Thermostable Enzyme Variants in the Lower Mevalonate Pathway Improve Isoprenoid Production by Cell-Free Biocatalysis

Sarnik, Sylvia A.; Martinsen, Mia R.; Korman, Tyler P.; Schwartz, Daniel K.; Kaar, Joel L.; Bomble, Yannick J.

doi:10.1021/acssuschemeng.5c03763

Thermostable Enzyme Variants in the Lower Mevalonate Pathway Improve Isoprenoid Production by Cell-Free Biocatalysis

Journal Article · Wed Aug 06 00:00:00 EDT 2025 · ACS Sustainable Chemistry & Engineering

DOI:https://doi.org/10.1021/acssuschemeng.5c03763· OSTI ID:2587729

Sarnik, Sylvia A. ^[1]; Martinsen, Mia R. ^[2]; Korman, Tyler P. ^[3]; ^[4]; ^[4]; ^[5]

University of Colorado, Boulder, CO (United States); National Renewable Energy Laboratory (NREL), Golden, CO (United States)
National Renewable Energy Laboratory (NREL), Golden, CO (United States); Colorado School of Mines, Golden, CO (United States)
Exozymes, Inc., Monrovia, CA (United States)
University of Colorado, Boulder, CO (United States)
National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Cell-free biocatalysis is a rapidly evolving field with great potential for sustainably producing valuable chemicals. Some challenges in cell-free biocatalysis include reaction longevity, enzyme stability, and the cost of the biocatalysts. Here, the challenge of enzyme instability was addressed by employing thermophilic enzymes to improve the productivity of the lower mevalonate pathway, using limonene as an example isoprenoid product. The Classical mesophilic mevalonate pathway was compared to a newly assembled set of thermophilic enzymes comprising the Archaea I mevalonate pathway. The thermophilic pathway enzymes were thermostable to at least 60 °C and exhibited a 6× longer operating lifetime at 22 °C. Thus, despite lower initial activity rates at ambient temperature, the thermophilic pathway was longer-lived and resulted in a more productive cell-free reaction overall, achieving 1.7× higher yield of limonene compared to using enzymes from mesophiles. Moreover, the thermostable pathway retained activity for longer with the challenge of solvent exposure, namely, ethanol and isoprenol, which broadens the scope of accessible substrates and/or products in cell-free reactions employing this pathway. Altogether, we showed significant improvement in the stability and productivity of the lower mevalonate pathway, which will enable more efficient cell-free biosynthesis of isoprenoid products. This represents a valuable strategy to increase the robustness of cell-free systems by carefully sourcing biocatalysts from thermophilic organisms, which have proven to be resilient to challenges unique to cell-free reaction systems.

View Accepted Manuscript (DOE)

Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Transportation Office. Bioenergy Technologies Office

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 2587729

Report Number(s):: NREL/JA--2700-93773

Journal Information:: ACS Sustainable Chemistry & Engineering, Journal Name: ACS Sustainable Chemistry & Engineering Journal Issue: 32 Vol. 13; ISSN 2168-0485

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Thermostable Enzyme Variants in the Lower Mevalonate Pathway Improve Isoprenoid Production by Cell-Free Biocatalysis

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