Skip to main content
SpringerLink
Log in

Enhanced production of l-sorbose in an industrial Gluconobacter oxydans strain by identification of a strong promoter based on proteomics analysis

  • Genetics and Molecular Biology of Industrial Organisms
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Gluconobacter oxydans is capable of rapidly incomplete oxidation of many sugars and alcohols, which means the strain has great potential for industrial purposes. Strong promoters are one of the essential factors that can improve strain performance by overexpression of specific genes. In this study, a pipeline for screening strong promoters by proteomics analysis was established. Based on the procedure, a new strong promoter designated as P B932_2000 was identified in G. oxydans WSH-003. The promoter region was characterized based on known genome sequence information using BPROM. The strength of P B932_2000 was further assessed by analysis of enhanced green fluorescent protein (egfp) expression and comparison with egfp expression by two commonly used strong promoters, P E. coli_tufB and P G. oxydans_tufB . Both quantitative real-time PCR and fluorescence intensities for egfp gene expression showed that P B932_2000 promoter is stronger than the other two. Overexpression of d-sorbitol dehydrogenase (sldh) by P B932_2000 in G. oxydans WSH-003 enhanced the titer and productivity of l-sorbose synthesis from d-sorbitol by 12.0 % and 33.3 %, respectively. These results showed that proteomics analysis is an efficient way to identify strong promoters. The isolated promoter P B932_2000 could further facilitate the metabolic engineering of G. oxydans.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Anderson NL, Anderson NG (1998) Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis 19:1853–1861

    Article  CAS  PubMed  Google Scholar 

  2. Battey AS, Schaffner DW (2001) Modelling bacterial spoilage in cold-filled ready to drink beverages by Acinetobacter calcoaceticus and Gluconobacter oxydans. J Appl Microbiol 91:237–247

    Article  CAS  PubMed  Google Scholar 

  3. Blackstock WP, Weir MP (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol 17:121–127

    Article  CAS  PubMed  Google Scholar 

  4. Bryksin AV, Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 48:463–465

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene-expression. Science 263:802–805

    Article  CAS  PubMed  Google Scholar 

  6. Cui Z, Zhang X, Zhang Z, Li S (2004) Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter. Biotechnol Lett 26(14):1115–1118

    Article  CAS  PubMed  Google Scholar 

  7. De Muynck C, Pereira C, Naessens M, Parmentier S, Soetaert W, Vandamme E (2007) The genus Gluconobacter oxydans: comprehensive overview of biochemistry and biotechnological applications. Crit Rev Biotechnol 27:147–171

    Article  PubMed  Google Scholar 

  8. Fukui K, Koseki C, Yamamoto Y, Nakamura J, Sasahara A, Yuji R, Hashiguchi K, Usuda Y, Matsui K, Kojima H, Abe K (2011) Identification of succinate exporter in Corynebacterium glutamicum and its physiological roles under anaerobic conditions. J Biotechnol 154:25–34

    Article  CAS  PubMed  Google Scholar 

  9. Gao L, Hu Y, Liu J, Du G, Zhou J, Chen J (2014) Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-l-gulonic acid from d-sorbitol. Metab Eng 24:30–37

    Article  CAS  PubMed  Google Scholar 

  10. Gao L, Zhou J, Liu J, Du G, Chen J (2012) Draft genome sequence of Gluconobacter oxydans WSH-003, a strain that is extremely tolerant of saccharides and alditols. J Bacteriol 194(16):4455–4456

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Gatgens C, Degner U, Bringer-Meyer S, Herrmann U (2007) Biotransformation of glycerol to dihydroxyacetone by recombinant Gluconobacter oxydans DSM 2343. Appl Microbiol Biotechnol 76:553–559

    Article  PubMed  Google Scholar 

  12. Gupta A, Singh VK, Qazi GN, Kumar A (2001) Gluconobacter oxydans: its biotechnological application. J Mol Microbiol Biotechnol 3:445–456

    CAS  PubMed  Google Scholar 

  13. He J, Thomas K, Leif JJ (2012) Comparative proteome analysis of Saccharomyces cerevisiae: a global overview of in vivo targets of the yeast activator protein 1. BMC Genom 13:230

    Article  Google Scholar 

  14. Ikeda M, Mizuno Y, Awane S, Hayashi M, Mitsuhashi S, Takeno S (2011) Identification and application of a different glucose uptake system that functions as an alternative to the phosphotransferase system in Corynebacterium glutamicum. Appl Microbiol Biotechnol 90:1443–1451

    Article  CAS  PubMed  Google Scholar 

  15. Ji AG, Gao PJ (2001) Substrate selectivity of Gluconobacter oxydans for production of 2,5-diketo-d-gluconic acid and synthesis of 2-keto-l-gulonic acid in a multienzyme system. Appl Biochem Biotechnol 94:213–223

