A refactored biosynthetic pathway for the production of glycosylated microbial sunscreens†

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Sıla Arsın, Maija Pollari, Endrews Delbaje, Jouni Jokela, Matti Wahlsten, Perttu Permi and David Fewer
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引用次数: 0

Abstract

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 (C22H36N2O15) as the dominant chemical variant in a series of MAAs from Nostoc 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 mysABCJ1D1G1H biosynthetic gene cluster in Escherichia coli (E. coli) led to the production of 450 Da monoglycosylated-palythine-Thr (C18H30N2O11). 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 MysD1 and MysD2 enzymes displayed a preference for Thr and Ser, respectively. Furthermore, one of the four glycosyltransferases identified, MysG1, was active in E. coli 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.

Abstract Image

Abstract Image

生产糖基化微生物防晒剂的重构生物合成途径。
类霉菌素氨基酸(MAAs)是一种水溶性无色次级代谢物,具有很高的消光系数,可用作微生物防晒剂。类 MAA 共用一个环己亚胺发色团,该发色团通过氨基酸替换和糖分子的连接而变得多样化。MAAs化学多样性的基因和酶基础在很大程度上仍未得到探索。在此,我们报告了一系列结构独特的 MAAs,并证明了从湖泊沉积物中分离出的蓝藻具有不寻常的分支生物合成途径。我们采用高分辨率液相色谱-质谱(HR-LCMS)分析和核磁共振(NMR)光谱相结合的方法,鉴定出二糖基化萼氨酸-Ser(C22H36N2O15)是来自 Nostoc sp.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 MAA 生物合成的遗传基础。我们确定了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,在大肠杆菌(E. coli)中直接途径克隆和异源表达完整的 mysABCJ 1 D 1 G 1 H 生物合成基因簇可产生 450 Da 单糖基化萼氨酰-Thr (C18H30N2O11)。我们在重构的合成途径中重建了两个遥远的生物合成基因簇的组合,并在异源宿主中表达了它们。这些结果表明,MysD1 和 MysD2 酶分别偏好 Thr 和 Ser。此外,鉴定出的四种糖基转移酶之一 MysG1 在大肠杆菌中具有活性,可催化六糖分子与萼氨酸-Thr 中间体的连接。这些结果首次揭示了 MAAs 糖基化的酶学基础,并证明了 MysD 酶的同族拷贝如何能够同时生物合成特定的化学变体,从而增加微生物防晒剂家族的结构变化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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