Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery
Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery
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触发遗传难治性细菌沉默基因簇的表达导致巩膜酸的发现
DOI：
10.1101/265645
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Alberti F
中科院分区：
文献类型：
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作者：
Alberti F
在这项研究中，我们报告了一种新的微生物天然产物的快速表征，该产物是由沉默的基因簇的合理去阻遏产生的。使用一组保守的5个调控基因作为查询，搜索基因组数据库并鉴定非典型生物合成基因簇(BGC)。通过基于酵母的同源重组，从遗传上难以解决的菌核链霉菌菌株中获得了一个20kb的BGC，并将其导入到有效的异源宿主中。然后利用CRISPR/Cas9介导的基因组编辑来合理地灭活关键的转录抑制物，并触发一类前所未有的杂交天然产物的产生，例如(2-(苯甲酰氧基)-乙酰基)-L-脯氨酸，称为sclera酸。随后几轮CRISPR/Cas9介导的基因缺失提供了一种生物合成基因突变株的选择，这导致了一条可行的巩膜酸组装的生物合成途径。还制备了红景天酸和关键的生物合成中间体的合成标准，以确认我们提出的化学结构。巩膜酸的组装包括两个独特的缩合反应，由一个NRPS模块和一个ATP-GRAP酶催化，分别将一个脯氨酸和一个苯甲酰残基连接到一个稀有的羟乙基-ACP中间体的两端。红景天酸对结核分枝杆菌表现出中等的抑制活性，对癌症相关代谢酶烟酰胺N-甲基转移酶(NNMT)也有抑制作用。
In this study, we report the rapid characterisation of a novel microbial natural product resulting from the rational derepression of a silent gene cluster. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable Streptomyces sclerotialus bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-L-proline, named scleric acid. Subsequent rounds of CRISPR/Cas9-mediated gene deletions afforded a selection of biosynthetic gene mutant strains which led to a plausible biosynthetic pathway for scleric acid assembly. Synthetic standards of scleric acid and a key biosynthetic intermediate were also prepared to confirm the chemical structures we proposed. The assembly of scleric acid involves two unique condensation reactions catalysed by a single NRPS module and an ATP-grasp enzyme that link a proline and a benzoyl residue to each end of a rare hydroxyethyl-ACP intermediate, respectively. Scleric acid was shown to exhibit moderate inhibition activity against Mycobacterium tuberculosis, as well as inhibition of the cancer-associated metabolic enzyme nicotinamide N-methyltransferase (NNMT).