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Engineering of an oleaginous bacterium for the production of fatty acids and fuels

Nature Chemical Biology volume 15, pages 721–729 (2019)Cite this article

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Abstract

Production of free fatty acids (FFAs) and derivatives from renewable non-food biomass by microbial fermentation is of great interest. Here, we report the development of engineered Rhodococcus opacus strains producing FFAs, fatty acid ethyl esters (FAEEs) and long-chain hydrocarbons (LCHCs). Culture conditions were optimized to produce 82.9 g l−1 of triacylglycerols from glucose, and an engineered strain with acyl-coenzyme A (CoA) synthetases deleted, overexpressing three lipases with lipase-specific foldase produced 50.2 g l−1 of FFAs. Another engineered strain with acyl-CoA dehydrogenases deleted, overexpressing lipases, foldase, acyl-CoA synthetase and heterologous aldehyde/alcohol dehydrogenase and wax ester synthase produced 21.3 g l−1 of FAEEs. A third engineered strain with acyl-CoA dehydrogenases and alkane-1 monooxygenase deleted, overexpressing lipases, foldase, acyl-CoA synthetase and heterologous acyl-CoA reductase, acyl-ACP reductase and aldehyde deformylating oxygenase produced 5.2 g l−1 of LCHCs. Metabolic engineering strategies and engineered strains developed here may help establish oleaginous biorefinery platforms for the sustainable production of chemicals and fuels.

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Fig. 1: The Kennedy pathway of TAG biosynthesis and fed-batch culture of the wild-type strain.
Fig. 2: Metabolic engineering strategies for the production of FFAs from TAGs and fed-batch cultures of engineered fatty acids-producing R. opacus strains expressing different lipases together with in vitro lipase activities.
Fig. 3: Metabolic engineering strategies and fed-batch cultures of engineered R. opacus strains for the production of FAEEs.
Fig. 4: Metabolic engineering strategies and fed-batch cultures of engineered R. opacus strains for the production of LCHCs.

Data availability

Data pertaining to this study are contained within the published paper and its Supplementary Information, or are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea (grant nos NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) to S.Y.L.

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Authors and Affiliations

  1. Metabolic and Biomolecular Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea

    Hye Mi Kim, Tong Un Chae, So Young Choi, Won Jun Kim & Sang Yup Lee

  2. BioInformatics Research Center, KAIST, Daejeon, Republic of Korea

    Sang Yup Lee

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Contributions

S.Y.L. generated the idea. H.M.K. and S.Y.L. designed the project. H.M.K. performed experiments. H.M.K., T.U.C., S.Y.C. and S.Y.L. analyzed the data. W.J.K. performed in silico simulations. H.M.K., T.U.C., S.Y.C. and S.Y.L. wrote the manuscript.

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Correspondence to Sang Yup Lee.

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Competing interests

S.Y.L. declares competing financial interests, as the strains and processes described in this paper are of commercial interest and are patents filed including, but not limited to, KR101546885B1, US9322004B2, KR101334981B1 and WO2011155799A2 for potential commercialization.

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Supplementary information

Supplementary Information

Supplementary Tables 1–8, Supplementary Figures 1–23, Supplementary Notes 1 and 2

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Supplementary Dataset 1

Supplementary Dataset 1 (Raw data of fed-batch fermentations).

Supplementary Dataset 2

Supplementary Dataset 2 (Metabolic reactions for genome-scale modeling).

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Kim, H.M., Chae, T.U., Choi, S.Y. et al. Engineering of an oleaginous bacterium for the production of fatty acids and fuels. Nat Chem Biol 15, 721–729 (2019). https://doi.org/10.1038/s41589-019-0295-5

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