Neoh_2024_Microb.Cell.Fact_23_52

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaC(BP-M-CPF4)) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaC(BP-M-CPF4) through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaC(BP-M-CPF4) were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaC(BP-M-CPF4) mutants were evaluated in C. necator mutant PHB(-)4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaC(BP-M-CPF4) showed a reduction in PHA content and cell dry weight except for PhaC(BP-M-CPF4) G8. PhaC(BP-M-CPF4) G8 and A27 showed an improved weight-average molecular weight (M(w)) of PHA produced due to lower expression of the truncated PhaC(BP-M-CPF4). Transformants harbouring PhaC(BP-M-CPF4) G8, A27, and T74 showed a reduction in the number of granules. PhaC(BP-M-CPF4) G8 produced higher M(w) PHA in mostly single larger PHA granules with comparable production as the full-length PhaC(BP-M-CPF4). CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, M(w), and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher M(w) in mostly single larger granules.