Whole-cell double oxidation of n-heptane

Müller CA, Dennig A, Welters T, Winkler T, Ruff AJ, Hummel W, Gröger H, Schwaneberg U (2014)
Journal of biotechnology 191: 196-204.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
Es wurden keine Dateien hochgeladen. Nur Publikationsnachweis!
Müller, Christina A.; Dennig, Alexander; Welters, Tim; Winkler, Till; Ruff, Anna Joelle; Hummel, WernerUniBi; Gröger, HaraldUniBi; Schwaneberg, Ulrich
Abstract / Bemerkung
Biocascades allow one-pot synthesis of chemical building blocks omitting purification of reaction intermediates and expenses for downstream processing. Here we show the first whole cell double oxidation of n-heptane to produce chiral alcohols and heptanones. The concept of an artificial operon for co-expression of a monooxygenase from Bacillus megaterium (P450 BM3) and an alcohol dehydrogenase (RE-ADH) from Rhodococcus erythropolis is reported and compared to the widely used two-plasmid or Duet-vector expression systems. Both catalysts are co-expressed on a polycistronic constructs (single mRNA) that reduces recombinant DNA content and metabolic burden for the host cell, therefore increasing growth rate and expression level. Using the artificial operon system, the expression of P450 BM3 reached 81mgg(-1) cell dry weight. In addition, in situ cofactor regeneration through the P450 BM3/RE-ADH couple was enhanced by coupling to glucose oxidation by E. coli. Under optimized reaction conditions the artificial operon system displayed a product formation of 656mgL(-1) (5.7mM) of reaction products (heptanols+heptanones), which is 3-fold higher than the previously reported values for an in vitro oxidation cascade. In conjunction with the high product concentrations it was possible to obtain ee values of >99% for (S)-3-heptanol. Coexpression of a third alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) in the same host yielded complete oxidation of all heptanol isomers. Introduction of a second ADH enabled further to utilize both cofactors in the host cell (NADH and NADPH) which illustrates the simplicity and modular character of the whole cell oxidation concept employing an artificial operon system.
Journal of biotechnology
Page URI


Müller CA, Dennig A, Welters T, et al. Whole-cell double oxidation of n-heptane. Journal of biotechnology. 2014;191:196-204.
Müller, C. A., Dennig, A., Welters, T., Winkler, T., Ruff, A. J., Hummel, W., Gröger, H., et al. (2014). Whole-cell double oxidation of n-heptane. Journal of biotechnology, 191, 196-204. doi:10.1016/j.jbiotec.2014.06.001
Müller, Christina A., Dennig, Alexander, Welters, Tim, Winkler, Till, Ruff, Anna Joelle, Hummel, Werner, Gröger, Harald, and Schwaneberg, Ulrich. 2014. “Whole-cell double oxidation of n-heptane”. Journal of biotechnology 191: 196-204.
Müller, C. A., Dennig, A., Welters, T., Winkler, T., Ruff, A. J., Hummel, W., Gröger, H., and Schwaneberg, U. (2014). Whole-cell double oxidation of n-heptane. Journal of biotechnology 191, 196-204.
Müller, C.A., et al., 2014. Whole-cell double oxidation of n-heptane. Journal of biotechnology, 191, p 196-204.
C.A. Müller, et al., “Whole-cell double oxidation of n-heptane”, Journal of biotechnology, vol. 191, 2014, pp. 196-204.
Müller, C.A., Dennig, A., Welters, T., Winkler, T., Ruff, A.J., Hummel, W., Gröger, H., Schwaneberg, U.: Whole-cell double oxidation of n-heptane. Journal of biotechnology. 191, 196-204 (2014).
Müller, Christina A., Dennig, Alexander, Welters, Tim, Winkler, Till, Ruff, Anna Joelle, Hummel, Werner, Gröger, Harald, and Schwaneberg, Ulrich. “Whole-cell double oxidation of n-heptane”. Journal of biotechnology 191 (2014): 196-204.

6 Zitationen in Europe PMC

Daten bereitgestellt von Europe PubMed Central.

