Abstract
Polyketides represent a diverse class of natural products that serve as major sources of medicinal compounds. Their biosynthesis is generally catalyzed by multimodular polyketide synthase comprising functional domains, such as a β-ketosynthase, an acyltransferase, and an acyl carrier protein. These domains mediate the elongation of polyketide chains via decarboxylative Claisen-like condensation. A reductive loop comprising β-ketoreductase (KR), dehydratase (DH), and enoyl reductase domains converts the β-keto group into a hydroxy group, alkene, and alkane, respectively. Particularly, the KR domains are pivotal in determining the stereochemical configurations of the hydroxy and methyl groups on the macrolide backbone and are classified into A1, A2, B1, B2, and C types. In this study, we performed a reductive loop exchange using pikromycin PKS, PikAIII module 5 (PikAIII-M5) as a template. The PikAIII-M5 was derived from the pikromycin biosynthetic gene cluster of the pikromycin-producing Streptomyces sp. AM4900. Next, we constructed a chimeric enzyme by replacing the KR domain of PikAIII-M5 with a DH-KR di-domain derived from PikAI module 2 (PikAI-M2), followed by the artificial addition of a thioesterase domain derived from PikAIV module 6. Thereafter, we evaluated the enzymatic activity of the construct using various chemically synthesized N-acetylcysteamine substrate analogs. This demonstrated that the chimeric module enzyme catalyzed the formation of (2R,3R,4S)-3-hydroxy-2,4-dimethylheptanoic acid, indicating that the KR domain of PikAI-M2 is a B1-type. These findings offer insights into the unresolved classification of KR domains that do not strictly conform to the Caffrey motif.