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Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor

Abstract

The thermostable NAD+-dependent alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH) was exploited with regard to the biocatalytic synthesis of ω-oxo lauric acid methyl ester (OLAMe), a key intermediate for biobased polyamide 12 production, from the corresponding long-chain alcohol. Recombinant BsADH was produced in Escherichia coli as a homogeneous tetrameric enzyme and showed high activity towards the industrially relevant substrate ω-hydroxy lauric acid methyl ester (HLAMe) with K M = 86 μM and 44 U mg−1. The equilibrium constant for HLAMe oxidation to the aldehyde (OLAMe) with NAD+ was determined as 2.16 × 10−3 from the kinetic parameters of the BsADH-catalyzed forward and reverse reactions. Since BsADH displayed limited stability under oxidizing conditions, the predominant oxidation-prone residue Cys257 was mutated to Leu based on sequence homology with related enzymes and computational simulation. This substitution resulted in an improved BsADH variant exhibiting prolonged stability and an elevated inactivation temperature. Semi-preparative biocatalysis at 60 °C using the stabilized enzyme, employing butyraldehyde for in situ cofactor regeneration with only catalytic amounts of NAD+, yielded up to 23 % conversion of HLAMe to OLAMe after 30 min. In contrast to other oxidoreductases, no overoxidation to the dodecanoic diacid monomethyl ester was detected. Thus, the mutated BsADH offers a promising biocatalyst for the selective oxidation of fatty alcohols to yield intermediates for industrial polymer production.

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References

  • Alberty RA (1953) The relationship between Michaelis constants, maximum velocities and the equilibrium constant for an enzyme-catalyzed reaction. J Am Chem Soc 75:1928–1932

    CAS  Article  Google Scholar 

  • Altschul SF, Gish W, Miller W, Meyers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    CAS  Article  PubMed  Google Scholar 

  • Auld DS, Bergman T (2008) Medium- and short-chain dehydrogenase/reductase gene and protein families. Cell Mol Life Sci 65:3961–3970

    CAS  Article  PubMed  Google Scholar 

  • Benson S, Shapiro J (1976) Plasmid-determined alcohol dehydrogenase activity in alkane-utilizing strains of Pseudomonas putida. J Bacteriol 126:794–798

    PubMed Central  CAS  PubMed  Google Scholar 

  • Bommarius AS, Paye MF (2013) Stabilizing biocatalysts. Chem Soc Rev 42:6534–6565

    CAS  Article  PubMed  Google Scholar 

  • Cannio R, Rossi M, Bartolucci S (1994) A few amino acid substitutions are responsible for the higher thermostability of a novel NAD+-dependent bacillar alcohol dehydrogenase. Eur J Biochem 222:345–352

    CAS  Article  PubMed  Google Scholar 

  • Cassimjee KE, Branneby C, Abedi V, Wells A, Berglund P (2010) Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration. Chem Commun 46:5569–5571

    CAS  Article  Google Scholar 

  • Ceccarelli C, Liang Z-X, Strickler M, Prehna G, Goldstein BM, Klinman JP, Bahnson BJ (2004) Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: insight into thermostability and cofactor binding. Biochemistry 43:5266–5277

    CAS  Article  PubMed  Google Scholar 

  • Chao C-C, Ma Y-S, Stadtman ER (1997) Modification of protein surface hydrophobicity and methionine oxidation by oxidative systems. Proc Natl Acad Sci U S A 94:2969–2974

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA

    Google Scholar 

  • Fiorentino G, Cannio R, Rossi M, Bartolucci S (1998) Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements. Protein Eng 11:925–930

    CAS  Article  PubMed  Google Scholar 

  • Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Geueke B, Riebel B, Hummel W (2003) NADH oxidase from Lactobacillus brevis: a new catalyst for the regeneration of NAD. Enzyme Microb Tech 32:205–211

    CAS  Article  Google Scholar 

  • Grant C, Woodley JM, Baganz F (2011) Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli. Enzym Microb Tech 48:480–486

