Engineering DoxA activity through the manipulation of associated electron transport partners
Ilomäki, Mikael (2021)
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Doxorubicin (DXR) is an anthracycline produced by some strains of the bacterium Streptomyces peucetius subsp. caesius. It is one of the most commonly used drugs in the treatment of several different types of cancer. It is produced as a result of a complicated biosynthetic pathway. The cytochrome P450 enzyme (CYP450) DoxA is responsible for the final three reaction steps in the biosynthesis. The final step, hydroxylation of daunorubicin (DNR) to DXR, is much less efficient than the preceding two steps, which causes low amounts of DXR to be produced in relation to DNR. This makes large-scale production of DXR difficult and a way to achieve complete biosynthetic conversion of DNR to DXR would be hugely beneficial. This thesis project aimed to find ways to improve DXR biosynthesis by studying the effect of different redox systems on the activity of DoxA. Preliminary data showed that overproduction of DXR might be related to the expression of the ferredoxins X4 and X5. In addition to these, the proteins SFX, SFR, PDX and PDR from other organisms, as well as a fusion of the proteins YkuN and Fpr, and finally, a fusion of DoxA and an RhF-domain, were used to try to improve DoxA activity. The proteins were produced in Escherichia coli, and the proteins were purified by affinity chromatography. In vitro activity assays with DoxA and the redox proteins were done to measure the activity of DoxA. The proteins from S. peucetius did not cause any activity at all, leading to the conclusions that incompatible pairs had probably been chosen. The unnatural redox systems supported DoxA activity to different extents, with PDX and PDR being the best system identified. Several functioning redox systems that can be used in future research were established in this project, and they all provide valuable insights into redox engineering as a whole and as a way to improve the biosynthesis of DXR.