Refactoring the Partial Photosynthesis Gene Cluster for the Biosynthesis of the Key Pigment, Bacteriochlorophyll a

In prokaryotes, the genes responsible for complex biological functions are often physically clustered on the chromosome for co-regulation. As a goal for synthetic biology, it is important to design and build manipulable gene clusters for the further construction of biological systems with higher hierarchy and useful biological functions. In order to achieve this goal, microbial photosynthesis machinery was chosen as a target for refactoring. Taking the purple non-sulfur bacterium Rhodobacter sphaeroides 2.4.1 as an example, almost all the genes responsible for photosynthesis are clustered on the chromosome spanning in an approximately 40 kb region. We are using a systematic approach to re-design this photosynthesis gene cluster by rebuilding it from the bottom-up using synthetic and well-characterized genetic parts. Focusing on the biosynthetic operons for the key pigment bacteriochlorophyll a (BChl a) biosynthesis, all the essential genes will be chosen for knock-out and complementation, with the exception of regulatory genes. An architecture with mono-cistronic expression of each gene will be adopted for the construction of the synthetic gene cluster. Each gene is being designed to be under the control of tunable genetic parts, including promoter, ribosome binding site, transcription terminator and insulator. The codons for the genes are adjusted to be as different as possible from the wild type genes, thus excluding possible internal regulation sequences. Finally, a simplified environment sensor will be implemented in a genetic circuit to regulate the gene expression profile. The synthetic gene cluster for the biosynthesis of BChl a may provide a modular platform for further combinatorial biosynthesis of BChl a derivatives, which may expand the chemical space for additional and novel photosynthetic pigments. Via this refactoring process, this gene cluster may also be easily transferred to other non-photosynthetic microorganisms, facilitating the engineering of artificial photosystems.