1887

Abstract

was engineered for the production of even- and odd-chain fatty acids (FAs) by fermentation. Co-production of thiolase, hydroxybutyryl-CoA dehydrogenase, crotonase and -enoyl-CoA reductase from a synthetic operon allowed the production of butyrate, hexanoate and octanoate. Elimination of native fermentation pathways by genetic deletion (Δ, Δ, Δ, Δ, Δ) helped eliminate undesired by-products and increase product yields. Initial butyrate production rates were high (0.7 g l h) but quickly levelled off and further study suggested this was due to product toxicity and/or acidification of the growth medium. Results also showed that endogenous thioesterases significantly influenced product formation. In particular, deletion of the thioesterase gene substantially increased hexanoate production while decreasing the production of butyrate. was also engineered to co-produce enzymes for even-chain FA production (described above) together with a coenzyme B-dependent pathway for the production of propionyl-CoA, which allowed the production of odd-chain FAs (pentanoate and heptanoate). The B-dependent pathway used here has the potential to allow the production of odd-chain FAs from a single growth substrate (glucose) in a more energy-efficient manner than the prior methods.

Funding
This study was supported by the:
  • National Science Foundation (Award EEC-0813570)
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2014-07-01
2024-03-28
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References

  1. Baek J. M., Mazumdar S., Lee S. W., Jung M. Y., Lim J. H., Seo S. W., Jung G. Y., Oh M. K. ( 2013). Butyrate production in engineered Escherichia coli with synthetic scaffolds. Biotechnol Bioeng 110:2790–2794 [View Article][PubMed]
    [Google Scholar]
  2. Bobik T. A., Xu Y., Jeter R. M., Otto K. E., Roth J. R. ( 1997). Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase. J Bacteriol 179:6633–6639[PubMed]
    [Google Scholar]
  3. Bond-Watts B. B., Bellerose R. J., Chang M. C. Y. ( 2011). Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat Chem Biol 7:222–227 [View Article][PubMed]
    [Google Scholar]
  4. Choi K., Jeon B. S., Kim B. C., Oh M. K., Um Y., Sang B. I. ( 2013). In situ biphasic extractive fermentation for hexanoic acid production from sucrose by megasphaera elsdenii NCIMB 702410. Appl Biochem Biotechnol 171:1094–1107 [View Article][PubMed]
    [Google Scholar]
  5. Clomburg J. M., Gonzalez R. ( 2011). Metabolic engineering of Escherichia coli for the production of 1,2-propanediol from glycerol. Biotechnol Bioeng 108:867–879 [View Article][PubMed]
    [Google Scholar]
  6. Clomburg J. M., Vick J. E., Blankschien M. D., Rodríguez-Moyá M., Gonzalez R. ( 2012). A synthetic biology approach to engineer a functional reversal of the β-oxidation cycle. ACS Synth Biol 1:541–554 [View Article][PubMed]
    [Google Scholar]
  7. Colby G. D., Chen J. S. ( 1992). Purification and properties of 3-hydroxybutyryl-coenzyme A dehydrogenase from Clostridium beijerinckii (“Clostridium butylicum”) NRRL B593. Appl Environ Microbiol 58:3297–3302[PubMed]
    [Google Scholar]
  8. Dekishima Y., Lan E. I., Shen C. R., Cho K. M., Liao J. C. ( 2011). Extending carbon chain length of 1-butanol pathway for 1-hexanol synthesis from glucose by engineered Escherichia coli. J Am Chem Soc 133:11399–11401 [View Article][PubMed]
    [Google Scholar]
  9. Dellomonaco C., Fava F., Gonzalez R. ( 2010). The path to next generation biofuels: successes and challenges in the era of synthetic biology. Microb Cell Fact 9:3 [View Article][PubMed]
    [Google Scholar]
  10. Dellomonaco C., Clomburg J. M., Miller E. N., Gonzalez R. ( 2011). Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476:355–359 [View Article][PubMed]
