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

Abstract

Abstract 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 (PhaCBP-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 PhaCBP-M-CPF4 through N-terminal truncation. Results The N-terminal truncated mutants of PhaCBP-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 PhaCBP-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 PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (M w) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher M w PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-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.

Keywords

• Polyhydroxyalkanoate synthase
• Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
• N-terminal truncation
• Molecular weights
• phaC gene expression
• PHA granule morphology