Among the 99 basic parts, CYP107D1 (Olep) is the most important, which comes from the oleandomycin synthesis pathway of Streptomyces antibioticus. We optimized oleP gene and added a histidine tag to the C terminus. Besides, many other basic parts have also been designed to construct our project.

A series of composite parts have been designed this year, and most of them are useful for our project. And our favorite composite part is T7-RBS4-GFP11-linker-Olep, Based on the bimolecular fluorescence complementation technique, a novel redox chaperone sensor was constructed to measure the interaction force between Olep and the redox chaperone. The sensor uses a superfolded green fluorescent protein (sfGFP), and sfGFP is divided into GFP1-10 and GFP-11. The interaction between GFP1-10 and GFP11 pulls down the distance in space. Olep was fused to the C terminus of GFP-11.

This year, our research focused on enhancing the soluble heterologous expression of Olep in E. coli. To achieve this, we conducted experiments using different plasmids with varying copy numbers. Additionally, we introduced a lytic tag at the N terminus of Olep. In order to optimize Olep's performance, we also investigated and modified redox chaperones that are better suited for Olep, aiming to enhance their catalytic activity. Our team utilized bimolecular fluorescence complementation technology to split sfGFP into GFP1-10 and GFP11, and then fused them with Olep and the redox chaperone involved in electron interaction. Through this process, we were able to screen and modify the redox chaperone of the P450 enzyme. Furthermore, our research revealed that redox chaperones are crucial for facilitating electron transport and activating oxygen molecules in P450 enzymes. Overall, our findings highlight the essential role of redox chaperones in electron transport within these enzymes.