N-terminal truncation of PhaC_BP-M-CPF4 and its effect on PHA production

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.

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.

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.