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Improving the Clostridium acetobutylicum butanol fermentation by engineering the strain for co-production of riboflavin

  • Original Paper
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Journal of Industrial Microbiology & Biotechnology

Abstract

Solvent-producing clostridia are well known for their capacity to use a wide variety of renewable biomass and agricultural waste materials for biobutanol production. To investigate the possibility of co-production of a high value chemical during biobutanol production, the Clostridium acetobutylicum riboflavin operon ribGBAH was over-expressed in C. acetobutylicum on Escherichia coli–Clostridium shuttle vector pJIR750. Constructs that either maintained the original C. acetobutylicum translational start codon or modified the start codons of ribG and ribB from TTG to ATG were designed. Riboflavin was successfully produced in both E. coli and C. acetobutylicum using these plasmids, and riboflavin could accumulate up to 27 mg/l in Clostridium culture. Furthermore, the C. acetobutylicum purine pathway was modified by over-expression of the Clostridium purF gene, which encodes the enzyme PRPP amidotransferase. The function of the plasmid pJaF bearing C. acetobutylicum purF was verified by its ability to complement an E. coli purF mutation. However, co-production of riboflavin with biobutanol by use of the purF over-expression plasmid was not improved under the experimental conditions examined. Further rational mutation of the purF gene was conducted by replacement of amino acid codons D302 V and K325Q to make it similar to the feedback-resistant enzymes of other species. However, the co-expression of ribGBAH and purFC in C. acetobutylicum also did not improve riboflavin production. By buffering the culture pH, C. acetobutylicum ATCC 824(pJpGN) could accumulate more than 70 mg/l riboflavin while producing 190 mM butanol in static cultures. Riboflavin production was shown to exert no effect on solvent production at these levels.

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References

  1. Alvira P, Tomas-Pejo E, Ballesteros M, Negro MJ (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 101:4851–4861

    Article  PubMed  CAS  Google Scholar 

  2. Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJ, Hanai T, Liao JC (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 10:305–311

    Article  PubMed  CAS  Google Scholar 

  3. Bacher A, Eberhardt S, Eisenreich W, Fischer M, Herz S, Illarionov B, Kis K, Richter G (2001) Biosynthesis of riboflavin. Vitam Horm 61:1–49

    Article  PubMed  CAS  Google Scholar 

  4. Bannam TL, Rood JI (1993) Clostridium perfringens-Escherichia coli shuttle vectors that carry single antibiotic resistance determinants. Plasmid 29:233–235

    Article  PubMed  CAS  Google Scholar 

  5. Beesch SC (1953) Acetone-butanol fermentation of starches. Appl Microbiol 1:85–95

    PubMed  CAS  Google Scholar 

  6. Burgess C, O’Connell-Motherway M, Sybesma W, Hugenholtz J, van Sinderen D (2004) Riboflavin production in Lactococcus lactis: potential for in situ production of vitamin-enriched foods. Appl Environ Microbiol 70:5769–5777

    Article  PubMed  CAS  Google Scholar 

  7. Claassen PA, Budde MA, Lopez-Contreras AM (2000) Acetone, butanol and ethanol production from domestic organic waste by solventogenic clostridia. J Mol Microbiol Biotechnol 2:39–44

    PubMed  CAS  Google Scholar 

  8. Clark SW, Bennett GN, Rudolph FB (1989) Isolation and characterization of mutants of Clostridium acetobutylicum ATCC 824 deficient in acetoacetyl-coenzyme A: acetate/butyrate:coenzyme A-transferase (EC 2.8.3.9) and in other solvent pathway enzymes. Appl Environ Microbiol 55:970–976

    PubMed  CAS  Google Scholar 

  9. Demain AL (1972) Riboflavin oversynthesis. Annu Rev Microbiol 26:369–388

    Article  PubMed  CAS  Google Scholar 

  10. Durre P (2007) Biobutanol: an attractive biofuel. Biotechnol J 2:1525–1534

    Article  PubMed  Google Scholar 

  11. Durre P (2008) Fermentative butanol production: bulk chemical and biofuel. Ann N Y Acad Sci 1125:353–362

    Article  PubMed  Google Scholar 

  12. Ezeji TC, Qureshi N, Blaschek HP (2007) Butanol production from agricultural residues: impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnol Bioeng 97:1460–1469

