Chen Yi-Jr, Tsai Pei-Chien, Hsu Chun-Hua, Lee Chia-Yin
Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan; Department of Nursing, Chang Gung University of Science and Technology, Tao-Yuan 33333, Taiwan.
Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan.
Enzyme Microb Technol. 2014 Mar 5;56:60-6. doi: 10.1016/j.enzmictec.2014.01.005. Epub 2014 Jan 16.
This study describes protein model of type II Pseudomonas putida GPo1 synthase (PhaC1(Pp)) and using single or multiple points mutagenesis to identify the beneficial amino acid residues that change the PHA accumulation and the substrate chain-length specificity of type II PHA synthase. The P. putida GPp104 PHA⁻ was used as a host for evaluating the substrate specificity and PHA yield of the mutated PhaC1(Pp). The evolved PhaC1(Pp) were coexpressed with β-ketothiolase (phbA(Re)) and the acetoacetyl-CoA reductase (phbB(Re)) to supply sufficient short-chain length (R)-3-hydroxyacyl-CoA as a substrate. A single point mutation at L484V remarkably enhanced the monomer ratio of (R)-3-hydroxybutyrate in a PHA accumulation experiment. Saturation mutagenesis experiment at 484 concluded that Val is the most favorable amino acid in PhaC1(Pp) for incorporating (R)-3-hydroxybutyrate unit synthesis. In addition, a single mutation at Q481M, S482G and A547V obviously increased PHA yields. Q481M and S482G enhanced the (R)-3-hydroxyhexanoate monomer composition in the PHA accumulation by P. putida GPp104 PHA⁻. This is the first data that spotlighted the important effect of Leu484 on substrate specificity of PHA synthase and Ala547 on the PHA accumulation.
本研究描述了II型恶臭假单胞菌GPo1合酶（PhaC1(Pp)）的蛋白质模型，并通过单点或多点诱变来鉴定可改变II型PHA合酶的PHA积累和底物链长特异性的有益氨基酸残基。恶臭假单胞菌GPp104 PHA⁻被用作宿主，以评估突变的PhaC1(Pp)的底物特异性和PHA产量。进化后的PhaC1(Pp)与β-酮硫解酶（phbA(Re)）和乙酰乙酰辅酶A还原酶（phbB(Re)）共表达，以提供足够的短链长度(R)-3-羟基酰基辅酶A作为底物。在PHA积累实验中，L484V处的单点突变显著提高了(R)-3-羟基丁酸的单体比例。在484位点的饱和诱变实验得出结论，Val是PhaC1(Pp)中用于掺入(R)-3-羟基丁酸单元合成的最有利氨基酸。此外，Q481M、S482G和A547V处的单点突变明显提高了PHA产量。Q481M和S482G提高了恶臭假单胞菌GPp104 PHA⁻在PHA积累中(R)-3-羟基己酸的单体组成。这是首次突出亮氨酸484对PHA合酶底物特异性以及丙氨酸547对PHA积累重要作用的数据。
