Methods Enzymol. 2026;730:75-90. doi: 10.1016/bs.mie.2025.11.001. Epub 2025 Nov 27.

ABSTRACT

Type III polyketide synthases (T3PKSs) are a versatile family of enzymes that generate structurally diverse natural products with ecological and clinical importance. A major contributor to their broad product scope is their inherent substrate promiscuity. Accurately predicting the substrate scope of T3PKSs supports their application in biocatalysis and may offer clues about their biological roles. In this chapter, we present a machine learning-based workflow for predicting substrate specificity in fungal T3PKSs. We provide step-by-step protocols for (1) predicting the substrate scope of new T3PKSs, and (2) selecting suitable T3PKSs for the transformation of specific substrates. In addition, we provide instructions on re-training the machine learning model with custom enzymatic activity data. The workflow includes instructions for generating high-quality figures for publication.

PMID:42177064 | DOI:10.1016/bs.mie.2025.11.001