Applied Microbiology and Biotechnology

, Volume 100, Issue 2, pp 719–728 | Cite as

Synthetic operon for (R,R)-2,3-butanediol production in Bacillus subtilis and Escherichia coli

  • Rafael R. de Oliveira
  • Wayne L. Nicholson
Applied Genetics and Molecular Biotechnology


To reduce dependence on petroleum, an alternative route to production of the chemical feedstock 2,3-butanediol (2,3-BD) from renewable lignocellulosic sources is desirable. In this communication, the genes encoding the pathway from pyruvate to 2,3-BD (alsS, alsD, and bdhA encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase, respectively) from Bacillus subtilis were engineered into a single tricistronic operon under control of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible Pspac promoter in a shuttle plasmid capable of replication and expression in either B. subtilis or Escherichia coli. We describe the construction and performance of a shuttle plasmid carrying the IPTG-inducible synthetic operon alsSDbdhA coding for 2,3-BD pathway capable of (i) expression in two important representative model microorganisms, the gram-positive B. subtilis and the gram-negative E. coli; (ii) increasing 2,3-BD production in B. subtilis; and (iii) successfully introducing the B. subtilis 2,3-BD pathway into E. coli. The synthetic alsSDbdhA operon constructed using B. subtilis native genes not only increased the 2,3-BD production in its native host but also efficiently expressed the pathway in the heterologous organism E. coli. Construction of an efficient shuttle plasmid will allow investigation of 2,3-BD production performance in related organisms with industrial potential for production of bio-based chemicals.


2,3-Butanediol Bacillus subtilis Butanediol dehydrogenase Escherichia coli Shuttle plasmid 



