Engineering Yarrowia lipolytica as a yeast cell factory for the de novo production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

The oleaginous yeast <i>Yarrowia lipolytica</i> presents a promising chassis for the production of biodegradable plastic polyhydroxyalkanoates (PHAs) due to its advantages in precursor supply capabilities and cost-effective substrate utilization. However, <i>Y. lipolytica</i> has primarily been engineered for the synthesis of homopolymeric PHAs, such as poly (3-hydroxybutyrate) (P3HB), which have limited material properties and application potential. In this study, the succinate dehydrogenase-deficient <i>Y. lipolytica</i> strains were utilized as hosts for the de novo biosynthesis of copolymer poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). By employing a strategy of subcellular partitioning, poly (4-hydroxybutyrate) (P4HB) was synthesized in the cytoplasm using 4-hydrobutyrate (4HB) derived from mitochondrial sources as a monomer. We then introduced the biosynthetic pathway for 3-hydroxybutyryl-CoA to facilitate P34HB accumulation. Importantly, the ratio of 4HB monomer in P34HB could be adjusted from 9.17 mol% to 45.26 mol% by varying medium components and carbon sources. Under fed-batch fermentation conditions in a 5 L bioreactor, the engineered strain achieved a P34HB titer of 18.61 g/L representing 19.18 % of cell dry weight (CDW). This study not only demonstrates the potential of <i>Y. lipolytica</i> for P34HB synthesis, but also expands the monomer pool available for PHAs in yeast cell factories.