title: Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins.
abstract: NADPH-dependent oxidoreductases are useful catalysts for the production of chiral synthons. However, preparative applications of oxidoreductases require efficient methods for in situ regeneration of the expensive nicotinamide cofactors. An advantageous method for cofactor regeneration is the construction of bifunctional fusion proteins composed of two enzymes, one catalysing the reduction reaction and the other one mediating the recycling of cofactors. Herein, we describe the in-frame fusion between an NADP+-accepting mutant of FDH (formate dehydrogenase) from Mycobacterium vaccae N10 and KR [3-ketoacyl-(acyl-carrier-protein) reductase] from Synechococcus sp. strain PCC 7942. The generation of linker insertion mutants led to a fusion protein exhibiting 100 and 80% of the enzymatic activities of native KR and FDH respectively. Escherichia coli cells expressing the fusion protein showed an approx. 2-fold higher initial reaction rate in the production of chiral alcohols than cells expressing the enzymes separately. The application of the engineered fusion protein in whole-cell bioreduction of pentafluoroacetophenone resulted in a substrate conversion of 99.97% with an excellent enantiomeric excess of 99.9% (S)-1-(pentafluorophenyl)ethanol.
authors.affiliation: Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Hölsch
authors.forename: Dirk
authors.initials: D
authors.lastname: Weuster-Botz
mesh_terms: D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000098:Acetophenones / Q000737:chemistry / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000438:Alcohols / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000378:metabolism; D007659:Ketones / Q000737:chemistry* / Q000378:metabolism*; D009161:Mycobacterium / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D000098:Acetophenones; D000438:Alcohols; D003067:Coenzymes; D007659:Ketones; D011993:Recombinant Fusion Proteins; D009249:NADP; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D005560:Formate Dehydrogenases