Structural insights and rational engineering strategies for modular polyketide synthases: A review.

Polyketide synthases (PKSs) are among the most complex enzymatic systems in nature, responsible for synthesizing a broad array of polyketides including antibiotics, antifungal agents, and immunosuppressants. Cis-Acyltransferase (AT) PKSs, distinguished by their multiple modules, diverse catalytic domains, and engineering flexibility, hold significant promise for synthetic biology and natural product discovery. However, even minor alterations to individual domains can propagate structural and functional changes throughout the assembly line, posing a major challenge to the rational design of cis-AT PKSs for diverse polyketides production. This review explores the multidimensionality of cis-AT PKS design, with a focus on the selecting modular building blocks and designing catalytic domains based on the structural and mechanistic insights. Modifications to acyltransferases, ketosynthases, and ketoreductase-dehydratase-enoylreductases can fine-tune substrate specificity and stereochemical complexity, while engineering of the thioesterase domain enables controlled hydrolysis or cyclization for precise polyketide tailoring. Key future directions in cis-AT engineering are also highlighted. Collectively, these insights support the adaptation of cis-AT PKS systems to enhance product yields and expand the repertoire of accessible polyketides. This review provides a systematic overview of cis-AT PKS architecture and engineering strategies, offering a valuable resource for researchers in the field.