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.
Subscribe to Journal
Get full journal access for 1 year
only $8.67 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Biermann, U., Bornscheuer, U., Meier, M. A. R., Metzger, J. O. & Schäfer, H. J. Oils and fats as renewable raw materials in chemistry. Angew. Chem. Int. Ed. 50, 3854–3871 (2011).
Sarria, S., Kruyer, N. S. & Peralta-Yahya, P. Microbial synthesis of medium-chain chemicals from renewables. Nat. Biotechnol. 35, 1158–1166 (2017).
Hernández Lozada, N. J. et al. Highly active C8-acyl-ACP thioesterase variant isolated by a synthetic selection strategy. ACS Synth. Biol. 7, 2205–2215 (2018).
Fargione, J., Hill, J., Tilman, D., Polasky, S. & Hawthorne, P. Land clearing and the biofuel carbon debt. Science 319, 1235–1238 (2008).
Pfleger, B. F., Gossing, M. & Nielsen, J. Metabolic engineering strategies for microbial synthesis of oleochemicals. Metab. Eng. 29, 1–11 (2015).
Töpfer, R., Martini, N. & Schell, J. Modification of plant lipid synthesis. Science 268, 681–686 (1995).
Jing, F. et al. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity. BMC Biochem. 12, 44 (2011).
Val, D., Banu, G., Seshadri, K., Lindqvist, Y. & Dehesh, K. Re-engineering ketoacyl synthase specificity. Structure 8, 565–566 (2000).
Joshi, A. K., Witkowski, A., Berman, H. A., Zhang, L. & Smith, S. Effect of modification of the length and flexibility of the acyl carrier protein–thioesterase interdomain linker on functionality of the animal fatty acid synthase. Biochemistry 44, 4100–4107 (2005).
Gajewski, J., Pavlovic, R., Fischer, M., Boles, E. & Grininger, M. Engineering fungal de novo fatty acid synthesis for short chain fatty acid production. Nat. Commun. 8, 14650 (2017).
Zhu, Z. et al. Expanding the product portfolio of fungal type I fatty acid synthases. Nat. Chem. Biol. 13, 360–362 (2017).
Gajewski, J. et al. Engineering fatty acid synthases for directed polyketide production. Nat. Chem. Biol. 13, 363–365 (2017).
Torella, J. P. et al. Tailored fatty acid synthesis via dynamic control of fatty acid elongation. Proc. Natl. Acad. Sci. USA 110, 11290–11295 (2013).
Xu, P., Qiao, K., Ahn, W. S. & Stephanopoulos, G. Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals. Proc. Natl. Acad. Sci. USA 113, 10848–10853 (2016).
Leber, C., Choi, J. W., Polson, B. & Da Silva, N. A. Disrupted short chain specific β-oxidation and improved synthase expression increase synthesis of short chain fatty acids in Saccharomyces cerevisiae. Biotechnol. Bioeng. 113, 895–900 (2016).
Heil, C. S., Wehrheim, S. S., Paithankar, K. S. & Grininger, M. Fatty acid biosynthesis: chain-length regulation and control. ChemBioChem 20, 2298–2321 (2019).
Dellomonaco, C., Clomburg, J. M., Miller, E. N. & Gonzalez, R. Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355–359 (2011).
Felnagle, E. A., Chaubey, A., Noey, E. L., Houk, K. N. & Liao, J. C. Engineering synthetic recursive pathways to generate non-natural small molecules. Nat. Chem. Biol. 8, 518–526 (2012).
Yuzawa, S. et al. Short-chain ketone production by engineered polyketide synthases in Streptomyces albus. Nat. Commun. 9, 4569 (2018).
Teixeira, P. G., Siewers, V. & Nielsen, J. Quantitative in vivo evaluation of the reverse β-oxidation pathway for fatty acid production in Saccharomyces cerevisiae. Preprint at http://www.biorxiv.org/content/10.1101/201616v1 (2017).
Boehringer, D., Ban, N. & Leibundgut, M. 7.5-Å cryo-EM structure of the mycobacterial fatty acid synthase. J. Mol. Biol. 425, 841–849 (2013).
Zhou, Y. J. et al. Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories. Nat. Commun. 7, 11709 (2016).
Qiao, K., Wasylenko, T. M., Zhou, K., Xu, P. & Stephanopoulos, G. Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism. Nat. Biotechnol. 35, 173–177 (2017).
Yu, T. et al. Reprogramming yeast metabolism from alcoholic fermentation to lipogenesis. Cell 174, 1549–1558 (2018).
Eriksen, D. T., HamediRad, M., Yuan, Y. & Zhao, H. An orthogonal fatty acid biosynthetic pathway improves fatty acid ethyl ester production in Saccharomyces cerevisiae. ACS Synth. Biol. 4, 808–814 (2015).