Biocatalytic Oxidation Reactions: A Chemist's Perspective.
Dong J, Fernández-Fueyo E, Hollmann F, Paul CE, Pesic M, Schmidt S, Wang Y, Younes S, Zhang W., Angew Chem Int Ed Engl 57(30), 2018
PMID: 29573076
A novel cytochrome P450 mono-oxygenase from Streptomyces platensis resembles activities of human drug metabolizing P450s.
Worsch A, Eggimann FK, Girhard M, von Bühler CJ, Tieves F, Czaja R, Vogel A, Grumaz C, Sohn K, Lütz S, Kittelmann M, Urlacher VB., Biotechnol Bioeng 115(9), 2018
PMID: 29943426
Whole-Cell Biocatalysts for Stereoselective C-H Amination Reactions.
Both P, Busch H, Kelly PP, Mutti FG, Turner NJ, Flitsch SL., Angew Chem Int Ed Engl 55(4), 2016
PMID: 26689856
A whole cell biocatalyst for double oxidation of cyclooctane.
Müller CA, Weingartner AM, Dennig A, Ruff AJ, Gröger H, Schwaneberg U., J Ind Microbiol Biotechnol 43(12), 2016
PMID: 27771781

48 References

Daten bereitgestellt von Europe PubMed Central.

Evaluation of Alcaligenes eutrophus cells as an NADH regenerating catalyst in organic-aqueous two-phase system.
Andersson M, Holmberg H, Adlercreutz P., Biotechnol. Bioeng. 57(1), 1998
PMID: 10099181
On oxygen limitation in a whole cell biocatalytic Baeyer-Villiger oxidation process.
Baldwin CV, Woodley JM., Biotechnol. Bioeng. 95(3), 2006
PMID: 16862597
Cytochromes P450 as versatile biocatalysts.
Bernhardt R., J. Biotechnol. 124(1), 2006
PMID: 16516322
Redox biocatalysis and metabolism: molecular mechanisms and metabolic network analysis.
Blank LM, Ebert BE, Buehler K, Buhler B., Antioxid. Redox Signal. 13(3), 2010
PMID: 20059399
Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method.
Blanusa M, Schenk A, Sadeghi H, Marienhagen J, Schwaneberg U., Anal. Biochem. 406(2), 2010
PMID: 20646988
Catalytic, mild, and selective oxyfunctionalization of linear alkanes: current challenges.
Bordeaux M, Galarneau A, Drone J., Angew. Chem. Int. Ed. Engl. 51(43), 2012
PMID: 22996726
Industrial methods for the production of optically active intermediates.
Breuer M, Ditrich K, Habicher T, Hauer B, Kesseler M, Sturmer R, Zelinski T., Angew. Chem. Int. Ed. Engl. 43(7), 2004
PMID: 14767950
Alkane C–H activation and functionalization with homogeneous transition metal catalysts: a century of progress – a new millennium in prospect
Crabtree, J. Chem. Soc. Dalton Trans. (), 2001
Directed evolution of P450 BM3 into a p-xylene hydroxylase
Dennig, ChemCatChem 4(), 2012
Plasmid effects on Escherichia coli metabolism.
Diaz Ricci JC, Hernandez ME., Crit. Rev. Biotechnol. 20(2), 2000
PMID: 10890453
Bioconversion of n-octane to octanoic acid by a recombinant Escherichia coli cultured in a two-liquid phase bioreactor.
Favre-Bulle O, Schouten T, Kingma J, Witholt B., Biotechnology (N.Y.) 9(4), 1991
PMID: 1367010
Metabolic load and heterologous gene expression.
Glick BR., Biotechnol. Adv. 13(2), 1995
PMID: 14537822
Practical asymmetric enzymatic reduction through discovery of a dehydrogenase-compatible biphasic reaction media.
Groger H, Hummel W, Buchholz S, Drauz K, Nguyen TV, Rollmann C, Husken H, Abokitse K., Org. Lett. 5(2), 2003
PMID: 12529133
Enzyme-mediated oxidations for the chemist
Hollmann, Green Chem. 13(), 2011
New alcohol dehydrogenases for the synthesis of chiral compounds.
Hummel W., Adv. Biochem. Eng. Biotechnol. 58(), 1997
PMID: 9103913
[The operon: a group of genes with expression coordinated by an operator. C.R.Acad. Sci. Paris 250 (1960) 1727-1729].
Jacob F, Perrin D, Sanchez C, Monod J, Edelstein S., C. R. Biol. 328(6), 2005
PMID: 15999435
Reengineering CelA2 cellulase for hydrolysis in aqueous solutions of deep eutectic solvents and concentrated seawater
Lehmann, Green Chem. 14(), 2012
Whole-cell biocatalysis in organic media
León, Enzyme Microb. Technol. 23(), 1998
Two-liquid-phase biocatalytic reactions
Lilly, J. Chem. Tech. Biotechnol. 32(), 1982
Catalytic hydroxylation in biphasic systems using CYP102A1 mutants
Maurer, Adv. Synth. Catal. 347(), 2005
In vitro double oxidation of n-heptane with direct cofactor regeneration
Müller, Adv. Synth. Catal. 355(), 2013

