Structure-Activity Predictions From Computational Mining of Protein Databases to Assist Modular Design of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are naturally produced by pro- and eukaryotes and are promising alternatives to antibiotics to fight multidrug-resistant microorganisms. However, despite thousands of AMP entries in respective databases, predictions about their structure-activity relationships are still limited. Similarly, common or dissimilar properties of AMPs that have evolved in different taxonomic groups are nearly unknown. We leveraged data entries for 10,987 peptides currently listed in the three antimicrobial peptide databases APD, DRAMP and DBAASP to aid structure-activity predictions. However, this number reduced to 3,828 AMPs that we could use for computational analyses, due to our stringent quality control criteria. The analysis uncovered a strong bias towards AMPs isolated from amphibians (1,391), whereas only 35 AMPs originate from fungi (0.9%), hindering evolutionary analyses on the origin and phylogenetic relationship of AMPs. The majority (62%) of the 3,828 AMPs consists of less than 40 amino acids but with a molecular weight higher than 2.5 kDa, has a net positive charge and shares a hydrophobic character. They are enriched in glycine, lysine and cysteine but are depleted in glutamate, aspartate and methionine when compared with a peptide set of the same size randomly selected from the UniProt database. The AMPs that deviate from this pattern (38%) can be found in different taxonomic groups, in particular in Gram-negative bacteria. Remarkably, the γ-core motif claimed so far as a unifying structural signature in cysteine-stabilised AMPs is absent in nearly 90% of the peptides, questioning its relevance as a prerequisite for antimicrobial activity. The disclosure of AMPs pattern and their variation in producing organism groups extends our knowledge of the structural diversity of AMPs and will assist future peptide screens in unexplored microorganisms. Structural design of peptide antibiotic drugs will benefit using natural AMPs as lead compounds. However, a reliable and statistically balanced database is missing which leads to a large knowledge gap in the AMP field. Thus, thorough evaluation of the available data, mitigation of biases and standardised experimental setups need to be implemented to leverage the full potential of AMPs for drug development programmes in the clinics and agriculture.

Quantitative phenotypic screens of Aspergillus niger mutants in solid and liquid culture.

This protocol describes procedures for quantifying Aspergillus niger growth in both solid and liquid culture. Firstly, by comparing radial growth between mutant and progenitor isolates on solid agar supplemented with sublethal stressors, susceptibility coefficients can be calculated. Secondly, analysis of macromorphological growth types in liquid culture allows full quantification of how a gene of interest affects submerged growth. By combining these assays, an extensive and quantitative dataset of how a gene of interest impacts growth in this fungus is possible. For complete details on the use and execution of this protocol, please refer to Cairns et al. (2019)1 and Cairns et al. (2022).2.

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.

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.

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.

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

[This corrects the article DOI: 10.1186/s40694-019-0081-x.].