Engineering Photosynthetic Electron Transfer for Increased Partitioning of Reducing Power to Cytochrome P450-Dependent Reactions

Photosynthetic organisms are promising metabolic engineering chassis. Photosynthesis allows high resource use efficiencies, and many plants can be cultivated cheaply at industrial scale. Therefore, engineering these hosts is of great relevance for future sustainable biomolecule production. Heterologous P450s can be targeted to plant thylakoid membranes, where the photosynthetic apparatus functionally substitutes native reductases via the endogenous electron carrier ferredoxin. But because ferredoxin acts as a distribution hub for photosynthetic reducing power, relying on this heavily contested source of reductant limits the photosynthesis-dependent P450 output that can be achieved. To overcome this limitation, we investigated the ability of alternative electron carriers- three plant ferredoxins and a flavodoxin-like protein - to increase photosynthesis-driven P450 activity. We also tested the electron carriers in vivo by fusing them to the P450 and transiently expressing these fusions targeted to chloroplasts in tobacco. Steady state kinetics reveals only modest differences in electron transfer rates between the carriers and CYP79A1 in vitro. However, the flavodoxin-like carrier plant P450 reductase outperforms the three ferredoxins when FNR is present to compete for reduced ferredoxin, likely because its FMN redox potential renders delivery of electrons to endogenous enzymes unfavourable. In vivo investigation via transient expression in tobacco show three of four CYP79A1-carrier fusions able to sequester more photosynthetic reducing power than the unfused P450. Notably fusion with the flavodoxin-like carrier yields 25-fold higher production on a per protein basis. This study demonstrates that non-native electron carriers can be used to construct novel electron transfer chains that alleviate the problem of endogenous competition. The effectiveness of such chains is governed by hey factors such as redox potential and carrier-photosystem I affinity. This demonstrates that electron transfer chain engineering and P450-carrier fusion can boosts photosynthesis-driven P450 activity and sets out concrete design parameters for future electron carrier engineering.