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

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

Quantification and regulation of pellet morphology in streptomyces hygroscopicus var; geldanus cultures

Streptomyces hygroscopicus var. geldanus, an actinomycete, produces a range of antibiotic compounds, one of which, Geldanamycin, is currently under investigation for its potential anticancer properties. Filamentous microbes, both bacterial and fungal, possess the ability to grow in a range of morphological forms in submerged fermentations. Secondary metabolite production by filamentous microorganisms is often dependent on the morphology of biomass aggregates within the culture and therefore morphological regulation in such fermentations is desirable in order to produce specific compounds of interest. Morphological assessment of pellet formation and growth necessitated the development of an image analysis technique to allow high throughput morphological characterisation of microbial fermentation broths. An assessment of environmental and nutritional conditions for culturing S. hygroscopicus , concentrating specifically on the impact of nutrient broth composition, spore loading density and shear rate on the morphological and physiological profiles of the organism, was undertaken. The formation of pelleted biomass in submerged fermentations occurs as a result of spore aggregation, hyphal aggregation or entanglement; each of which was identified as a potential morphological control strategy. The microbial polysaccharide xanthan gum was used to artificially regulate the apparent viscosity and hence the rheological characteristics of S. hygroscopicus fermentations broths and succeeded in controlling particle collision, aggregation and hence pellet formation. Control of broth surface tension through the introduction of surfactants, facilitates regulation of hydrophobic particle interaction, thus enabling regulation of hyphal aggregation and hence pellet formation in S. hygroscopicus cultures. Furthermore, we have identified the production of a previously unreported biosurfactant which appears to have significant effect on the morphology of the organism. Preliminary purification and characterisation of this molecules and an assessment of its usefulness as a morphological control agent in Streptomyces fermentations has been conducted

Relationship between mycelial morphology, cell wall composition and product formation of rhizopus arrhizus

Pelleting of hyphae of Rhizopus arrhizus commences 9-10 h after inoculation, and not at a pregermination stage as for other fungi. The anionic polymers carboxymethylcellulose (CMC) and Carbopol-934 prevent pelleting, causing dispersal of growth of most species of Rhizopus, including R. arrhizus, when used as media supplements. The non-ionic polymer methylcellulose also dispersed growth, but less effectively. The specific growth rate of mycelia in control (unsupplemented) and in polymer-supplemented media was similar until 24 h, when growth became restricted in control medium. Polymer supplementation promoted increased biomass production, but in the case of Carbopol, this increase may be partly due to the tightly bound polymer, as indicated by scanning electron microscopy and biomass studies, particularly at low pH's. Hyphal extension zones and branch points of mycelia from Carbopol-supplemented media at low pH did not fluoresce when treated with Calcofluor White, unlike those from control, CMC- or Carbopol-supplemented media at higher pH values. The cell walls of dispersed mycelia from viscous media had a higher concentration of hexosamines (34-35%) and a lower concentration of protein (7-6%) compared to the cell walls of pelleted (24% hexosamines/18% protein) or less finely dispersed mycelia (23% hexosamines/13% protein). Results from acid/alkali extraction indicate that a greater proportion of glucuronan is held in non-glucosamine linkages in walls from pelleted than from dispersed mycelia. The analysis of the chitin component of the cell walls by X-ray diffraction demonstrated less crystallinity in the cell walls isolated from Carbopol-supplemented media compared to the other cell wall types examined. The presence of solid or semi-solid medium components were necessary to promote production of glucoamylase by R. arrhizus. The presence of Carbopol inhibited glucoamylase production. It was also shown to inhibit fumaric acid production when mycelia were grown in' its presence and when used in the biotransformation media using pre-grown mycelia. Dispersed filamentous mycelia from CMC-supplemented medium produced the highest yields of fumaric acid in batch culture and in biotransformation studies. However in pH controlled fermenters, where higher aeration/agitation rates prevented densley-packed pelleting, the control mycelia produced the highest yields. Dispersed mycelia from CMC-supplemented medium also had the highest specific capacity to convert progesterone to 1la-hydroxyprogesterone. 48 h congealed mycelia from control medium manifested a low metabolic rate and poor rates of biotransformation

Tailoring fungal morphology of Aspergillus niger MYA 135 by altering the hyphal morphology and the conidia adhesion capacity: biotechnological applications