We express our thanks to Dr. K.T. Shanmugam for expert assistance and discussion. We also thank Jeff Richards and the late Dr. Lanfang Levine for their excellent technical support with HPLC. This work was supported by grant FLA-MCS-04602 from the US Department of Agriculture, administered through the Florida Agriculture Experiment Station to W.L.N., and support from the UF Department of Microbiology and Cell Science to R.R.O.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aguilera RF (2014) Production costs of global conventional and unconventional petroleum. Energy Policy 64:134–140CrossRefGoogle Scholar
  2. Akhtar P, Anand SP, Watkins SC, Khan SA (2009) The tubulin-like RepX protein encoded by the pXO1 plasmid forms polymers in vivo in Bacillus anthracis. J Bacteriol 191(8):2493–2500. doi: 10.1128/JB.00027-09 PubMedPubMedCentralCrossRefGoogle Scholar
  3. Anderson TD, Miller JI, Fierobe HP, Clubb RT (2013) Recombinant Bacillus subtilis that grows on untreated plant biomass. Appl Environ Microbiol 79(3):867–876. doi: 10.1128/AEM.02433-12 PubMedPubMedCentralCrossRefGoogle Scholar
  4. Antelmann H, Tjalsma H, Voigt B, Ohlmeier S, Bron S, Dijl JM, Hecker M (2001) A proteomic view on genome-based signal peptide predictions. Genome Res 11:1484–1502PubMedCrossRefGoogle Scholar
  5. Anvari M, Pahlavanzadeh H, Vasheghani-Farahani E, Khayati G (2009) In situ recovery of 2,3-butanediol from fermentation by liquid-liquid extraction. J Ind Microbiol Biotechnol 36(2):313–317. doi: 10.1007/s10295-008-0501-z PubMedCrossRefGoogle Scholar
  6. Band L, Henner DJ (1984) Bacillus subtilis requires a stringent Shine-Dalgarno region for gene expression. DNA 3(1):17–21PubMedCrossRefGoogle Scholar
  7. Biswas R, Yamaoka M, Nakayama H, Kondo T, Yoshida K, Bisaria VS, Kondo A (2012) Enhanced production of 2,3-butanediol by engineered Bacillus subtilis. Appl Microbiol Biotechnol 94:651–658PubMedCrossRefGoogle Scholar
  8. Bokinskya G, Peralta-Yahyaa PP, Georgea A, Holmesa BM, Steena EJ, Dietricha J, Leea TS, Tullman-Erceka D, Voigtg CA, Simmonsa BA, Keasling JD (2011) Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. Proc Natl Acad Sci U S A 108(50):19949–19954CrossRefGoogle Scholar
  9. Boylan RJ, Brooks D, Young FE, Mendelson NH (1972) Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid. J Bacteriol 110(1):281–290PubMedPubMedCentralGoogle Scholar
  10. Celińska E, Grajek W (2009) Biotechnological production of 2,3-butanediol—current state and prospects. Biotechnol Adv 27(6):715–725. doi: 10.1016/j.biotechadv.2009.05.002 PubMedCrossRefGoogle Scholar
  11. Cruz Ramos H, Hoffmann T, Marino M, Nedjari H, Presecan-Siedel E, Dreesen O, Glaser P, Jahn D (2000) Fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression. J Bacteriol 182(11):3072–3080PubMedPubMedCentralCrossRefGoogle Scholar
  12. Cutting SM, Vander Horn PB (1990) Genetic analysis. In: Harwood CR, Cutting SM (eds) Molecular biological methods for Bacillus. John Wiley and Sons, Sussex, pp 27–74Google Scholar
  13. Dittmar M (2012) Nuclear energy: status and future limitations. Energy 37:35–40CrossRefGoogle Scholar
  14. Dong H, Nilsson L, Kurland CG (1995) Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction. J Bacteriol 177(6):1497–1504PubMedPubMedCentralGoogle Scholar
  15. Fu J, Wang Z, Chen T, Liu W, Shi T, Wang G, Tang YJ, Zhao X (2014) NADH plays the vital role for chiral pure D-(-)-2,3-butanediol production in Bacillus subtilis under limited oxygen conditions. Biotechnol Bioeng 111(10):2126–2131. doi: 10.1002/bit.25265 PubMedCrossRefGoogle Scholar
  16. Gerber L, Maréchal F (2012) Environomic optimal configurations of geothermal energy conversion systems: application to the future construction of enhanced geothermal systems in Switzerland. Energy 45(1):908–923CrossRefGoogle Scholar
  17. Gundlach L, Burfeindt B, Mahrt J, Willig F (2012) Dynamics of ultrafast photoinduced heterogeneous electron transfer, implications for recent solar energy conversion scenarios. Chem Phys Lett 545:35–39CrossRefGoogle Scholar
  18. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4):557–580PubMedCrossRefGoogle Scholar
