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Expanding ester biosynthesis in Escherichia coli

Abstract

To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l−1). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters.

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Figure 1: Enzymatic ester synthesis by combining alcohols and acyl-CoAs.
Figure 2: Constructing acetate ester synthesis pathways in E. coli.
Figure 3: Tetradecyl acetate production from glucose in E. coli.
Figure 4: Constructing higher chain ester biosynthesis pathway in E. coli.

References

  1. Beekwilder, J. et al. Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant Physiol. 135, 1865–1878 (2004).

    Article  CAS  Google Scholar 

  2. Verstrepen, K.J. et al. Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters. Appl. Environ. Microbiol. 69, 5228–5237 (2003).

    Article  CAS  Google Scholar 

  3. Iwasaki, T., Maegawa, Y., Ohshima, T. & Mashima, K. in Kirk-Othmer Encyclopedia of Chemical Technology Vol. 10, 497–516 (John Wiley & Sons, New Jersey, 2012).

  4. Liu, Y.J., Lotero, E. & Goodwin, J.G. Effect of water on sulfuric acid catalyzed esterification. J. Mol. Catal. Chem. 245, 132–140 (2006).

    Article  CAS  Google Scholar 

  5. Nelson, D.L. & Cox, M.M. Lehninger Principles of Biochemistry 5th edn. (Sara Tenney, New York, 2008).

  6. Stergiou, P.Y. et al. Advances in lipase-catalyzed esterification reactions. Biotechnol. Adv. 31, 1846–1859 (2013).

    Article  CAS  Google Scholar 

  7. Dhake, K.P., Thakare, D.D. & Bhanage, B.M. Lipase: a potential biocatalyst for the synthesis of valuable flavour and fragrance ester compounds. Flavour Fragrance J. 28, 71–83 (2013).

    Article  CAS  Google Scholar 

  8. Rabinovitch-Deere, C.A., Oliver, J.W., Rodriguez, G.M. & Atsumi, S. Synthetic biology and metabolic engineering approaches to produce biofuels. Chem. Rev. 113, 4611–4632 (2013).

    Article  CAS  Google Scholar 

  9. Bornscheuer, U.T. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012).

    Article  CAS  Google Scholar 

  10. Fujii, T. et al. Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene. Appl. Environ. Microbiol. 60, 2786–2792 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Rowland, O. & Domergue, F. Plant fatty acyl reductases: enzymes generating fatty alcohols for protective layers with potential for industrial applications. Plant Sci. 193–194, 28–38 (2012).

    Article  Google Scholar 

  12. Rontani, J.F., Bonin, P.C. & Volkman, J.K. Production of wax esters during aerobic growth of marine bacteria on isoprenoid compounds. Appl. Environ. Microbiol. 65, 221–230 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Lenneman, E.M., Ohlert, J.M., Palani, N.P. & Barney, B.M. Fatty alcohols for wax esters in Marinobacter aquaeolei VT8: two optional routes in the wax biosynthesis pathway. Appl. Environ. Microbiol. 79, 7055–7062 (2013).

    Article  CAS  Google Scholar 

  14. Reiser, S. & Somerville, C. Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme A reductase. J. Bacteriol. 179, 2969–2975 (1997).

    Article  CAS  Google Scholar 

  15. Holtzapple, E. & Schmidt-Dannert, C. Biosynthesis of isoprenoid wax ester in Marinobacter hydrocarbonoclasticus DSM 8798: identification and characterization of isoprenoid coenzyme A synthetase and wax ester synthases. J. Bacteriol. 189, 3804–3812 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Stöveken, T., Kalscheuer, R., Malkus, U., Reichelt, R. & Steinbuchel, 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).

    Article  Google Scholar 

  18. Atsumi, S., Hanai, T. & Liao, J.C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–89 (2008).

    Article  CAS  Google Scholar 

  19. Shen, C.R. et al. Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl. Environ. Microbiol. 77, 2905–2915 (2011).

    Article  CAS  Google Scholar 

  20. Bond-Watts, B.B., Bellerose, R.J. & Chang, M.C. Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat. Chem. Biol. 7, 222–227 (2011).

    Article  CAS  Google Scholar 

  21. Inokuma, K., Liao, J.C., Okamoto, M. & Hanai, T. Improvement of isopropanol production by metabolically engineered Escherichia coli using gas stripping. J. Biosci. Bioeng. 110, 696–701 (2010).

    Article  CAS  Google Scholar 

  22. 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).

    Article  CAS  Google Scholar 

  23. Saerens, S.M. et al. The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzymes with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity. J. Biol. Chem. 281, 4446–4456 (2006).

    Article  CAS  Google Scholar 

  24. Lilly, M. et al. The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates. Yeast 23, 641–659 (2006).

    Article  CAS  Google Scholar 

  25. Mason, A.B. & Dufour, J.P. Alcohol acetyltransferases and the significance of ester synthesis in yeast. Yeast 16, 1287–1298 (2000).

    Article  CAS  Google Scholar 

  26. Fujii, T., Yoshimoto, H. & Tamai, Y. Acetate ester production by Saccharomyces cerevisiae lacking the ATF1 gene encoding the alcohol acetyltransferase. J. Ferment. Bioeng. 81, 538–542 (1996).

    Article  CAS  Google Scholar 

  27. Howard, T.P. et al. Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. Proc. Natl. Acad. Sci. USA 110, 7636–7641 (2013).

