Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture
Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture
复制标题
DOI：
10.1186/s40694-019-0081-x
复制
发表时间：
2019-11-09
影响因子：
--
通讯作者：
Meyer, Vera
中科院分区：
文献类型：
--
作者：
Cairns, Timothy C.;Feurstein, Claudia;Meyer, Vera
背景丝状真菌细胞工厂被用于生产大量的蛋白质、酶和有机酸。蛋白质分泌和丝状生长在菌丝顶端紧密耦合。此外，这两个过程都需要来自柠檬酸循环的ATP和氨基酸前体。尽管有机酸生产和蛋白质分泌/丝状生长之间存在这种相互联系，但在真菌中很少有研究确定可能同时影响这三个过程的基因。结果我们在黑曲霉细胞工厂中应用了一种新的全球共表达网络的筛选，以确定可能同时影响大体形态和蛋白质/有机酸发酵的候选基因。这些基因被预测编码高尔基体定位的Arfa GTPase激活蛋白(GAP，AgEB)和Arfa鸟嘌呤核苷酸交换因子(GEF SecG和GeaB)，并与柠檬酸循环基因共表达。因此，我们使用基于CRISPR的基因组编辑来将可滴定的Tet-on表达系统置于AgeB、secG和geaB的上游。功能分析表明，ageB和geaB对于丝状细胞的早期生长是必需的，而secG是必不可少的。接下来，在蛋白质或有机酸生产的条件下，在深层培养过程中滴定基因表达。ARFA调节剂在球团形成过程中发挥着不同的、依赖于培养的作用。值得注意的是，ageb或geaB的表达水平对蛋白质分泌有重大影响，而secG是必不可少的。相反，每个预测的ARFA调节子的表达减少导致生长介质中没有柠檬酸。最后，任何一种GEF的滴定表达都会导致培养上清液中草酰乙酸浓度的增加。结论在工业应用中，高尔基体在调节有机酸滴度方面可能发挥了被低估的作用，并且在黑曲霉中依赖于SecG、GeaB和AgEB。这些数据可能导致丝状真菌菌株优化的新途径，以提高蛋白质和有机酸滴度。
BackgroundFilamentous fungal cell factories are used to produce numerous proteins, enzymes, and organic acids. Protein secretion and filamentous growth are tightly coupled at the hyphal tip. Additionally, both these processes require ATP and amino acid precursors derived from the citric acid cycle. Despite this interconnection of organic acid production and protein secretion/filamentous growth, few studies in fungi have identified genes which may concomitantly impact all three processes. ResultsWe applied a novel screen of a global co-expression network in the cell factory Aspergillus niger to identify candidate genes which may concomitantly impact macromorphology, and protein/organic acid fermentation. This identified genes predicted to encode the Golgi localized ArfA GTPase activating protein (GAP, AgeB), and ArfA guanine nucleotide exchange factors (GEFs SecG and GeaB) to be co-expressed with citric acid cycle genes. Consequently, we used CRISPR-based genome editing to place the titratable Tet-on expression system upstream of ageB, secG, and geaB in A. niger. Functional analysis revealed that ageB and geaB are essential whereas secG was dispensable for early filamentous growth. Next, gene expression was titrated during submerged cultivations under conditions for either protein or organic acid production. ArfA regulators played varied and culture-dependent roles on pellet formation. Notably, ageB or geaB expression levels had major impacts on protein secretion, whereas secG was dispensable. In contrast, reduced expression of each predicted ArfA regulator resulted in an absence of citric acid in growth media. Finally, titrated expression of either GEFs resulted in an increase in oxaloacetic acid concentrations in supernatants. ConclusionOur data suggest that the Golgi may play an underappreciated role in modulating organic acid titres during industrial applications, and that this is SecG, GeaB and AgeB dependent in A. niger. These data may lead to novel avenues for strain optimization in filamentous fungi for improved protein and organic acid titres.