Advertisement

Branched-chain amino acid catabolism of Thermoanaerobacter pseudoethanolicus reveals potential route to branched-chain alcohol formation

  • 54 Accesses

Abstract

The fermentation of branched-chain amino acids (BCAAs) to branched-chain fatty acids (BCFAs) and branched-chain alcohols (BCOHs) is described using Thermoanaerobacter pseudoethanolicus. BCAAs were not degraded without an electron scavenging system but were degraded to a mixture of their BCFA (major) and BCOH (minor) when thiosulfate was added to the culture. Various environmental parameters were investigated using isoleucine as the substrate which ultimately demonstrated that at higher liquid–gas phase ratios the formation of 2-methyl-1-butanol from isoleucine achieved a maximal titer of 3.4 mM at a 1:1 liquid–gas ratio suggesting that higher partial pressure of hydrogen influences the BCOH/BCFA ratio but did not increase further with higher L–G phase ratios. Alternately, increasing the thiosulfate concentration decreased the BCOH to BCFA ratio. Kinetic monitoring of BCAA degradation revealed that the formation of BCOHs occurs slowly after the onset of BCFA formation. 13C2-labeled studies of leucine confirmed the production of a mixture of 3-methyl-1-butyrate and 3-methyl-1-butanol, while experiments involving 13C1-labeled 3-methyl-1-butyrate in fermentations containing leucine demonstrated that the carboxylic acid is reduced to the corresponding alcohol. Thus, the role of carboxylic acid reduction is likely of importance in the production of BCOH formation during the degradation of BCAA such as leucine.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Andreesen JR, Bahl H, Gottschalk G (1989) Introduction to the physiology and biochemistry of the genus Clostridium. In: Minton NP, Clarke DJ (eds) Clostridia. Plenum Press, New York, pp 27–62

  2. Barker HA (1939) The use of glutamic acid for the isolation and identification of Clostridium cochlearium and Cl. tetanomorphum. Arch Microbiol 10:376–389

  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  4. Bryant FO, Wiegel J, Ljungdahl LG (1988) Purification and properties of primary and secondary alcohol dehydrogenases from Thermoanaerobacter ethanolicus. Appl Environ Microbiol 54:460–465

  5. Bsharat O, Musa MM, Vieille C et al (2017) Asymmetric reduction of substituted 2-tetralones by Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase. ChemCatChem 9:1487–1493. https://doi.org/10.1002/cctc.201601618

  6. Burdette D, Zeikus JG (1994) Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2° Adh) as a bifunctional alcohol dehydrogenase-acetyl-CoA reductive thioes. Biochem J 302:163–170

  7. Burdette DS, Jung S-H, Shen G-J et al (2002) Physiological function of alcohol dehydrogenases and long-chain (C30) fatty acids in alcohol tolerance of Thermoanaerobacter ethanolicus. Appl Environ Microbiol 68:1914–1918. https://doi.org/10.1128/AEM.68.4.1914-1918.2002

  8. Chades T, Scully SM, Ingvadottir EM, Orlygsson J (2018) Fermentation of mannitol extracts from brown macro algae by thermophilic clostridia. Front Microbiol 9:1–13. https://doi.org/10.3389/fmicb.2018.01931

  9. Cline JD (1969) Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol Oceanogr 14:454–458

  10. Elsden SR, Hilton MG (1978) Volatile acid production from threonine, valine, leucine and isoleucine by clostridia. Arch Microbiol 117:165–172. https://doi.org/10.1007/BF00402304

  11. Elsden SR, Hilton MG (1979) Amino acid utalization patterns in clostridial taxonomy. Arch Microbiol 123:137–141

  12. Eschenlauer SCP, Mckain N, Walker ND et al (2002) Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen. Appl Environ Microbiol 68:4925–4931. https://doi.org/10.1128/AEM.68.10.4925

  13. Fardeau ML, Patel BKC, Magot M, Ollivier B (1997) Utilization of serine, leucine, isoleucine, and valine by Thermoanaerobacter brockii in the presence of thiosulfate or Methanobacterium sp. as electron accepters. Anaerobe 3:405–410. https://doi.org/10.1006/anae.1997.0126

  14. Faudon C, Fardeau ML, Heim J et al (1995) Peptide and amino acid oxidation in the presence of thiosulfate by members of the genus Thermoanaerobacter. Curr Microbiol 31:152–157

  15. Firkins JL, Yu Z, Morrison M (2007) Ruminal nitrogen metabolism: perspectives for integration of microbiology and nutrition for dairy. J Dairy Sci 90:E1–E16. https://doi.org/10.3168/jds.2006-518