    Article  CAS  Google Scholar 

  28. Lin, F., Das, D., Lin, X.N. & Marsh, E.N. Aldehyde-forming fatty acyl-CoA reductase from cyanobacteria: expression, purification and characterization of the recombinant enzyme. FEBS J. 280, 4773–4781 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Mooney, B.P., Miernyk, J.A. & Randall, D.D. The complex fate of α-ketoacids. Annu. Rev. Plant Biol. 53, 357–375 (2002).

    Article  CAS  Google Scholar 

  31. Rodriguez, G.M. & Atsumi, S. Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity. Microb. Cell Fact. 11, 90 (2012).

    Article  CAS  Google Scholar 

  32. de la Plaza, M., Fernandez de Palencia, P., Pelaez, C. & Requena, T. Biochemical and molecular characterization of α-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis. FEMS Microbiol. Lett. 238, 367–374 (2004).

    CAS  PubMed  Google Scholar 

  33. Bastian, S. et al. Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli. Metab. Eng. 13, 345–352 (2011).

    Article  CAS  Google Scholar 

  34. Baez, A., Cho, K.M. & Liao, J.C. High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal. Appl. Microbiol. Biotechnol. 90, 1681–1690 (2011).

    Article  CAS  Google Scholar 

  35. Atsumi, S. et al. Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes. Appl. Microbiol. Biotechnol. 85, 651–657 (2010).

    Article  CAS  Google Scholar 

  36. Connor, M.R., Cann, A.F. & Liao, J.C. 3-Methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation. Appl. Microbiol. Biotechnol. 86, 1155–1164 (2010).

    Article  CAS  Google Scholar 

  37. Belas, R. et al. Bacterial bioluminescence: isolation and expression of the luciferase genes from Vibrio harveyi. Science 218, 791–793 (1982).

    Article  CAS  Google Scholar 

  38. Meighen, E.A. Bacterial bioluminescence: organization, regulation, and application of the lux genes. FASEB J. 7, 1016–1022 (1993).

    Article  CAS  Google Scholar 

  39. Ferrández, A., Garcia, J.L. & Diaz, E. Genetic characterization and expression in heterologous hosts of the 3-(3-hydroxyphenyl)propionate catabolic pathway of Escherichia coli K-12. J. Bacteriol. 179, 2573–2581 (1997).

    Article  Google Scholar 

  40. Lee, S.J., Ko, J.H., Kang, H.Y. & Lee, Y. Coupled expression of MhpE aldolase and MhpF dehydrogenase in Escherichia coli. Biochem. Biophys. Res. Commun. 346, 1009–1015 (2006).

    Article  CAS  Google Scholar 

  41. Hester, K., Luo, J., Burns, G., Braswell, E.H. & Sokatch, J.R. Purification of active E1α2β2 of Pseudomonas putida branched-chain-oxoacid dehydrogenase. Eur. J. Biochem. 233, 828–836 (1995).

    Article  CAS  Google Scholar 

  42. Hester, K.L., Luo, J. & Sokatch, J.R. Purification of Pseudonomas Putida branched-chain keto acid dehydrogenase E1 component. Methods Enzymol. 324, 129–138 (2000).

    Article  CAS  Google Scholar 

  43. Alonso-Gutierrez, J. et al. Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metab. Eng. 19, 33–41 (2013).

    Article  CAS  Google Scholar 

  44. Park, Y.C., Shaffer, C.E. & Bennett, G.N. Microbial formation of esters. Appl. Microbiol. Biotechnol. 85, 13–25 (2009).

    Article  CAS  Google Scholar 

  45. van den Berg, C., Heeres, A.S., Van der Wielen, L.A. & Straathof, A.J. Simultaneous Clostridial fermentation, lipase-catalyzed esterification, and ester extraction to enrich diesel with butyl butyrate. Biotechnol. Bioeng. 110, 137–142 (2013).

    Article  CAS  Google Scholar 

  46. Machado, H.B., Dekishima, Y., Luo, H., Lan, E.I. & Liao, J.C. A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols. Metab. Eng. 14, 504–511 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by University of California–Davis startup fund and the Hellman fellowship to S.A. G.M.R. is supported by a US National Institutes of Health Biotechnology Training Grant Fellowship (T32-GM008799) and a Sloan Fellowship. Y.T. is supported by Japan Society for the Promotion of Science postdoctoral fellowship for research abroad. We would like to thank R. Luu and R.E. Parales (University of California–Davis) for graciously providing genomic DNA from P. putida g7 and M. Kato and S.-J. Lin (University of California–Davis) for providing genomic DNA from S. cerevisiae BY4742. We also thank M.D. Toney and C.A. Rabinovitch-Deere (University of California–Davis) for critical reading of the manuscript. Finally, we thank S. Desai for technical assistance with HPLC analysis and for constructing pAL603.

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G.M.R., Y.T. and S.A. designed research; G.M.R. and Y.T. performed the experiments; G.M.R., Y.T. and S.A. analyzed data; and G.M.R., Y.T. and S.A. wrote the paper.

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Correspondence to Shota Atsumi.

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Rodriguez, G., Tashiro, Y. & Atsumi, S. Expanding ester biosynthesis in Escherichia coli. Nat Chem Biol 10, 259–265 (2014). https://doi.org/10.1038/nchembio.1476

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