Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids

Holmes, Eric C.; Bleem, Alissa C.; Johnson, Christopher W.; Beckham, Gregg T.

doi:10.1016/j.ymben.2024.10.011

Title: Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids

Journal Article · 28 October 2024 · Metabolic Engineering

DOI: https://doi.org/10.1016/j.ymben.2024.10.011 · OSTI ID:2477614

Holmes, Eric C. ^[1]; Bleem, Alissa C. ^[1]; Johnson, Christopher W. ^[1];

^[1]

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

Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, in this study, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.

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

Sponsoring Organization:: USDOE Laboratory Directed Research and Development (LDRD) Program

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 2477614

Report Number(s):: NREL/JA--2A00-91435; MainId:93213; UUID:071bb607-0037-4cd6-8f5c-78e39ae70c55; MainAdminId:74122

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering Vol. 86; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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09 BIOMASS FUELS
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
PHB biosynthesis
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volatile fatty acids
waste-derived feedstock

Title: Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids

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