Masoud Tourang

High production cost is one of the major factors that limit the market growth of polyhydroxyalkanoates (PHAs) as a biopolymer. Improving PHA synthesis performance and utilizing low-grade feedstocks are two logical strategies for reducing costs. As an oleaginous yeast, Y. lipolytica has a high carbon flux through acetyl-CoA (the main PHB precursor), which makes it a desired cell factory for PHB biosynthesis. In the current study, two different metabolic pathways (NBC and ABC) were introduced into Y. lipolytica PO1f for synthesizing PHB. Compared to the ABC pathway, the NBC pathway, which includes NphT7 to redirect the lipogenesis pathway and catalyze acetoacetyl-CoA synthesis in a more energy-favored reaction, led to PHB accumulation of up to 11% of cell dry weight (CDW), whereas the ABC pathway resulted in non-detectable accumulations of PHB. Further modifications of the strain with the NBC pathway through peroxisomal compartmentalization and gene dose overexpression reached 41% PHB of CDW and a growth rate of 0.227 h⁻¹. A low growth rate was observed with acetate as the sole source of carbon and energy or glucose as the sole substrate at high concentrations. Using a co-substrate strategy helped overcoming the inhibitory and toxic effects of both substrates. Cultivating the engineered strain in the optimal co-substrate condition predicted by response surface methodology (RSM) led to 83.4 g/L of biomass concentration and 31.7 g/L of PHB. These results offer insight into a more cost-effective production of PHB with engineered Y. lipolytica.

High production cost is one of the major factors that limit the market growth of polyhydroxyalkanoates (PHAs) as a biopolymer. Improving PHA synthesis performance and utilizing low grade feedstock are two logical strategies for reducing the cost. As an oleaginous yeast, Y. lipolytica has a high carbon flux through acetyl-CoA (main PHB precursor) which makes it a desired cell factory for PHB biosynthesis. Two different metabolic pathways introduced into Y. lipolytica PO1f in this study for enhancing PHB biosynthesis, along with glucose and acetate as co-substrates. Results showed that NBC pathway led to more than 11% PHB accumulation, while there was non-detectable PHB accumulations using ABC pathway. Further modifications were done using peroxisomal compartmentalization and gene-dose overexpression. Final strain showed up to 41% PHB of CDW with a growth rate of 0.227 h-1. Low growth rate was observed using acetate or glucose as sole carbon and energy source. The co-substrate strategy showed compensatory effect on overcoming inhibitory and toxic effects of both substrates at high concentrations. Cultivating the engineered strain in optimal co-substrate condition predicted by RSM led to 83.4 g/L biomass concentration and 31.7 g/L PHB.