Reference
[1] Kang W, Ma T, Liu M, et al. Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux[J]. Nature Communications, 2019, 10(1).
Team:DUT China/Contribution
Contribution
Our contribution to iGEM will mainly show two aspects on this webpage. On the one hand, it is a supplement to the existing parts content. The supplementary parts content is as follows; on the other hand, the peptides that can be used to construct enzyme complexes, RIDD and RIAD introduce the iGEM community to provide a new idea for other teams to construct enzyme complexes in the future and combines our literature research and practical experience to give some suggestions.
| Parts number | Supplementary summary |
|---|---|
| BBa_K2010000 | The registry part page has been supplemented with a directed evolution of an enzyme that efficiently degrades PET plastic, and we used this enzyme to construct an enzyme complex. |
| BBa_K2013003 | The registry part page has supplemented new data collected from laboratory experiments by Ailurus vecTM and the literature content of the fusion expression of MHETase and PETase. |
Studies have shown discoveries in the interaction of RIAD-RIDD dimer or trimer (two RIDD with one RIAD) peptides. These two types of peptides have a strong binding affinity to each other. Inspired by this discovery, we proposed the idea that IsPETase and MHETase can be fused with RIDD and RIAD, respectively so that closer physical contact with the active sites of these two enzymes can significantly accelerate PET degradation through intermediate transmission. We further accelerate the rate of enzyme degradation without interfering with the structure of the enzyme active site or enzyme activity. The final result of the experiment confirmed our initial idea.
Through experiments, we understand the in-depth mechanism of action between each of the critical components and calculate the response of the intermediate product to the enzyme, which can help us better interpret our system.
Figure 1: Schematic diagram of this system [1] (The assembly of tri-enzyme units. E1, E2: enzymes; green and blue structure: RIDD dimer; black line: linker; pink structure: RIAD; one orange circle: cysteine; two orange circles: disulfide bond)
To apply our hybrid system to other enzyme systems, we have established a modular design that uses our modules based on an in-depth understanding of the mechanism of action. We will give several points for those who want to use this RIDD-RIAD dimer or trimer peptides system for enzyme complexes. There are several necessary conditions for using this molecular system:
1. The enzyme upstream of the production chain must be a rate-limiting enzyme.
2. Characterize and calculate the combination result of the product and the enzyme. If the product has an inhibitory effect on the enzyme in the previous step, the enzyme system can play a more significant role.
3. Consider the influence of the sequence of enzymes on the activity, even though the impact of our backbone on the movement of the enzyme has been minimized.
We hope our experience and tips can help them who are interested in modular enzyme assembly or complexes.