The oleaginous yeast Rhodosporidium toruloides engineered for biomass hydrolysate-derived (E)-⍺-bisabolene production.

The oleaginous yeast R. toruloides has been exploited for many bioproducts, including several terpenes, owing to its oleaginous nature and biomass inhibitor tolerance. Here, we built upon previous (E)-⍺-bisabolene work by iteratively stacking the complete mevalonate pathway from Saccharomyces cerevisiae onto a multicopy bisabolene synthase parent strain. Metabolomics and proteomics verified heterologous pathway expression and identified metabolic bottlenecks at three intermediate steps, with candidate feedback-resistant mevalonate kinases screening improving titers 15%. Subtle differences in codon optimization, and preliminary attenuation of completing flux toward lipids resulted in 6-fold, 7-fold higher titers relative to controls, respectively. Media optimization led to modest improvements, with zinc identified as the most promising at 10% titer improvement. Ultimately, high-performance strains were cultivated with corn-stover biomass hydrolysate in microtiter plates at 300 g/L total sugar, achieving 20.8 g/L bisabolene, the highest reported titer in the literature. A 2 L glucose minimal medium bioreactor achieved 19.3 g/L bisabolene and a literature-high productivity of 0.11 g/L/h.