Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Growth Conditions
2.2. Plasmid Design and Construction
2.3. Deepwell Plate Cultivation
2.4. Shake-Flask Cultivation
2.5. Purification of Strep-Tagged Hox Proteins
2.6. Western Blot Analysis
2.7. Spectroscopic Analysis and Activity Assays of RH
3. Results
3.1. Construction of Expression Vectors for Heterologous Hydrogenase Production in E. coli
3.2. Screening of Inducer Concentrations for HoxBC Overproduction in E. coli
3.3. Low Inducer Concentrations and Low Temperatures Support Production of Soluble RH
3.4. Optimization of Expression Time
3.5. Fed-Batch Like Cultivation for Heterologous RH Production in E. coli
3.6. Upscalable RH Production in Shake Flask in Batch and Fed-Batch Cultivations
3.7. Biochemical and Spectroscopic Characterization of the Recombinant HoxBC and HoxC Proteins
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
- Greening, C.; Biswas, A.; Carere, C.R.; Jackson, C.J.; Taylor, M.C.; Stott, M.B.; Cook, G.M.; Morales, S.E. Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival. ISME J. 2016, 10, 761–777. [Google Scholar] [CrossRef] [Green Version]
- Fritsch, J.; Lenz, O.; Friedrich, B. Structure, function and biosynthesis of O2-tolerant hydrogenases. Nat. Rev. Microbiol. 2013, 11, 106–114. [Google Scholar] [CrossRef]
- Friedrich, B.; Fritsch, J.; Lenz, O. Oxygen-tolerant hydrogenases in hydrogen-based technologies. Curr. Opin. Biotechnol. 2011, 22, 358–364. [Google Scholar] [CrossRef] [PubMed]
- Preissler, J.; Reeve, H.A.; Zhu, T.; Nicholson, J.; Urata, K.; Lauterbach, L.; Wong, L.L.; Vincent, K.A.; Lenz, O. Dihydrogen-Driven NADPH Recycling in Imine Reduction and P450-Catalyzed Oxidations Mediated by an Engineered O2-Tolerant Hydrogenase. ChemCatChem 2020, 12, 4853–4861. [Google Scholar] [CrossRef]
- Lu, Y.; Koo, J. O2 sensitivity and H2 production activity of hydrogenases—A review. Biotechnol. Bioeng. 2019, 116, 3124–3135. [Google Scholar] [CrossRef]
- Vignais, P.M.; Billoud, B. Occurrence, Classification, and Biological Function of Hydrogenases: An Overview. Chem. Rev. 2007, 107, 4206–4272. [Google Scholar] [CrossRef]
- Lubitz, W.; Ogata, H.; Rüdiger, O.; Reijerse, E. Hydrogenases. Chem. Rev. 2014, 114, 4081–4148. [Google Scholar] [CrossRef]
- Peters, J.W.; Schut, G.J.; Boyd, E.S.; Mulder, D.W.; Shepard, E.M.; Broderick, J.B.; King, P.W.; Adams, M.W.W. [FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation. Biochim. Biophys. Acta-Mol. Cell Res. 2015, 1853, 1350–1369. [Google Scholar] [CrossRef] [Green Version]
- Lacasse, M.J.; Zamble, D.B. [NiFe]-hydrogenase maturation. In Biochemistry; American Chemical Society: Washington, DC, USA, 2016; pp. 1689–1701. [Google Scholar]
- Lenz, O.; Lauterbach, L.; Frielingsdorf, S.; Friedrich, B. Oxygen-tolerant hydrogenases and their biotechnological potential. In Biohydrogen; De Gruyter: Berlin, Germany; München, Germany; Boston, MA, USA, 2015; pp. 61–96. [Google Scholar]
- Lenz, O.; Friedrich, B. A novel multicomponent regulatory system mediates H2 sensing in Alcaligenes eutrophus. Proc. Natl. Acad. Sci. USA 1998, 95, 12474–12479. [Google Scholar] [CrossRef] [Green Version]