    Article  CAS  PubMed  Google Scholar 

  16. Jopcik M, Bauer M, Moravcikova J, Boszoradova E, Matusikova I, Libantova J (2013) Plant tissue-specific promoters can drive gene expression in Escherichia coli. Plant Cell Tissue Organ Cult 113:387–396

    Article  CAS  Google Scholar 

  17. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM (1995) 4 new derivatives of the broad-host-range cloning vector PBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176

    Article  CAS  PubMed  Google Scholar 

  18. Lee JS, An G, Friesen JD, Fill NP (1981) Location of the tufB promoter of E. coli: cotranscription of tufB with four transfer RNA genes. Cell 25:251–258

    Article  CAS  PubMed  Google Scholar 

  19. Lu L, Wei L, Zhu K, Wei D, Hua Q (2012) Combining metabolic engineering and adaptive evolution to enhance the production of dihydroxyacetone from glycerol by Gluconobacter oxydans in a low-cost way. Bioresour Technol 117:317–324

    Article  CAS  PubMed  Google Scholar 

  20. Matsushita K, Toyama H, Adachi O (1994) Respiratory chains and bioenergetics of acetic acid bacteria. Adv Microb Physiol 36:247–301

    CAS  PubMed  Google Scholar 

  21. Merfort M, Herrmann U, Bringer-Meyer S, Sahm H (2006) High-yield 5-keto-d-gluconic acid formation is mediated by soluble and membrane-bound gluconate-5-dehydrogenases of Gluconobacter oxydans. Appl Microbiol Biotechnol 73:443–451

    Article  CAS  PubMed  Google Scholar 

  22. Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255–262

    Article  CAS  PubMed  Google Scholar 

  23. Pátek M, Nešvera J (2012) Promoters and plasmid vectors of Corynebacterium glutamicum. In: Yukawa H, Inui M (eds) Biology and biotechnology of Corynebacterium glutamicum. Springer, Berlin, pp 51–88

    Google Scholar 

  24. Patek M, Holatko J, Busche T, Kalinowski J, Nesvera J (2013) Corynebacterium glutamicum promoters: a practical approach. Microb Biotechnol 6:103–117

    Article  PubMed Central  PubMed  Google Scholar 

  25. Patterson GH, Knobel SM, Sharif WD, Kain SR, Piston DW (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73:2782–2790

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Peters B, Junker A, Brauer K, Muhlthaler B, Kostner D, Mientus M, Liebl W, Ehrenreich A (2013) Deletion of pyruvate decarboxylase by a new method for efficient markerless gene deletions in Gluconobacter oxydans. Appl Microbiol Biotechnol 97:2521–2530

    Article  CAS  PubMed  Google Scholar 

  27. Prust C, Hoffmeister M, Liesegang H, Wiezer A, Fricke WF, Ehrenreich A, Gottschalk G, Deppenmeier U (2005) Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. Nat Biotechnol 23:195–200

    Article  CAS  PubMed  Google Scholar 

  28. Rhodius VA, Mutalik VK (2010) Predicting strength and function for promoters of the Escherichia coli alternative sigma factor, σE. Proc Natl Acad Sci USA 107(7):2854–2859

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Richard G (2010) Prokaryotic transcription. In: Kebs Jocelyn E, Kilpatrick Stephen T, Goldstein Elliott S (eds) Lewein’s gene X. Jones and Bartlett Publishers, Massachusetts, pp 514–515

    Google Scholar 

  30. Saito Y, Ishii Y, Hayashi H, Imao Y, Akashi T (1997) Cloning genes coding for l-sorbose and l-sorbosone dehydrogenase from Gluconobacter oxydans and microbial production of 2-keto-l-gulonate, a precursor of l-ascorbic acid, in a recombinant G. oxydans strain. Appl Environ Microbiol 63:454–460

    CAS  PubMed Central  PubMed  Google Scholar 

  31. Schleyer U, Bringer-Meyer S, Sahm H (2008) An easy cloning and expression vector system for Gluconobacter oxydans. Int J Food Microbiol 125:91–95

    Article  CAS  PubMed  Google Scholar 

  32. Schmid R, Gerloff DL (2004) Functional properties of the alternative NADH:ubiquinone oxidoreductase from E. coli through comparative 3-D modelling. FEBS Lett 578:163–168

    Article  CAS  PubMed  Google Scholar 

  33. Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, Sittka A, Chabas S, Reiche K, Hackermuller J, Reinhardt R, Stadler PF, Vogel J (2010) The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 464(7286):250–255

    Article  CAS  PubMed  Google Scholar 

  34. Shi L, Li K, Zhang H, Liu X, Lin J, Wei D (2014) Identification of a novel promoter gHp0169 for gene expression in Gluconobacter oxydans. J Biotechnol 175:69–74

