Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230:: genes required for halogenation
Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230:: genes required for halogenation
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DOI：
10.1099/mic.0.26319-0
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发表时间：
2004-01-01
期刊：
影响因子：
2.8
通讯作者：
Vining, LC
中科院分区：
文献类型：
--
作者：
Piraee, M;White, RL;Vining, LC
从委内瑞拉链霉菌ISP 5230基因组DNA文库的一个片段中检测到5个ORF，通过与从代表已知卤化酶序列的共有序列的引物扩增的IPCIR产物杂交。测序和功能分析表明，ORFs 11和12（而不是ORFs 13-15）延伸了染色体上氯霉素（Cm）生物合成的部分特征基因簇。ORF 11（cmlK）或ORF 12（cmkS）的破坏和插入失活基因的接合转移到S.委内瑞拉产的突变菌株VS 1111和VS 1112，每一种都产生一系列类似的Cm类似物，其中未卤代的酰基取代了Cm的二氯乙酰基取代基。1H-1-NMR确定，在破碎的菌株中的主要代谢产物是α-N-丙酰基类似物。CmlK的序列牵连的蛋白质在腺苷酸化，并参与卤化推断从生物合成的类似物的cmlK破坏的突变体。在生成二氯乙酰基取代基的作用得到支持的部分恢复Cm生物合成时，将克隆拷贝的cmlK的反式引入VS 1111。突变体的互补还表明，cmlK的失活，而不是破坏cmlS表达的极性效应干扰了二氯乙酰基的生物合成。推测的CmlS序列类似于FADH（2）依赖性卤化酶的序列。将cmlK或cmlS接合转移到S中。委内瑞拉的cml-2是一种氯化缺陷型菌株，其具有遗传定位于Cm生物合成基因簇的突变，其不与cml-2损伤互补，这表明除了cmlK和cmlS之外还需要一个或多个基因来组装二氯乙酰基取代基。ORF 13的插入失活不影响Cm的产生，ORF 14和ORF 15的产物与天蓝色链霉菌A3（2）蛋白相匹配，该蛋白在Cm的生物合成中缺乏可能的功能。因此，cmlS似乎标记基因簇的下游末端。
Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a IPCIR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmkS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. H-1-NMR established that the principal metabolite in the disrupted strains was the alpha-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH(2)-dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.