- Brecht, M.; van Gastel, M.; Buhrke, T.; Friedrich, B.; Lubitz, W. Direct detection of a hydrogen ligand in the [NiFe] center of the regulatory H2-sensing hydrogenase from Ralstonia e utropha in its reduced state by HYSCORE and ENDOR spectroscopy. J. Am. Chem. Soc. 2003, 125, 13075–13083. [Google Scholar] [CrossRef]
- Buhrke, T.; Brecht, M.; Lubitz, W.; Friedrich, B. The H2-sensor of Ralstonia eutropha: Biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site. JBIC J. Biol. Inorg. Chem. 2002, 7, 897–908. [Google Scholar] [CrossRef]
- Caserta, G.; Lorent, C.; Ciaccafava, A.; Keck, M.; Breglia, R.; Greco, C.; Limberg, C.; Hildebrandt, P.; Cramer, S.P.; Zebger, I.; et al. The large subunit of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha—A minimal hydrogenase? Chem. Sci. 2020, 11, 5453–5465. [Google Scholar] [CrossRef]
- Ash, P.A.; Liu, J.; Coutard, N.; Heidary, N.; Horch, M.; Gudim, I.; Simler, T.; Zebger, I.; Lenz, O.; Vincent, K.A. Electrochemical and infrared spectroscopic studies provide insight into reactions of the NiFe regulatory hydrogenase from Ralstonia eutropha with O2 and CO. J. Phys. Chem. B 2015, 119, 13807–13815. [Google Scholar] [CrossRef]
- Roncaroli, F.; Bill, E.; Friedrich, B.; Lenz, O.; Lubitz, W.; Pandelia, M.-E. Cofactor composition and function of a H2-sensing regulatory hydrogenase as revealed by Mössbauer and EPR spectroscopy. Chem. Sci. 2015, 6, 4495–4507. [Google Scholar] [CrossRef] [Green Version]
- Horch, M.; Schoknecht, J.; Mroginski, M.A.; Lenz, O.; Hildebrandt, P.; Zebger, I. Resonance Raman Spectroscopy on [NiFe] hydrogenase provides structural insights into catalytic intermediates and reactions. J. Am. Chem. Soc. 2014, 136, 9870–9873. [Google Scholar] [CrossRef]
- Caserta, G.; Pelmenschikov, V.; Lorent, C.; Tadjoung Waffo, A.F.; Katz, S.; Lauterbach, L.; Schoknecht, J.; Wang, H.; Yoda, Y.; Tamasaku, K.; et al. Hydroxy-bridged resting states of a [NiFe]-hydrogenase unraveled by cryogenic vibrational spectroscopy and DFT computations. Chem. Sci. 2021, 12, 2189–2197. [Google Scholar] [CrossRef]
- Bernhard, M.; Buhrke, T.; Bleijlevens, B.; De Lacey, A.L.; Fernandez, V.M.; Albracht, S.P.J.; Friedrich, B. The H2 Sensor of Ralstonia eutropha. J. Biol. Chem. 2001, 276, 15592–15597. [Google Scholar] [CrossRef] [Green Version]
- Buhrke, T.; Löscher, S.; Lenz, O.; Schlodder, E.; Zebger, I.; Andersen, L.K.; Hildebrandt, P.; Meyer-Klaucke, W.; Dau, H.; Friedrich, B.; et al. Reduction of unusual iron-sulfur clusters in the H2-sensing regulatory Ni-Fe hydrogenase from Ralstonia eutropha H16. J. Biol. Chem. 2005, 280, 19488–19495. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lenz, O.; Zebger, I.; Hamann, J.; Hildebrandt, P.; Friedrich, B. Carbamoylphosphate serves as the source of CN−, but not of the intrinsic CO in the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha. FEBS Lett. 2007, 581, 3322–3326. [Google Scholar] [CrossRef] [Green Version]
- Fan, Q.; Neubauer, P.; Lenz, O.; Gimpel, M. Heterologous hydrogenase overproduction systems for biotechnology—An overview. Int. J. Mol. Sci. 2020, 21, 5890. [Google Scholar] [CrossRef]
- Kleihues, L.; Lenz, O.; Bernhard, M.; Buhrke, T.; Friedrich, B. The H2 sensor of Ralstonia eutropha is a member of the subclass of regulatory [NiFe] hydrogenases. J. Bacteriol. 2000, 182, 2716–2724. [Google Scholar] [CrossRef] [Green Version]
