College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, China.
J Appl Microbiol. 2012 Feb;112(2):353-62. doi: 10.1111/j.1365-2672.2011.05206.x. Epub 2012 Jan 3.
In this study, we explored the possibility of construction of a 'universal targeting vector' by Red/ET recombination to inactivate L gene encoding 3-amino-5-hydroxybenzoic acid (AHBA)-oxidoreductase in AHBA biosynthetic gene cluster to facilitate the detection of ansamycins production in actinomycetes.
Based on the conserved regions of linked AHBA synthase (K), oxidoreductase (L) and phosphatase (M) gene clusters, degenerate primers were designed and PCR was performed to detect KLM gene clusters within 33 AHBA synthase gene-positive actinomycetes strains. Among them, 22 KLM gene cluster-positive strains were identified. A 'universal targeting vector' was further constructed using the 50-nt homologous sequences chosen from four strains internal L gene in KLM gene clusters through Red/ET recombination. The L gene from nine of the KLM gene cluster-positive actinomycetes strains was inactivated by insertion of a kanamycin (Km) resistance marker into its internal region from the 'universal targeting vector'. By comparison of the metabolites produced in parent strains with those in L gene-inactivated mutants, we demonstrated the possible ansamycins production produced by these strains. One strain (4089) was proved to be a geldanamycin producer. Three strains (3-20, 7-32 and 8-32) were identified as potential triene-ansamycins producers. Another strain (3-27) was possible to be a streptovaricin C producer. Strains 24-100 and 4-124 might be served as ansamitocin-like producers.
The results confirmed the feasibility that a 'universal targeting vector' could be constructed through Red/ET recombination using the conserved regions of KLM gene clusters to detect ansamycins production in actinomycetes.
The 'universal targeting vector' provides a rapid approach in certain degree to detect the potential ansamycin producers from the 22 KLM gene cluster-positive actinomycetes strains.
本研究通过 Red/ET 重组探索构建“通用靶向载体”的可能性，以灭活编码 3-氨基-5-羟基苯甲酸（AHBA）-氧化还原酶的 L 基因，从而促进放线菌中 ansamycin 产生的检测。
基于连接的 AHBA 合酶（K）、氧化还原酶（L）和磷酸酶（M）基因簇的保守区域，设计了简并引物，并进行 PCR 以检测 33 株 AHBA 合酶阳性放线菌菌株中的 KLM 基因簇。其中，鉴定出 22 株 KLM 基因簇阳性株。通过 Red/ET 重组，从 KLM 基因簇内的四个菌株内部 L 基因中选择 50nt 同源序列，进一步构建了“通用靶向载体”。通过将卡那霉素（Km）抗性标记物插入其内部区域，从“通用靶向载体”中失活了 9 株 KLM 基因簇阳性放线菌菌株的 L 基因。通过比较亲本菌株和 L 基因失活突变体产生的代谢产物，我们证明了这些菌株可能产生 ansamycin。一株（4089）被证明是格尔德霉素产生菌。三株（3-20、7-32 和 8-32）被鉴定为潜在的三烯-ansamycin 产生菌。另一株（3-27）可能是链道霉素 C 产生菌。菌株 24-100 和 4-124 可能是放线菌素样物质产生菌。
结果证实，通过 Red/ET 重组使用 KLM 基因簇的保守区域构建“通用靶向载体”以检测放线菌中 ansamycin 产生的可行性。
“通用靶向载体”为从 22 株 KLM 基因簇阳性放线菌菌株中快速筛选潜在的 ansamycin 产生菌提供了一种方法。
