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Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression

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Abstract

Acetic acid bacteria are used in biotechnology due to their ability to incompletely oxidize a great variety of carbohydrates, alcohols, and related compounds in a regio- and stereo-selective manner. These reactions are catalyzed by membrane-bound dehydrogenases (mDHs), often with a broad substrate spectrum. In this study, the promoters of six mDHs of Gluconobacter oxydans 621H were characterized. The constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of the inositol dehydrogenase were used to construct a shuttle vector system for the fully functional expression of mDHs in the multi-deletion strain G. oxydans BP.9 that lacks its mDHs. This system was used to express each mDH of G. oxydans 621H, in order to individually characterize the substrates, they oxidize. From 55 tested compounds, the alcohol dehydrogenase oxidized 30 substrates and the polyol dehydrogenase 25. The substrate spectrum of alcohol dehydrogenase overlapped largely with the aldehyde dehydrogenase and partially with polyol dehydrogenase. Thus, we were able to resolve the overlapping substrate spectra of the main mDHs of G. oxydans 621H. The described approach could also be used for the expression and detailed characterization of substrates used by mDHs from other acetic acid bacteria or a metagenome.

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References

  • Adachi O, Moonmangmee D, Toyama H, Yamada M, Shinagawa E, Matsushita K (2003) New developments in oxidative fermentation. Appl Microbiol Biotechnol 60(6):643–653

    Article  CAS  PubMed  Google Scholar 

  • Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41(3):459–72

  • Condon C, FitzGerald RJ, O'Gara F (1991) Conjugation and heterologous gene expression in Gluconobacter oxydans ssp suboxydans. FEMS Microbiol Lett 80(2–3):173–177

    Article  CAS  Google Scholar 

  • De Muynck C, Pereira CS, Naessens M, Parmentier S, Soetaert W, Vandamme EJ (2007) The genus Gluconobacter oxydans: comprehensive overview of biochemistry and biotechnological applications. Crit Rev Biotechnol 27(3):147–171

    Article  CAS  PubMed  Google Scholar 

  • Deppenmeier U, Ehrenreich A (2009) Physiology of acetic acid bacteria in light of the genome sequence of Gluconobacter oxydans. J Mol Microbiol Biotechnol 16(1–2):69–80

    Article  CAS  PubMed  Google Scholar 

  • Deppenmeier U, Hoffmeister M, Prust C (2002) Biochemistry and biotechnological applications of Gluconobacter strains. Appl Microbiol Biotechnol 60(3):233–242

    Article  CAS  PubMed  Google Scholar 

  • Fukaya M, Okumura H, Masai H, Uozumi T, Beppu T (1985) Development of a host-vector system for Gluconobacter-Suboxydans. Agric Biol Chem 49(8):2407–2411

    CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Holscher T, Weinert-Sepalage D, Gorisch H (2007) Identification of membrane-bound quinoprotein inositol dehydrogenase in Gluconobacter oxydans ATCC 621H. Microbiology 153(Pt 2):499–506

    Article  PubMed  Google Scholar 

  • Kallnik V, Meyer M, Deppenmeier U, Schweiger P (2010) Construction of expression vectors for protein production in Gluconobacter oxydans. J Biotechnol 150(4):460–465

    Article  CAS  PubMed  Google Scholar 

  • Kawai S, Goda-Tsutsumi M, Yakushi T, Kano K, Matsushita K (2013) Heterologous overexpression and characterization of a flavoprotein-cytochrome c complex fructose dehydrogenase of Gluconobacter japonicus NBRC3260. Appl Environ Microbiol 79(5):1654–1660

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kostner D, Peters B, Mientus M, Liebl W, Ehrenreich A (2013) Importance of codB for new codA-based markerless gene deletion in Gluconobacter strains. Appl Microbiol Biotechnol 97(18):8341–8349

    Article  CAS  PubMed  Google Scholar 

  • Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop II RM, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176

  • Merfort M, Herrmann U, Bringer-Meyer S, Sahm H (2006a) 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(2):443–451

    Article  CAS  PubMed  Google Scholar 

  • Merfort M, Herrmann U, Ha SW, Elfari M, Bringer-Meyer S, Gorisch H, Sahm H (2006b) Modification of the membrane-bound glucose oxidation system in Gluconobacter oxydans significantly increases gluconate and 5-keto-d-gluconic acid accumulation. Biotechnol J 1(5):556–563

    Article  CAS  PubMed  Google Scholar 

  • Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 352–355

    Google Scholar 

  • Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260(3):289–298

    Article  CAS  PubMed  Google Scholar 

  • Okumura H, Uozumi T, Beppu T (1985) Construction of a plasmid vector and genetic transformation system for Acetobacter aceti. Biol Chem 49:1011–1017

    CAS  Google Scholar 

  • Peters B, Junker A, Brauer K, Muhlthaler B, Kostner D, Mientus M, Liebl W, Ehrenreich A (2013a) Deletion of pyruvate decarboxylase by a new method for efficient markerless gene deletions in Gluconobacter oxydans. Appl Microbiol Biotechnol 97(6):2521–2530

