Chemical and Biological Processing Group, Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA, 99352, USA.
Appl Microbiol Biotechnol. 2020 May;104(9):3981-3992. doi: 10.1007/s00253-020-10418-0. Epub 2020 Mar 11.
The filamentous fungus Aspergillus terreus has been successfully used for industrial production of itaconic acid (IA) for many years. The IA biosynthesis pathway has recently been characterized at a molecular genetic level as an IA gene cluster by a clone-based transcriptomic approach. The cluster consists of four genes, including genes for cis-aconitic acid decarboxylase (cadA), a predicted transcription factor (tf), a mitochondrial organic acid transporter (mttA) and an MFS (major facilitator superfamily) type transporter (mfsA). In this research, we performed expressed sequence tag (EST) analysis and systematic gene deletions to further investigate the role of those genes during IA biosynthesis in A. pseudoterreus ATCC32359. EST analysis showed a similar expression pattern among those four genes that were distinct from neighboring genes and further confirmed that they belong to the same biosynthesis cluster. Systematic gene deletion analysis demonstrated that tf, cadA, mttA and mfsA genes in the cluster are essential for IA production; deletion of any of them will either completely abolish the IA production or dramatically decrease the amount of IA produced. The tf gene plays a regulatory role in this cluster. Deletion of tf led to decreased expression levels of cadA, mttA and mfsA. More importantly, a significant amount of aconitic acid was detected in the cadA deletion strain but not in the other deletion strains. Therefore, by deleting only one gene, the cadA, we established a novel microbial host for the production of aconitic acid and other value-added chemicals from sugars in lignocellulosic biomass.
丝状真菌土曲霉多年来一直成功地用于异柠檬酸（IA）的工业生产。最近，通过基于克隆的转录组学方法，从分子遗传学水平上对 IA 生物合成途径进行了特征描述，确定其为一个 IA 基因簇。该簇由四个基因组成，包括顺乌头酸脱羧酶（cadA）、一个预测的转录因子（tf）、一个线粒体有机酸转运蛋白（mttA）和一个 MFS（主要易化超家族）型转运蛋白（mfsA）。在这项研究中，我们进行了表达序列标签（EST）分析和系统基因缺失，以进一步研究这些基因在 A. pseudoterreus ATCC32359 中异柠檬酸生物合成过程中的作用。EST 分析表明，这四个基因的表达模式相似，与相邻基因明显不同，进一步证实它们属于同一生物合成簇。系统基因缺失分析表明，簇中的 tf、cadA、mttA 和 mfsA 基因对于 IA 生产是必需的；缺失其中任何一个基因都会完全或显著降低 IA 的产量。tf 基因在该簇中起调节作用。缺失 tf 导致 cadA、mttA 和 mfsA 的表达水平降低。更重要的是，在 cadA 缺失菌株中检测到大量的乌头酸，但在其他缺失菌株中没有检测到。因此，通过只删除一个基因 cadA，我们为利用木质纤维素生物质中的糖生产乌头酸和其他有价值的化学品建立了一个新型的微生物宿主。
