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

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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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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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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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Stephanopoulos, G. Challenges in engineering microbes for biofuels production. Science 315, 801–804 (2007).

  2. 2.

    Choi, Y. J. & Lee, S. Y. Microbial production of short-chain alkanes. Nature 502, 571–574 (2013).

  3. 3.

    Peralta-Yahya, P. P., Zhang, F. Z., del Cardayre, S. B. & Keasling, J. D. Microbial engineering for the production of advanced biofuels. Nature 488, 320–328 (2012).

  4. 4.

    Peralta-Yahya, P. P. & Keasling, J. D. Advanced biofuel production in microbes. Biotechnol. J. 5, 147–162 (2010).

  5. 5.

    Lee, S. Y., Kim, H. M. & Cheon, S. Metabolic engineering for the production of hydrocarbon fuels. Curr. Opin. Biotechnol. 33, 15–22 (2015).

  6. 6.

    Certik, M. & Shimizu, S. Biosynthesis and regulation of microbial polyunsaturated fatty acid production. J. Biosci. Bioeng. 87, 1–14 (1999).

  7. 7.

    Tee, T. W., Chowdhury, A., Maranas, C. D. & Shanks, J. V. Systems metabolic engineering design: fatty acid production as an emerging case study. Biotechnol. Bioeng. 111, 849–857 (2014).

  8. 8.

    Lu, X., Vora, H. & Khosla, C. Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab. Eng. 10, 333–339 (2008).

  9. 9.

    Xu, P., Gu, Q., Wang, W., Wong, L., Bower, A. G., Collins, C. H. & Koffas, M. A. Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat. Commun. 4, 1409 (2013).

  10. 10.

    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).

  11. 11.

    Zhou, Y. J. et al. Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories. Nat. Commun. 7, 11709 (2016).

  12. 12.

    Ruffing, A. M. Improved free fatty acid production in cyanobacteria with Synechococcus sp. PCC 7002 as Host. Front. Bioeng. Biotechnol. 2, 17 (2016).

  13. 13.

    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).

  14. 14.

    Blazeck, J. et al. Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat. Commun. 5, 3131 (2014).

  15. 15.

    Xua, P., Qiaoa, K., Ahna, 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).

  16. 16.

    Amaro, R. L., Dulermo, R., Niehus, X. & Nicaud, J. M. Combining metabolic engineering and process optimization to improve production and secretion of fatty acids. Metab. Eng. 38, 38–46 (2016).

  17. 17.

    Kurosawa, K., Boccazzi, P., de Almeida, N. M. & Sinskey, A. J. High-cell-density batch fermentation of Rhodococcus opacus PD630 using a high glucose concentration for triacylglycerol production. J. Biotechnol. 147, 212–218 (2010).

  18. 18.

    Alvarez, H. M., Kalscheuer, R. & Steinbüchel, A. Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126. Appl. Microbiol. Biotechnol. 54, 218–223 (2000).

  19. 19.

    Tucker, D. L., Tucker, N. & Conway, T. Gene expression profiling of the pH response in Escherichia coli. J. Bacteriol. 23, 6551–6558 (2002).

  20. 20.

    Alvarez, H. M., Silva, R. A., Herrero, M., Hernández, M. A. & Villalba, M. S. Metabolism of triacylglycerols in Rhodococcus species: insights from physiology and molecular genetics. J. Mol. Biochem. 2, 69–78 (2012).

  21. 21.

    Gupta, R., Gupta, N. & Rathi, P. Bacterial lipases: an overview of production, purification and biochemical properties. Appl. Microbiol. Biotechnol. 64, 763–781 (2004).

  22. 22.

    Parish, T., Mahenthiralingam, E., Draper, P., Davis, E. O. & Colston, M. J. Regulation of the inducible acetamidase gene of Mycobacterium smegmatis. Microbiology 143, 2267–2276 (1997).

  23. 23.

    Chen, Y. et al. Integrated omics study delineates the dynamics of lipid droplets in Rhodococcus opacus PD630. Nucleic Acids Res. 42, 1052–1064 (2014).

  24. 24.

    Kranen, E., Detzel, C., Weber, T. & Jose, J. Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs. Microb. Cell. Fact. 13, 19 (2014).

