Metabolic engineering of Saccharomyces cerevisiae for polyhydroxybutyrate production

Doctoral thesis, 2013

Establishing industrial biotechnology for the production of chemical compounds from the biosynthetic pathway has received a significant boost with the implementation of metabolic engineering. At present, metabolic engineering in Saccharomyces cerevisiae gains significant advantages of integration of knowledge acquired through a long history of use and data acquisition from novel –omics technologies hence enabling the development of a tailor-made S. cerevisiae with desired features for various industrial applications. With regard to environmentally friendly (eco-friendly) materials, engineering of biodegradable polyhydroxybutyrate (PHB) producing microbes has been studied as a potential alternative to petroleum-based thermoplastics. Heterologous expression of the bacterial PHB biosynthesis pathway in S. cerevisiae involves the utilization of acetyl-CoA, an intermediate of the central carbon metabolism, as precursor and NADPH, a redox cofactor used during anabolic metabolism, as a required cofactor for the catalyzing enzymes in the PHB biosynthesis pathway. Provision of acetyl-CoA and NADPH by alteration of the endogenous pathways and/or implementation of a heterologous gene/pathway was investigated with the aim to improve PHB production in S. cerevisiae. Since the specific growth rate and the type of carbon source (fermentable/non-fermentable) influence cell physiology and affect the growth of S. cerevisiae, PHB production was examined at different specific growth rates on different carbon sources. Overexpression of genes in the native ethanol degradation pathway and heterologous expression of a phosphoketolase pathway from Aspergillus nidulans aiming to increase the production of cytosolic acetyl-coA and chromosomal integration of gapN from Streptococcus mutans to enhance the availability of NADPH were evaluated for their possibility to promote PHB production in S. cerevisiae. The enhancement of acetyl-CoA and NADPH either by the combined strategies of the ethanol degradation pathway and gapN or utilization of the phosphoketolase pathway resulted in the improved PHB content from 4 mg/gDW in the reference strain to approximately 28 mg/gDW. It is difficult for S. cerevisiae to compete with other natural PHB producers like Ralstonia eutropha which benefit from native enzymes for the biosynthesis or with the engineered E. coli since the metabolism in S. cerevisiae is more complex and involves compartmentalization and shuttle systems for precursor and redox balancing. However, the strategies employed in this study involve both engineering of the central carbon and redox metabolism and it demonstrated that it is possible to substantially improve PHB production. Furthermore, the applied strategies may well be suitable also for improving the production of other chemicals, derived from acetyl-CoA and requires NADPH for its biosynthesis.