Long-chain N-acyl amino acid antibiotics isolated from heterologously expressed environmental DNA
Long-chain N-acyl amino acid antibiotics isolated from heterologously expressed environmental DNA
复制标题
DOI：
10.1021/ja002990u
复制
发表时间：
2000-12-27
影响因子：
15
通讯作者：
Clardy, J
中科院分区：
文献类型：
--
作者：
Brady, SF;Clardy, J
极少数的土壤微生物可以很容易地用标准技术培养，大约0.1%到1.0%，1产生一系列壮观的生物活性天然产物。未培养的大多数可能产生具有与培养的微生物相似的化学多样性和生物活性的天然产物。为了获得由未培养的微生物产生的天然产物，2构建了直接从土壤样品中提取的DNA（环境DNA，eDNA）的粘粒文库，并筛选了生物活性小分子的产生。3其中一个活性粘粒克隆，CSL 12，产生一系列长链N-酰基-L-酪氨酸抗生素。长链N-酰基氨基酸是细菌天然产物的一个不断增长的家族，其生物合成基因尚未被鉴定。对克隆在CSL 12中的eDNA的分析表明，单个开放阅读框（ORF）负责这些抗生素的产生，从而导致我们认为是第一个长链N-酰基氨基酸生物合成基因的鉴定。在这篇文章中，我们报告了这些新的天然产物抗生素的特征和长链N-酰基氨基酸合酶的序列。一个连续的两种抗生素的选择方案被用来识别，然后恢复粘粒克隆产生抗菌活性。eDNA的粘粒文库最初是在含有卡那霉素的LB平板上选择的，并在30 ℃下孵育2-4天.然后用含有卡那霉素抗性枯草芽孢杆菌的顶层琼脂覆盖成熟菌落。在30 ℃下再孵育24小时后，在B中产生生长抑制区的菌落。通过将从活性菌落中挑选的细菌划线到含有氨苄青霉素（50 μg/mL）的LB平板上来回收指示产生抗菌活性的枯草菌苔。第二个选择是氨苄青霉素，删除了B。枯草杆菌测定菌株，并允许直接从测定板回收抗菌命中物。在筛选的约70万个克隆中，发现了65个具有抗菌活性的菌落。由于我们对小分子抗生素最感兴趣，因此从小规模培养的活性克隆的乙酸乙酯提取物进行了抗菌活性测定。选择产生非常活性的有机提取物的克隆之一CSL 12用于进一步表征。将纯化的CSL 12粘粒转化E. coli中的活性成分，表明克隆的eDNA是引起所观察到的活性的原因。枯草芽孢杆菌从在LB（30 μg/mL卡那霉素）中于30 ℃生长60小时的中和培养物中获得粗乙酸乙酯提取物.乙酸乙酯萃取物通过正相快速色谱法（CHCl 3：MeOH分步梯度，含0.1% HOAc）进行分配，然后将从硅胶柱洗脱的活性物质通过反相LC-CN快速色谱法（CH 3CN：H2O分步梯度，含0.1%三乙胺）进一步分配。通过反相HPLC从从第二个快速柱洗脱的抗菌活性物质中分离出十三种通常命名为CSL 12-A至CSL 12-M的相关化合物。4来自CSL 12的3.5kb BamH I亚克隆CSL 12的乙酸乙酯提取物的活性物质。1，产生相同的反相HPLC迹线，并且该亚克隆用于我们的表征研究的剩余部分。
The tiny minority of soil microorganisms that can easily be cultured with standard techniques, roughly 0.1 to 1.0%, 1 produce a spectacular array of biologically active natural products. The uncultured majority likely produces natural products with chemical diversity and biological activity similar to that of cultured microorganisms. To access the natural products produced by uncultured microorganisms, 2 a cosmid library of DNA extracted directly from soil samples (environmental DNA, eDNA) was constructed and screened for the production of biologically active small molecules. 3 One of the active cosmid clones, CSL12, produces a series of long-chain N-acyl-L-tyrosine antibiotics. Long-chain N-acyl amino acids are a growing family of bacterial natural products for which no biosynthesis genes have yet been identified. Analysis of the eDNA cloned in CSL12 indicated that a single open reading frame (ORF) was responsible for the production of these antibiotics and thus led to the identification of what we believe to be the first long-chain N-acyl amino acid biosynthesis gene. In this communication we report the characterization of these new natural product antibiotics and the sequence for a long-chain N-acyl amino acid synthase. A sequential two antibiotic selection scheme was used to identify and then recover cosmid clones that produce antibacterial activities. The cosmid library of eDNA was originally selected on LB plates containing kanamycin and allowed to incubate at 30 C for 2-4 days. The mature colonies were then overlayed with top agar containing kanamycin resistant Bacillus subtilis. After an additional 24 h of incubation at 30 C, colonies that produced zones of growth inhibition in the B. subtilis lawn, indicating the production of antibacterial activity, were recovered by streaking bacteria picked from the active colonies onto LB plates containing ampicillin (50 μg/mL). The second selection, on ampicillin, removes the B. subtilis assay strain and allows for the recovery of antibacterial hits directly from the assay plates. Of the approximately 700 000 clones screened, 65 antibacterial active colonies were found. Since we were most interested in small molecule antibiotics, ethyl acetate extracts from small-scale cultures of the active clones were assayed for antibacterial activity. One of the clones that produced a very active organic extract, CSL12, was chosen for further characterization. When the purified cosmid from CSL12 was retransformed into E. coli, it continued to confer antibacterial activity indicating that the cloned eDNA was responsible for the observed activity.The active constituents in the ethyl acetate extract from cultures of CSL12 were isolated using a bioassay-guided fractionation against B. subtilis. Crude ethyl acetate extracts were obtained from neutralized cultures grown in LB (30 μg/mL of kanamycin) at 30 C for 60 h. The ethyl acetate extract was partitioned by normal phase flash chromatography (CHCl3: MeOH step gradient with 0.1% HOAc) and the active material that eluted from the silica column was then further partitioned by reversed-phase LC-CN flash chromatography (CH3CN: H2O step gradient with 0.1% triethylamine). Thirteen related compounds trivially named CSL12-A through CSL12-M were isolated by reversed-phase HPLC from the antibacterial active material that eluted from the second flash column. 4 The active material from the ethyl acetate extract of a 3.5 kb BamH I subclone of CSL12, CSL12. 1, produced an identical reversed-phase HPLC trace, and this subclone was used for the remainder of our characterization studies.