Exploring Fungal Polyketide C-Methylation through Combinatorial Domain Swaps

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Polyketide biosynthesis involves complex, iterative reactions catalyzed by multidomain polyketide synthases (PKSs).
• Fungal PKSs exhibit iterative mechanisms where domains act on multiple enzyme-tethered intermediates.
• C-methylation is a key modification in polyketide structures, influencing their properties.

Purpose of the Study:

• To investigate the role of C-methyltransferase (CMeT) domains in controlling polyketide C-methylation patterns in fungi.
• To understand how different methylation patterns are installed during nonreducing PKS biosynthesis.
• To explore the potential for engineering biosynthetic pathways through targeted C-methylation.

Main Methods:

• Surveyed a collection of C-methyltransferase (CMeT) domains from nonreducing fungal PKSs.
• Performed in vitro assays to assess CMeT activity and substrate specificity.
• Analyzed the interplay between CMeT domains and other PKS domains.

Main Results:

• C-methyltransferase (CMeT) domains primarily control the site-specificity of polyketide C-methylation.
• CMeTs can effectively intercept and methylate intermediates from noncognate nonreducing PKS domains.
• Methylation patterns are determined by a competition between CMeT activity and ketosynthase-catalyzed chain extension.

Conclusions:

• The CMeT domain is the key determinant of C-methylation patterns in fungal polyketide biosynthesis.
• Understanding these mechanisms enables the potential for targeted C-C bond formation in engineered pathways.
• This research provides insights for synthetic biology applications in creating novel polyketide structures.