Haushalter, R. W. et al. Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production. Metab. Eng. 30, 1–6 (2015).
Jarboe, L. R., Liu, P. & Royce, L. A. Engineering inhibitor tolerance for the production of biorenewable fuels and chemicals. Curr. Opin. Chem. Eng. 1, 38–42 (2011).
Legras, J. L. et al. Activation of two different resistance mechanisms in Saccharomyces cerevisiae upon exposure to octanoic and decanoic acids. Appl. Environ. Microbiol. 76, 7526–7535 (2010).
Borrull, A., López-Martínez, G., Poblet, M., Cordero-Otero, R. & Rozès, N. New insights into the toxicity mechanism of octanoic and decanoic acids on Saccharomyces cerevisiae. Yeast 32, 451–460 (2015).
Liu, P. et al. Membrane stress caused by octanoic acid in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 97, 3239–3251 (2013).
Bell, G. H. The action of monocarboxylic acids on Candida tropicalis growing on hydrocarbon substrates. Antonie Leeuwenhoek 37, 385–400 (1971).
McDonough, V., Stukey, J. & Cavanagh, T. Mutations in erg4 affect the sensitivity of Saccharomyces cerevisiae to medium-chain fatty acids. Biochim. Biophys. Acta 1581, 109–118 (2002).
Zhu, Z. et al. Enabling the synthesis of medium chain alkanes and 1-alkenes in yeast. Metab. Eng. 44, 81–88 (2017).
Mukhopadhyay, A. Tolerance engineering in bacteria for the production of advanced biofuels and chemicals. Trends Microbiol. 23, 498–508 (2015).
Keasling, J. D. Manufacturing molecules through metabolic engineering. Science 330, 1355–1358 (2010).
Tan, Z., Black, W., Yoon, J. M., Shanks, J. V. & Jarboe, L. R. Improving Escherichia coli membrane integrity and fatty acid production by expression tuning of FadL and OmpF. Microb. Cell Fact. 16, 38 (2017).
Wu, J. et al. Improving medium chain fatty acid production in Escherichia coli by multiple transporter engineering. Food Chem. 272, 628–634 (2019).
Dunlop, M. J. et al. Engineering microbial biofuel tolerance and export using efflux pumps. Mol. Syst. Biol. 7, 487 (2011).
Fisher, M. A. et al. Enhancing tolerance to short-chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non-native substrate n-butanol. ACS Synth. Biol. 3, 30–40 (2014).
Portnoy, V. A., Bezdan, D. & Zengler, K. Adaptive laboratory evolution—harnessing the power of biology for metabolic engineering. Curr. Opin. Biotechnol. 22, 590–594 (2011).
Royce, L. A. et al. Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity. Metab. Eng. 29, 180–188 (2015).
Lee, J. W. et al. Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat. Chem. Biol. 8, 536–546 (2012).
Grininger, M. Perspectives on the evolution, assembly and conformational dynamics of fatty acid synthase type I (FAS I) systems. Curr. Opin. Struct. Biol. 25, 49–56 (2014).
Yu, T. et al. Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals. Nat. Commun. 8, 15587 (2017).
González-Ramos, D. et al. A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations. Biotechnol. Biofuels 9, 173 (2016).
Albertsen, M., Bellahn, I., Krämer, R. & Waffenschmidt, S. Localization and function of the yeast multidrug transporter Tpo1p. J. Biol. Chem. 278, 12820–12825 (2003).
Sá-Correia, I., dos Santos, S. C., Teixeira, M. C., Cabrito, T. R. & Mira, N. P. Drug:H+ antiporters in chemical stress response in yeast. Trends Microbiol. 17, 22–31 (2009).
Fletcher, E. et al. Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments. Metab. Eng. 39, 19–28 (2017).
Tong, J., Manik, M. K., Yang, H. & Im, Y. J. Structural insights into nonvesicular lipid transport by the oxysterol binding protein homologue family. Biochim. Biophys. Acta 1861, 928–939 (2016).
Jessop-Fabre, M. M. et al. EasyClone-MarkerFree: a vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR–Cas9. Biotechnol. J. 11, 1110–1117 (2016).
Rodriguez, S. et al. ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae. Microb. Cell Fact. 15, 48 (2016).
Westfall, P. J. et al. Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc. Natl. Acad. Sci. USA 109, E111–E118 (2012).
Blazeck, J., Garg, R., Reed, B. & Alper, H. S. Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters. Biotechnol. Bioeng. 109, 2884–2895 (2012).
Hoshida, H., Kondo, M., Kobayashi, T., Yarimizu, T. & Akada, R. 5′-UTR introns enhance protein expression in the yeast Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 101, 241–251 (2017).
