Arsın Sıla, Pollari Maija, Delbaje Endrews, Jokela Jouni, Wahlsten Matti, Permi Perttu, Fewer David
Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki 00014 Helsinki Finland
Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki 00014 Helsinki Finland.
RSC Chem Biol. 2024 Aug 20;5(10):1035-44. doi: 10.1039/d4cb00128a.
Mycosporine-like amino acids (MAAs) are a family of water-soluble and colorless secondary metabolites, with high extinction coefficients, that function as microbial sunscreens. MAAs share a cyclohexinimine chromophore that is diversified through amino acid substitutions and attachment of sugar moieties. The genetic and enzymatic bases for the chemical diversity of MAAs remain largely unexplored. Here we report a series of structurally distinct MAAs and evidence for an unusual branched biosynthetic pathway from a cyanobacterium isolated from lake sediment. We used a combination of high-resolution liquid chromatography-mass spectrometry (HR-LCMS) analysis and nuclear magnetic resonance (NMR) spectroscopy to identify diglycosylated-palythine-Ser (CHNO) as the dominant chemical variant in a series of MAAs from sp. UHCC 0302 that contained either Ser or Thr. We obtained a complete 9.9 Mb genome sequence to gain insights into the genetic basis for the biosynthesis of these structurally distinct MAAs. We identified MAA biosynthetic genes encoded at two locations on the circular chromosome. Surprisingly, direct pathway cloning and heterologous expression of the complete biosynthetic gene cluster in () led to the production of 450 Da monoglycosylated-palythine-Thr (CHNO). We reconstructed combinations of the two distant biosynthetic gene clusters in refactored synthetic pathways and expressed them in the heterologous host. These results demonstrated that the MysD and MysD enzymes displayed a preference for Thr and Ser, respectively. Furthermore, one of the four glycosyltransferases identified, MysG, was active in and catalysed the attachment of a hexose moiety to the palythine-Thr intermediate. Together these results provide the first insights into the enzymatic basis for glycosylation of MAAs and demonstrates how paralogous copies of the MysD enzymes allow the simultaneous biosynthesis of specific chemical variants to increase the structural variation in this family of microbial sunscreens.
类菌孢素氨基酸（MAAs）是一类水溶性无色次生代谢产物，具有高消光系数，起微生物防晒剂的作用。MAAs共有一个环己亚胺发色团，该发色团通过氨基酸取代和糖基部分的连接而多样化。MAAs化学多样性的遗传和酶学基础在很大程度上仍未被探索。在此，我们报告了一系列结构不同的MAAs，以及从湖泊沉积物中分离出的一种蓝细菌存在异常分支生物合成途径的证据。我们结合使用高分辨率液相色谱 - 质谱（HR-LCMS）分析和核磁共振（NMR）光谱，以鉴定双糖基化 - 岩沙海葵毒素 - 丝氨酸（CHNO）是来自含有丝氨酸或苏氨酸的UHCC 0302菌株的一系列MAAs中的主要化学变体。我们获得了完整的9.9 Mb基因组序列，以深入了解这些结构不同的MAAs生物合成的遗传基础。我们鉴定出在环状染色体上两个位置编码的MAA生物合成基因。令人惊讶的是，直接对完整生物合成基因簇进行途径克隆并在（）中进行异源表达，产生了450 Da的单糖基化 - 岩沙海葵毒素 - 苏氨酸（CHNO）。我们在重构的合成途径中重建了两个远距离生物合成基因簇的组合，并在异源宿主中表达它们。这些结果表明，MysD和MysD酶分别对苏氨酸和丝氨酸表现出偏好。此外，鉴定出的四种糖基转移酶之一MysG在（）中具有活性，并催化己糖部分连接到岩沙海葵毒素 - 苏氨酸中间体上。这些结果共同首次揭示了MAAs糖基化的酶学基础，并证明了MysD酶的旁系同源拷贝如何允许同时生物合成特定的化学变体，以增加这类微生物防晒剂家族的结构多样性。
