Directed Evolution of Sequence-Regulating Polyhydroxyalkanoate Synthase to Synthesize a Medium-Chain-Length?Short-Chain-Length (MCL?SCL) Block Copolymer
Directed Evolution of Sequence-Regulating Polyhydroxyalkanoate Synthase to Synthesize a Medium-Chain-Length?Short-Chain-Length (MCL?SCL) Block Copolymer
复制标题
序列调节聚羟基脂肪酸酯合成酶的定向进化合成中链长短链长（MCL？SCL）嵌段共聚物
DOI：
10.1021/acs.biomac.1c01480
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发表时间：
2022
影响因子：
6.2
通讯作者：
Matsumoto Ken’ichiro
中科院分区：
文献类型：
--
作者：
Phan Hien Thi;Hosoe Yumi;Guex Maureen;Tomoi Masayoshi;Tomita Hiroya;Zinn Manfred;Matsumoto Ken’ichiro
序列调节聚羟基烷酸合成酶PhaCAR是由鱼子气单胞菌和真营养拉尔斯氏菌的PHAC组成的嵌合酶。它从单体底物的混合物中自发合成了短链(SCl，poly(2-hydroxybutyrate)-b-poly(3-hydroxybutyrate)C5)嵌段共聚物≤[P(2HB)-b-P(3HB)]。在这项研究中，PhaCAR的定向进化被用来提高其向中链长度(MCL，C6-12)单体3-羟基己酰(3HHx)-辅酶A(CoA)的活性。在大肠杆菌P(3HB-co-3HHx)产生的基础上进行随机诱变和选择，发现有益突变N149D和F314L增加了3HHx组分。与催化中心C315相邻的314位的定点饱和突变表明，F314H合成了P(3HHx)均聚物。与亲本相比，F314H突变体对3HHx-CoA的活性增强，而对3HB-CoA的活性降低。AlphaFold2预测的PhaCAR的三级结构为有益突变的机制提供了洞察力。此外，这一发现使得合成了一种新的PHA嵌段共聚物P(3HHx)-b-P(2HB)。溶剂分级表明聚合物链段之间存在共价键。这种新型的MCL-SCL嵌段共聚物极大地扩展了PHA嵌段共聚物的分子设计范围。
Sequence-regulating polyhydroxyalkanoate synthase PhaCARis a chimeric enzyme comprising PhaCs fromAeromonas caviaeandRalstonia eutropha(Cupriavidus necator). It spontaneously synthesizes a short-chain-length (SCL, ≤C5) block copolymer poly(2-hydroxybutyrate)-b-poly(3-hydroxybutyrate) [P(2HB)-b-P(3HB)] from a mixture of monomer substrates. In this study, directed evolution of PhaCARwas performed to increase its activity toward a medium-chain-length (MCL, C6–12) monomer, 3-hydroxyhexanoyl (3HHx)-coenzyme A (CoA). Random mutagenesis and selection based on P(3HB-co-3HHx) production inEscherichia colifound that beneficial mutations N149D and F314L increase the 3HHx fraction. The site-directed saturation mutagenesis at position 314, which is adjacent to the catalytic center C315, demonstrated that F314H synthesizes the P(3HHx) homopolymer. The F314H mutant exhibited increased activity toward 3HHx-CoA compared with the parent enzyme, whereas the activity toward 3HB-CoA decreased. The predicted tertiary structure of PhaCARby AlphaFold2 provided insight into the mechanism of the beneficial mutations. In addition, this finding enabled the synthesis of a new PHA block copolymer, P(3HHx)-b-P(2HB). Solvent fractionation indicated the presence of a covalent linkage between the polymer segments. This novel MCL–SCL block copolymer considerably expands the range of the molecular design of PHA block copolymers.