A quantitative image analysis pipeline for the characterization of filamentous fungal morphologies as a tool to uncover targets for morphology engineering: a case study using aplD in Aspergillus niger

Background Fungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains. Results In this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to identify a putative gamma-adaptin encoding gene (aplD) that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN. Conclusion The MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.DFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

How a fungus shapes biotechnology: 100 years of Aspergillus niger research

In 1917, a food chemist named James Currie made a promising discovery: any strain of the filamentous mould Aspergillus niger would produce high concentrations of citric acid when grown in sugar medium. This tricarboxylic acid, which we now know is an intermediate of the Krebs cycle, had previously been extracted from citrus fruits for applications in food and beverage production. Two years after Currie’s discovery, industrial-level production using A. niger began, the biochemical fermentation industry started to flourish, and industrial biotechnology was born. A century later, citric acid production using this mould is a multi-billion dollar industry, with A. niger additionally producing a diverse range of proteins, enzymes and secondary metabolites. In this review, we assess main developments in the field of A. niger biology over the last 100 years and highlight scientific breakthroughs and discoveries which were influential for both basic and applied fungal research in and outside the A. niger community. We give special focus to two developments of the last decade: systems biology and genome editing. We also summarize the current international A. niger research community, and end by speculating on the future of fundamental research on this fascinating fungus and its exploitation in industrial biotechnology.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berli

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

Background: Filamentous 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. Results: We 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. Conclusion: Our 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.TU Berlin, Open-Access-Mittel - 201

Tet-on, or tet-off, that is the question: advanced conditional gene expression in Aspergillus

In Aspergillus, controlled gene expression is often achieved using the reverse tetracycline-controlled transactivator (rtTA) dependent Tet-on system, whereby transcription is activated in a titratable manner by addition of the tetracycline derivative doxycycline. The complementary Tet-off system utilises the tetracycline-controlled transactivator (tTA) component to quantitatively reduce gene expression. In this study, we utilised a synthetic biological approach to engineer highly optimised Tet-off conditional expression systems in Aspergillus niger and Aspergillus fumigatus. Steps for delivery of these tools include utilising codon optimised cassette components, testing several promoters for improved genetic stability and validating two modified luciferase reporters for highly accurate measurements of gene expression. The Tet-off cassettes developed in this study enable facile and quantitative functional analysis, as validated by Tet-off analysis of genes involved in chitin synthesis and cell wall polarity in A. niger, and para-aminobenzoic acid synthesis in A. fumigatus. We also used a racAG18V dominant allele to demonstrate that Tet-off in A. niger enables gene over-expression and downregulation in a single isolate. Additionally, we used the improved luciferase reporters to show that the Tet-off cassette in A. niger enables quantification of gene oscillations. In order to demonstrate that synthetic biological approaches developed here are broadly applicable to engineering transcriptional circuits in filamentous fungi, we used our strategy for improving cassette stability by promoter replacement in the A. niger Tet-on system, which resulted in a modified Tet-on cassette with higher stability in recipient genomes

Updating genome annotation for the microbial cell factory Aspergillus niger using gene co-expression networks

A significant challenge in our understanding of biological systems is the high number of genes with unknown function in many genomes. The fungal genus Aspergillus contains important pathogens of humans, model organisms, and microbial cell factories. Aspergillus niger is used to produce organic acids, proteins, and is a promising source of new bioactive secondary metabolites. Out of the 14,165 open reading frames predicted in the A. niger genome only 2% have been experimentally verified and over 6,000 are hypothetical. Here, we show that gene co-expression network analysis can be used to overcome this limitation. A meta-analysis of 155 transcriptomics experiments generated co-expression networks for 9,579 genes (∼65%) of the A. niger genome. By populating this dataset with over 1,200 gene functional experiments from the genus Aspergillus and performing gene ontology enrichment, we could infer biological processes for 9,263 of A. niger genes, including 2,970 hypothetical genes. Experimental validation of selected co-expression sub-networks uncovered four transcription factors involved in secondary metabolite synthesis, which were used to activate production of multiple natural products. This study constitutes a significant step towards systems-level understanding of A. niger, and the datasets can be used to fuel discoveries of model systems, fungal pathogens, and biotechnology.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität BerlinEC/FP7/607332/EU/Quantitative Biology for Fungal Secondary Metabolite Producers/QuantFun

A Library of Aspergillus niger Chassis Strains for Morphology Engineering Connects Strain Fitness and Filamentous Growth With Submerged Macromorphology

