Polyhydroxyalkanoate production in yeast Saccharomyces cerevisiae

This thesis was inspired by the global need to find replacements for non-biodegradable plastic materials originating from fossil sources. Microbially produced polyhydroxyalkanoates (PHAs) are diverse group of fully biobased and biodegradable polyesters with interesting properties for many applications. However, wide use of PHAs is still hindered by their limited mechanical properties and relatively high costs of sugar feedstocks. This thesis explored possibilities to use lignocellulose derived cellobiose as a carbon source for PHA production in yeast Saccharomyces cerevisiae. In addition, it focused on polymerization of a 2-hydroxacid (D-lactic acid) and controlling PHA copolymer structure in yeast S. cerevisiae with synthetic biology. These approaches contribute to the wider themes of using lignocellulose-based carbon sources for PHA production and improving PHA polymer properties for use in even wider range of different applications in future.

Polymerization of 2-hydroxyacids in S. cerevisiae was shown for the first time in Publication 1. As an example, D-lactic was polymerized into poly(D-lactic acid) (PDLA) and copolymer poly(D-lactate-co-3-hydroxybutyrate) [P(LA-3HB)]. D-lactic acid was produced in vivo by ex-ressing ldhA gene from Leuconostoc mesenteroides. Expressed pathways included also either PHA synthase gene phaC1437Ps6-19 from Pseudomonas sp. MBEL 6-19 or phaC1Pre from Pseudomonas resinovorans, both with same four amino acid changes E130D, S325T, S477G, and Q481K. The strains were supplied also with propionyl-CoA transferase genes, either pctMe from Megasphaera elsdenii or engineered pct540Cp from Clostridium propionicum, and with PHB pathway genes phaA, phaB1, and phaC1 from Cupriavidus necator. The highest PDLA accumulation level was 0.73% of the cell dry weight (CDW) and the highest P(LA-3HB) accumulation 3.6% of CDW. Formed polymers had small molecular weights (Mw) of less than 7 kDa and 25 kDa, respectively.

The 2-hydroxyacid polymerization was improved in Publication 2 by controlling D-lactic acid production in vivo by adjusting expression of ldhA gene with doxycycline-based Tet-On system. Increase in D-lactic acid production improved PLDA and P(LA-3HB) accumulation in the cells to 5.2% and 19% of CDW, respectively. Adjustable ldhA expression allowed also control of D-lactic acid content in the copolymer from 6 to 92 mol%.

In Publication 3, S. cerevisiae was engineered to utilize cellobiose for poly(3-hydroxybutyrate) (PHB) production. Cellobiose uptake was enabled by expressing cellodextrin transporter gene CDT-1 from Neurospora crassa and either cellobiose phosphorylase gene cbp from Ruminococcus flavefaciens or β-glucosidase gene GH1-1 from N. crassa. Both pathways allowed accumulation of high molecular weight PHB (Mw 450-500 kDa) in S. cerevisiae. The strains expressing GH1-1 consumed cellobiose faster than strains expressing cbp, which lead also to faster growth and PHB accumulation in GH1-1 strains. However, both strains accumulated more PHB on cellobiose (as % of CDW and per consumed sugar) in comparison to control strain grown on glucose. The highest PHB accumulation levels of 13.4±0.9% and 18.5±3.9% PHB of CDW for cbp and GH1-1 strains, respectively, were obtained in pH controlled (pH 6) bioreactor experiments.