Donadio Stefano, Monciardini Paolo, Alduina Rosa, Mazza Paola, Chiocchini Claudia, Cavaletti Linda, Sosio Margherita, Puglia Anna Maria
Biosearch Italia, via R. Lepetit 34, 21040 Gerenzano(VA), Italy.
J Biotechnol. 2002 Nov 13;99(3):187-98. doi: 10.1016/s0168-1656(02)00209-2.
Soil microbes represent an important source of biologically active compounds. These molecules present original and unexpected structure and are selective inhibitors of their molecular targets. At Biosearch Italia, discovery of new bioactive molecules is mostly carried out through the exploitation of a proprietary strain collection of over 50000 strains, mostly unusual genera of actinomycetes and uncommon filamentous fungi. A critical element in a drug discovery based on microbial extracts is the isolation of unexploited groups of microorganisms that are at the same time good producers of secondary metabolites. Molecular genetics can assist in these efforts. We will review the development and application of molecular methods for the detection of uncommon genera of actinomycetes in soil DNA and for the rapid dereplication of actinomycete isolates. The results indicate a substantial presence in many soils of the uncommon genera and a large diversity of isolated actinomycetes. However, while uncommon actinomycete strains may provide an increased chance of yielding novel structures, their genetics and physiology are poorly understood. To speed up their manipulation, we have developed vectors capable of stably maintaining large segments of actinomycete DNA in Escherichia coli and of integrating site specifically in the Streptomyces genome. These vectors are suitable for the reconstruction of gene clusters from smaller segment of cloned DNA, the preparation of large-insert libraries from unusual actinomycete strains and the construction of environmental libraries.
土壤微生物是生物活性化合物的重要来源。这些分子具有独特且意想不到的结构，是其分子靶点的选择性抑制剂。在意大利生物搜索公司，新生物活性分子的发现主要通过利用一个拥有超过50000个菌株的专有菌株库来进行，这些菌株大多是不常见的放线菌属和罕见的丝状真菌。基于微生物提取物的药物发现中的一个关键要素是分离未被开发利用但同时又是次生代谢产物良好生产者的微生物群体。分子遗传学可以助力这些工作。我们将综述用于检测土壤DNA中不常见放线菌属以及放线菌分离株快速去重复的分子方法的发展与应用。结果表明，许多土壤中大量存在不常见的放线菌属，且分离出的放线菌具有丰富的多样性。然而，尽管不常见的放线菌菌株可能提供更多产生新结构的机会，但其遗传学和生理学却知之甚少。为了加速对它们的操作，我们开发了能够在大肠杆菌中稳定维持大片段放线菌DNA并能特异性整合到链霉菌基因组中的载体。这些载体适用于从克隆DNA的较小片段重建基因簇、从不常见放线菌菌株制备大插入片段文库以及构建环境文库。
