title: Screening and Engineering the Synthetic Potential of Carboxylating Reductases from Central Metabolism and Polyketide Biosynthesis.
abstract: Carboxylating enoyl-thioester reductases (ECRs) are a recently discovered class of enzymes. They catalyze the highly efficient addition of CO2 to the double bond of α,β-unsaturated CoA-thioesters and serve two biological functions. In primary metabolism of many bacteria they produce ethylmalonyl-CoA during assimilation of the central metabolite acetyl-CoA. In secondary metabolism they provide distinct α-carboxyl-acyl-thioesters to vary the backbone of numerous polyketide natural products. Different ECRs were systematically assessed with a diverse library of potential substrates. We identified three active site residues that distinguish ECRs restricted to C4 and C5-enoyl-CoAs from highly promiscuous ECRs and successfully engineered a selected ECR as proof-of-principle. This study defines the molecular basis of ECR reactivity, allowing for predicting and manipulating a key reaction in natural product diversification.
authors.affiliation: Biochemistry and Synthetic Biology of Microbial Metabolism Group, Max-Planck-Institute for terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043 Marburg (Germany).|Institute of Microbiology, Eidgenössisch Technische Hochschule (ETH) Zürich, Vladimir-Prelog-Weg 4, 8050 Zürich (Switzerland).
authors.forename: Dominik M
authors.initials: DM
authors.lastname: Peter
authors.affiliation: Biochemistry and Synthetic Biology of Microbial Metabolism Group, Max-Planck-Institute for terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043 Marburg (Germany).|Institute of Microbiology, Eidgenössisch Technische Hochschule (ETH) Zürich, Vladimir-Prelog-Weg 4, 8050 Zürich (Switzerland).
authors.forename: Lennart
authors.initials: L
authors.lastname: Schada von Borzyskowski
authors.affiliation: Institute of Microbiology, Eidgenössisch Technische Hochschule (ETH) Zürich, Vladimir-Prelog-Weg 4, 8050 Zürich (Switzerland).
authors.forename: Patrick
authors.initials: P
authors.lastname: Kiefer
authors.affiliation: Institute of Microbiology, Eidgenössisch Technische Hochschule (ETH) Zürich, Vladimir-Prelog-Weg 4, 8050 Zürich (Switzerland).
authors.forename: Philipp
authors.initials: P
authors.lastname: Christen
authors.affiliation: Institute of Microbiology, Eidgenössisch Technische Hochschule (ETH) Zürich, Vladimir-Prelog-Weg 4, 8050 Zürich (Switzerland).
authors.forename: Julia A
authors.initials: JA
authors.lastname: Vorholt
authors.affiliation: Biochemistry and Synthetic Biology of Microbial Metabolism Group, Max-Planck-Institute for terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043 Marburg (Germany). toerb@mpi-marburg.mpg.de.|Institute of Microbiology, Eidgenössisch Technische Hochschule (ETH) Zürich, Vladimir-Prelog-Weg 4, 8050 Zürich (Switzerland). toerb@mpi-marburg.mpg.de.
authors.forename: Tobias J
authors.initials: TJ
authors.lastname: Erb
mesh_terms: D008958:Models, Molecular; D015394:Molecular Structure; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000378:metabolism*; D061065:Polyketides / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering
chemical_list: D061065:Polyketides; D044925:Oxidoreductases Acting on CH-CH Group Donors; C434934:trans-2-enoyl-CoA reductase (NADPH)
keywords: CO2 fixation; biotechnology; crotonyl-CoA carboxylase/reductase; natural product engineering; polyketides