Data from: Expansion and diversification of the MSDIN family of cyclic peptide genes in the poisonous agarics Amanita phalloides and A. bisporigera

Background: The cyclic peptide toxins of Amanita mushrooms, such as α-amanitin and phalloidin, are encoded by the “MSDIN” gene family and ribosomally biosynthesized. Based on partial genome sequence and PCR analysis, some members of the MSDIN family were previously identified in Amanita bisporigera, and several other members are known from other species of Amanita. However, the complete complement in any one species, and hence the genetic capacity for these fungi to make cyclic peptides, remains unknown.

Results: Draft genome sequences of two cyclic peptide-producing mushrooms, the “Death Cap” A. phalloides and the “Destroying Angel” A. bisporigera, were obtained. Each species has ~30 MSDIN genes, most of which are predicted to encode unknown cyclic peptides. Some MSDIN genes were duplicated in one or the other species, but only three were common to both species. A gene encoding cycloamanide B, a previously described nontoxic cyclic heptapeptide, was also present in A. phalloides, but genes for antamanide and cycloamanides A, C, and D were not. In A. bisporigera, RNA expression was observed for 20 of the MSDIN family members. Based on their predicted sequences, novel cyclic peptides were searched for by LC/MS/MS in extracts of A. phalloides. The presence of two cyclic peptides, named cycloamanides E and F with structures cyclo(SFFFPVP) and cyclo(IVGILGLP), was thereby demonstrated. Of the MSDIN genes reported earlier from another specimen of A. bisporigera, 9 of 14 were not found in the current genome assembly. Differences between previous and current results for the complement of MSDIN genes and cyclic peptides in the two fungi probably represents natural variation among geographically dispersed isolates of A. phalloides and among the members of the poorly defined A. bisporigera species complex. Both A. phalloides and A. bisporigera contain two prolyl oligopeptidase genes, one of which (POPB) is probably dedicated to cyclic peptide biosynthesis as it is in Galerina marginata.

Conclusion: The MSDIN gene family has expanded and diverged rapidly in Amanita section Phalloideae. Together, A. bisporigera and A. phalloides are predicted to have the capacity to make more than 50 cyclic hexa-, hepta-, octa-, nona- and decapeptides.

背景：鹅膏属（Amanita）蘑菇的环肽毒素，如α-鹅膏毒肽（α-amanitin）和毒伞肽（phalloidin），由"MSDIN"基因家族编码，且经核糖体途径完成生物合成。此前基于部分基因组序列与聚合酶链式反应（PCR）分析，研究人员已在双孢鹅膏（Amanita bisporigera）中鉴定出MSDIN家族的部分成员，同时从其他鹅膏属物种中也获知该家族的若干成员。但目前尚无任何一个鹅膏物种中MSDIN家族完整成员组成的相关报道，因此这类真菌合成环肽的遗传潜力仍未明确。

结果：本研究获取了两种产环肽蘑菇——"死亡帽"毒鹅膏（A. phalloides）与"毁灭天使"双孢鹅膏（A. bisporigera）的草图基因组序列。两个物种均携带约30个MSDIN基因，其中大多数被预测编码功能未知的环肽。部分MSDIN基因在其中一个物种中发生了复制，但仅有3个基因在两个物种中均存在。毒鹅膏中还存在一个编码非毒性环七肽cycloamanide B的基因，该物质此前已有报道，但未发现编码抗鹅膏肽（antamanide）以及cycloamanides A、C、D的基因。在双孢鹅膏中，研究人员检测到20个MSDIN家族成员的RNA表达。基于预测的肽序列，本研究通过液相色谱-串联质谱（LC/MS/MS）在毒鹅膏的提取物中搜寻新型环肽，最终确认了两种环肽的存在，分别命名为cycloamanides E和F，其结构分别为cyclo(SFFFPVP)与cyclo(IVGILGLP)。相较于此前从另一株双孢鹅膏标本中报道的MSDIN基因，本次基因组组装中未找到其中14个里的9个。两种真菌在MSDIN家族基因组成与环肽种类上的新旧研究结果差异，大概率反映了地理分布不同的毒鹅膏分离株之间，以及定义尚不明确的双孢鹅膏物种复合群内成员间的自然变异。毒鹅膏与双孢鹅膏均携带两种脯氨酰寡肽酶基因，其中一个（POPB）可能与环肽生物合成专门相关，正如边缘盔孢伞（Galerina marginata）中的情况。

结论：MSDIN基因家族在鹅膏属毒鹅膏组（Amanita section Phalloideae）中经历了快速的扩张与分化。综合来看，双孢鹅膏与毒鹅膏被预测具备合成超过50种六肽、七肽、八肽、九肽及十肽环肽的遗传能力。