Submerged fermentation using filamentous fungal cell factories is used to produce a diverse portfolio of useful molecules, including food, medicines, enzymes, and platform chemicals. Depending on strain background and abiotic culture conditions, different macromorphologies are formed during fermentation, ranging from dispersed hyphal fragments to approximately spherical pellets several millimetres in diameter. These macromorphologies are known to have a critical impact on product titres and rheological performance of the bioreactor. Pilot productivity screens in different macromorphological contexts is technically challenging, time consuming, and thus a significant limitation to achieving maximum product titres. To address this bottleneck, we developed a library of conditional expression mutants in the organic, protein, and secondary metabolite cell factory Aspergillus niger. Thirteen morphology-associated genes transcribed during fermentation were placed via CRISPR-Cas9 under control of a synthetic Tet-on gene switch. Quantitative analysis of submerged growth reveals that these strains have distinct and titratable macromorphologies for use as chassis during strain engineering programs. We also used this library as a tool to quantify how pellet formation is connected with strain fitness and filamentous growth. Using multiple linear regression modelling, we predict that pellet formation is dependent largely on strain fitness, whereas pellet Euclidian parameters depend on fitness and hyphal branching. Finally, we have shown that conditional expression of the putative kinase encoding gene pkh2 can decouple fitness, dry weight, pellet macromorphology, and culture heterogeneity. We hypothesize that further analysis of this gene product and the cell wall integrity pathway in which it is embedded will enable more precise engineering of A. niger macromorphology in future

The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Raw data have been deposited in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE54810. Funding: This work was supported in part by grants to EMB from the MRC (G0501164) and BBSRC (BB/G009619/1), to EMB and NDR from the Wellcome Trust (WT093596MA), to MB from Imperial College London (Division of Investigative Sciences PhD Studentship), to HH from the ERA-NET PathoGenoMics project TRANSPAT, Austrian Science Foundation (FWF I282-B09), to SGF from the National Institutes of Health, USA (R01AI073829). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper.

The EUROFUNG network is a virtual centre of multidisciplinary expertise in the field of fungal biotechnology. The first academic-industry Think Tank was hosted by EUROFUNG to summarise the state of the art and future challenges in fungal biology and biotechnology in the coming decade. Currently, fungal cell factories are important for bulk manufacturing of organic acids, proteins, enzymes, secondary metabolites and active pharmaceutical ingredients in white and red biotechnology. In contrast, fungal pathogens of humans kill more people than malaria or tuberculosis. Fungi are significantly impacting on global food security, damaging global crop production, causing disease in domesticated animals, and spoiling an estimated 10 % of harvested crops. A number of challenges now need to be addressed to improve our strategies to control fungal pathogenicity and to optimise the use of fungi as sources for novel compounds and as cell factories for large scale manufacture of bio-based products. This white paper reports on the discussions of the Think Tank meeting and the suggestions made for moving fungal bio(techno)logy forward

Beyond the biosynthetic gene cluster paradigm: Genome-wide coexpression networks connect clustered and unclustered transcription factors to secondary metabolic pathways

Fungal secondary metabolites are widely used as therapeutics and are vital components of drug discovery programs. A major challenge hindering discovery of novel secondary metabolites is that the underlying pathways involved in their biosynthesis are transcriptionally silent under typical laboratory growth conditions, making it difficult to identify the transcriptional networks that they are embedded in. Furthermore, while the genes participating in secondary metabolic pathways are typically found in contiguous clusters on the genome, known as biosynthetic gene clusters (BGCs), this is not always the case, especially for global and pathway-specific regulators of pathways’ activities. To address these challenges, we used 283 genome-wide gene expression data sets of the ascomycete cell factory Aspergillus niger generated during growth under 155 different conditions to construct two gene coexpression networks based on Spearman’s correlation coefficients (SCCs) and on mutual rank-transformed Pearson’s correlation coefficients (MR-PCCs). By mining these networks, we predicted six transcription factors, named MjkA to MjkF, to regulate secondary metabolism in A. niger. Overexpression of each transcription factor using the Tet-On cassette modulated the production of multiple secondary metabolites. We found that the SCC and MR-PCC approaches complemented each other, enabling the delineation of putative global (SCC) and pathway-specific (MR-PCC) transcription factors. These results highlight the potential of coexpression network approaches to identify and activate fungal secondary metabolic pathways and their products. More broadly, we argue that drug discovery programs in fungi should move beyond the BGC paradigm and focus on understanding the global regulatory networks in which secondary metabolic pathways are embedded.DFG, 404295023, Etablierung eines innovativen Ko-Kultivierungssystems zur Hochdurchsatzidentifizierung von antimikrobiellen WirkstoffenEC/FP7/607332/EU/Quantitative Biology for Fungal Secondary Metabolite Producers/QUANTFUN