Fusion Protein

Introduction

Although there are many metabolic regulation aspects as well as bioreactor considerations to the nootkatone production system, the fusion protein is the “Bell of the Ball”. The two enzymes not contained in Saccharomyces cerevisiae, Valencene Synthase (CnVS) and Valencene Oxidase (CYP706M1), are necessary for the production of nootkatone are fused and put under the control of a strong promoter for optimal expression.

Protein Folding

Both protein sequences were pulled from GenBank (GenBank ID: JX040471.1 and AGJ03150.1 Synthase and Oxidase Respectively). Each sequence was then converted to a 3D model using protein folding and homology predicting software, RaptorX and intFOLD to ensure the predicted fold was accurate. Once generated, the top-ranking models for both proteins were compared to the opposing software and only minor differences were observed in the models.

Linker Determination

Linker Region Determination

Using the 3D models, a linker region was placed between the two proteins to fuse them. The optimal region for linker placement was determined to be the N-terminal region of Valencene Synthase and then C-terminal region of Valencene Oxidase. This is because Valencene Synthase is observed to have a long the C-terminal region with no function nor secondary structure, serving as an optimal site to be linked. Additionally, the N-terminal and the C-terminal regions of Valencene Oxidase both appeared to be equally exposed to solvent and equally available to have a linker attachment so for simplicity, the C-terminal end of the protein was chosen.

Linker Determination

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Using literature published by Meng et al., 2020 (1), who expressed a very similar fusion protein, our team evaluated all linker regions used in the paper to determine the optimal linker to use in our fusion protein. The linker tested in their paper were as follows:

-GSG-

-GSGGGGS-

-GGGGS-

The GSGGGGS linker was found to yield the highest fusion protein expression when expressing a fusion protein of FPP Synthase and Valencene Synthase at about 11mg/L and thus, was used to create our fusion protein.

Protein Construct

The two proteins were linked in the manner outlined above using CHIMERA (2).

Figure 1 - 3D model of the Valencene Synthase and Valencene Oxidase proteins designed for expression in Saccharomyces cerevisiae cells. The chimeric protein was created and optimized for expression in yeast cells and linked using a glycine/serine linker attached to the N-terminal end of the valencene synthase and the C-terminal end of the valencene oxidase.

Figure 2 - Valencene oxidase model created with intFOLD and RaptorX via homology modelling and energy minimization.

Our model created from the sequence deposited on GenBank by Cankar et al., 2014.

Figure 3 - Valencene synthase model created with intFOLD and RaptorX via homology modelling and energy minimization.

Our model created from the sequence deposited on GenBank by Beekwilder et al., 2012.

Figure 4 - Valencene oxidase predicted ligand binding domain via intFOLD. The predicted active site of valencene oxidase which catalyzes the conversion of valencene to nootkatone in the final step of the biosynthesis process.

Figure 5 - Valencene synthase predicted ligand binding domain via intFOLD. The predicted active site of the valencene synthase enzyme which catalyzes the conversion of FPP to valencene.

References

1. Meng, X., Liu, H., Xu, W., Zhang, W., Wang, Z., and Liu, W. (2020) Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone. Microb. Cell Fact. 10.1186/S12934-020-1295-6

2. EF, P., TD, G., CC, H., GS, C., DM, G., EC, M., and TE, F. (2004) UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612

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Team:Queens Canada/fusionProtein