title: Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids.
abstract: BACKGROUND
  De novo production of multi-hydroxylated diterpenoids is challenging due to the lack of efficient redox systems.
  
  
  RESULTS
  In this study a new reductase/ferredoxin system from Streptomyces afghaniensis (AfR·Afx) was identified, which allowed the Escherichia coli-based production of the trihydroxylated diterpene cyclooctatin, a potent inhibitor of human lysophospholipase. This production system provides a 43-fold increase in cyclooctatin yield (15 mg/L) compared to the native producer. AfR·Afx is superior in activating the cylcooctatin-specific class I P450s CotB3/CotB4 compared to the conventional Pseudomonas putida derived PdR·Pdx model. To enhance the activity of the PdR·Pdx system, the molecular basis for these activity differences, was examined by molecular engineering.
  
  
  CONCLUSIONS
  We demonstrate that redox system engineering can boost and harmonize the catalytic efficiency of class I hydroxylase enzyme cascades. Enhancing CotB3/CotB4 activities also provided for identification of CotB3 substrate promiscuity and sinularcasbane D production, a functionalized diterpenoid originally isolated from the soft coral Sinularia sp.
authors.affiliation: Division of Industrial Biocatalysis, Technical University of Munich, Lichtenberg Str. 4, 85748, Garching, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Görner
authors.affiliation: Division of Industrial Biocatalysis, Technical University of Munich, Lichtenberg Str. 4, 85748, Garching, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Schrepfer
authors.affiliation: Division of Industrial Biocatalysis, Technical University of Munich, Lichtenberg Str. 4, 85748, Garching, Germany.
authors.forename: Veronika
authors.initials: V
authors.lastname: Redai
authors.affiliation: Chair of Bioinformatics, Technical University of Munich, Boltzmannstraße 3, 85748, Garching, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Wallrapp
authors.affiliation: Division of Structural Biology, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 6, 14195, Berlin, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Loll
authors.affiliation: Chair of Biochemistry, Technical University of Munich, Lichtenberg Str. 4, 85748, Garching, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eisenreich
authors.affiliation: Chair of Biotechnology, Technical University of Munich, Lichtenbergstr. 4, 85748, Garching, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Haslbeck
authors.affiliation: Division of Industrial Biocatalysis, Technical University of Munich, Lichtenberg Str. 4, 85748, Garching, Germany. brueck@tum.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Brück
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D006900:Hydroxylation; D062105:Molecular Docking Simulation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004224:Diterpenes; D005288:Ferredoxins; C078947:cyclooctatin; D010088:Oxidoreductases
keywords: Cyclooctatin; Diterpene biosynthesis; Redox system; Sinularcasbane D; Streptomyces afghaniensis