Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

Kirby, James; Geiselman, Gina M.; Yaegashi, Junko; Kim, Joonhoon; Zhuang, Xun; Tran-Gyamfi, Mary Bao; Prahl, Jan-Philip; Sundstrom, Eric R.; Gao, Yuqian; Munoz, Nathalie; Burnum-Johnson, Kristin E.; Benites, Veronica T.; Baidoo, Edward E. K.; Fuhrmann, Anna; Seibel, Katharina; Webb-Robertson, Bobbie-Jo M.; Zucker, Jeremy; Nicora, Carrie D.; Tanjore, Deepti; Magnuson, Jon K.; Skerker, Jeffrey M.; Gladden, John M.

doi:10.1186/s13068-021-01950-w

Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

Journal Article · Wed Apr 21 00:00:00 EDT 2021 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/s13068-021-01950-w· OSTI ID:2325570

Kirby, James; Geiselman, Gina M.; Yaegashi, Junko; Kim, Joonhoon; Zhuang, Xun; Tran-Gyamfi, Mary Bao; Prahl, Jan-Philip; Sundstrom, Eric R.; Gao, Yuqian; Munoz, Nathalie; Burnum-Johnson, Kristin E.; Benites, Veronica T.; Baidoo, Edward E. K.; Fuhrmann, Anna; Seibel, Katharina; Webb-Robertson, Bobbie-Jo M.; Zucker, Jeremy; Nicora, Carrie D.; Tanjore, Deepti; Magnuson, Jon K. more »

Abstract Background

Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides , is a promising host for the production of terpenes from lignocellulosic hydrolysates.

Results

This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides . The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement.

Conclusions

This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2325570

Alternate ID(s):: OSTI ID: 1826711
OSTI ID: 1828023
OSTI ID: 1784623

Journal Information:: Biotechnology for Biofuels, Journal Name: Biotechnology for Biofuels Journal Issue: 1 Vol. 14; ISSN 1754-6834

Publisher:: Springer Science + Business MediaCopyright Statement

Country of Publication:: Netherlands

Language:: English

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Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

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Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

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