    Article  PubMed  CAS  Google Scholar 

  13. Ezeji TC, Qureshi N, Blaschek HP (2004) Butanol fermentation research: upstream and downstream manipulations. Chem Rec 4:305–314

    Article  PubMed  CAS  Google Scholar 

  14. Fassbinder F, Kist M, Bereswill S (2000) Structural and functional analysis of the riboflavin synthesis genes encoding GTP cyclohydrolase II (ribA), DHBP synthase (ribBA), riboflavin synthase (ribC), and riboflavin deaminase/reductase (ribD) from Helicobacter pylori strain P1. FEMS Microbiol Lett 191:191–197

    Article  PubMed  CAS  Google Scholar 

  15. Gamage J, Lam H, Zhang ZS (2010) Bioethanol production from lignocellulosic biomass, a review. J Biobased Mater Bioenergy 4:3–11

    Article  CAS  Google Scholar 

  16. Gelfand MS, Mironov AA, Jomantas J, Kozlov YI, Perumov DA (1999) A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes. Trends Genet 15:439–442

    Article  PubMed  CAS  Google Scholar 

  17. Green EM, Boynton ZL, Harris LM, Rudolph FB, Papoutsakis ET, Bennett GN (1996) Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824. Microbiology 142:2079–2086

    Article  PubMed  CAS  Google Scholar 

  18. Hickey JR (1945) Production of riboflavin by butyl alcohol producing bacteria. US patent 2425280

  19. Hümbelin M, Griesser V, Keller T, Schurter W, Haiker M, Hohmann HP, Ritz H, Richter G, Bacher A, van Loon APGM (1999) GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production. J Ind Microbiol Biotechnol 22:1–7

    Article  Google Scholar 

  20. Yatsishyn VY, Fedorovich DV, Sibirnyi AA (2009) The microbial synthesis of flavin nucleotides: a review. Appl Biochem Biotechnol 45:133–142

    Google Scholar 

  21. Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H, Yamamoto S, Okino S, Suzuki N, Yukawa H (2008) Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biotechnol 77:1305–1316

    Article  PubMed  CAS  Google Scholar 

  22. Jimenez A, Santos MA, Pompejus M, Revuelta JL (2005) Metabolic engineering of the purine pathway for riboflavin production in Ashbya gossypii. Appl Environ Microbiol 71:5743–5751

    Article  PubMed  CAS  Google Scholar 

  23. Johnson JL, Toth J, Santiwatanakul S, Chen JS (1997) Cultures of “Clostridium acetobutylicum” from various collections comprise Clostridium acetobutylicum, Clostridium beijerinckii, and two other distinct types based on DNA–DNA reassociation. Int J Syst Bacteriol 47:420–424

    Article  PubMed  CAS  Google Scholar 

  24. Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524

    PubMed  CAS  Google Scholar 

  25. Knorr B, Schlieker H, Hohmann H-P, Weuster-Botz D (2007) Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis. Biochem Eng J 33:263–274

    Article  CAS  Google Scholar 

  26. Lee JY, Jang YS, Lee J, Papoutsakis ET, Lee SY (2009) Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production. Biotechnol J 4:1432–1440

    Article  PubMed  CAS  Google Scholar 

  27. Leviton A (1946) The microbiological synthesis of riboflavin—a theory concerning its inhibition. J Am Chem Soc 68:835–840

    Article  PubMed  CAS  Google Scholar 

  28. Leviton A, Whittier EO (1950) The utilization of whey in the microbiological synthesis of riboflavin. J Dairy Sci 33:402

    Google Scholar 

  29. Martinez I, Zhu J, Lin H, Bennett GN, San KY (2008) Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways. Metab Eng 10:352–359

    Article  PubMed  CAS  Google Scholar 

  30. Marx H, Mattanovich D, Sauer M (2008) Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris. Microb Cell Fact 7:23

    Article  PubMed  Google Scholar 

  31. Mermelstein LD, Papoutsakis ET (1993) In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 59:1077–1081

    PubMed  CAS  Google Scholar 

  32. Mermelstein LD, Welker NE, Bennett GN, Papoutsakis ET (1992) Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. Biotechnology (NY) 10:190–195

    Article  CAS  Google Scholar 

  33. Muchmore CR, Krahn JM, Kim JH, Zalkin H, Smith JL (1998) Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Protein Sci 7:39–51

