Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany.
J Appl Microbiol. 2010 Sep;109(3):1067-75. doi: 10.1111/j.1365-2672.2010.04732.x.
Natural rubber (poly-[cis-1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA is a novel type of dihaem dioxygenase with unknown cleavage mechanism of the rubber carbon backbone. Analysis of mutant RoxA after mutagenesis could be a way to investigate the function of selected amino acids of RoxA during catalysis. Unfortunately, expression of functional RoxA in recombinant Escherichia coli or in recombinant γ-Proteobacteria such as Pseudomonas putida was not possible in our hands. Therefore, expression of recombinant RoxA in the homologous host, Xanthomonas, was performed.
A transformation system via electroporation was established, and a conjugation system was optimized for Xanthomonas sp. Inactivation of the chromosomal roxA gene by insertional mutagenesis resulted in inability of Xanthomonas sp. to produce active RoxA and to utilize rubber as a sole source of carbon and energy. When an intact copy of roxA was cloned under control of a rhamnose-inducible promoter in a broad host range vector and was transferred to Xanthomonas sp., high expression levels of functional RoxA in the presence of rhamnose were obtained.
Purification of recombinantly expressed RoxA was simplified because of drastically shortened fermentation times and because separation of RoxA from remaining rubber latex particles was not necessary with rhamnose-induced cultures. About 6 mg purified RoxA were obtained from 1 l of cell-free culture fluid. Purified recombinant RoxA was highly active and revealed comparable spectral properties as RoxA purified from the wild type. The results of our study are the methodical basis for molecular biological manipulation in Xanthomonas sp. and will simplify investigation into the biochemical mechanisms by which rubber can be biodegraded in the environment by this novel extracellular dihaem dioxygenase RoxA.
从黄单胞菌中分离出的橡胶氧合酶 A（RoxA）可将天然橡胶（聚-[顺式-1,4-异戊二烯]）切割成 12-氧代-4,8-二甲基十三碳-4,8-二烯-1-醛。RoxA 是一种新型的双血红素双加氧酶，其橡胶碳主链的断裂机制尚不清楚。对诱变后突变 RoxA 的分析可以作为研究 RoxA 催化过程中选定氨基酸功能的一种方法。不幸的是，我们无法在重组大肠杆菌或重组γ-变形菌（如假单胞菌）中表达功能性 RoxA。因此，在同源宿主黄单胞菌中表达重组 RoxA。
通过电穿孔建立了转化系统，并对黄单胞菌进行了优化。通过插入诱变使染色体 roxA 基因失活，导致黄单胞菌无法产生活性 RoxA，也无法将橡胶作为唯一的碳源和能源。当 roxA 完整基因在广泛宿主范围载体的鼠李糖诱导启动子控制下克隆并转移到黄单胞菌中时，在存在鼠李糖的情况下可获得功能性 RoxA 的高表达水平。
由于发酵时间大大缩短，并且由于不需要用鼠李糖诱导的培养物分离 RoxA 与剩余的橡胶乳胶颗粒，因此简化了重组表达 RoxA 的纯化。从 1 L 无细胞培养物中获得约 6 mg 纯化的 RoxA。纯化的重组 RoxA 具有很高的活性，显示出与从野生型中纯化的 RoxA 相当的光谱特性。我们的研究结果为黄单胞菌中的分子生物学操作提供了方法学基础，并将简化对环境中新型细胞外双血红素双加氧酶 RoxA 可生物降解橡胶的生化机制的研究。
