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首页> 外文期刊>Applied Microbiology >Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis
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Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis

机译：辣椒素生物合成中脱氧糖部分的形成和附着以及基因簇的组装

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Caprazamycins are antimycobacterials produced by Streptomyces sp. MK730-62F2. Previously, cosmid cpzLK09 was shown to direct the biosynthesis of caprazamycin aglycones, but not of intact caprazamycins. Sequence analysis of cpzLK09 identified 23 genes involved in the formation of the caprazamycin aglycones and the transfer and methylation of the sugar moiety, together with genes for resistance, transport, and regulation. In this study, coexpression of cpzLK09 in Streptomyces coelicolor M512 with pRHAM, containing all the required genes for dTDP-l-rhamnose biosynthesis, led to the production of intact caprazamycins. In vitro studies showed that Cpz31 is responsible for the attachment of the l-rhamnose to the caprazamycin aglycones, generating a rare acylated deoxyhexose. An l-rhamnose gene cluster was identified elsewhere on the Streptomyces sp. MK730-62F2 genome, and its involvement in caprazamycin formation was demonstrated by insertional inactivation of cpzDIII . The l-rhamnose subcluster was assembled with cpzLK09 using Red/ET-mediated recombination. Heterologous expression of the resulting cosmid, cpzEW07, led to the production of caprazamycins, demonstrating that both sets of genes are required for caprazamycin biosynthesis. Knockouts of cpzDI and cpzDV in the l-rhamnose subcluster confirmed that four genes, cpzDII , cpzDIII , cpzDIV , and cpzDVI , are sufficient for the biosynthesis of the deoxysugar moiety. The presented recombineering strategy may provide a useful tool for the assembly of biosynthetic building blocks for heterologous production of microbial compounds.
机译：卡帕霉素是由链霉菌属产生的抗分枝杆菌。 MK730-62F2。以前，粘粒cpzLK09可以指导辣椒素苷元的生物合成，但不能指导完整的辣椒素。 cpzLK09的序列分析确定了23个基因与辣椒素糖苷配基的形成以及糖部分的转移和甲基化，以及抗性，转运和调控的基因。在这项研究中，cpzLK09在coelicolor链霉菌M512中与pRHAM共表达，其中包含dTDP-1-鼠李糖生物合成的所有必需基因，导致完整的辣椒素产生。体外研究表明，Cpz31负责将l-鼠李糖连接到辣椒素糖苷配基上，生成一种罕见的酰化脱氧己糖。在链霉菌属（Streptomyces sp。）的其他地方鉴定了l-鼠李糖基因簇。通过cpzDIII的插入失活证明了MK730-62F2基因组及其参与辣椒素的形成。使用Red / ET介导的重组将l-鼠李糖亚簇与cpzLK09组装在一起。所得粘粒cpzEW07的异源表达导致了卡扎霉素的产生，表明卡扎霉素的生物合成需要两组基因。在l-鼠李糖亚簇中敲除cpzDI和cpzDV证实，四个基因cpzDII，cpzDIII，cpzDIV和cpzDVI足以用于脱氧糖部分的生物合成。提出的重组策略可为组装用于微生物化合物异源生产的生物合成构件提供有用的工具。

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