School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou, 510006, China; Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou, 510006, China.
Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen, AB24 3UE, Scotland, UK; School of Forensic and Applied Sciences, Faculty of Science & Technology, University of Central Lancashire, Preston, Lancashire, PR1 2HE, UK.
Microbiol Res. 2021 Jul;248:126735. doi: 10.1016/j.micres.2021.126735. Epub 2021 Mar 3.
Although the mechanisms of regulating secondary metabolism by LaeA remains unclear, the synthesis of many secondary metabolites (SMs) in Aspergilli could be activated by LaeA mutation. In our previous sutdy, RNA-seq data has showed that the transcriptional level of many SM backbone genes could be upregulated by overexpressing LaeA. Herein, we analyzed the chemical profile of activated secondary metabolites in the variant of A. niger FGSC A1279 by overexpressing LaeA (OElaeA). 14 compounds were activated in A. niger FGSC A1279 OElaeA variant in the WATM medium. Chemical workup of organic extracts of the culture broth from the A. niger OElaeA mutant identified three pure compounds, flaviolin, orlandin and kotanin. The structures of these compounds were confirmed by HR-ESIMS, 1D/2D NMR, and computer assisted structure elucidation (CASE). Based on homologous alignment and comparison of literatures, the biosynthetic gene cluster (fla) of flaviolin was identified. The in vivo function of the backbone gene, flaA, encoding a multidomain non-reducing polyketide synthase (SAT-KS-AT-PT-ACP), was verified via gene knockout and chemical analysis. Finally, a biosynthetic model for fungal flaviolin was proposed.
虽然 LaeA 调节次级代谢的机制尚不清楚，但 LaeA 突变可以激活许多曲霉次级代谢物 (SMs) 的合成。在我们之前的研究中，RNA-seq 数据表明，过表达 LaeA 可以上调许多 SM 骨干基因的转录水平。在此，我们通过过表达 LaeA (OElaeA) 分析了 A. niger FGSC A1279 变体中激活的次级代谢物的化学特征。在 WATM 培养基中，A. niger FGSC A1279 OElaeA 变体中激活了 14 种化合物。从 A. niger OElaeA 突变体的培养物的有机提取物的化学处理中鉴定出三种纯化合物，即 flaviolin、orlandin 和 kotanin。这些化合物的结构通过高分辨率电喷雾电离质谱 (HR-ESIMS)、1D/2D NMR 和计算机辅助结构解析 (CASE) 得到确认。基于同源性比对和文献比较，鉴定了 flaviolin 的生物合成基因簇 (fla)。通过基因敲除和化学分析验证了编码多结构域非还原聚酮合酶 (SAT-KS-AT-PT-ACP) 的骨干基因 flaA 的体内功能。最后，提出了真菌 flaviolin 的生物合成模型。