- Winter, G.; Buhrke, T.; Lenz, O.; Jones, A.K.; Forgber, M.; Friedrich, B. A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit. FEBS Lett. 2005, 579, 4292–4296. [Google Scholar] [CrossRef] [Green Version]
- Baer, R.; Bankier, A.T.; Biggin, M.D.; Deininger, P.L.; Farrell, P.J.; Gibson, T.J.; Hatfull, G.; Hudson, G.S.; Satchwell, S.C.; Séguin, C.; et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 1984, 310, 207–211. [Google Scholar] [CrossRef] [PubMed]
- Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: New York, NY, USA, 1989. [Google Scholar]
- Neubauer, P.; Šiurkus, J.; Panula-Perälä, J.; Neubauer, A.; Ukkonen, K.; Krause, M.; Meyer, D.; Pelzer, S.; Eck, J.; Tegel, H.; et al. EnBase™–MTP based high-cell-density fermentation for high-throughput and high-content screening. New Biotechnol. 2009, 25, S161. [Google Scholar] [CrossRef]
- Kraft, M.; Knüpfer, U.; Wenderoth, R.; Kacholdt, A.; Pietschmann, P.; Hock, B.; Horn, U. A dual expression platform to optimize the soluble production of heterologous proteins in the periplasm of Escherichia coli. Appl. Microbiol. Biotechnol. 2007, 76, 1413–1422. [Google Scholar] [CrossRef]
- Preis, H.; Eckart, R.A.; Gudipati, R.K.; Heidrich, N.; Brantl, S. CodY activates transcription of a small RNA in Bacillus subtilis. J. Bacteriol. 2009, 191, 5446–5457. [Google Scholar] [CrossRef] [Green Version]
- Kang, D.; Gho, Y.S.; Suh, M.; Kang, C. Highly sensitive and fast protein detection with Coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis [5]. Bull. Korean Chem. Soc. 2002, 23, 1511–1512. [Google Scholar]
- Gimpel, M.; Heidrich, N.; Mäder, U.; Krügel, H.; Brantl, S. A dual-function sRNA from B. subtilis: SR1 acts as a peptide encoding mRNA on the gapA operon. Mol. Microbiol. 2010, 76, 990–1009. [Google Scholar] [CrossRef] [Green Version]
- Lenz, O.; Lauterbach, L.; Frielingsdorf, S. O2-tolerant [NiFe]-hydrogenases of Ralstonia eutropha H16: Physiology, molecular biology, purification, and biochemical analysis. In Methods in Enzymology; Elsevier Inc.: Amsterdam, The Netherlands, 2018; Volume 613, pp. 117–151. [Google Scholar]
- Buhrke, T.; Lenz, O.; Porthun, A.; Friedrich, B. The H2-sensing complex of Ralstonia eutropha: Interaction between a regulatory [NiFe] hydrogenase and a histidine protein kinase. Mol. Microbiol. 2004, 51, 1677–1689. [Google Scholar] [CrossRef] [Green Version]
- Caserta, G.; Lorent, C.; Pelmenschikov, V.; Schoknecht, J.; Yoda, Y.; Hildebrandt, P.; Cramer, S.P.; Zebger, I.; Lenz, O. In Vitro Assembly as a Tool to Investigate Catalytic Intermediates of [NiFe]-Hydrogenase. ACS Catal. 2020, 10, 13890–13894. [Google Scholar] [CrossRef]
- Grodberg, J.; Dunn, J.J. OmpT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J. Bacteriol. 1988, 170, 1245–1253. [Google Scholar] [CrossRef] [Green Version]
- Gottesman, S. Proteases and their targents in Escherichia coli. Annu. Rev. Genet. 1996, 30, 465–506. [Google Scholar] [CrossRef]
- Catherine, H. Schein; Mathieu, H.M. Noteborn. Formation of soluble recombinant proteins in Escherichia Coli is favored by lower growth temperature. Nat. Biotechnol. 1988, 6, 291–294. [Google Scholar]
