Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, USA.
Mycosynthetix, Inc., Hillsborough, NC, USA.
J Antibiot (Tokyo). 2021 Aug;74(8):496-507. doi: 10.1038/s41429-021-00432-3. Epub 2021 Jun 21.
Resorcylic acid lactones (RALs) with a cis-enone moiety, represented by hypothemycin (1) and (5Z)-7-oxozeaenol (2), are fungal secondary metabolites with irreversible inhibitory activity against protein kinases, with particularly selective activity for inhibition of TAK1 (transforming growth factor beta-activated kinase 1). Gram-scale quantities of these compounds were needed as feedstock for semi-synthesizing RAL-analogues in a step-economical fashion. To do so, this study had three primary goals: identifying fungi that biosynthesized 1 and 2, enhancing their production by optimizing the fermentation conditions on the lab scale, and developing straight forward purification processes. After evaluating 536 fungal extracts via an in-house dereplication protocol, three strains were identified as producing cis-enone RALs (i.e., MSX78495, MSX63935, MSX45109). Screening these fungal strains on three grain-based media revealed enhanced production of 1 by strain MSX78495 on oatmeal medium, while rice medium increased the biosynthesis of 2 by strain MSX63935. Furthermore, the purification processes were improved, moving away from HPLC purification to utilizing two to four cycles of resuspension and centrifugation in small volumes of organic solvents, generating gram-scale quantities of these metabolites readily. In addition, studying the chemistry profiles of strains MSX78495 and MSX63935 resulted in the isolation of ten other RALs (3-12), two radicinin analogues (13-14), and six benzopyranones (15-20), with 19 and 20 being newly described chlorinated benzopyranones.
顺式-烯酮结构的雷锁酸内酯（RALs），以 Hypothemycin（1）和（5Z）-7-氧代玉米烯醇（2）为代表，是具有不可逆抑制蛋白激酶活性的真菌次级代谢产物，对 TAK1（转化生长因子β激活激酶 1）具有特别选择性的抑制活性。这些化合物的克级数量是半合成 RAL 类似物的必要原料，以经济的方式进行。为此，本研究有三个主要目标：鉴定生物合成 1 和 2 的真菌，通过优化实验室规模的发酵条件来提高它们的产量，以及开发简单的纯化工艺。在通过内部去重复协议评估了 536 种真菌提取物后，有 3 株被鉴定为产生顺式-烯酮 RALs（即 MSX78495、MSX63935 和 MSX45109）。在三种基于谷物的培养基上筛选这些真菌菌株，发现菌株 MSX78495 在燕麦培养基上可增强 1 的产生，而菌株 MSX63935 在大米培养基上可增加 2 的生物合成。此外，还改进了纯化工艺，从 HPLC 纯化转向利用少量有机溶剂进行两到四个循环的重悬和离心，可轻松获得克级数量的这些代谢产物。此外，研究菌株 MSX78495 和 MSX63935 的化学成分特征导致分离出另外 10 种 RALs（3-12）、两种雷地辛类似物（13-14）和 6 种苯并吡喃酮（15-20），其中 19 和 20 是新描述的氯化苯并吡喃酮。