    [Google Scholar]
  11. Desbois A. P., Smith V. J. ( 2010). Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. Appl Microbiol Biotechnol 85:1629–1642 [View Article][PubMed]
    [Google Scholar]
  12. Fischer C. R., Tseng H. C., Tai M., Prather K. L., Stephanopoulos G. ( 2010). Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli. Appl Microbiol Biotechnol 88:265–275 [View Article][PubMed]
    [Google Scholar]
  13. Hartmanis M. G., Stadtman T. C. ( 1982). Isolation of a selenium-containing thiolase from Clostridium kluyveri: identification of the selenium moiety as selenomethionine. Proc Natl Acad Sci U S A 79:4912–4916 [View Article][PubMed]
    [Google Scholar]
  14. Jang Y. S., Woo H. M., Im J. A., Kim I. H., Lee S. Y. ( 2013). Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid. Appl Microbiol Biotechnol 97:9355–9363 [View Article][PubMed]
    [Google Scholar]
  15. Jarboe L. R., Grabar T. B., Yomano L. P., Shanmugan K. T., Ingram L. O. ( 2007). Development of ethanologenic bacteria. Adv Biochem Eng Biotechnol 108:237–261[PubMed]
    [Google Scholar]
  16. Jeon B. S., Kim B. C., Um Y., Sang B. I. ( 2010). Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1. Appl Microbiol Biotechnol 88:1161–1167 [View Article][PubMed]
    [Google Scholar]
  17. Jiang L., Wang J., Liang S., Wang X., Cen P., Xu Z. ( 2010). Production of butyric acid from glucose and xylose with immobilized cells of Clostridium tyrobutyricum in a fibrous-bed bioreactor. Appl Biochem Biotechnol 160:350–359 [View Article][PubMed]
    [Google Scholar]
  18. Johnson C. L., Pechonick E., Park S. D., Havemann G. D., Leal N. A., Bobik T. A. ( 2001). Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene. J Bacteriol 183:1577–1584 [View Article][PubMed]
    [Google Scholar]
  19. Leal N. A., Havemann G. D., Bobik T. A. ( 2003). PduP is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2. Arch Microbiol 180:353–361 [View Article][PubMed]
    [Google Scholar]
  20. Lennen R. M., Pfleger B. F. ( 2012). Engineering Escherichia coli to synthesize free fatty acids. Trends Biotechnol 30:659–667 [View Article][PubMed]
    [Google Scholar]
  21. Lim J. H., Seo S. W., Kim S. Y., Jung G. Y. ( 2013). Refactoring redox cofactor regeneration for high-yield biocatalysis of glucose to butyric acid in Escherichia coli. Bioresour Technol 135:568–573 [View Article][PubMed]
    [Google Scholar]
  22. Lu X. F., Vora H., Khosla C. ( 2008). Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab Eng 10:333–339 [View Article][PubMed]
    [Google Scholar]
  23. Machado H. B., Dekishima Y., Luo H., Lan E. I., Liao J. C. ( 2012). A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols. Metab Eng 14:504–511 [View Article][PubMed]
    [Google Scholar]
  24. Martin C. H., Dhamankar H., Tseng H. C., Sheppard M. J., Reisch C. R., Prather K. L. J. ( 2013). A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone. Nat Commun 4:1414 [View Article][PubMed]
    [Google Scholar]
  25. Mayer K. M., Shanklin J. ( 2007). Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach. BMC Plant Biol 7:1 [View Article][PubMed]
    [Google Scholar]
  26. McMahon M. D., Prather K. L. ( 2014). Functional screening and in vitro analysis reveal thioesterases with enhanced substrate specificity profiles that improve short-chain fatty acid production in Escherichia coli. Appl Environ Microbiol 80:1042–1050 [View Article][PubMed]
    [Google Scholar]
  27. Nunn W. D., Simons R. W., Egan P. A., Maloy S. R. ( 1979). Kinetics of the utilization of medium and long chain fatty acids by mutant of Escherichia coli defective in the fadL gene. J Biol Chem 254:9130–9134[PubMed]
    [Google Scholar]