- Carrió, M.M.; Villaverde, A. Localization of chaperones DnaK and GroEL in bacterial inclusion bodies. J. Bacteriol. 2005, 187, 3599–3601. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Vasina, J.A.; Baneyx, F. Recombinant protein expression at low temperatures under the transcriptional control of the major Eschenchia coli cold shock promoter cspA. Appl. Environ. Microbiol. 1996, 62, 1444–1447. [Google Scholar] [CrossRef] [Green Version]
- Yamanaka, K. Cold shock response in Escherichia coli. J. Mol. Microbiol. Biotechnol. 1999, 1, 193–202. [Google Scholar]
- Warnecke, T.; Gill, R.T. Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications. Microb. Cell Factories 2005, 4, 25. [Google Scholar] [CrossRef] [Green Version]
- Sezonov, G.; Joseleau-Petit, D.; D’Ari, R. Escherichia coli physiology in Luria-Bertani broth. J. Bacteriol. 2007, 189, 8746–8749. [Google Scholar] [CrossRef] [Green Version]
- Losen, M.; Frölich, B.; Pohl, M.; Büchs, J. Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures. Biotechnol. Prog. 2004, 20, 1062–1068. [Google Scholar] [CrossRef]
- Panula-Perälä, J.; Šiurkus, J.; Vasala, A.; Wilmanowski, R.; Casteleijn, M.G.; Neubauer, P. Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks. Microb. Cell Factories 2008, 7, 31. [Google Scholar] [CrossRef] [Green Version]
- Krause, M.; Ukkonen, K.; Haataja, T.; Ruottinen, M.; Glumoff, T.; Neubauer, A.; Neubauer, P.; Vasala, A. A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb. Cell Fact. 2010, 9, 11. [Google Scholar] [CrossRef] [Green Version]
- Ongey, E.L.; Giessmann, R.T.; Fons, M.; Rappsilber, J.; Adrian, L.; Neubauer, P. Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli. Front. Microbiol. 2018, 9, 1688. [Google Scholar] [CrossRef]
- Glazyrina, J.; Materne, E.; Hillig, F.; Neubauer, P.; Junne, S. Two-compartment method for determination of the oxygen transfer rate with electrochemical sensors based on sulfite oxidation. Biotechnol. J. 2011, 6, 1003–1008. [Google Scholar] [CrossRef]
- Strandberg, L.; Enfors, S.O. Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli. Appl. Environ. Microbiol. 1991, 57, 1669–1674. [Google Scholar] [CrossRef] [Green Version]
- Baneyx, F.; Mujacic, M. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 2004, 22, 1399–1407. [Google Scholar] [CrossRef]
- Guisbert, E.; Yura, T.; Rhodius, V.A.; Gross, C.A. Convergence of Molecular, Modeling, and Systems Approaches for an Understanding of the Escherichia coli Heat Shock Response. Microbiol. Mol. Biol. Rev. 2008, 72, 545–554. [Google Scholar] [CrossRef] [Green Version]
- Evans, C.R.; Fan, Y.; Ling, J. Increased mistranslation protects E. coli from protein misfolding stress due to activation of a RpoS-dependent heat shock response. FEBS Lett. 2019, 593, 3220–3227. [Google Scholar] [CrossRef] [Green Version]
- Chesshyre, J.A.; Hipkiss, A.R. Low temperatures stabilize interferon a-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli. Appl. Microbiol. Biotechnol. 1989, 31, 158–162. [Google Scholar] [CrossRef]
- Neubauer, P.; Fahnert, B.; Lilie, H.; Villaverde, A. Protein inclusion bodies in recombinant bacteria. In Inclusions in Prokaryotes; Springer: Berlin/Heidelberg, Germany, 2006; pp. 237–292. [Google Scholar]