    Article  CAS  PubMed  Google Scholar 

  • Peters B, Mientus M, Kostner D, Junker A, Liebl W, Ehrenreich A (2013b) Characterization of membrane-bound dehydrogenases from Gluconobacter oxydans 621H via whole-cell activity assays using multideletion strains. Appl Microbiol Biotechnol 97(14):6397–6412

    Article  CAS  PubMed  Google Scholar 

  • 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(2):195–200

    Article  CAS  PubMed  Google Scholar 

  • Saida F (2007) Overview on the expression of toxic gene products in Escherichia coli. Curr Protoc Protein Sci 5(5):19

    PubMed  Google Scholar 

  • Saito Y, Ishii Y, Hayashi H, Imao Y, Akashi T, Yoshikawa K, Noguchi Y, Soeda S, Yoshida M, Niwa M, Hosoda J, Shimomura K (1997) Cloning of genes coding for l-sorbose and l-sorbosone dehydrogenases 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(2):454–460

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Schleyer U, Bringer-Meyer S, Sahm H (2008) An easy cloning and expression vector system for Gluconobacter oxydans. Int J Food Microbiol 125(1):91–95

    Article  CAS  PubMed  Google Scholar 

  • Schweiger P, Gross H, Deppenmeier U (2010) Characterization of two aldo-keto reductases from Gluconobacter oxydans 621H capable of regio- and stereoselective alpha-ketocarbonyl reduction. Appl Microbiol Biotechnol 87(4):1415–1426

    Article  CAS  PubMed  Google Scholar 

  • Schweiger P, Gross H, Zeiser J, Deppenmeier U (2013) Asymmetric reduction of diketones by two Gluconobacter oxydans oxidoreductases. Appl Microbiol Biotechnol 97(8):3475–3484

    Article  CAS  PubMed  Google Scholar 

  • Schweiger P, Volland S, Deppenmeier U (2007) Overproduction and characterization of two distinct aldehyde-oxidizing enzymes from Gluconobacter oxydans 621H. J Mol Microbiol Biotechnol 13(1–3):147–155

    Article  CAS  PubMed  Google Scholar 

  • Shinagawa E, Toyama H, Matsushita K, Tuitemwong P, Theeragool G, Adachi O (2006) A novel type of formaldehyde-oxidizing enzyme from the membrane of Acetobacter sp. SKU 14. Biosci Biotechnol Biochem 70(4):850–857

    Article  CAS  PubMed  Google Scholar 

  • Shinjoh M, Hoshino T (1995) Development of a stable shuttle vector and a conjugative transfer system for Gluconobacter oxydans. J Ferment Bioeng 79(2):95–99

    Article  CAS  Google Scholar 

  • Takemura H, Kondo K, Horinouchi S, Beppu T (1993) Induction by ethanol of alcohol dehydrogenase activity in Acetobacter pasteurianus. J Bacteriol 175(21):6857–6866

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tillett D, Neilan BA (1999) Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites. Nucleic Acids Res 27(19):e26

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tonouchi N, Sugiyama M, Yokozeki K (2003) Construction of a vector plasmid for use in Gluconobacter oxydans. Biosci Biotechnol Biochem 67(1):211–213

    Article  CAS  PubMed  Google Scholar 

  • Trcek J, Raspor P, Teuber M (2000) Molecular identification of Acetobacter isolates from submerged vinegar production, sequence analysis of plasmid pJK2-1 and application in the development of a cloning vector. Appl Microbiol Biotechnol 53(3):289–295

    Article  CAS  PubMed  Google Scholar 

  • Voss J, Ehrenreich A, Liebl W (2010) Characterization and inactivation of the membrane-bound polyol dehydrogenase in Gluconobacter oxydans DSM 7145 reveals a role in meso-erythritol oxidation. Microbiology 156(Pt 6):1890–1899

    Article  CAS  PubMed  Google Scholar 

  • Wach A (1996) PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12(3):259–265

    Article  CAS  PubMed  Google Scholar 

  • Zhang L, Lin J, Ma Y, Wei D, Sun M (2010) Construction of a novel shuttle vector for use in Gluconobacter oxydans. Mol Biotechnol 46(3):227–233

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Lehrstuhl für Mikrobiologie Technische Universität München, Emil-Ramann-Str. 4, 85354, Freising, Germany

    Markus Mientus, David Kostner, Björn Peters, Wolfgang Liebl & Armin Ehrenreich

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  1. Markus Mientus
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  2. David Kostner
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  3. Björn Peters
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  4. Wolfgang Liebl
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  5. Armin Ehrenreich
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Correspondence to Armin Ehrenreich.

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Funding

This study was funded by the Bavarian State Ministry of the Environment and Consumer Protection via the project association “BayBiotech” (http://www.baybiotech.de) (grant number TLK01U-69038).

Conflict of interest

Markus Mientus declares that he has no conflict of interest. David Kostner declares that he has no conflict of interest. Björn Peters declares that he has no conflict of interest. Wolfgang Liebl declares that he has no conflict of interest. Armin Ehrenreich declares that he has no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Mientus, M., Kostner, D., Peters, B. et al. Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression. Appl Microbiol Biotechnol 101, 3189–3200 (2017). https://doi.org/10.1007/s00253-016-8069-4

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  • DOI: https://doi.org/10.1007/s00253-016-8069-4

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