Genetic Reprogramming of the Ergot Alkaloid Pathway of Metarhizium brunneum
Genetic Reprogramming of the Ergot Alkaloid Pathway of Metarhizium brunneum
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DOI：
10.1128/aem.01251-20
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发表时间：
2020-10-01
影响因子：
4.4
通讯作者：
Panaccione, Daniel G.
中科院分区：
文献类型：
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作者：
Davis, Kyle A.;Sampson, Jessi K.;Panaccione, Daniel G.
麦角生物碱是重要的特殊真菌代谢物，可用于制造治疗神经系统疾病和失调的有效药物。麦角酸（LA）和二氢麦角酸（DHLA）是药物半合成中理想的先导化合物，但通常是麦角生物碱和二氢麦角酸生物碱途径中的短暂中间体。之前与Neosartorya fumigata的工作证明了生产这些化合物作为途径最终产物的策略，但它们的产率（产物状态与前体状态相对的分子百分比）很低。此外，麦角生物碱通常保留在真菌中，而不是被分泌。我们使用集群规则间隔短回文重复(CRISPR)-CRISPR相关蛋白9 （Cas9）和异源表达方法在棕绿绿僵菌（Metarhizium brunneum）中设计这些化合物，代表来自不同真菌谱系的替代表达宿主。LA的相对产率为86.9%，DHLA的相对产率为72.8%，远高于先前设计菌株的产率（分别为2.6%和2.0%）。测定了这些生物碱的分泌量，平均有98.4%的LA和87.5%的DHLA分泌到生长培养基中；两者的分泌量均显著高于烟谷奈瑟菌衍生物的分泌量（均低于5.6%）。我们使用类似的方法在M. brunneum中设计了一种新的双氢麦角生物碱，并通过高效液相色谱-质谱（LC-MS）分析，暂时确定它是麦角酸a-羟乙基酰胺（dihydro-LAH）的二氢化形式。这些菌株的工程化为在更适合其生产的真菌中生产新的和具有重要药用意义的化学物质提供了一种策略。从LA或DHLA中提取的麦角生物碱是许多药物的基础，用于治疗痴呆、偏头痛、高泌乳素血症和其他疾病。然而，从天然来源提取麦角生物碱效率低，化学合成成本高。控制和重定向真菌中麦角生物碱合成的能力可能使这些重要化学物质的生产更有效，并促进新衍生物的研究。我们的研究结果表明，绿僵菌可以有效地产生和分泌LA和DHLA，也可以产生一种以前在自然界中没有发现的DHLA的新型衍生物。双氢麦角生物碱的工程，包括一个新的物种，是很重要的，因为很少有这些化合物的天然来源已知。我们的方法建立了一个平台，利用M. brunneum研究其他麦角生物碱的生产，特别是那些分类为麦角酸酰胺和二氢麦角生物碱。
Ergot alkaloids are important specialized fungal metabolites that are used to make potent pharmaceuticals for neurological diseases and disorders. Lysergic acid (LA) and dihydrolysergic acid (DHLA) are desirable lead compounds for pharmaceutical semisynthesis but are typically transient intermediates in the ergot alkaloid and dihydroergot alkaloid pathways. Previous work with Neosartorya fumigata demonstrated strategies to produce these compounds as pathway end products, but their percent yield (percentage of molecules in product state as opposed to precursor state) was low. Moreover, ergot alkaloids in N. fumigata are typically retained in the fungus as opposed to being secreted. We used clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) and heterologous expression approaches to engineer these compounds in Metarhizium brunneum, representing an alternate expression host from a different lineage of fungi. The relative percent yields of LA (86.9%) and DHLA (72.8%) were much higher than those calculated here for previously engineered strains of N. fumigata (2.6% and 2.0%, respectively). Secretion of these alkaloids also was measured, with averages of 98.4% of LA and 87.5% of DHLA being secreted into the growth medium; both values were significantly higher than those measured for the N. fumigata derivatives (both of which were less than 5.6% secreted). We used a similar approach to engineer a novel dihydroergot alkaloid in M. brunneum and, through high-performance liquid chromatographymass spectrometry (LC-MS) analyses, provisionally identified it as the dihydrogenated form of lysergic acid a-hydroxyethylamide (dihydro-LAH). The engineering of these strains provides a strategy for producing novel and pharmaceutically important chemicals in a fungus more suitable for their production.IMPORTANCE Ergot alkaloids derived from LA or DHLA are the bases for numerous pharmaceuticals with applications in the treatment of dementia, migraines, hyperprolactinemia, and other conditions. However, extraction of ergot alkaloids from natural sources is inefficient, and their chemical synthesis is expensive. The ability to control and redirect ergot alkaloid synthesis in fungi may allow more efficient production of these important chemicals and facilitate research on novel derivatives. Our results show that Metarhizium brunneum can be engineered to efficiently produce and secrete LA and DHLA and, also, to produce a novel derivative of DHLA not previously found in nature. The engineering of dihydroergot alkaloids, including a novel species, is important because very few natural sources of these compounds are known. Our approach establishes a platform with which to use M. brunneum to study the production of other ergot alkaloids, specifically those classified as lysergic acid amides and dihydroergot alkaloids.