. Next, we wanted to investigate if it was possible to utilize the knowledge gained in the initial study to produce flavokermesic acid, which is a precursor of the industrial colorant carminic acid (Chapter 4 - Manuscript II). The pathway to produce flavokermesic acid consisting of a type III PKS and two cyclases had previously been expressed in Nicotiana benthamiana and Aspergillus nidulans. The aim of this study was to investigate if utilizing S. cerevisiaeas a cell factory production host was feasible, as well as investigating if fusion of biosynthetic enzymes would limit shunt products of the pathway, and thereby improve the production of flavokermesic acid. It was found that S. cerevisiae was able to produce 52 mg flavokermesic acid per liter of medium if the biosynthetic enzymes were not fused, while all versions of the fused biosynthetic enzymes were found to have reduced levels of flavokermesic acid, compared to the non-fused version. Two more tailoring steps are needed in order to synthesize carminic acid from flavokermesic acid, but the work presented here shows the potential of the platform to create a polyketide scaffold, which can be developed into an industrially important compound. For the platform to be fully developed the incorporation of tailoring enzymes to alter the polyketide scaffolds are needed. To gain insight into a natural system, evolved to generate a high chemical diversity of polyketides, investigation of the pgl1gene cluster in the fungal genus Fusarium was conducted (Chapter 5 -Manuscript III). The investigation had two aims. The first being to investigate the diversity of the biosynthetic gene cluster throughout the genus of Fusarium, and the second being to propose a biosynthetic model for the chemical diversity thought to be derived form the pgl1 biosynthetic cluster. The genes in the biosynthetic gene cluster were found to be highly conserved across the genus while the topology of the cluster was found to be less conserved, especially in the species complexes solani, decemcellulare and buxicola. It was proposed that these species within these species complexes could contain more biosynthetic activities in the pgl1 cluster than other species in the Fusariumgenus. This was an interesting hypothesis as the biosynthetic steps needed to create the chemodiversity found in the putative biosynthetic pathway, included many more enzymatic activities than what is included in currently identified biosynthetic cluster. This could either mean that more genes are implicated in the biosynthetic pathway, that some of the needed conversions happen spontaneously, or that the biosynthetic enzymes are promiscuous and therefore have low substrate specificity and may have several enzymatic specificities. In summary, this PhD project has investigated the possibilities of creating a programmable platform for production of aromatic polyketides in S. cerevisiae, which allow control of chain length and folding pattern. Furthermore it has investigated what enzymatic machinery is employed in nature to derivatize a given polyketide scaffold. This work lays the foundation for further development of the platform to expand polyketide diversity with longer polyketide chains and cyclases to direct more of the folding of the polyketide chain, and furthermore, to attempt introduction of tailoring enzymes to fully develop the chemodiversity, which is possible to create with the platform.