    Article  PubMed  CAS  Google Scholar 

  34. Nair RV, Green EM, Watson DE, Bennett GN, Papoutsakis ET (1999) Regulation of the sol locus genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 by a putative transcriptional repressor. J Bacteriol 181:319–330

    PubMed  CAS  Google Scholar 

  35. Pridham TG (1946) Microbial synthesis of riboflavin. Econ Bot 6:185–205

    Article  Google Scholar 

  36. Qureshi N, Blaschek HP (2001) ABE production from corn: a recent economic evaluation. J Ind Microbiol Biotechnol 27:292–297

    Article  PubMed  CAS  Google Scholar 

  37. Sambrook J, Russel, David W (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New york

    Google Scholar 

  38. Sauer U, Hatzimanikatis V, Hohmann HP, Manneberg M, van Loon AP, Bailey JE (1996) Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis. Appl Environ Microbiol 62:3687–3696

    PubMed  CAS  Google Scholar 

  39. Scotcher MC, Bennett GN (2005) SpoIIE regulates sporulation but does not directly affect solventogenesis in Clostridium acetobutylicum ATCC 824. J Bacteriol 187:1930–1936

    Article  PubMed  CAS  Google Scholar 

  40. Scotcher MC, Bennett GN (2008) Activity of abrB310 promoter in wild type and spo0A-deficient strains of Clostridium acetobutylicum. J Ind Microbiol Biotechnol 35:743–750

    Article  PubMed  CAS  Google Scholar 

  41. Shimaoka M, Takenaka Y, Kurahashi O, Kawasaki H, Matsui H (2007) Effect of amplification of desensitized purF and prs on inosine accumulation in Escherichia coli. J Biosci Bioeng 103:255–261

    Article  PubMed  CAS  Google Scholar 

  42. Smith JL, Zaluzec EJ, Wery JP, Niu L, Switzer RL, Zalkin H, Satow Y (1994) Structure of the allosteric regulatory enzyme of purine biosynthesis. Science 264:1427–1433

    Article  PubMed  CAS  Google Scholar 

  43. Stahmann KP, Revuelta JL, Seulberger H (2000) Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production. Appl Microbiol Biotechnol 53:509–516

    Article  PubMed  CAS  Google Scholar 

  44. Steen EJ, Chan R, Prasad N, Myers S, Petzold CJ, Redding A, Ouellet M, Keasling JD (2008) Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microb Cell Fact 7:36

    Article  PubMed  Google Scholar 

  45. Switzer RL, Ruppen ME, Bernlohr DA (1982) Inactivation of glutamine: 5-phosphoribosyl 1-pyrophosphate amidotransferase in Bacillus subtilis—oxidation of an essential Fe-S center precedes selective degradation. Biochem Soc Trans 10:322–324

    PubMed  CAS  Google Scholar 

  46. Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS (2002) Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation. Nucleic Acids Res 30:3141–3151

    Article  PubMed  CAS  Google Scholar 

  47. Zalkin H, Smith JL, Switzer RL (2001) Deregulation of glutamine PRPP amidotransferase activity. US patent 6,204,041

  48. Zhao YS, Hindorff LA, Chuang A, Monroe-Augustus M, Lyristis M, Harrison ML, Rudolph FB, Bennett GN (2003) Expression of a cloned cyclopropane fatty acid synthase gene reduces solvent formation in Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 69:2831–2841

    Article  PubMed  CAS  Google Scholar 

  49. Zhou G, Smith JL, Zalkin H (1994) Binding of purine nucleotides to two regulatory sites results in synergistic feedback inhibition of glutamine 5-phosphoribosylpyrophosphate amidotransferase. J Biol Chem 269:6784–6789

    PubMed  CAS  Google Scholar 

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Acknowledgments

This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2006-35504-17294. Also, we would like to thank Mary Harrison for her assistance for this research.

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  1. Department of Biochemistry and Cell Biology, MS 140, Rice University, 6100 Main Street, Houston, TX, 77005-1892, USA

    Xianpeng Cai & George N. Bennett

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  1. Xianpeng Cai
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Correspondence to George N. Bennett.

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Cai, X., Bennett, G.N. Improving the Clostridium acetobutylicum butanol fermentation by engineering the strain for co-production of riboflavin. J Ind Microbiol Biotechnol 38, 1013–1025 (2011). https://doi.org/10.1007/s10295-010-0875-6

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  • DOI: https://doi.org/10.1007/s10295-010-0875-6

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