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

Abstract

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 ...

Disruption or reduced expression of the orotidine-5′-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger

“…3). Consistent with the previous studies [31][32][33][34], Tet-on system is a useful method to functionally analyze essential genes, enabling analyses of null, loss-of-function, and over-expression in a single strain by addition of the inducer Dox. After co-transformation, ten randomly selected primary transformants were subcultured on MM plates, MM supplemented with 20 µg/mL Dox and MM supplemented with uridine.…”

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

“…4a and a summary of growth phenotypes given in Table 2. Additionally, pellet Euclidian parameters (maximum diameter, area, solidity, and aspect ratio, see methods) were quantified using the automated Morphology of Pelleted and Dispersed growth (MPD) image analysis pipeline [37] and used to determine the dimensionless morphology number (MN [38]), which generates a value between 0 (a theoretical one-dimensional line) and 1 (a perfect round sphere, Fig. 4b).

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The mechanisms of hyphal pellet formation mediated by polysaccharides, α-1,3-glucan and galactosaminogalactan, in Aspergillus species

“…Recently, Jeennor et al [69] reported that an A. oryzae mutant defective in the ags1 (agsB) gene showed an increase in lipid productivity in submerged flask and 5-L reactor cultivation in comparison with the parental strain. Recently developed software enables automatic characterization of hyphal macromorphology including hyphal area, pellet diameter, aspect ratio, and solidity [70]. Applying this software for characterization of α-1,3-glucan mutant hyphae could be a new approach to understand the relationship between fungal cell wall composition and morphogenesis.…”