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 chromatography-mass spectrometry (LC-MS) analyses, provisionally identified it as the dihydrogenated form of lysergic acid α-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.

中文翻译：

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梅毒根的麦角生物碱途径的遗传重编程。

麦角生物碱是重要的专门真菌代谢产物，可用于制备有效的神经疾病和病症药物。麦角酸（LA）和二氢麦角酸（DHLA）是药物半合成的理想先导化合物，但通常是麦角生物碱和二氢麦角生物碱途径中的过渡中间体。先前与Neosartorya fumigata的合作证明了将这些化合物作为途径最终产物生产的策略，但是它们的产率（分子处于产物状态而不是前体状态的百分比）较低。此外，熏蒸猪笼草的麦角生物碱通常被保留在真菌中而不是被分泌。我们使用群集规则间隔开的短回文重复序列（CRISPR）-CRISPR相关蛋白9（Cas9）和异源表达方法来设计在这些化合物绿僵brunneum，表示从不同谱系的真菌的替代表达宿主。LA（86.9％）和DHLA（72.8％）的相对百分率比此处以前计算的熏蒸猪笼草菌株的相对百分率高得多（分别为2.6％和2.0％）。还测量了这些生物碱的分泌，平均有98.4％的LA和87.5％的DHLA被分泌到生长培养基中。这两个值均显着高于烟熏猪笼草的测量值衍生物（两者均少于5.6％分泌）。我们采用了类似的方法在布鲁氏杆菌中设计了一种新型的二氢麦角生物碱，并通过高效液相色谱-质谱（LC-MS）分析，暂时将其鉴定为麦角酸α-羟乙酰胺（二氢-LAH）的二氢形式。 ）。这些菌株的工程化提供了在真菌中生产更适合其生产的新型和药学上重要的化学药品的策略。