title: Whole-cell double oxidation of n-heptane.
abstract: 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.
authors.affiliation: Institute of Biotechnology, RWTH Aachen, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Müller
authors.affiliation: Institute of Biotechnology, RWTH Aachen, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dennig
authors.affiliation: Institute of Biotechnology, RWTH Aachen, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Welters
authors.affiliation: Institute of Molecular Enzyme Technology at the Heinrich-Heine-University of Düsseldorf, Research Centre Jülich, Stetternicher Forst, 52426 Jülich, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Winkler
authors.affiliation: Institute of Biotechnology, RWTH Aachen, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Anna Joelle
authors.initials: AJ
authors.lastname: Ruff
authors.affiliation: Institute of Molecular Enzyme Technology at the Heinrich-Heine-University of Düsseldorf, Research Centre Jülich, Stetternicher Forst, 52426 Jülich, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Hummel
authors.affiliation: Faculty of Chemistry, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Gröger
authors.affiliation: Institute of Biotechnology, RWTH Aachen, Worringerweg 3, 52074 Aachen, Germany. Electronic address: u.schwaneberg@biotec.rwth-aachen.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D000426:Alcohol Dehydrogenase / Q000096:biosynthesis* / Q000378:metabolism; D000473:Alkanes / Q000737:chemistry / Q000378:metabolism; D001410:Bacillus megaterium / Q000201:enzymology; D002384:Catalysis; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D006536:Heptanes / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000378:metabolism; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology; D014867:Water / Q000737:chemistry / Q000378:metabolism
chemical_list: D000473:Alkanes; D006536:Heptanes; D014867:Water; C028618:n-heptane; D009249:NADP; D006899:Mixed Function Oxygenases; D000426:Alcohol Dehydrogenase; D010100:Oxygen
keywords: Alcohol dehydrogenase; Cascade reaction; Directed evolution; Double oxidation; Monooxygenase