Rational design of chimeric polyhydroxyalkanoate synthases suggests a conserved N-terminal domain-to-LID region network influencing 3HHx incorporation and granule morphology.

The global microplastic crisis and the suboptimal material properties of biodegradable polyhydroxyalkanoates (PHAs) necessitate the engineering of efficient PHA synthases (PhaCs). However, the narrow substrate specificity of natural PhaCs limits the production of tailored PHA copolymers. Here we report a bioinformatics-guided strategy for the rational design of functional chimeric PhaCs, achieving a 100% success rate in generating 16 active chimeras from two complementary class I enzymes (high-activity PhaCCs and high-3HHx-incorporating PhaCBP-M-CPF4). The resulting chimeras, notably PhaCCSBP1, PhaCCSBP3, PhaCCSBP4, and PhaCCSBP5, combine high PHA yield, preserved 3HHx incorporation (up to 13.3 mol%), and a single-granule morphology ideal for downstream processing. Domain swapping and truncation experiments indicate that the catalytic domain primarily influences 3HHx incorporation and granule morphology, whereas the N-terminal domain (NTD) plays a modulatory role in both 3HHx incorporation and granule morphology. Structural inspection of the 3D model suggests a conserved hydrogen bond network between the NTD and the LID region of the CAP subdomain, formed by the last three α-helices of the NTD; disruption of this network is proposed to impair LID mobility and selectively exclude bulky 3-hydroxyhexanoate-CoA (3HHx-CoA). This work provides a predictive framework for chimerization of PhaCs, identifies a potential allosteric engineering target, and a mechanistic hypothesis for substrate gating.