Kim, C.-W. et al. Induced polymerization of mammalian acetyl-CoA carboxylase by MIG12 provides a tertiary level of regulation of fatty acid synthesis. Proc. Natl. Acad. Sci. USA 107, 9626–9631 (2010).
Hunkeler, M., Stuttfeld, E., Hagmann, A., Imseng, S. & Maier, T. The dynamic organization of fungal acetyl-CoA carboxylase. Nat. Commun. 7, 11196 (2016).
Wei, J. et al. A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation. Cell Discov. 2, 16044 (2016).
Shi, S., Chen, Y., Siewers, V. & Nielsen, J. Improving production of malonyl coenzyme A-derived metabolites by abolishing Snf1-dependent regulation of Acc1. mBio 5, e01130-14 (2014).
Choi, J. W. & Da Silva, N. A. Improving polyketide and fatty acid synthesis by engineering of the yeast acetyl-CoA carboxylase. J. Biotechnol. 187, 56–59 (2014).
Besada-Lombana, P. B., Fernandez-Moya, R., Fenster, J. & Da Silva, N. A. Engineering Saccharomyces cerevisiae fatty acid composition for increased tolerance to octanoic acid. Biotechnol. Bioeng. 114, 1531–1538 (2017).
Sumper, M., Riepertinger, C., Lynen, F. & Oesterhelt, D. Die synthese verschiedener carbonsäuren durch den multienzymkomplex der fettsäuresynthese aus hefe und die erklärung ihrer bildung. Eur. J. Biochem. 10, 377–387 (1969).
Zhu, Z. et al. A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nat. Commun. 3, 1112 (2012).
Swarbrick, C. M. D., Perugini, M. A., Cowieson, N. & Forwood, J. K. Structural and functional characterization of TesB from Yersinia pestis reveals a unique octameric arrangement of hotdog domains. Acta Crystallogr. D 71, 986–995 (2015).
Scharnewski, M., Pongdontri, P., Mora, G., Hoppert, M. & Fulda, M. Mutants of Saccharomyces cerevisiae deficient in acyl-CoA synthetases secrete fatty acids due to interrupted fatty acid recycling. FEBS J. 275, 2765–2778 (2008).
Runguphan, W. & Keasling, J. D. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metab. Eng. 21, 103–113 (2014).
Scherrer, R. A. & Howard, S. M. Use of distribution coefficients in quantitative structure–activity relations. J. Med. Chem. 20, 53–58 (1977).
Ghosh, A. et al. 13C metabolic flux analysis for systematic metabolic engineering of S. cerevisiae for overproduction of fatty acids. Front. Bioeng. Biotechnol. 4, 76 (2016).
Xiao, Y., Bowen, C. H., Liu, D. & Zhang, F. Exploiting nongenetic cell-to-cell variation for enhanced biosynthesis. Nat. Chem. Biol. 12, 339–344 (2016).
Schweizer, E. & Hofmann, J. Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems. Microbiol. Mol. Biol. Rev. 68, 501–517 (2004).
Swinnen, S., Thevelein, J. M. & Nevoigt, E. Genetic mapping of quantitative phenotypic traits in Saccharomyces cerevisiae. FEMS Yeast Res. 12, 215–227 (2012).
Qi, Y., Liu, H., Chen, X. & Liu, L. Engineering microbial membranes to increase stress tolerance of industrial strains. Metab. Eng. 53, 24–34 (2019).
Meynial-Salles, I., Dorotyn, S. & Soucaille, P. A new process for the continuous production of succinic acid from glucose at high yield, titer, and productivity. Biotechnol. Bioeng. 99, 129–135 (2008).
Nielsen, J. & Keasling, J. D. Engineering cellular metabolism. Cell 164, 1185–1197 (2016).
Shao, Z., Zhao, H. & Zhao, H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 37, e16 (2009).
Gietz, R. D. & Schiestl, R. H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34 (2007).
Jensen, N. B. et al. EasyClone: method for iterative chromosomal integration of multiple genes Saccharomyces cerevisiae. FEMS Yeast Res. 14, 238–248 (2014).
Jenjaroenpun, P. et al. Complete genomic and transcriptional landscape analysis using third-generation sequencing: a case study of Saccharomyces cerevisiae CEN.PK113-7D. Nucleic Acids Res. 46, e38 (2018).
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.
V.S. and J.N. are shareholders in Biopetrolia AB. All other authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
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
Fusing α and β subunits of the fungal fatty acid synthase leads to improved production of fatty acids
Scientific Reports (2020)
Bioaldehydes and beyond: Expanding the realm of bioderived chemicals using biogenic aldehydes as platforms
Current Opinion in Chemical Biology (2020)
Trends in Biotechnology (2020)
Structure-guided reshaping of the acyl binding pocket of ‘TesA thioesterase enhances octanoic acid production in E. coli
Metabolic Engineering (2020)