Niamsiri Nuttawee, Delamarre Soazig C, Kim Young-Rok, Batt Carl A
Department of Food Science, Stocking Hall, Cornell University, Ithaca, New York 14853, USA.
Appl Environ Microbiol. 2004 Nov;70(11):6789-99. doi: 10.1128/AEM.70.11.6789-6799.2004.
PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.
聚羟基脂肪酸酯合酶（PHA synthase）是参与聚羟基脂肪酸酯（PHA）生物合成的关键酶。我们采用组合遗传策略构建独特的嵌合II类PHA合酶，获得了一些具有改进催化特性的新型嵌合体。为构建嵌合PHA合酶，我们从食油假单胞菌（Pseudomonas oleovorans）构建了一个缺失内部540 bp片段的合成phaC基因（phaC1Po）。使用从土壤中分离的基因组DNA生成随机扩增的PCR产物（使用基于缺失内部片段侧翼保守phaC序列的引物创建），并替换540 bp内部区域。嵌合基因在真养产碱菌（Ralstonia eutropha）的PHA阴性菌株PHB(-)4（DSM 541）中表达。在筛选PHA产生的1478个重组克隆中，我们获得了五个不同的嵌合phaC1Po基因，它们产生的PHA比天然phaC1Po更多。嵌合体S1-71、S4-8、S5-58、S3-69和S3-44的体内活性水平分别提高了1.3倍、1.4倍、2.0倍、2.1倍和3.0倍。所有突变体介导合成的PHA中3-羟基辛酸的摩尔分数略有增加；然而，所有情况下PHA的重均分子量（Mw）几乎保持不变。基于DNA序列分析，各种phaC片段似乎起源于荧光假单胞菌（Pseudomonas fluorescens）和金黄色假单胞菌（Pseudomonas aureofaciens）。氨基酸序列分析表明，嵌合蛋白与野生型phaC1Po有17至20个氨基酸差异，且这些差异集中在五个嵌合克隆的相同位置。基于该酶与稻瘟病菌脂肪酶的同源性开发的PhaC1Po穿线模型表明，活性嵌合体中发现的氨基酸取代大多位于蛋白质模型表面。因此，我们的组合基因工程策略被证明在提高PHA合酶的催化活性方面具有广泛的用途。
