Graduate School of Chemical Sciences and Engineering, Hokkaido University, N13W8, Kitaku, Sapporo, 060-8628, Japan.
Department of Engineering, Hokkaido University, N13W8, Kitaku, Sapporo, 060-8628, Japan.
Microb Cell Fact. 2022 May 14;21(1):84. doi: 10.1186/s12934-022-01811-7.
Polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized by PHA synthases. Naturally occurring PHA copolymers possess a random monomer sequence. The development of PhaC, a unique sequence-regulating PHA synthase, has enabled the spontaneous biosynthesis of PHA block copolymers. PhaC 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 and the monomer sequences of the resulting copolymers to identify the determinants of the monomer sequence. PhaC is a class I PHA synthase that is thought to incorporate long-main-chain hydroxyalkanoates (LMC HAs, > C 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.
Recombinant Escherichia coli harboring PhaC, 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. 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.
PhaC possesses a wide substrate scope towards 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates. 3HB or 3HP units are essential for polymer synthesis using PhaC. 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 and pave the way for designing PHA block copolymers.
聚羟基脂肪酸酯（PHA）是由 PHA 合酶合成的微生物聚酯。天然存在的 PHA 共聚物具有随机的单体序列。独特的序列调节 PHA 合酶 PhaC 的开发，使 PHA 嵌段共聚物的自发生物合成成为可能。PhaC 合成了嵌段共聚物聚（2-羟基丁酸酯）-b-聚（3-羟基丁酸酯）[P（2HB）-b-P（3HB）]和无规共聚物，聚（3HB-co-3-羟基己酸酯），表明单体的组合决定了单体序列。因此，在这项研究中，我们探索了 PhaC 的底物范围和所得共聚物的单体序列，以确定单体序列的决定因素。PhaC 是一种 I 类 PHA 合酶，被认为可以掺入长主链羟基烷酸酯（LMC HAs，主链中> C）。因此，我们测试了 LMC 单体 4-羟基-2-甲基丁酸酯（4H2MB）、5-羟基戊酸酯（5HV）和 6-羟基己酸酯（6HHx）以及 2HB、3HB 和 3-羟基丙酸酯（3HP）。
含有 PhaC、CoA 转移酶和 CoA 连接酶基因的重组大肠杆菌被用于 PHA 生产。培养基中含有单体前体 2HB、3HB、3HP、4H2MB、5HV 和 6HHx，单独或组合使用。结果仅获得了 3HB 和 3HP 的均聚物。此外，PhaC 随机共聚 3HB 和 3HP。通过 PhaC 生产了含有高达 2.9 mol% 4H2MB、4.8 mol% 5HV 或 1.8 mol% 6HHx 的 3HB 为基础的二元共聚物 P（3HB-co-LMC HA）s。共聚的差示扫描量热法分析表明，P（3HB-co-LMC HA）s 具有无规序列。相比之下，将 3HP 和 2HB 结合在一起会诱导 P（3HP）-b-P（2HB）的合成。同样，合成了含有 P（2HB）段的嵌段共聚物 P（3HB-co-LMC HA）-b-P（2HB）s。未获得 LMC HAs 和 2HB 的二元共聚物，表明 3HB 或 3HP 单元对于聚合物合成是必不可少的。
PhaC 对 2-、3-、4-、5-和 6-羟基烷酸酯具有广泛的底物范围。对于 PhaC 介导的聚合物合成，3HB 或 3HP 单元是必不可少的。2HB 单体的存在是合成嵌段共聚物（如 P（3HP）-b-P（2HB）和 P（3HB-co-LMC HA）-b-P（2HB）s）的关键。不含 2HB 单元的共聚物具有无规序列。本研究结果为 PhaC 序列调节机制提供了新的见解，并为设计 PHA 嵌段共聚物铺平了道路。