  19. Hobert O (2002) PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques 32(4):728–730PubMedGoogle Scholar
  20. Ji XJ, Huang H, Ouyang PK (2011) Microbial 2,3-butanediol production: a state-of-the-art review. Biotechnol Adv 29(3):351–364. doi: 10.1016/j.biotechadv.2011.01.007 PubMedCrossRefGoogle Scholar
  21. Joseph P, Fantino JR, Herbaud ML, Denizot F (2001) Rapid orientated cloning in a shuttle vector allowing modulated gene expression in Bacillus subtilis. FEMS Microbiol Lett 205:91–97PubMedCrossRefGoogle Scholar
  22. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessieres P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S, Bruschi CV, Caldwell B, Capuano V, Carter NM, Choi SK, Codani JJ, Connerton IF, Cummings NJ, Daniel RA, Denziot F, Devine KM, Düsterhöft A, Ehrlich SD, Emmerson PT, Entian KD, Errington J, Fabret C, Ferrari E, Foulger D, Fritz C, Fujita M, Fuma S, Galizzi A, Galleron N, Ghim SY, Glaser P, Goffeau A, Golightly EJ, Grandi G, Guiseppi G, Guy BJ, Haga K, Haiech J, Harwood CR, Hènaut A, Hilbert H, Holsappel S, Hosono S, Hullo MF, Itaya M, Joris B, Karamata D, Kasahara Y, Klaerr-Balnchard M, Klein C, Kobayashi Y, Koetter P, Konigstein G, Krogh S, Kumano M, Kurita K, Lapidus A, Lardinois S, Lauber J, Lazarevic V, Lee SM, Levine A, Liu H, Masuda S, Mauël C, Médigue C, Medina N, Mellado RP, Mizuno M, Moestl D, Nakai S, Noback M, Noone D, O'Reilly M, Ogawa K, Ogiwara A, Oudega B, Park SH, Parro V, Pohl TM, Portelle D, Porwollik S, Prescott AM, Presecan E, Pujic P, Purnelle B, Rapaport G, Rey M, Reynolds S, Rieger M, Rivolta C, Rocha E, Roche B, Rose M, Sadaie Y, Sato T, Scanlan E, Schleich S, Schroeter R, Scoffone F, Sekiguchi J, Sekowska A, Seror SJ, Serror P, Shin BS, Soldo B, Sorokin A, Tacconi E, Takagi T, Takahashi H, Takemaru K, Takeuchi M, Tamakoshi A, Tanaka T, Terpstra P, Togoni A, Tosato V, Uchiyama S, Vandebol M, Vannier F, Vassarotti A, Viari A, Wambutt R, Wedler H, Weitzenegger T, Winters P, Wipat A, Yamamoto H, Yamane K, Yasumoto K, Yata K, Yoshida K, Yoshikawa HF, Zumstein E, Yoshikawa H, Danchin A (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390(6657):249–256. doi: 10.1038/36786
  23. Lumbreras S, Ramos A (2012) Offshore wind farm electrical design: a review. Wind Energy 10:1002–1498Google Scholar
  24. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  25. Moet GJ, Jones RN, Biedenbach DJ, Stilwell MG, Fritsche TR (2007) Contemporary causes of skin and soft tissue infections in North america, Latin america, and Europe: report from the SENTRY Antimicrobial Surveillance Program (1998–2004). Ann Clin Microbiol Antimicrob 57:7–13Google Scholar
  26. Murray J, King D (2012) Oil’s tipping point has passed. Nature 481:433–435PubMedCrossRefGoogle Scholar
  27. Nakano MM, Zuber P (1998) Anaerobic growth of a “strict aerobe” (Bacillus subtilis). Annu Rev Microbiol 52:165–190. doi: 10.1146/annurev.micro.52.1.165 PubMedCrossRefGoogle Scholar
  28. Nicholson WL (2008) The Bacillus subtilis ydjL (bdhA) gene encodes acetoin reductase/2,3-butanediol dehydrogenase. Appl Environ Microbiol 74(22):6832–6838. doi: 10.1128/aem.00881-08 PubMedPubMedCentralCrossRefGoogle Scholar
  29. Oliveira RR, Nicholson WL (2013) The LysR-type transcriptional regulator (LTTR) AlsR indirectly regulates expression of the Bacillus subtilis bdhA gene encoding 2,3-butanediol dehydrogenase. Appl Microbiol Biotechnol 97(16):7307–7316. doi: 10.1007/s00253-013-4871-4 PubMedCrossRefGoogle Scholar
  30. Otero JM, Panagiotou G, Olsson L (2007) Fueling industrial biotechnology growth with bioethanol. Adv Biochem Eng Biotechnol 108:1–40. doi: 10.1007/10_2007_071 PubMedGoogle Scholar
  31. Palsson BO, Fathi-Afshar S, Rudd DF, Lightfoot EN (1981) Biomass as a source of chemical feedstocks: an economic evaluation. Science 213(4507):513–517. doi: 10.1126/science.213.4507.513 PubMedCrossRefGoogle Scholar
  32. Pauly M, Keegstra K (2010) Plant cell wall polymers as precursors for biofuels. Curr Opin Plant Biol 13:305–312PubMedCrossRefGoogle Scholar
  33. Petit MA, Ehrlich SD (2000) The NAD-dependent ligase encoded by yerG is an essential gene of Bacillus subtilis. Nucleic Acids Res 28(23):4642–4648PubMedPubMedCentralCrossRefGoogle Scholar