    CAS  Article  Google Scholar 

  • Grant C, da Silva Damas Pinto AC, Lui H-P, Woodley JM, Baganz F (2012) Tools for characterizing the whole-cell bio-oxidation of alkanes at microscale. Biotechnol Bioeng 109:2179–2189

    CAS  Article  PubMed  Google Scholar 

  • Gröger H (2014) Hydroxy functionalization of non-activated C–H and C=C bonds: new perspectives for the synthesis of alcohols through biocatalytic processes. Angew Chem Int Ed Engl 53:3067–3069

    Article  PubMed  Google Scholar 

  • Guagliardi A, Matilde M, Ingram I, De Rosa M, Rossi M, Bartolucci S (1996) Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70°C. Int J Biochem Cell Biol 28:239–246

    CAS  Article  PubMed  Google Scholar 

  • Guterl JK, Garbe D, Carsten J, Steffler F, Sommer B, Reisse S, Philipp A, Haack M, Ruhmann B, Koltermann A, Kettling U, Bruck T, Sieber V (2012) Cell-free metabolic engineering: production of chemicals by minimized reaction cascades. ChemSusChem 5:2165–2172

    CAS  Article  PubMed  Google Scholar 

  • Hanekom AJ, Hofmeyr JHS, Snoep JL, Rohwer JM (2006) Experimental evidence for allosteric modifier saturation as predicted by the bi-substrate Hill equation. Syst Biol (Stevenage) 153:342–345

    CAS  Article  Google Scholar 

  • Henry CS, Broadbelt LJ, Hatzimanikatis V (2007) Thermodynamics-based metabolic flux analysis. Biophys J 92:1792–1805

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Kara S, Schrittwieser JH, Hollmann F, Ansorge-Schumacher MB (2014) Recent trends and novel concepts in cofactor-dependent biotransformations. Appl Microbiol Biotechnol 98:1517–1529

    CAS  Article  PubMed  Google Scholar 

  • Keinan E, Hafeli EK, Seth KK, Lamed R (1986) Thermostable enzymes in organic synthesis. 2. Asymmetric reduction of ketones with alcohol dehydrogenase from Thermoanaerobium brockii. J Am Chem Soc 108:162–169

    CAS  Article  Google Scholar 

  • Kirmair L, Skerra A (2014) Biochemical analysis of recombinant AlkJ from Pseudomonas putida reveals a membrane-associated, flavin adenine dinucleotide-dependent dehydrogenase suitable for the biosynthetic production of aliphatic aldehydes. Appl Environ Microbiol 80:2468–2477

    PubMed Central  Article  PubMed  Google Scholar 

  • Koch DJ, Chen MM, van Beilen JB, Arnold FH (2009) In vivo evolution of butane oxidation by terminal alkane hydroxylases AlkB and CYP153A6. Appl Environ Microbiol 75:337–344

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Lerchner A, Achatz S, Rausch C, Haas T, Skerra A (2013) Coupled enzymatic alcohol-to-amine conversion of isosorbide using engineered transaminases and dehydrogenases. ChemCatChem 5:3374–3383

    CAS  Article  Google Scholar 

  • Li S, Schöneich C, Borchardt RT (1995) Chemical instability of protein pharmaceuticals: mechanisms of oxidation and strategies for stabilization. Biotechnol Bioeng 48:490–500

    CAS  Article  PubMed  Google Scholar 

  • Littlechild JA, Guy MN, Isupov MN (2004) Hyperthermophilic dehydrogenase enzymes. Biochem Soc Trans 32:255–258

    CAS  Article  PubMed  Google Scholar 

  • Marshall SA, Lazar GA, Chirino AJ, Desjarlais JR (2003) Rational design and engineering of therapeutic proteins. Drug Discov Today 8:212–221

    CAS  Article  PubMed  Google Scholar 

  • May SW, Katopodis AG (1986) Oxygenation of alcohol and sulphide substrates by a prototypical non-haem iron monooxygenase: catalysis and biotechnological potential. Enzym Microb Tech 8:17–21

