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

Adamczyk, Paul A.; Hwang, Hee Jin; Chang, Ta-Hsuan; Gao, Yuqian; Baidoo, Edward E.K.; Kim, Joonhoon; Webb-Robertson, Bobbie-Jo M.; Flores, Javier E.; Quijano, Kirch Czarina; Burnet, Meagan C.; Munoz, Nathalie; Sundstrom, Eric; Gladden, John M.; Liu, Di

doi:10.1016/j.ymben.2025.02.014

Title: The oleaginous yeast Rhodosporidium toruloides engineered for biomass hydrolysate-derived (E)-α-bisabolene production

Journal Article · Mon Mar 03 00:00:00 UTC 2025 · Metabolic Engineering

DOI: https://doi.org/10.1016/j.ymben.2025.02.014 · OSTI ID:2550668

Adamczyk, Paul A. ^[1]; Hwang, Hee Jin ^[1]; Chang, Ta-Hsuan ^[2];

^[3]; Baidoo, Edward E.K. ^[4];

^[3]; Webb-Robertson, Bobbie-Jo M. ^[3]; Flores, Javier E. ^[3]; Quijano, Kirch Czarina ^[2]; Burnet, Meagan C. ^[3]; Munoz, Nathalie ^[3]; Sundstrom, Eric ^[2];

^[5];

^[1]

USDOE Agile BioFoundry, Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
USDOE Agile BioFoundry, Emeryville, CA (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
USDOE Agile BioFoundry, Emeryville, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
USDOE Agile BioFoundry, Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)

The oleaginous yeast Rhodosporidium 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)-a-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 competing 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 300g/L total sugar, achieving 20.8g/L bisabolene, the highest reported titer in the literature. A 2L glucose minimal medium bioreactor achieved 19.3 g/L bisabolene and a literature-high productivity of 0.11 g/L/h.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL); Sandia National Laboratories (SNL-CA), Livermore, CA (United States)

Sponsoring Organization:: US Department of Energy; USDOE National Nuclear Security Administration (NNSA); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO); USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23), Biological Systems Science Division (SC-23.2 )

Grant/Contract Number:: AC02-05CH11231; AC05-76RL01830; NA0003525

OSTI ID:: 2550668

Report Number(s):: PNNL-SA--210177

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering Vol. 90; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Title: The oleaginous yeast Rhodosporidium toruloides engineered for biomass hydrolysate-derived (E)-α-bisabolene production

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