  34. Podschun R, Ullmann U (1998) Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11(4):589–603PubMedPubMedCentralGoogle Scholar
  35. Qi G, Kang Y, Li L, Xiao A, Zhang S, Wen Z, Xu D, Chen S (2014) Deletion of meso-2,3-butanediol dehydrogenase gene budC for enhanced D-2,3-butanediol production in Bacillus licheniformis. Biotechnol Biofuels 7(1):16. doi: 10.1186/1754-6834-7-16 PubMedPubMedCentralCrossRefGoogle Scholar
  36. Rabaey K, Boon N, Siciliano SD, Verhaege M, Verstraete W (2004) Biofuel cells select for microbial consortia that self-mediate electron transfer. Environ Microbiol 70:5373–5382CrossRefGoogle Scholar
  37. Renna MC, Najimudin N, Winik LR, Zahler SA (1993) Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin. J Bacteriol 175(12):3863–3875PubMedPubMedCentralGoogle Scholar
  38. Rourke FO, Boyle F, Reynolds A (2010) Tidal energy update 2009. Appl Energy 87:398–409CrossRefGoogle Scholar
  39. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  40. Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17PubMedCrossRefGoogle Scholar
  41. Sharp PM, Cowe E, Higgins DG, Shields DC, Wolfe KH, Wright F (1988) Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity. Nucleic Acids Res 16(17):8207–8211PubMedPubMedCentralCrossRefGoogle Scholar
  42. Steubing B, Zah R, Ludwig C (2011) Life cycle assessment of SNG from wood for heating, electricity, and transportation. Biomass Bioenergy 35:2950–2960CrossRefGoogle Scholar
  43. Underwood SA, Zhou S, Causey TB, Yomano LP, Shanmugam KT, Ingram LO (2002) Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli. Appl Environ Microbiol 68:6263–6272PubMedPubMedCentralCrossRefGoogle Scholar
  44. Vagner V, Dervyn E, Ehrlich SD (1998) A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144:3097–3104PubMedCrossRefGoogle Scholar
  45. Van Dien S (2013) From the first drop to the first truckload: commercialization of microbial processes for renewable chemicals. Curr Opin Biotechnol 24:1061–1068PubMedCrossRefGoogle Scholar
  46. Villa DC, Angioni S, Quartarone E, Righetti PP, Mustarelli P (2013) New sulfonated PBIs for PEMFC application. Fuel Cells 13:98–103CrossRefGoogle Scholar
  47. Voloch M, Jansen NB, Ladisch MR, Tsao GT, Narayan R, Rodwell VW (1985) 2,3-Butanediol. In: Murray M-Y, Cooney CL, Humphrey AE (eds) Comprehensive biotechnology: the principles, applications and regulations of biotechnology in industry, agriculture and medicine. Pergamon Press, New York, pp 933–947Google Scholar
  48. Wang Q, Chen T, Zhao X, Chamu J (2012) Metabolic engineering of thermophilic Bacillus licheniformis for chiral pure D-2,3-butanediol production. Biotechnol Bioeng 109(7):1610–1621. doi: 10.1002/bit.24427 PubMedCrossRefGoogle Scholar
  49. Xiu ZL, Zeng AP (2008) Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol. Appl Microbiol Biotechnol 78(6):917–926. doi: 10.1007/s00253-008-1387-4 PubMedCrossRefGoogle Scholar
  50. Xu Y, Chu H, Gao C, Tao F, Zhou Z, Li K, Li L, Ma C, Xu P (2014) Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2,3-butanediol. Metab Eng 23:22–33PubMedCrossRefGoogle Scholar
  51. Yan Y, Lee C, Liao JC (2009) Enantioselective synthesis of pure (R, R)-2,3-butanediol in Escherichia coli with stereospecific secondary alcohol dehydrogenases. Org Biomol Chem 7:3914–3917PubMedCrossRefGoogle Scholar
  52. Yang T, Rao Z, Zhang X, Xu M, Xu Z, Yang ST (2013) Improved production of 2,3-butanediol in Bacillus amyloliquefaciens by over-expression of glyceraldehyde-3-phosphate dehydrogenase and 2,3-butanediol dehydrogenase. PLoS One 8(10):e76149. doi: 10.1371/journal.pone.0076149 PubMedPubMedCentralCrossRefGoogle Scholar
  53. Zhang X, Zhang R, Bao T, Yang T, Xu M, Li H, Xu Z, Rao Z (2013) Moderate expression of the transcriptional regulator AlsR enhances acetoin production by Bacillus subtilis. J Ind Microbiol Biotechnol 40:1067–1076PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Department of Microbiology and Cell ScienceUniversity of FloridaMerritt IslandUSA
  2. 2.IPR–PUCRSPorto AlegreBrazil

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