Combinatorial biosynthesis and the basis for substrate promiscuity in class I diterpene synthases

Area of Science:

• Biochemistry
• Enzymology
• Metabolic Engineering

Background:

• Terpene synthases (TSs) catalyze complex reactions, including the lysis of diphosphate esters, leading to diverse products like labdane-related diterpenes (LRDs).
• Previous work identified TSs with high substrate promiscuity but specific product outcomes, useful for combinatorial biosynthesis.
• Diterpene cyclases (DTCs) initiate LRD formation through bicyclization, creating structural variations.

Purpose of the Study:

• To investigate two novel diterpene synthases (DTSs) producing cis or trans endo-ene LRD derivatives for combinatorial biosynthesis.
• To determine if these DTSs exhibit substrate promiscuity analogous to previously studied enzymes.
• To explore the evolutionary basis for substrate promiscuity in DTSs.

Main Methods:

• Characterization of two DTSs, one plant-derived and one bacterial, that produce endo-ene LRD derivatives.
• Assessing substrate promiscuity and catalytic specificity of the examined DTSs.
• Phylogenetic analysis to correlate enzyme promiscuity with evolutionary origin.

Main Results:

• The bacterial trans-endo-ene forming DTS displayed significant substrate promiscuity with moderate catalytic specificity.
• Plant-derived DTS showed limited substrate promiscuity.
• Bacterial DTSs exhibited greater substrate promiscuity than plant DTSs, correlating with phylogenetic origin rather than reaction complexity.

Conclusions:

• Bacterial DTSs offer greater potential for combinatorial biosynthesis of LRDs due to higher substrate promiscuity.
• Phylogenetic origin, particularly in bacteria with less elaborated LRD metabolism, is a key factor influencing DTS substrate promiscuity.
• This research provides novel biosynthetic pathways to nearly 19 LRDs, showcasing the utility of combinatorial approaches.