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CsCCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production
Nan Liang1,2, Ming-Dong Yao1,2, Ying Wang1,2
1Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Journal of Agricultural and Food Chemistry
|September 23, 2021
Summary
Microbial synthesis of crocetin, a valuable saffron compound, was improved using engineered enzymes. This "hybrid-tunnel" approach enhanced enzyme efficiency, leading to higher yields for food and medicine applications.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Metabolic Engineering
Background:
- Crocetin, a high-value apocarotenoid from saffron, has significant applications in food and medicine.
- Current large-scale cultivation methods are insufficient to meet market demand for crocetin.
- Microbial synthesis presents a promising alternative for crocetin production.
Purpose of the Study:
- To engineer the Crocus sativus-derived carotenoid cleavage dioxygenase 2 (CsCCD2) enzyme for improved crocetin synthesis.
- To broaden the substrate specificity and enhance the catalytic efficiency of CsCCD2 for microbial crocetin production.
Main Methods:
- Utilized "hybrid-tunnel" engineering to create variants of the CsCCD2 enzyme.
- Employed in silico protein design to modify enzyme access tunnels.
- Conducted yeast-based fermentations and in vitro enzymatic assays to evaluate enzyme variants.
Main Results:
- Engineered variant S323A demonstrated a 5-fold increase in crocetin titer in yeast fermentation.
- S323A exhibited a 12.83-fold higher catalytic efficiency (kcat/Km) toward zeaxanthin compared to wild-type CsCCD2.
- Achieved a production of 107 mg/L crocetin in fed-batch fermentation, surpassing previous reports.
Conclusions:
- "Hybrid-tunnel" engineering is a viable strategy to enhance enzyme catalytic properties and substrate specificity.
- Optimized CsCCD2 variants can significantly improve microbial crocetin production.
- This approach holds potential for refining enzyme functions in various biotechnological applications.