  16. Fonknechten N, Chaussonnerie S, Tricot S et al (2010) Clostridium sticklandii, a specialist in amino acid degradation: revisiting its metabolism through its genome sequence. BMC Genom 11:555. https://doi.org/10.1186/1471-2164-11-555

  17. Gottschalk G (1986) Bacterial metabolism, 2nd edn. Springer, New York

  18. Harwood CS, Canale-Parola E (1981) Branched-chain amino acid fermentation by a marine spirochete: strategy for starvation survival. J Bacteriol 148:109–116

  19. Harwood CS, Canale-Parola E (1983) Spirochaeta isovalerica sp. nov., a marine anaerobe that forms branched-chain fatty acids as fermentation products. Int J Syst Bacteriol 33:573–579

  20. Hazelwood LA, Daran J-M, van Maris AJA et al (2008) The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Appl Environ Microbiol 74:2259–2266. https://doi.org/10.1128/AEM.02625-07

  21. He Q, Lokken PM, Chen S, Zhou J (2009) Characterization of the impact of acetate and lactate on ethanolic fermentation by Thermoanaerobacter ethanolicus. Bioresour Technol 100:5955–5965. https://doi.org/10.1016/j.biortech.2009.06.084

  22. Hitschler L, Kuntz M, Langschied F, Basen M (2018) Thermoanaerobacter species differ in their potential to reduce organic acids to their corresponding alcohols. Appl Microbiol Biotechnol 102:8465–8476. https://doi.org/10.1007/s00253-018-9210-3

  23. Kozianowski G, Canganella F, Rainey FA et al (1997) Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov. Extremophiles 1:171–182. https://doi.org/10.1007/s007920050031

  24. Lamed RJ, Zeikus JG (1981) Novel NADP-linked alcohol-aldehyde/ketone oxidoreductase in thermophilic ethanologenic bacteria. Biochem J 195:183–190

  25. Lee C, Saha BC, Zeikus JG (1990) Characterization of Thermoanaerobacter glucose isomerase in relation to saccharidase synthesis and development of single-step processes for sweetener production. Appl Environ Microbiol 56:2895–2901

  26. Leyer GJ, Johnson EA (1990) Repression of toxin production by tryptophan. Arch Microbiol 154:443–447

  27. Loll MJ, Bollag J-M (1983) Protein transformation in soil. Adv Agron 36:351–382

  28. McInerney MJ (1988) Anaerobic hydrolysis and fermentation of fats and proteins. In: Zehnder AJB (ed) Biology of anaerobic microorganisms. Wiley, New York, pp 373–415

  29. Mead GC (1971) The amino acid-fermenting clostridia. J Gen Microbiol 67:47–56. https://doi.org/10.1099/00221287-67-1-47

  30. Mitchell WJ (2001) Biology and physiololgy. In: Bahl H, Durre P (eds) Clostridia: biotechnology and medical applications. Wiley-VCH, Weinheim, pp 49–104

  31. Mitruka BM, Costilow RN (1967) Arginine and ornithine catabolism by Clostridium botulinum. J Bacteriol 93:295–301

  32. Musa MM, Phillips RS (2011) Recent advances in alcohol dehydrogenase-catalyzed asymmetric production of hydrophobic alcohols. Catal Sci Technol 1:1311–1323. https://doi.org/10.1039/c1cy00160d

  33. Musa MM, Bsharat O, Karume I et al (2018) Expanding the substrate specificity of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase by a dual site mutation. Eur J Org Chem 2018:798–805. https://doi.org/10.1002/ejoc.201701351

  34. Napora-Wijata K, Strohmeier GA, Winkler M (2014) Biocatalytic reduction of carboxylic acids. Biotechnol J 9:822–843. https://doi.org/10.1002/biot.201400012

  35. Onyenwoke RU, Kevbrin VV, Lysenko AM, Wiegel J (2007) Thermoanaerobacter pseudethanolicus sp. nov., a thermophilic heterotrophic anaerobe from Yellowstone National Park. Int J Syst Evol Microbiol 57:2191–2193. https://doi.org/10.1099/ijs.0.65051-0

  36. Orlygsson J, Baldursson SRB (2007) Phylogenetic and physiological studies of four hydrogen-producing thermoanareobes. Iceland Agric Sci 20:93–105

  37. Orlygsson J, Houwen FP, Svensson BH (1995) Thermophilic anaerobic amino acid degradation: deamination rates and end product formation. Appl Microbiol Biotechnol 43:235–241

  38. Reid SJ, Stutz HE (2005) Nitrogen assimilation in clostridia. In: Durre P (ed) Handbook of clostridia. CRC Press, Boca Raton, pp 239–260