- Messenger, S.L.; Green, J. FNR-mediated regulation of hyp expression in Escherichia coli. FEMS Microbiol. Lett. 2003, 228, 81–86. [Google Scholar] [CrossRef] [Green Version]
- Eitinger, T.; Mandrand-Berthelot, M.A. Nickel transport systems in microorganisms. Arch. Microbiol. 2000, 173, 1–9. [Google Scholar] [CrossRef]
- Unden, G.; Achebach, S.; Holighaus, G.; Tran, H.Q.; Wackwitz, B.; Zeuner, Y. Control of FNR function of Escherichia coli by O2 and reducing conditions. J. Mol. Microbiol. Biotechnol. 2002, 4, 263–268. [Google Scholar]
- Pinske, C.; Bönn, M.; Krüger, S.; Lindenstrauß, U.; Sawers, R.G. Metabolic deficiences revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3). PLoS ONE 2011, 6, e22830. [Google Scholar] [CrossRef] [Green Version]
- Hartmann, S.; Frielingsdorf, S.; Caserta, G.; Lenz, O. A membrane-bound [NiFe]-hydrogenase large subunit precursor whose C-terminal extension is not essential for cofactor incorporation but guarantees optimal maturation. Microbiologyopen 2020, 9, 1197–1206. [Google Scholar] [CrossRef]
- Theodoratou, E.; Paschos, A.; Mintz-Weber, S.; Böck, A. Analysis of the cleavage site specificity of the endopeptidase involved in the maturation of the large subunit of hydrogenase 3 from Escherichia coli. Arch. Microbiol. 2000, 173, 110–116. [Google Scholar] [CrossRef] [PubMed]
- Senger, M.; Stripp, S.T.; Soboh, B. Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis. J. Biol. Chem. 2017, 292, 11670–11681. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Thomas, C.; Muhr, E.; Sawers, R.G. Coordination of synthesis and assembly of a modular membrane-associated [NiFe]-hydrogenase is determined by cleavage of the C-terminal peptide. J. Bacteriol. 2015, 197, 2989–2998. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Buhrke, T.; Bleijlevens, B.; Albracht, S.P.J.; Friedrich, B. Involvement of hyp gene products in maturation of the H2-sensing [NiFe] hydrogenase of Ralstonia eutropha. J. Bacteriol. 2001, 183, 7087–7093. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Shima, S.; Chen, D.; Xu, T.; Wodrich, M.D.; Fujishiro, T.; Schultz, K.M.; Kahnt, J.; Ataka, K.; Hu, X. Reconstitution of [Fe]-hydrogenase using model complexes. Nat. Chem. 2015, 7, 995–1002. [Google Scholar] [CrossRef] [PubMed]
- Artero, V.; Berggren, G.; Atta, M.; Caserta, G.; Roy, S.; Pecqueur, L.; Fontecave, M. From enzyme maturation to synthetic chemistry: The case of hydrogenases. Acc. Chem. Res. 2015, 48, 2380–2387. [Google Scholar] [CrossRef] [PubMed]
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Fan, Q.; Caserta, G.; Lorent, C.; Lenz, O.; Neubauer, P.; Gimpel, M. Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli. Microorganisms 2021, 9, 1195. https://doi.org/10.3390/microorganisms9061195
Fan Q, Caserta G, Lorent C, Lenz O, Neubauer P, Gimpel M. Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli. Microorganisms. 2021; 9(6):1195. https://doi.org/10.3390/microorganisms9061195
Chicago/Turabian StyleFan, Qin, Giorgio Caserta, Christian Lorent, Oliver Lenz, Peter Neubauer, and Matthias Gimpel. 2021. "Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli" Microorganisms 9, no. 6: 1195. https://doi.org/10.3390/microorganisms9061195