Current problems of filamentous fungi fermentations and their further successful developments as microbial cell factories are dependent on control fungal morphology. In this connection, this work explored new experimental procedures in order to quantitatively check the potential of some culture conditions to induce a determined fungal morphology by altering both hyphal morphology and conidia adhesion capacity. The capacity of environmental conditions to modify hyphal morphology was evaluated by examining the influence of some culture conditions on the cell wall lytic potential of Aspergillus niger MYA 135. The relative value of the cell wall lytic potential was determined by measuring a cell wall lytic enzyme activity such as the mycelium-bound β-N-acetyl-Dglucosaminidase (Mb-NAGase). On the other hand, the quantitative value of conidia adhesion was considered as an index of its aggregation capacity. Concerning microscopic morphology, a highly negative correlation between the hyphal growth unit length (lHGU) and the specific Mb-NAGase activity was found (r = −0.915, P < 0.001). In fact, the environment was able to induce highly branched mycelia only under those culture conditions compatible with specific Mb-NAGase values equal to or higher than 190 U gdry.wt -1. Concerning macroscopic morphology, a low conidia adhesion capacity was followed by a dispersed mycelial growth. In fact, this study showed that conidia adhesion units per ml equal to or higher than 0.50 were necessary to afford pellets formation. In addition, it was also observed that once the pellet was formed the lHGU had an important influence on its final diameter. Finally, the biotechnological significance of such results was discussed as well.Fil: Colin, Veronica Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET- Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); Argentina;Fil: Baigori, Mario Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET- Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); Argentina; Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina;Fil: Pera, Licia Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET- Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); Argentina

Influence of enzyme location and culture rheology on glucose oxidase production and recovery by Aspergillus niger NRRL-3 and Penicillium sp. CBS120262 Madelyn Johnstone-Robertson.

Includes synopsis.Includes bibliographical references.The aim of this thesis is to establish and understand the location of glucose oxidase (GO) in fungi Aspergillus niger NRRL-3 and Penicillium sp. CBS120262 to inform the enzyme recovery. GO was chosen to be a model system and the outcomes obtained in this study could then be applied to other enzyme and microbial systems in which product location has been shown to be influenced by operating conditions

BTT 303/3 – Biochemical Engineering [Kejuruteraan Biokimia]- Jun 2014

Second Semester Examination Academic Session 2013/2014 June 2014 Duration: 3 hour

Rocking Aspergillus: morphology-controlled cultivation of Aspergillus niger in a wave-mixed bioreactor for the production of secondary metabolites

Background Filamentous fungi including Aspergillus niger are cell factories for the production of organic acids, proteins and bioactive compounds. Traditionally, stirred-tank reactors (STRs) are used to cultivate them under highly reproducible conditions ensuring optimum oxygen uptake and high growth rates. However, agitation via mechanical stirring causes high shear forces, thus affecting fungal physiology and macromorphologies. Two-dimensional rocking-motion wave-mixed bioreactor cultivations could offer a viable alternative to fungal cultivations in STRs, as comparable gas mass transfer is generally achievable while deploying lower friction and shear forces. The aim of this study was thus to investigate for the first time the consequences of wave-mixed cultivations on the growth, macromorphology and product formation of A. niger. Results We investigated the impact of hydrodynamic conditions on A. niger cultivated at a 5 L scale in a disposable two-dimensional rocking motion bioreactor (CELL-tainer®) and a BioFlo STR (New Brunswick®), respectively. Two different A. niger strains were analysed, which produce heterologously the commercial drug enniatin B. Both strains expressed the esyn1 gene that encodes a non-ribosomal peptide synthetase ESYN under control of the inducible Tet-on system, but differed in their dependence on feeding with the precursors d-2-hydroxyvaleric acid and l-valine. Cultivations of A. niger in the CELL-tainer resulted in the formation of large pellets, which were heterogeneous in size (diameter 300–800 μm) and not observed during STR cultivations. When talcum microparticles were added, it was possible to obtain a reduced pellet size and to control pellet heterogeneity (diameter 50–150 μm). No foam formation was observed under wave-mixed cultivation conditions, which made the addition of antifoam agents needless. Overall, enniatin B titres of about 1.5–2.3 g L−1 were achieved in the CELL-tainer® system, which is about 30–50% of the titres achieved under STR conditions. Conclusions This is the first report studying the potential use of single-use wave-mixed reactor systems for the cultivation of A. niger. Although final enniatin yields are not competitive yet with titres achieved under STR conditions, wave-mixed cultivations open up new avenues for the cultivation of shear-sensitive mutant strains as well as high cell-density cultivations.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berli