  28. Peralta-Yahya P. P., Zhang F., del Cardayre S. B., Keasling J. D. ( 2012). Microbial engineering for the production of advanced biofuels. Nature 488:320–328 [View Article][PubMed]
    [Google Scholar]
  29. Royce L. A., Liu P., Stebbins M. J., Hanson B. C., Jarboe L. R. ( 2013). The damaging effects of short chain fatty acids on Escherichia coli membranes. Appl Microbiol Biotechnol 97:8317–8327 [View Article][PubMed]
    [Google Scholar]
  30. Sambrook J., Russell D. ( 2001). Molecular Cloning: A Laboratory Manual, 3rd ed.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  31. Seedorf H., Fricke W. F., Veith B., Brüggemann H., Liesegang H., Strittmatter A., Miethke M., Buckel W., Hinderberger J. & other authors ( 2008). The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A 105:2128–2133 [View Article][PubMed]
    [Google Scholar]
  32. Seregina T. A., Shakulov R. S., Debabov V. G., Mironov A. S. ( 2010). Construction of a butyrate-producing E. coli strain without the use of heterologous genes. Appl Biochem Microbiol 46:745–754 [View Article]
    [Google Scholar]
  33. Shen C. R., Lan E. I., Dekishima Y., Baez A., Cho K. M., Liao J. C. ( 2011). Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl Environ Microbiol 77:2905–2915 [View Article][PubMed]
    [Google Scholar]
  34. Sliwkowski M. X., Hartmanis M. G. ( 1984). Simultaneous single-step purification of thiolase and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri. Anal Biochem 141:344–347 [View Article][PubMed]
    [Google Scholar]
  35. Sliwkowski M. X., Stadtman T. C. ( 1985). Incorporation and distribution of selenium into thiolase from Clostridium kluyveri. J Biol Chem 260:3140–3144[PubMed]
    [Google Scholar]
  36. Steen E. J., Kang Y., Bokinsky G., Hu Z., Schirmer A., McClure A., Del Cardayre S. B., Keasling J. D. ( 2010). Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463:559–562 [View Article][PubMed]
    [Google Scholar]
  37. Tseng H. C., Prather K. L. ( 2012). Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways. Proc Natl Acad Sci U S A 109:17925–17930 [View Article][PubMed]
    [Google Scholar]
  38. Tseng H. C., Harwell C. L., Martin C. H., Prather K. L. J. ( 2010). Biosynthesis of chiral 3-hydroxyvalerate from single propionate-unrelated carbon sources in metabolically engineered E. coli. Microb Cell Fact 9:96 [View Article][PubMed]
    [Google Scholar]
  39. von Hugo H., Schoberth S., Madan V. K., Gottschalk G. ( 1972). Coenzyme specificity of dehydrogenases and fermentation of pyruvate by clostridia. Arch Mikrobiol 87:189–202 [View Article][PubMed]
    [Google Scholar]
  40. Waterson R. M., Hill R. L. ( 1972). Enoyl coenzyme A hydratase (crotonase). Catalytic properties of crotonase and its possible regulatory role in fatty acid oxidation. J Biol Chem 247:5258–5265[PubMed]
    [Google Scholar]
  41. Wei D., Liu X., Yang S. T. ( 2013). Butyric acid production from sugarcane bagasse hydrolysate by Clostridium tyrobutyricum immobilized in a fibrous-bed bioreactor. Bioresour Technol 129:553–560 [View Article][PubMed]
    [Google Scholar]
  42. Zhang C. H., Yang H., Yang F. X., Ma Y. J. ( 2009). Current progress on butyric acid production by fermentation. Curr Microbiol 59:656–663 [View Article][PubMed]
    [Google Scholar]
  43. Zhang X., Li M., Agrawal A., San K. Y. ( 2011). Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases. Metab Eng 13:713–722 [View Article][PubMed]
    [Google Scholar]
  44. Zhu H., Gonzalez R., Bobik T. A. ( 2011). Coproduction of acetaldehyde and hydrogen during glucose fermentation by Escherichia coli.. Appl Environ Microbiol 77:6441–6450 [View Article][PubMed]
    [Google Scholar]
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