Plants produce a multitude of specialized metabolites with use in the medicinal, fragrance and flavor industries. Cytochromes P450 (P450s) are key enzymes in biosynthetic pathways of these metabolites[1]. P450s are difficult to express heterologously in active form, and due to their requirement for reducing power in the form of NADPH their use for in vitro and whole-cell production is complicated. We recently showed that plant P450s can be expressed in chloroplasts of tobacco plants and in cyanobacteria, where they will insert into the thylakoid membrane and that photosynthesis can support P450 catalytic activity independent of NADPH through the action ferredoxin [3,4]. In the current study, we report the fusion of ferredoxin with the plant P450 CYP79A1, which catalyzes the initial step of the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows the cytochrome P450 enzyme to obtain electrons for catalysis directly from photosynthesis by interacting with photosystem I. Furthermore, the electrons captured by the fused ferredoxin domain are directed more effectively towards the P450 in competition with other ferredoxin requiring enzymes [2]. As a result, it partially overcomes the problem of competition for reduced ferredoxin by electron sinks coupled to endogenous metabolic pathways. The ferredoxin-P450 fusion enzyme obtains reducing power solely from its fused ferredoxin, but maintains a similar level of catalytic activity compared to unfused CYP79A1 at in vivo concentrations of soluble ferredoxin. This demonstrates that electron transfer from photosystem I to CYP79A1 is greatly accelerated as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for increased partitioning of photosynthetic reducing power towards P450-dependent biosynthesis of important natural products. This approach has high potential for stably engineering cyanobacteria that enables high-level cytochrome P450-dependent production of high-value natural products in a light-driven manner.
References:
1. Lassen, LM, Zygadlo Nielsen A, Friis Ziersen BE; Gnanasekaran T, Møller BL, Jensen PE (2014). Redirecting photosynthetic electron flow into light-driven synthesis of alternative products including high-value bioactive natural compounds. ACS Synth. Biol. 3(1): 1–12. DOI: 10.1111/ppl.12108
2. Mellor S, Nielsen A, Burow M, Motawia M, Jakubauskas D, Møller BL, Jensen PE (2016). Fusion of ferredoxin and cytochrome P450 enables direct light-driven biosynthesis. ACS Chemical Biology, DOI: 10.1021/acschembio.6b00190.
3. Wlodarczyka A, Gnanasekarana T, Nielsen AZ, Zulu NN, Mellor SB, Thøfner JFB, Olsen CE, Mottawie MS, Burow M, Luckner M, Pribil M, Feussner I, Møller BL, and Jensen PE (2016). Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803. Metabolic Engineering 33: 1-11. doi: 10.1016/j.ymben.2015.10.009.
4. Zygadlo Nielsen A, Friis Ziersen BE, Jensen K, Lassen LM, Olsen CE, Møller BL, Jensen PE (2013). Redirecting photosynthetic reducing power towards bioactive natural product synthesis. ACS Synthetic Biology 2: 308−315. dx.doi.org/10.1021/sb300128r.
