Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaC(AR).
Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaC(AR).
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使用序列调节聚羟基链烷酸酯合酶PhaC（AR）生产由2-、3-、4-、5-和6-羟基链烷酸酯组成的无规和嵌段共聚物的通用脂肪族聚酯生物合成系统。
DOI：
10.1186/s12934-022-01811-7
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发表时间：
2022-05-14
影响因子：
6.4
通讯作者：
中科院分区：
文献类型：
--
作者：
聚羟基链烷酸酯 (PHA) 是由 PHA 合酶合成的微生物聚酯。天然存在的 PHA 共聚物具有随机单体序列。 PhaCAR（一种独特的序列调节 PHA 合酶）的开发使得 PHA 嵌段共聚物的自发生物合成成为可能。 PhaCAR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)，这表明单体的组合决定了单体序列。因此，在本研究中，我们探索了 PhaCAR 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。 PhaCAR 是一种 I 类 PHA 合酶，被认为掺入长主链羟基链烷酸酯（LMC HA，主链中 > C3）。因此，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)。携带 PhaCAR、CoA 转移酶和 CoA 连接酶基因的重组大肠杆菌用于 PHA 生产。该介质含有单独或组合的单体前体2HB、3HB、3HP、4H2MB、5HV和6HHx。结果，仅获得了3HB和3HP的均聚物。此外，3HB和3HP是通过PhaCAR随机共聚的。生产出含有高达 2.9 mol% 4H2MB、4.8 mol% 5HV 或 1.8 mol% 6HHx 的基于 3HB 的二元共聚物 P(3HB-co-LMC HA)。共聚物的差示扫描量热分析表明P(3HB-co-LMC HA)具有随机序列。相反，结合 3HP 和 2HB 诱导 P(3HP)-b-P(2HB) 的合成。类似地，合成了含有P(2HB)链段的嵌段共聚物P(3HB-co-LMC HA)-b-P(2HB)s。没有获得 LMC HA 和 2HB 的二元共聚物，表明 3HB 或 3HP 单元对于聚合物合成至关重要。 PhaCAR 对 2-、3-、4-、5- 和 6- 羟基链烷酸酯具有广泛的底物范围。 3HB 或 3HP 单元对于使用 PhaCAR 进行聚合物合成至关重要。 2HB单体的存在是合成嵌段共聚物的关键，例如P(3HP)-b-P(2HB)和P(3HB-co-LMC HA)-b-P(2HB)s。不含 2HB 单元的共聚物具有随机序列。这项研究的结果提供了对 PhaCAR 序列调控机制的见解，并为设计 PHA 嵌段共聚物铺平了道路。在线版本包含可在 10.1186/s12934-022-01811-7 获取的补充材料。
Polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized by PHA synthases. Naturally occurring PHA copolymers possess a random monomer sequence. The development of PhaCAR, a unique sequence-regulating PHA synthase, has enabled the spontaneous biosynthesis of PHA block copolymers. PhaCAR 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 PhaCAR and the monomer sequences of the resulting copolymers to identify the determinants of the monomer sequence. PhaCAR is a class I PHA synthase that is thought to incorporate long-main-chain hydroxyalkanoates (LMC HAs, > C3 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 PhaCAR, 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 PhaCAR. 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. PhaCAR possesses a wide substrate scope towards 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates. 3HB or 3HP units are essential for polymer synthesis using PhaCAR. 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 PhaCAR and pave the way for designing PHA block copolymers. The online version contains supplementary material available at 10.1186/s12934-022-01811-7.
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影响因子：
5
作者：
Danko M;Mosnáčková K;Vykydalová A;Kleinová A;Puškárová A;Pangallo D;Bujdoš M;Mosnáček J
通讯作者：
Mosnáček J
影响因子：
2.8
作者：
Goto, Saki;Hokamura, Ayaka;Matsusaki, Hiromi
通讯作者：
Matsusaki, Hiromi
影响因子：
6.2
作者：
Matsumoto, Ken'ichiro;Takase, Kazuma;Taguchi, Seiichi
通讯作者：
Taguchi, Seiichi
影响因子：
6.2
作者：
Matsumoto, Ken'ichiro;Terai, Satsuki;Taguchi, Seiichi
通讯作者：
Taguchi, Seiichi
影响因子：
5
作者：
Kato, M;Bao, HJ;Doi, Y
通讯作者：
Doi, Y