Halogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC – the first predicted dimetal-carboxylate halogenase to be characterized – was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis. Homologs of CylC are also found in other characterized cyanobacterial secondary metabolite biosynthetic gene clusters. Due to its novelty in biological catalysis, selectivity and ability to perform C-H activation, this halogenase class is of considerable fundamental and applied interest. The study of CylC-like enzymes will provide insights into substrate scope, mechanism and catalytic partners, and will also enable engineering these biocatalysts for similar or additional C-H activating functions. Still, little is known regarding the diversity and distribution of these enzymes. In this study, we used both genome mining and PCR-based screening to explore the genetic diversity of CylC homologs and their distribution in bacteria. While we found non-cyanobacterial homologs of these enzymes to be rare, we identified a large number of genes encoding CylC-like enzymes in publicly available cyanobacterial genomes and in our in-house culture collection of cyanobacteria. Genes encoding CylC homologs are widely distributed throughout the cyanobacterial tree of life, within biosynthetic gene clusters of distinct architectures (combination of unique gene groups). These enzymes are found in a variety of biosynthetic contexts, which include fatty-acid activating enzymes, type I or type III polyketide synthases, dialkylresorcinol-generating enzymes, monooxygenases or Rieske proteins. Our study also reveals that dimetal-carboxylate halogenases are among the most abundant types of halogenating enzymes in the phylum Cyanobacteria. Our data show that dimetal-carboxylate halogenases are widely distributed throughout the Cyanobacteria phylum and that BGCs encoding CylC homologs are diverse and mostly uncharacterized. This work will help guide the search for new halogenating biocatalysts and natural product scaffolds.

中文翻译：

---

蓝藻中二金属羧酸卤化酶的分布和多样性

卤化是天然产物中反复出现的特征，尤其是来自海洋生物的产物。卤化酶作用于其底物的选择性使卤化酶成为生物催化剂开发的有趣目标。最近，CylC——第一个被预测的二金属羧酸盐卤化酶被证明在环环烷生物合成过程中区域选择性和立体选择性地将氯原子安装到未活化的碳中心上。在其他特征化的蓝藻次级代谢物生物合成基因簇中也发现了 CylC 的同源物。由于其在生物催化、选择性和执行 CH 活化方面的新颖性，这种卤化酶类具有相当大的基础和应用兴趣。CylC 样酶的研究将提供对底物范围的见解，机制和催化伙伴，并且还将能够对这些生物催化剂进行工程设计以实现类似或额外的 CH 激活功能。尽管如此，人们对这些酶的多样性和分布知之甚少。在这项研究中，我们使用基因组挖掘和基于 PCR 的筛选来探索 CylC 同源物的遗传多样性及其在细菌中的分布。虽然我们发现这些酶的非蓝藻同源物很少见，但我们在公开可用的蓝藻基因组和我们内部的蓝藻培养物收藏中鉴定了大量编码 CylC 样酶的基因。编码 CylC 同源物的基因广泛分布在整个蓝藻生命树中，在不同结构的生物合成基因簇内（独特基因组的组合）。这些酶存在于各种生物合成环境中，包括脂肪酸激活酶、I 型或 III 型聚酮合酶、二烷基间苯二酚生成酶、单加氧酶或 Rieske 蛋白。我们的研究还表明，二金属羧酸盐卤化酶是蓝藻门中最丰富的卤化酶类型之一。我们的数据显示，二金属羧酸盐卤化酶广泛分布在整个蓝藻门中，并且编码 CylC 同源物的 BGC 是多种多样的，而且大多没有特征。这项工作将有助于指导寻找新的卤化生物催化剂和天然产物支架。我们的研究还表明，二金属羧酸盐卤化酶是蓝藻门中最丰富的卤化酶类型之一。我们的数据显示，二金属羧酸盐卤化酶广泛分布在整个蓝藻门中，并且编码 CylC 同源物的 BGC 是多种多样的，而且大多没有特征。这项工作将有助于指导寻找新的卤化生物催化剂和天然产物支架。我们的研究还表明，二金属羧酸盐卤化酶是蓝藻门中最丰富的卤化酶类型之一。我们的数据显示，二金属羧酸盐卤化酶广泛分布在整个蓝藻门中，并且编码 CylC 同源物的 BGC 是多种多样的，而且大多没有特征。这项工作将有助于指导寻找新的卤化生物催化剂和天然产物支架。