Application of Synthetic Biology for Biopolymer Production using Saccharomyces cerevisiae

Abstract: Plastics are versatile, cheap and durable materials that are omnipresent in modern society. Since most of them are derived from crude oil and are not biodegradable, their production leads to the depletion of fossil fuels and the accumulation of enormous amounts of plastic waste that pollutes ecosystems worldwide. For this reason, the European Union and other organisations are investing in research aimed at developing eco-friendly alternatives such as bioplastics.Poly-3-D-hydroxybutyrate (PHB) is a type of biodegradable bioplastic that is naturally synthesized and used by specific microorganisms as an energy source and for carbon storage under stressful environmental conditions. However, these micro-organisms are not well suited for growth in biomass hydrolysates. Baker’s yeast, Saccharomyces cerevisiae, might represent an interesting host for PHB production since it is a well-known industrial platform for the effective conversion of various carbon sources into key precursor metabolites, and has high tolerance to low pH and fermentation inhibitors that are present in biomass hydrolysates. The aim of the present work was to use synthetic biology tools to “rewire” S. cerevisiae metabolism, and to investigate whether it can be used for the efficient production of PHB from xylose feedstock.Recombinant S. cerevisiae strains carrying the oxido-reductive xylose pathway from Scheffersomyces stipitis were engineered for heterologous gene expression of the biosynthetic PHB pathway from Cupriavidus necator. This enabled PHB production from xylose as the sole carbon source, although the production was low and oxygen-dependent. Further improvements were achieved by modification of the cofactor balance through the introduction of alternative enzymes with different cofactor requirements. The introduction of a xylose reductase variant with increased affinity for NADH cofactor enabled better redox homeostasis, allowing PHB production from xylose under anaerobic conditions. PHB biosynthesis was also improved by the substitution of the NADPH-dependent acetoacetyl-CoA reductase by an NADH-dependent counterpart.As the availability of cytosolic acetyl-CoA, which is the precursor of PHB, remained a major challenge, several strategies were tested to redirect the carbon flux towards acetyl-CoA, such as the use of alternative pathways and the downregulation of the ethanol route. However, the production of PHB could only be improved under aerobic conditions due to tight cell regulation at the cytosolic NADH and acetyl-CoA levels.The xylose-fermentation capabilities of different Spathaspora species were ex¬plored in parallel, with the aim of finding better enzymes for further improvement of anaerobic xylose fermentation by S. cerevisiae. The xylose reductase gene XYL1.2 from Sp. passalidarum was found to encode an enzyme with a high affinity for NADH. When transferred to S. cerevisiae, xylose fermentation and ethanol production were improved under anaerobic conditions.This work demonstrates how the S. cerevisiae genome can be reprogrammed for PHB production from sugars derived from lignocellulosic biomass. However, further modification of the metabolism will be necessary before industrial implementation is possible.