Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids

Abstract

BackgroundDe novo production of multi-hydroxylated diterpenoids is challenging due to the lack of efficient redox systems.ResultsIn 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0487-6) contains supplementary material, which is available to authorized users.

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…56 Although again, the operon contains no redox partners for the P450s, it has been shown that heterologous co-expression of a reductase/ferredoxin system from Streptomyces afghaniensis led to more efficient in vitro oxidation than the putidaredoxin/reductase system. 57 These results are consistent with a common reductase/ferredoxin system coupling to a number of P450s in Streptomyces.…”

Isolation and Investigation of Natural Rare Earth Metal Chelating Agents From Calothrix brevissima - A Step Towards Unraveling the Mechanisms of Metal Biosorption

“…After HPLC data evaluation, the remaining sample material was processed with a trimethylsilyl (TMS) derivatization for subsequent GC-MS based identification of the carbohydrate constituents. For this, a modified version of a previously described protocol was used ( Gorner et al, 2016 ). 50 µL of pyridine were added to each sample.…”

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems

“…Specifically, codon optimization and the truncation of distinct domains which are responsible for, e.g., membrane localization can improve the enzyme activity. To date, identifying the necessary sequence segment for soluble expression in the bacterial host alongside with finding the optimal redox partner for P450 enzymes is still a matter of empirical work [ 44 , 91 – 92 ]. Furthermore, integration of every additional enzyme to the production system will eventually result in a significant decrease in the final yield, which makes very complex biosyntheses involving multiple oxygenation steps challenging [ 93 ].…”