Gross Frank, Gottschalk Daniela, Müller Rolf
Institut für Pharmazeutische Biologie, Technische Universität Carolo-Wilhelmina, Mendelssohnstrasse 1, 38106 Braunschweig, Germany.
Appl Microbiol Biotechnol. 2005 Jul;68(1):66-74. doi: 10.1007/s00253-004-1836-7. Epub 2005 Jan 6.
We demonstrate the ability of Pseudomonas putida KT2440, Pseudomonas syringae pv. tomato DC3000 and Pseudomonas stutzeri DSM10701 to posttranslationally activate carrier protein (CP) domains of various polyketide synthases, nonribosomal peptide synthetases, and fatty acid synthase by their intrinsic phosphopantetheinyl transferase. The apo-form is modified to the holo-form of the CP by attaching a phosphopantetheine moiety from coenzymeA to a conserved serine residue. The coding regions of the respective domains were cloned in order to generate C-terminal fusions with intein-chitin. The constructs were subcloned into a broad host range vector and transferred into the three pseudomonad hosts. The resulting recombinant pseudomonad strains were cultivated and each fusion protein was purified by affinity chromatography. Each purified CP was analysed using MALDI/TOF for the expected mass increase. Of the seven CPs tested, six could be purified from P. putida, which was chosen as the general host strain. Out of the six domains, five were completely activated, whereas only 5% of the protein of the sixth domain was in holo-form. Four domains were also expressed in the other hosts.
我们证明了恶臭假单胞菌KT2440、丁香假单胞菌番茄致病变种DC3000和施氏假单胞菌DSM10701能够通过其内在的磷酸泛酰巯基乙胺基转移酶对各种聚酮合酶、非核糖体肽合成酶和脂肪酸合酶的载体蛋白（CP）结构域进行翻译后激活。通过将辅酶A的磷酸泛酰巯基乙胺部分连接到保守的丝氨酸残基上，无辅基形式被修饰为CP的全酶形式。克隆了各个结构域的编码区，以产生与内含肽-几丁质的C端融合体。将构建体亚克隆到广泛宿主范围的载体中，并转移到三种假单胞菌宿主中。培养所得的重组假单胞菌菌株，并用亲和色谱法纯化每种融合蛋白。使用基质辅助激光解吸电离/飞行时间质谱（MALDI/TOF）分析每种纯化的CP，以确定预期的质量增加。在测试的七个CP中，有六个可以从恶臭假单胞菌中纯化出来，恶臭假单胞菌被选为通用宿主菌株。在这六个结构域中，有五个被完全激活，而第六个结构域只有5%的蛋白质处于全酶形式。另外四个结构域也在其他宿主中表达。
