Identification, characterisation and recombinant expression of flavonoid 3',5'-hydroxylases and cytochrome P450 reductases from Vaccinium species

Flavonoid 3,5-hydroxylases (F3'5'Hs) play a key role in biosynthesis of blue coloured anthocyanin complexes in plants. Thus these proteins have potential application in the development of a natural blue coloured food dye using microbial cell factories. However, F3'5'Hs are membrane proteins that require a redox partner, NADPH-cytochrome P450 reductase (CPR). The aim of the research was to identify F3'5'H sequences from Vaccinium species plants and express the respective proteins in yeast to test their potential in biotechnological production of precursors of anthocyanins. In this study, novel coding DNA sequences of F3'5'Hs from Vaccinium myrtillus and Vaccinium uliginosum, and two CPRs from V. myrtillus were identified and characterised. The newly obtained proteins and F3'5'H from Vaccinium corymbosum and CPR from Helianthus annuus were expressed in Pichia pastoris. Addition of DMSO into the culture medium increased production of F3'5'Hs and CPRs. A truncated form of V. corymbosum F3'5'H, that lacked the predicted first N-terminal alpha helix, expressed at higher level compared to the full-length protein. Vaccinium F3'5'Hs were combined with different CPRs and substrates to identify which CPR acts as a redox partner for F3'5'Hs and which substrates are preferred. Unfortunately, only substrates but not the products could be detected, indicating that the recombinant F3'5'Hs were inactive. Therefore, despite progress in protein expression, P. pastoris was not a suitable host for producing Vaccinium F3'5'Hs.