  25. 25.

    Steen, E. J. et al. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463, 559–562 (2010).

  26. 26.

    Holland-Staley, C. A., Lee, K., Clark, D. P. & Cunningham, P. R. Aerobic activity of Escherichia coli alcohol dehydrogenase is determined by a single amino acid. J. Bacteriol. 182, 6049–6054 (2000).

  27. 27.

    Barney, B. M., Wahlen, B. D., Garner, E., Wei, J. S. & Seefeldt, L. C. Differences in substrate specificities of five bacterial wax ester synthases. Appl. Environ. Microb. 78, 5734–5745 (2012).

  28. 28.

    Stöveken, T., Kalscheuer, R., Malkus, U., Reichelt, R. & Steinbüchel, A. The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J. Bacteriol. 187, 1369–1376 (2005).

  29. 29.

    Na, D., Lee, S. & Lee, D. Mathematical modeling of translation initiation for the estimation of its efficiency to computationally design mRNA sequences with desired expression levels in prokaryotes. BMC Syst. Biol. 4, 71 (2010).

  30. 30.

    Schirmer, A., Rude, M. A., Li, X. Z., Popova, E. & del Cardayre, S. B. Microbial biosynthesis of alkanes. Science 329, 559–562 (2010).

  31. 31.

    Harger, M. et al. Expanding the product profile of a microbial alkane biosynthetic pathway. ACS Synth. Biol. 2, 59–62 (2013).

  32. 32.

    Lui, R. et al. Metabolic engineering of fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli. Metab. Eng. 22, 10–21 (2014).

  33. 33.

    Holder, J. W. et al. Comparative and functional genomics of Rhodococcus opacus PD630 for biofuels development. PLoS Genet. 7, 9 (2011).

  34. 34.

    Jamshidi, N. & Palsson, B. Ø. Investigating the metabolic capabilities of Mycobacterium tuberculosis H37Rv using the in silico strain iNJ661 and proposing alternative drug targets. BMC Syst. Biol. 1, 26 (2007).

  35. 35.

    Lee, S. Y. & Kim, H. U. Systems strategies for developing industrial microbial strains. Nat. Biotechnol. 33, 1061–1072 (2015).

  36. 36.

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

  37. 37.

    Hänisch, J., Wältermann, M., Robenek, H. & Steinbüchel, A. The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins. Microbiology 152, 3271–3280 (2006).

  38. 38.

    Park, S. H. et al. Metabolic engineering of Corynebacterium glutamicum for l-arginine production. Nat. Commun. 5, 4618 (2014).

  39. 39.

    Kalscheuer, R., Arenskotter, M. & Steinbüchel, A. Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids). Appl. Microbiol. Biotechnol. 52, 508–515 (1999).

  40. 40.

    Waltermann, M., Luftmann, H., Baumeister, D., Kalscheuer, R. & Steinbüchel, A. Rhodococcus opacus strain PD630 as a new source of high-value single-cell oil? Isolation and characterization of triacylglycerols and other storage lipids. Microbiology 146, 1143–1149 (2000).

  41. 41.

    Cai, S. F. et al. Identification of the haloarchaeal phasin (PhaP) that functions in polyhydroxyalkanoate accumulation and granule formation in Haloferax mediterranei. Appl. Environ. Microb. 78, 1946–1952 (2012).

  42. 42.

    Tian, J. M. et al. Analysis of transient polyhydroxybutyrate production in Wautersia eutropha H16 by quantitative Western analysis and transmission electron microscopy. J. Bacteriol. 187, 3825–3832 (2005).

  43. 43.

    Weber, T. et al. antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 43, 237–243 (2015).

  44. 44.

    Kumar, N. & Skolnick, J. EFICAz2.5: application of a high-precision enzyme function predictor to 396 proteomes. Bioinformatics 28, 2687–2688 (2012).

  45. 45.

    King, Z. A. et al. BiGG Models: a platform for integrating, standardizing and sharing genome-scale models. Nucleic Acids Res. 4, 515–525 (2015).

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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.

Author information

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.

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.

Correspondence to Sang Yup Lee.

Supplementary information

Supplementary Information

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

Reporting Summary

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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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.