Aspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited. In this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5′-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors. In this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.

中文翻译：

---

Orotidine-5'-脱羧酶基因pyrG的表达中断或表达降低可增加柠檬酸的产生：黑曲霉中可回收基因组编辑过程中的一个新发现

黑曲霉是一种丝状真菌，用于全球柠檬酸的大部分生产。基因组编辑的最新发展现在使生物技术人员能够设计和优化黑曲霉。然而，目前，用于使工业黑曲霉分离物中的柠檬酸效价最大化的遗传线索是有限的。在这项研究中，我们尝试设计两个柠檬酸黑曲霉生产分离株WT-D和D353，以作为未来高通量基因组工程的平台菌株。因此，我们使用基因组编辑功能来同时破坏编码Orotidine-5'-脱羧酶（pyrG）和非同源末端连接成分（kusA）的基因，从而可以使用pyrG选择/计数器选择系统，并提高同源重组率， 分别。在常规筛选这些pyrG突变菌株的过程中，我们意外地观察到了2。与祖先对照相比，柠檬酸产量增加了17倍，这表明尿苷/嘧啶合成的抑制作用可能会增加柠檬酸滴度。为了进一步检验该假设，将pyrG基因置于可滴定四环素的盒的控制下，这证实了该基因表达的降低提高了摇瓶和生物反应器发酵中柠檬酸的滴度。随后，我们进行了细胞内代谢组学分析，结果表明，pyrG破坏增强了糖酵解通量，并显着提高了柠檬酸盐及其前体的丰度。在这项研究中，我们提供了两个由于产生pyrG / kusA缺失而可进行高通量遗传操作的柠檬酸分离株。令人惊讶的是，我们首次证明了A。尼日尔pyrG是产生柠檬酸高产菌株的有前途的遗传先导。我们的数据支持以下假设：尿苷/嘧啶生物合成途径为菌株工程研究提供了未来的途径。