CLONING AND EXPRESSION IN ESCHERICHIA-COLI OF THE ALCALIGENES-EUTROPHUS H16 POLY-BETA-HYDROXYBUTYRATE BIOSYNTHETIC-PATHWAY
CLONING AND EXPRESSION IN ESCHERICHIA-COLI OF THE ALCALIGENES-EUTROPHUS H16 POLY-BETA-HYDROXYBUTYRATE BIOSYNTHETIC-PATHWAY
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DOI：
10.1128/jb.170.10.4431-4436.1988
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发表时间：
1988-10-01
影响因子：
3.2
通讯作者：
DENNIS, DE
中科院分区：
文献类型：
--
作者：
SLATER, SC;VOIGE, WH;DENNIS, DE
聚-β-克隆了真养产碱杆菌H16合成羟基丁酸（PHB）途径的基因，并在大肠杆菌中表达。最初，A。通过使用粘粒pVK 102构建真养型H16基因组文库，并通过测定途径的第一种酶β-β-葡聚糖酶来寻找编码PHB生物合成途径的粘粒克隆。酮硫解酶鉴定了6个酶阳性克隆。其中三个克隆表现出乙酰乙酰辅酶A还原酶活性，生物合成途径的第二个酶，并积累了PHB。在粘粒克隆中产生的PHB水平约为A.真养型对一个粘粒克隆进行亚克隆实验，并在5.2-内切酶KpnI-EcoRI片段上分离出PHB生物合成途径。将该片段克隆到多拷贝小载体中，可以指导大肠杆菌中的PHB合成。大肠杆菌的水平接近80%的细菌细胞干重。
The poly-.beta.-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, .beta.-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme ofthe biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.