Engineering class I terpene synthases for skeletal diversity: strategies and applications

Area of Science:

• Biochemistry
• Synthetic Biology
• Natural Product Chemistry

Background:

• Terpenoids are nature's largest and most diverse class of natural products.
• Terpene synthases (TSs) are key enzymes responsible for generating structural diversity in terpenoids.
• Applications span medicine, agriculture, and fragrance industries.

Purpose of the Study:

• To review strategies for engineering terpene synthases (TSs) to generate diverse terpene skeletons.
• To highlight key approaches in synthetic biology for terpene production.
• To explore future directions in expanding terpenoid chemical space.

Main Methods:

• Structure-guided design targeting active sites, water networks, and conserved motifs.
• Evolutionary methods using natural variation and phylogenetic insights.
• Mechanism-focused engineering and second-shell modifications.
• Semi-rational and random mutagenesis (e.g., alanine scanning, directed evolution) combined with computational modeling and high-throughput screening.

Main Results:

• Integration of structural biology, computational simulations, and diverse engineering techniques improves outcomes in TS engineering.
• Despite challenges like catalytic complexity and weak sequence-function correlations, progress is steadily being made.
• Advanced screening methods are crucial for identifying successful engineered variants.

Conclusions:

• Engineering TSs is a powerful strategy for accessing novel terpenoid chemical space.
• Future advances in machine learning, mechanistic understanding, and metabolic engineering integration will further enhance biotechnological exploration of terpenoids.
• Continued development of interdisciplinary approaches is vital for unlocking the full potential of TS engineering.