Ortiz, 2005
Translation initiation in Escherichia coli: sequences within the ribosome-binding site.
Ringquist S, Shinedling S, Barrick D, Green L, Binkley J, Stormo GD, Gold L., Mol. Microbiol. 6(9), 1992
PMID: 1375310
To get what we aim for – progress in diversity generation methods
Ruff, FEBS J. (), 2013
Determination of hydrocarbon-water partition coefficients from chromatographic data based on solution thermodynamics and theory
Schantz, J. Chromatogr. 391(), 1987
Whole-cell biocatalysis for selective and productive C–O functional group introduction and modification
Schrewe, Chem. Soc. Rev. (), 2013
Activation of Cminus signH Bonds by Metal Complexes.
Shilov AE, Shul'pin GB., Chem. Rev. 97(8), 1997
PMID: 11851481
Determinant of cistron specificity in bacterial ribosomes.
Shine J, Dalgarno L., Nature 254(5495), 1975
PMID: 803646
Directed evolution of a highly active Yersinia mollaretii phytase.
Shivange AV, Serwe A, Dennig A, Roccatano D, Haefner S, Schwaneberg U., Appl. Microbiol. Biotechnol. 95(2), 2011
PMID: 22159661
Direct oxidation of cycloalkanes to cycloalkanones with oxygen in water.
Staudt S, Burda E, Giese C, Muller CA, Marienhagen J, Schwaneberg U, Hummel W, Drauz K, Groger H., Angew. Chem. Int. Ed. Engl. 52(8), 2013
PMID: 23339093
Biocatalytic hydroxylation of n-butane with in situ cofactor regeneration at low temperature and under normal pressure
Staudt, Beilstein J. of Org. Chem. 8(), 2012
Directed evolution of oxygenases: screening systems, success stories and challenges.
Tee KL, Schwaneberg U., Comb. Chem. High Throughput Screen. 10(3), 2007
PMID: 17346119
Aqueous solubility and octanol-water partition coefficients of organic compounds at 298K
Tewari, J. Chem. Eng. Data 27(), 1982
Strategies for protein coexpression in Escherichia coli.
Tolia NH, Joshua-Tor L., Nat. Methods 3(1), 2006
PMID: 16369554
Cytochrome P450 monooxygenases: perspectives for synthetic application.
Urlacher VB, Eiben S., Trends Biotechnol. 24(7), 2006
PMID: 16759725

Weissermel, 2003
P450(BM3) (CYP102A1): connecting the dots.
Whitehouse CJ, Bell SG, Wong LL., Chem Soc Rev 41(3), 2011
PMID: 22008827
Biosynthesis of synthons in two-liquid-phase media.
Wubbolts MG, Favre-Bulle O, Witholt B., Biotechnol. Bioeng. 52(2), 1996
PMID: 18629897

Markieren/ Markierung löschen
Markierte Publikationen

Open Data PUB

Web of Science

Dieser Datensatz im Web of Science®

PMID: 24925696
PubMed | Europe PMC

Suchen in

Google Scholar