Exploring linker's sequence diversity to fuse carotene cyclase and hydroxylase for zeaxanthin biosynthesis

Fusion of catalytic domains can accelerate cascade reactions by bringing enzymes in close proximity. However, the design of a protein fusion and the choice of a linker are often challenging and lack of guidance. To determine the impact of linker parameters on fusion proteins, a library of linkers featuring various lengths, secondary structures, extensions and hydrophobicities was designed. Linkers were used to fuse the lycopene cyclase (crtY) and beta-carotene hydroxylase (crtZ) from Pantoea ananatis to create fusion proteins to produce zeaxanthin. The fusion efficiency was assessed by comparing the carotenoids content in a carotenoid-producer Escherichia coli strain. It was shown that in addition to the orientation of the enzymes and the size of the linker, the first amino acid of the linker is also a key factor in determining the efficiency of a protein fusion. The wide range of sequence diversity in our linker library enables the fine tuning of protein fusion and this approach can be easily transferred to other enzyme couples.

Automated summary

Fusion of catalytic domains can enhance cascade reactions, but designing a suitable fusion protein and choosing an appropriate linker is challenging. To study the impact of linker parameters on fusion proteins, a library of various linkers was created. Fusion proteins were obtained by fusing lycopene cyclase (crtY) and beta-carotene hydroxylase (crtZ) using the linkers, and their efficiency was evaluated. The results showed that in addition to enzyme orientation and linker size, the first amino acid of the linker plays a crucial role in determining fusion protein efficiency. The diverse linker library can be applied to fine-tune fusion proteins with other enzyme pairs.