    CAS  Article  Google Scholar 

  • Metzger JO, Bornscheuer U (2006) Lipids as renewable resources: current state of chemical and biotechnological conversion and diversification. Appl Microbiol Biotechnol 71:13–22

    CAS  Article  PubMed  Google Scholar 

  • Olaofe O, Fenner C, Gudiminchi RK, Smit M, Harrison S (2013) The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6. Microb Cell Factories 12:8

    CAS  Article  Google Scholar 

  • Opperman DJ, Reetz MT (2010) Towards practical Baeyer-Villiger-monooxygenases: design of cyclohexanone monooxygenase mutants with enhanced oxidative stability. ChemBioChem 11:2589–2596

    CAS  Article  PubMed  Google Scholar 

  • Otte KB, Kirtz M, Nestl BM, Hauer B (2013) Synthesis of 9-oxononanoic acid, a precursor for biopolymers. ChemSusChem 6:2149–2156

    CAS  Article  PubMed  Google Scholar 

  • Pennacchio A, Giordano A, Rossi M, Raia CA (2011) Asymmetric reduction of α-keto esters with Thermus thermophilus NADH-dependent carbonyl reductase using glucose dehydrogenase and alcohol dehydrogenase for cofactor regeneration. Eur J Org Chem 2011:4361–4366

    CAS  Article  Google Scholar 

  • Pennacchio A, Rossi M, Raia CA (2013) Synthesis of cinnamyl alcohol from cinnamaldehyde with Bacillus stearothermophilus alcohol dehydrogenase as the isolated enzyme and in recombinant E. coli cells. Appl Biochem Biotechnol 170:1482–1490

    CAS  Article  PubMed  Google Scholar 

  • Peretz M, Weiner LM, Burstein Y (1997) Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase. Protein Sci 6:1074–1083

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Perry LJ, Wetzel R (1987) The role of cysteine oxidation in the thermal inactivation of T4 lysozyme. Protein Eng 1:101–105

    CAS  Article  PubMed  Google Scholar 

  • Sambrook J and Russell DW (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

  • Sattler JH, Fuchs M, Tauber K, Mutti FG, Faber K, Pfeffer J, Haas T, Kroutil W (2012) Redox self-sufficient biocatalyst network for the amination of primary alcohols. Angew Chem Int Ed 51:9156–9159

    CAS  Article  Google Scholar 

  • Schaffer S, Haas T (2014) Biocatalytic and fermentative production of α,ω-bifunctional polymer precursors. Org Process Res Dev 18:752–766

    CAS  Article  Google Scholar 

  • Scheps D, Malca SH, Richter SM, Marisch K, Nestl BM, Hauer B (2013) Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct. Microb Biotechnol 6:694–707

    PubMed Central  CAS  PubMed  Google Scholar 

  • Schmidt TG, Skerra A (2007) The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc 2:1528–1535

    CAS  Article  PubMed  Google Scholar 

  • Schmitt J, Hess H, Stunnenberg HG (1993) Affinity purification of histidine-tagged proteins. Mol Biol Rep 18:223–230

    CAS  Article  PubMed  Google Scholar 

  • Schrewe M, Magnusson AO, Willrodt C, Bühler B, Schmid A (2011) Kinetic analysis of terminal and unactivated C-H bond oxyfunctionalization in fatty acid methyl esters by monooxygenase-based whole-cell biocatalysis. Adv Synth Catal 353:3485–3495

    CAS  Article  Google Scholar 

  • Schrewe M, Ladkau N, Bühler B, Schmid A (2013) Direct terminal alkylamino-functionalization via multistep biocatalysis in one recombinant whole-cell catalyst. Adv Synth Catal 355:1693–1697

    CAS  Article  Google Scholar 

  • Schrewe M, Julsing MK, Lange K, Czarnotta E, Schmid A, Bühler B (2014) Reaction and catalyst engineering to exploit kinetically controlled whole-cell multistep biocatalysis for terminal FAME oxyfunctionalization. Biotechnol Bioeng 111:1820–1830

