Engineering chimeric polyhydroxyalkanoate synthases for enhanced copolymerization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate): A promising biotechnological approach

The escalating health and environmental threats posed by microplastics and nanoplastics (MNPs) highlight the urgent need for sustainable alternatives like polyhydroxyalkanoates (PHAs), biodegradable polyesters synthesized by bacterial PHA synthases (PhaCs). However, natural PhaCs exhibit suboptimal substrate specificity and polymer heterogeneity, limiting industrial scalability. To address this, chimeric PhaCs were engineered by swapping N-terminal domains between PhaC from mangrove soil metagenome (PhaC; low 3-hydroxyhexanoate [3HHx] content, fewer but larger granules) and PhaC2 of Rhodococcus aetherivorans I24 (PhaC2; high 3HHx, numerous small granules). This strategy aimed to combine enhanced 3HHx incorporation with controlled granule morphology. Using structural predictions, chimeric enzymes were constructed and tested, revealing that the C-terminal domain retained compatibility with diverse N-terminal regions. The resulting chimeras exhibited improved PHA production, enhanced 3HHx incorporation, and optimized granule formation, overcoming historical challenges in chimeric enzyme design by avoiding β-strand interference. Among the chimeras, distinct strains achieved: (i) up to 200 % increase in PHA production; (ii) up to 45 mol% 3HHx incorporation; and (iii) optimized granule formation, approaching a single-granule-per-cell phenotype (mean count: 1.079) and a granule size increase of up to 7.2-fold (mean area: 1.272 µm). This approach provides a robust framework for tailoring PhaCs to produce high-performance copolymers. By elucidating domain compatibility, the study advances strategies in synthetic biology for creating modular enzymes with tailored functionalities, offering transformative potential in sustainable materials, protein engineering, and innovation in biodegradable plastics.