Sivashankari Ramamoorthi M, Mierzati Maierwufu, Miyahara Yuki, Mizuno Shoji, Nomura Christopher T, Taguchi Seiichi, Abe Hideki, Tsuge Takeharu
Department of Materials Science and Engineering, Tokyo Institute of Technology, Yokohama, Japan.
Department of Biological Sciences, College of Science, University of Idaho, Moscow, ID, United States.
Front Bioeng Biotechnol. 2023 Feb 21;11:1114946. doi: 10.3389/fbioe.2023.1114946. eCollection 2023.
Polyhydroxyalkanoate (PHA) synthases (PhaCs) are key enzymes in PHA polymerization. PhaCs with broad substrate specificity are attractive for synthesizing structurally diverse PHAs. In the PHA family, 3-hydroxybutyrate (3HB)-based copolymers are industrially produced using Class I PhaCs and can be used as practical biodegradable thermoplastics. However, Class I PhaCs with broad substrate specificities are scarce, prompting our search for novel PhaCs. In this study, four new PhaCs from the bacteria , , , and were selected a homology search against the GenBank database, using the amino acid sequence of PHA synthase (PhaC), a Class I enzyme with a wide range of substrate specificities, as a template. The four PhaCs were characterized in terms of their polymerization ability and substrate specificity, using as a host for PHA production. All the new PhaCs were able to synthesize P(3HB) in with a high molecular weight, surpassing PhaC. The substrate specificity of PhaCs was evaluated by synthesizing 3HB-based copolymers with 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate, 3-hydroxy-2-methylbutyrate, and 3-hydroxypivalate monomers. Interestingly, PhaC from (PhaC) exhibited relatively broad substrate specificity. PhaC was further engineered through site-directed mutagenesis, and the variant resulted in an enzyme with improved polymerization ability and substrate specificity.
聚羟基脂肪酸酯（PHA）合成酶（PhaCs）是PHA聚合反应中的关键酶。具有广泛底物特异性的PhaCs对于合成结构多样的PHA具有吸引力。在PHA家族中，基于3-羟基丁酸（3HB）的共聚物是使用I类PhaCs进行工业生产的，并且可以用作实用的可生物降解热塑性塑料。然而，具有广泛底物特异性的I类PhaCs很少，这促使我们寻找新型PhaCs。在本研究中，通过针对GenBank数据库进行同源性搜索，以具有广泛底物特异性的I类酶PHA合成酶（PhaC）的氨基酸序列作为模板，从细菌、、和中筛选出四种新的PhaCs。以作为PHA生产的宿主，对这四种PhaCs的聚合能力和底物特异性进行了表征。所有新的PhaCs都能够在中合成高分子量的聚（3HB），超过了PhaC。通过与3-羟基己酸、3-羟基-4-甲基戊酸、3-羟基-2-甲基丁酸和3-羟基新戊酸单体合成基于3HB的共聚物，评估了PhaCs的底物特异性。有趣的是，来自的PhaC（PhaC）表现出相对广泛的底物特异性。通过定点诱变对PhaC进行了进一步改造，该变体产生了一种聚合能力和底物特异性都有所提高的酶。
