Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture

Timothy C Cairns,Li Hui Zhang,Vera Meyer,Ping Zheng,Jibin Sun,Xiaomei Zheng,Claudia Feurstein

doi:10.1186/s40694-019-0081-x

Abstract

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.

Highlights

Filamentous fungal cell factories are used to produce numerous proteins, enzymes, and organic acids
We have previously demonstrated that A. niger arfA complements the ARF1/2 lethal phenotype in S. cerevisiae, it is a functional equivalent of Arf1/2 [21]
Multi‐gene co‐expression network analysis suggests the tricarboxylic acid (TCA) cycle and protein secretion are transcriptionally coupled with ageB, secG, and geaB We reasoned that A. niger gene co-expression networks, which we made publicly available at the data repository FungiDB [6, 10], could be mined for genes which are transcriptionally coupled with protein secretion and citric acid production

Summary

Introduction

Filamentous fungal cell factories are used to produce numerous proteins, enzymes, and organic acids. We conducted a meta-analysis of over 283 publicly available micro-array datasets, covering 155 different cultivation conditions of A. niger, after which co-expression networks were generated at an individual gene level [10] These co-expression networks can be used to generate novel hypotheses regarding gene function, based on the so called ‘guilt by association’ hypothesis, whereby genes with robust coexpression profiles over sufficiently diverse conditions can be hypothesised to be involved in similar, or the same, biological processes or pathways [11, 12]. Using this approach in our previous study, two so far unknown globally acting transcription factors MjkA and MjkB were identified, which likely control numerous natural product biosynthetic gene clusters in A. niger [10]

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Conclusion