Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids

Abstract

Medium-chain fatty acids (MCFAs; C6–C12) are valuable molecules used for biofuel and oleochemical production; however, it is challenging to synthesize these fatty acids efficiently using microbial biocatalysts due to the cellular toxicity of MCFAs. In this study, both the endogenous fatty acid synthase (FAS) and an orthogonal bacterial type I FAS were engineered for MCFA production in the yeast Saccharomyces cerevisiae. To improve cellular tolerance to toxic MCFAs, we performed directed evolution of the membrane transporter Tpo1 and strain adaptive laboratory evolution, which elevated the MCFA production by 1.3 ± 0.3- and 1.7 ± 0.2-fold, respectively. We therefore further engineered the highly resistant strain to augment the metabolic flux towards MCFAs. This multidimensional engineering of the yeast at the single protein/enzyme level, the pathway level and the cellular level, combined with an optimized cultivation process, resulted in the production of >1 g l−1 extracellular MCFAs—a more than 250-fold improvement over the original strain.

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Fig. 1: Engineering microbial FASI enzymes for MCFA synthesis.
Fig. 2: Engineering the membrane transporter Tpo1.
Fig. 3: Improving cell fitness via ALE.
Fig. 4: MCFAs produced by engineered yeast cell factories.

Data availability

All genomic sequences are available at NCBI under BioProject accession code PRJNA542834. The data that support the findings of this study are available from the corresponding author upon reasonable request. All plasmids and strains used in this study are available from the corresponding author under a material transfer agreement.

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Acknowledgements

This work was financially supported by the Novo Nordisk Foundation (grant no. NNF10CC1016517), Energimyndigheten, the Knut and Alice Wallenberg Foundation and the Swedish Foundation for Strategic Research. This project has received funding from the European Union’s Horizon 2020 Framework Programme for Research and Innovation (grant agreement no. 720824). We thank Y. J. Zhou, F. David and T. Yu for critical discussion, A. Hoffmeyer for genome sequencing, and the Chalmers Mass Spectrometry Infrastructure for assistance with metabolite analysis.

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Z.Z. and J.N. conceived this study. Z.Z. designed and performed most of the experiments, analysed the data and drafted the manuscript. Y.H. engineered the Tpo1 transporter and assisted with metabolite analysis and fermentation. V.S. and Y.C. assisted with data analysis and interpretation. P.G.T. participated in the ALE. R.P. analysed the genome sequencing data. All authors revised and approved the manuscript.

Corresponding author

Correspondence to Jens Nielsen.

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V.S. and J.N. are shareholders in Biopetrolia AB. All other authors declare no competing interests.

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Supplementary Figs. 1–18, Tables 1–5 and references.

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Supplementary Data 1

Genetic mutations during ALE.

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Zhu, Z., Hu, Y., Teixeira, P.G. et al. Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids. Nat Catal 3, 64–74 (2020). https://doi.org/10.1038/s41929-019-0409-1

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