Chai Yi, Shan Shiping, Weissman Kira J, Hu Shengbiao, Zhang Youming, Müller Rolf
Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Department of Pharmaceutical Biotechnology, Saarland University, Saarbrücken 66041, Germany.
Chem Biol. 2012 Mar 23;19(3):361-71. doi: 10.1016/j.chembiol.2012.01.007.
Although the tubulysin (tub) biosynthetic gene cluster has been located in two myxobacterial strains, it appears in both cases to be incomplete as obvious candidates for acyl transfer and oxidation functions are lacking. Here, we report the engineering of a heterologous expression system for the tub biosynthetic pathway from strain Cystobacter sp. SBCb004. The entire tub core cluster was reconstituted from two cosmids using Red/ET recombineering and heterologous expression achieved in strains Pseudomonas putida and Myxococcus xanthus. Availability of the heterologous expression system and the natural producer strain SBCb004 provided a platform for the functional investigation of various biosynthetic genes by targeted inactivation. In addition, BLAST analysis of SBCb004 genome data was used to identify multiple candidate monooxygenases, whose involvement in tubulysin assembly was evaluated using a combination of knockout mutagenesis and heterologous expression.
尽管在两种粘细菌菌株中已定位到微管溶素（tub）生物合成基因簇，但在这两种情况下它似乎都是不完整的，因为缺乏明显的酰基转移和氧化功能候选基因。在此，我们报道了来自囊肿杆菌属菌株SBCb004的tub生物合成途径的异源表达系统的构建。使用Red/ET重组工程从两个粘粒中重构了完整的tub核心簇，并在恶臭假单胞菌和黄色粘球菌菌株中实现了异源表达。异源表达系统和天然生产菌株SBCb004的可用性为通过靶向失活对各种生物合成基因进行功能研究提供了一个平台。此外，利用SBCb004基因组数据的BLAST分析来鉴定多个候选单加氧酶，通过敲除诱变和异源表达相结合的方法评估了它们在微管溶素组装中的作用。
