TY - JOUR
T1 - Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids
AU - Görner, Christian
AU - Schrepfer, Patrick
AU - Redai, Veronika
AU - Wallrapp, Frank
AU - Loll, Bernhard
AU - Eisenreich, Wolfgang
AU - Haslbeck, Martin
AU - Brück, Thomas
N1 - Publisher Copyright:
© 2016 The Author(s).
PY - 2016
Y1 - 2016
N2 - 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. Conclusion: 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.
AB - 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. Conclusion: 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.
KW - Cyclooctatin
KW - Diterpene biosynthesis
KW - Redox system
KW - Sinularcasbane D
KW - Streptomyces afghaniensis
UR - http://www.scopus.com/inward/record.url?scp=85007425772&partnerID=8YFLogxK
U2 - 10.1186/s12934-016-0487-6
DO - 10.1186/s12934-016-0487-6
M3 - Article
C2 - 27216162
AN - SCOPUS:85007425772
SN - 1475-2859
VL - 15
JO - Microbial Cell Factories
JF - Microbial Cell Factories
IS - 1
M1 - 86
ER -