    CAS  Article  PubMed  Google Scholar 

  • Shim E-J, Sang-Hoon J, Kwang-Hoon K (2003) Overexpression, purification and biochemical characterization of the thermostable NAD-dependent alcohol dehydrogenase from Bacillus stearo- thermophilus. J Microbiol Biotechnol 13:738–744

  • Skerra A (1994) Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151:131–135

    CAS  Article  PubMed  Google Scholar 

  • Slavica A, Dib I, Nidetzky B (2005) Single-site oxidation, cysteine 108 to cysteine sulfinic acid, in d-amino acid oxidase from Trigonopsis variabilis and its structural and functional consequences. Appl Environ Microbiol 71:8061–8068

  • Song J-W, Lee J-H, Bornscheuer UT, Park J-B (2014) Microbial synthesis of medium-chain α,ω-dicarboxylic acids and ω-aminocarboxylic acids from renewable long-chain fatty acids. Adv Synth Catal 356:1782–1788

    CAS  Article  Google Scholar 

  • Tiwari A, Bhat R (2006) Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions. Biophys Chem 124:90–99

    CAS  Article  PubMed  Google Scholar 

  • Tokuriki N, Stricher F, Serrano L and Tawfik DS (2008) How protein stability and new functions trade off. PLoS Comput Biol 4:e1000002.

  • Tufvesson P, Lima-Ramos J, Nordblad M, Woodley JM (2010) Guidelines and cost analysis for catalyst production in biocatalytic processes. Org Process Res Dev 15:266–274

    Article  Google Scholar 

  • Vagenende V, Yap MGS, Trout BL (2009) Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry 48:11084–11096

    CAS  Article  PubMed  Google Scholar 

  • van Beilen JB, Duetz WA, Schmid A, Witholt B (2003) Practical issues in the application of oxygenases. Trends Biotechnol 21:170–177

    Article  PubMed  Google Scholar 

  • van der Donk WA, Zhao H (2003) Recent developments in pyridine nucleotide regeneration. Curr Opin Biotechnol 14:421–426

    Article  PubMed  Google Scholar 

  • Vogt W (1995) Oxidation of methionyl residues in proteins: tools, targets, and reversal. Free Radic Biol Med 18:93–105

    CAS  Article  PubMed  Google Scholar 

  • Wu H, Tian C, Song X, Liu C, Yang D, Jiang Z (2013) Methods for the regeneration of nicotinamide coenzymes. Green Chem 1773-1789

  • Xia X, Longo LM, Blaber M (2015) Mutation choice to eliminate buried free cysteines in protein therapeutics. J Pharm Sci 104:566–576

    CAS  Article  PubMed  Google Scholar 

  • Xie M, Alonso H, Roujeinikova A (2011) An improved procedure for the purification of catalytically active alkane hydroxylase from Pseudomonas putida GPo1. Appl Biochem Biotechnol 165:823–831

    CAS  Article  PubMed  Google Scholar 

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Acknowledgments

The authors are grateful to Evonik Industries AG, Marl, Germany, for materials and equipment. L.K. wishes to thank Stefan Achatz for help with automated liquid handling, Irmgard Neumaier for technical assistance, Andreas Reichert for ESI-MS measurements, and Dr. Lukas Eisoldt for discussions.

Funding

This work was financially supported through the German Bundesministerium für Bildung und Forschung in frame of the project “BISON” (Grant No. 0316044) as well as the Federal State of Bavaria and the Deutsche Forschungsgemeinschaft by providing the robotic screening platform in frame of their Major Research Instrumentation Programme (Grant No. INST 95/1031-1).

Conflict of interest

The authors declare that they have no competing interests.

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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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Correspondence to Arne Skerra.

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Kirmair, L., Seiler, D.L. & Skerra, A. Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor. Appl Microbiol Biotechnol 99, 10501–10513 (2015). https://doi.org/10.1007/s00253-015-6930-5

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  • DOI: https://doi.org/10.1007/s00253-015-6930-5

Keywords

  • Biobased polymer
  • Biocatalysis
  • BsADH
  • Cofactor recycling
  • Enzyme engineering
  • Protein thermostability