In:
Microbial Cell Factories, Springer Science and Business Media LLC, Vol. 21, No. 1 ( 2022-12)
Abstract:
Polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized by PHA synthases. Naturally occurring PHA copolymers possess a random monomer sequence. The development of PhaC AR , a unique sequence-regulating PHA synthase, has enabled the spontaneous biosynthesis of PHA block copolymers. PhaC AR synthesizes both a block copolymer poly(2-hydroxybutyrate)- b -poly(3-hydroxybutyrate) [P(2HB)- b -P(3HB)], and a random copolymer, poly(3HB- co -3-hydroxyhexanoate), indicating that the combination of monomers determines the monomer sequence. Therefore, in this study, we explored the substrate scope of PhaC AR and the monomer sequences of the resulting copolymers to identify the determinants of the monomer sequence. PhaC AR is a class I PHA synthase that is thought to incorporate long-main-chain hydroxyalkanoates (LMC HAs, 〉 C 3 in the main [backbone] chain). Thus, the LMC monomers, 4-hydroxy-2-methylbutyrate (4H2MB), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HHx), as well as 2HB, 3HB, and 3-hydroxypropionate (3HP) were tested. Results Recombinant Escherichia coli harboring PhaC AR , CoA transferase and CoA ligase genes was used for PHA production. The medium contained the monomer precursors, 2HB, 3HB, 3HP, 4H2MB, 5HV, and 6HHx, either individually or in combination. As a result, homopolymers were obtained only for 3HB and 3HP. Moreover, 3HB and 3HP were randomly copolymerized by PhaC AR . 3HB-based binary copolymers P(3HB- co -LMC HA)s containing up to 2.9 mol% 4H2MB, 4.8 mol% 5HV, or 1.8 mol% 6HHx were produced. Differential scanning calorimetry analysis of the copolymers indicated that P(3HB- co -LMC HA)s had a random sequence. In contrast, combining 3HP and 2HB induced the synthesis of P(3HP)- b -P(2HB). Similarly, P(2HB) segment-containing block copolymers P(3HB- co -LMC HA)- b -P(2HB)s were synthesized. Binary copolymers of LMC HAs and 2HB were not obtained, indicating that the 3HB or 3HP unit is essential to the polymer synthesis. Conclusion PhaC AR possesses a wide substrate scope towards 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates. 3HB or 3HP units are essential for polymer synthesis using PhaC AR . The presence of a 2HB monomer is key to synthesizing block copolymers, such as P(3HP)- b -P(2HB) and P(3HB- co -LMC HA)- b -P(2HB)s. The copolymers that did not contain 2HB units had a random sequence. This study’s results provide insights into the mechanism of sequence regulation by PhaC AR and pave the way for designing PHA block copolymers.
Type of Medium:
Online Resource
ISSN:
1475-2859
DOI:
10.1186/s12934-022-01811-7
Language:
English
Publisher:
Springer Science and Business Media LLC
Publication Date:
2022
detail.hit.zdb_id:
2091377-1
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