Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803

• Expression of multiple genes using self-replicating expression vector in cyanobacteria
• Eukaryotic P450s can be expressed and inserted in cyanobacterial membranes.
• Cyanobacterial photosystem I and ferredoxin are efficient electron donors for plant P450s.
• Pathway intermediates and final products are excreted to the growth media.
• Engineered strain was grown in a photobioreactor using simple nutrient mixture.

Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66 mg/L of p-hydroxyphenylacetaldoxime and 5 mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3 mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.