  39. Schwartz AC, Schäfer R (1973) New amino acids, and heterocyclic compounds participating in the stickland reaction of Clostridium sticklandii. Arch Microbiol 276:267–276

  40. Scully SM (2019) Amino acid and related catabolism of Thermoanaerobacter species. University of Iceland, Reykjavík

  41. Scully SM, Orlygsson J (2014) Branched-chain alcohol formation from branched-chain amino acids by Thermoanaerobacter brockii and Thermoanaerobacter yonseiensis. Anaerobe 30:82–84. https://doi.org/10.1016/j.anaerobe.2014.09.003

  42. Scully SM, Orlygsson J (2015) Amino acid metabolism of Thermoanaerobacter strain AK90: the role of electron-scavenging systems in end product formation. J Amino Acids. https://doi.org/10.1155/2015/410492

  43. Scully SM, Orlygsson J (2019) Branched-chain amino acid catabolism of Thermoanaerobacter strain AK85 and the influence of culture conditions on branched-chain alcohol formation. Amino Acids. https://doi.org/10.1007/s00726-019-02744-z

  44. Scully SM, Iloranta P, Myllymaki P, Orlygsson J (2015) Branched-chain alcohol formation by thermophilic bacteria within the genera of Thermoanaerobacter and Caldanaerobacter. Extremophiles 19:809–818. https://doi.org/10.1007/s00792-015-0756-z

  45. Scully SM, Brown A, Ross AB, Orlygsson J (2019) Biotransformation of organic acids to their corresponding alcohols by Thermoanaerobacter pseudoethanolicus. Anaerobe 57:28–31. https://doi.org/10.1016/j.anaerobe.2019.03.004

  46. Speelmans G, de Vrij W, Konings WN (1989) Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus. J Bacteriol 171:3788–3795

  47. Stickland LH (1934) CCXXXII. Studies in the metabolism of the strict anaerobes (genus Clostridium). I. The chemical reactions by which Cl. sporogenes obtains its energy. Biochem J 28:1746–1759. https://doi.org/10.1042/bj0281746

  48. Strobl G, Feicht R, White H et al (2011) The tungsten-containing aldehyde oxidoreductase from Clostridium thermoaceticum and its complex with a viologen-accepting NADPH oxidoreductase. Biol Chem Hoppe Seyler 373:123–132. https://doi.org/10.1515/bchm3.1992.373.1.123

  49. Tarlera S, Muxí L, Soubes M, Stams AJ (1997) Caloramator proteoclasticus sp. nov., a new moderately thermophilic anaerobic proteolytic bacterium. Int J Syst Bacteriol 47:651–656

  50. Westley J (1987) Thiocyanate and thiosulfate. Methods Enzymol 143:22–25

  51. White H, Huber C, Feicht R, Simon H (1993) On a reversible molybdenum-containing aldehyde oxidoreductase from Clostridium formicoaceticum. Arch Microbiol 159:244–249. https://doi.org/10.1007/BF00248479

  52. Xue Y, Xu Y, Liu Y et al (2001) Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int J Syst Evol Microbiol 51:1335–1341. https://doi.org/10.1099/00207713-51-4-1335

  53. Yao S, Mikkelsen MJ (2010) Identification and overexpression of a bifunctional aldehyde/alcohol dehydrogenase responsible for ethanol production in Thermoanaerobacter mathranii. J Mol Microbiol Biotechnol 19:123–133. https://doi.org/10.1159/000321498

  54. Zeikus JG, Ben-Bassat A, Hegge PW (1980) Microbiology of methanogenesis in thermal, volcanic environments. J Bacteriol 143:432–440

  55. Zhou J, Shao X, Olson DG et al (2017) Determining the roles of the three alcohol dehydrogenases (AdhA, AdhB and AdhE) in Thermoanaerobacter ethanolicus during ethanol formation. J Ind Microbiol Biotechnol 44:745–757. https://doi.org/10.1007/s10295-016-1896-6

Download references

Acknowledgements

The authors are grateful for funding for this work from Landsvirkjun (NÝR-08 – 2015). The authors wish to thank Sigríður Jónsdóttir of the University of Iceland for her assistance with obtaining the NMR spectra and Eva María Ingvadóttir for her much appreciated editorial assistance.

Author information

Correspondence to Johann Orlygsson.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by H. Atomi.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 165 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Scully, S.M., Orlygsson, J. Branched-chain amino acid catabolism of Thermoanaerobacter pseudoethanolicus reveals potential route to branched-chain alcohol formation. Extremophiles 24, 121–133 (2020). https://doi.org/10.1007/s00792-019-01140-5

Download citation

Keywords

  • 2-Methyl-1-butanol
  • Carboxylic acid reduction
  • Thermophiles
  • Anaerobes