    Article  CAS  PubMed  Google Scholar 

  35. Siegl T, Tokovenko B, Myronovskyi M, Luzhetskyy A (2013) Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes. Metab Eng 19:98–106

    Article  CAS  PubMed  Google Scholar 

  36. Siekevitz M, Feinberg MB, Holbrook N, Wong-Staal F, Greene WC (1987) Activation of interleukin 2 and interleukin 2 receptor (Tac) promoter expression by the trans-activator (tat) gene product of human T-cell leukemia virus, type I. Proc Natl Acad Sci USA 84:5389–5393

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Singh BR, Al-Khedhairy AA, Alarifi SA, Musarrat J (2009) Regulatory elements in the 5′region of 16SrRNA gene of Bacillus sp. strain SJ-101. Bioinformation 3:375–380

    Article  PubMed Central  PubMed  Google Scholar 

  38. Soboleski MR, Oaks J, Halford WP (2005) Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. FASEB J 19:440–442

    CAS  PubMed Central  PubMed  Google Scholar 

  39. Solovyev V, Salamov A (2011) Automatic annotation of microbial genomes and metagenomic sequences. In: Li RW (ed) Metagenomics and its applications in agriculture, biomedicine and environmental studies. Nova Science Publishers, New York, pp 61–78

    Google Scholar 

  40. Solovyev VV, Shahmuradov IA, Salamov AA (2010) Identification of promoter regions and regulatory sites. Methods Mol Biol 674:57–83

    CAS  PubMed  Google Scholar 

  41. Srivastava S, Singh V, Kumar V, Verma PC, Srivastava R, Basu V, Gupta V, Rawat AK (2008) Identification of regulatory elements in 16S rRNA gene of Acinetobacter species isolated from water sample. Bioinformation 3:173–176

    Article  PubMed Central  PubMed  Google Scholar 

  42. Wang J, Ai X, Mei H, Fu Y, Chen B, Yu Z, He J (2013) High-throughput identification of promoters and screening of highly active promoter-5′-UTR DNA region with different characteristics from Bacillus thuringiensis. PLoS ONE 8(5):e62960

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Xu Y, Liu Q, Zhou L, Yang Z, Zhang Y (2008) Surface display of GFP by Pseudomonas syringae truncated ice nucleation protein in attenuated Vibrio anguillarum strain. Mar Biotechnol 10:701–708

    Article  CAS  PubMed  Google Scholar 

  44. Yang M (2013) Generation of an artificial double promoter for protein expression in Bacillus subtilis through a promoter trap system. PLoS ONE 8(2):e56321

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Yang X, Wei L, Lin J, Yin B, Wei D (2008) Membrane-bound pyrroloquinoline quinone-dependent dehydrogenase in Gluconobacter oxydans M5, responsible for production of 6-(2-hydroxyethyl) amino-6-deoxy-l-sorbose. Appl Environ Microbiol 74:5250–5253

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Zhou J, Du G, Chen J (2012) Metabolic engineering of microorganisms for vitamin C production. Subcell Biochem 64:241–259

    CAS  PubMed  Google Scholar 

  47. Zhou J, Wang K, Xu S, Wu J, Liu P, Du G, Li J, Chen J (2015) Identification of membrane proteins associated with phenylpropanoid tolerance and transport in Escherichia coli BL21. J Proteomics 113:15–28

    Article  CAS  PubMed  Google Scholar 

  48. Zhu Y, Liu J, Liu J, Du G, Zhou J, Chen J (2012) A high throughput method to screen companion bacterium for 2-keto-l-gulonic acid biosynthesis by co-culturing Ketogulonicigenium vulgare. Process Biochem 47:1428–1432

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the National High Technology Research and Development Program of China (863 Program, 2012AA022103), the Major State Basic Research Development Program of China (973 Program, 2014CB745103), the Major Program of National Natural Science Foundation of China (21390204), the Program for New Century Excellent Talents in University (NCET-12-0876), the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (FANEDD, 201256), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the 111 Project (111-2-06).

Author information

Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China

    Yudong Hu, Hui Wan, Jianghua Li & Jingwen Zhou

  2. Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China

    Jianghua Li & Jingwen Zhou

Authors
  1. Yudong Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hui Wan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jianghua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jingwen Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jingwen Zhou.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 15 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hu, Y., Wan, H., Li, J. et al. Enhanced production of l-sorbose in an industrial Gluconobacter oxydans strain by identification of a strong promoter based on proteomics analysis. J Ind Microbiol Biotechnol 42, 1039–1047 (2015). https://doi.org/10.1007/s10295-015-1624-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-015-1624-7

Keywords

Search

Navigation