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.

DETECCIÓN DE HONGOS TOXIGÉNICOS EN LA CADENA PRODUCTIVA DEL MAÍZ (Zea maíz L.)

Se ha determinado la presencia de hongos toxigénicos en los principales productos de la cadena productiva del maíz que son tierra de cultivo, choclo, maíz, maíz pelado y harina de maíz. Los cuales proceden de tres provincias del Departamento de Junín (Huancayo, Concepción y Chupaca). El objetivo de este trabajo fue cuantificar, aislar e identificar los hongos toxigénicos presentes en esta cadena, para proponer medidas de control y evitar su proliferación. Los hongos identificados fueron Penicillium oxalicum, P. viridicatum y P. digitatum en casi todo el estudio, Fusarium roseum y F. moniliforme especialmente en la tierra de cultivo; Aspergillus niger , Rizophus stolonifer y levaduras, especialmente en la harina de maíz. De ellos, Los hongos toxigénicos identificados fueron: F. moniliforme F. roseum, P. viridicatum y A. Níger, y los hongos que causan podredumbre fueron P. digitatum y P. oxalicum. La incidencia de hongos varió de 10 a 4,5 x 105 ufc/g siendo el promedio 4,2 x 104 ufc/g en toda la cadena. También se ha determinado que el maíz pelado y la harina de maíz presentan alto número de hongos especialmente aquellos procedentes de Huancayo, lo que indica que debe hacer un mejor manejo postcosecha y comercialización, ya que estos productos se expenden a granel con mayor riesgo de contaminación.

Integration of Aspergillus niger transcriptomic profile with metabolic model identifies potential targets to optimise citric acid production from lignocellulosic hydrolysate

Background Citric acid is typically produced industrially by Aspergillus niger-mediated fermentation of a sucrose-based feedstock, such as molasses. The fungus Aspergillus niger has the potential to utilise lignocellulosic biomass, such as bagasse, for industrial-scale citric acid production, but realising this potential requires strain optimisation. Systems biology can accelerate strain engineering by systematic target identification, facilitated by methods for the integration of omics data into a high-quality metabolic model. In this work, we perform transcriptomic analysis to determine the temporal expression changes during fermentation of bagasse hydrolysate and develop an evolutionary algorithm to integrate the transcriptomic data with the available metabolic model to identify potential targets for strain engineering. Results The novel integrated procedure matures our understanding of suboptimal citric acid production and reveals potential targets for strain engineering, including targets consistent with the literature such as the up-regulation of citrate export and pyruvate carboxylase as well as novel targets such as the down-regulation of inorganic diphosphatase. Conclusions In this study, we demonstrate the production of citric acid from lignocellulosic hydrolysate and show how transcriptomic data across multiple timepoints can be coupled with evolutionary and metabolic modelling to identify potential targets for further engineering to maximise productivity from a chosen feedstock. The in silico strategies employed in this study can be applied to other biotechnological goals, assisting efforts to harness the potential of microorganisms for bio-based production of valuable chemicals.

Harnessing the Phytase Production Potential of Soil-Borne Fungi from Wastewater Irrigated Fields Based on Eco-Cultural Optimization under Shake Flask Method

Indigenous fungi present in agricultural soils could have synchronized their inherent potentials to the local climatic conditions. Therefore, the fungi resident in the untreated wastewater irrigated agricultural field might develop their potential for producing various enzymes to handle the induced full organic load from domestic wastewater and toxic chemicals from the textile industry. Around 53 various fungal isolates were grown and separated from the soil samples from these sites through soil dilution, soil-culture plate, and soil-culture plate methods. All the purified fungi were subjected to a phosphatase production test, and only 13 fungal strains were selected as phosphatase producers. Among them, only five fungi identified as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium purourogenum, and Mucor rouxii based on morphological similarities, showing higher phosphate solubilizing indices, were utilized for eco-cultural fine-tuning to harness their full production potential under shake flask (SF) method. Among various media, orchestral tuning, 200 µM sodium phytate as substrate with 1.5 mL of inoculum size of the fungi, pH 7, temperature 30 °C, glucose, and ammonium nitrate as carbon and nitrogen additive with seven days of incubation were found to be the most appropriate cultural conditions to harness the phytase production potential of the selected fungi. Aspergillus niger and Aspergillus flavus showed initial phytase activity (5.2 Units/mL, 4.8 Units/mL) and phytase specific activity (2.85, 2.65 Units/mL per mg protein) during screening to be enhanced up to 17 ± 0.033 (Units/mL), 16 ± 0.033 (Units/mL) and (13 ± 0.012), 10 ± 0.066 (Units/mL per mg protein), respectively, with the above-mentioned conditions. The phytase enzyme produced from these fungi were found to be almost stable for a wide range of pH (4–8); temperature (20–60 °C); insensitive to Ca2+ and Mg2+ ions, and EDTA, Ni2+, and Ba2+ inhibitors but highly sensitive to Mn2+, Cu2+, and Zn2+ ions, and Co2+, Cr3+, Al3+, Fe2+ and Ag1+ inhibitors. It was suggested that both phytase-producing strains of A. niger and A. flavus or their crude phytase enzymes might be good candidates for application in soils to release phosphates from phytate and a possible valuable substitute of phosphate fertilizers.

Optimization of the decolorization conditions of Rose Bengal by using Aspergillus niger TF05 and a decolorization mechanism

Aspergillus niger TF05 was applied to decolorize Rose Bengal dye. The effects of carbon source, nitrogen source, metal ion and spore concentration on Rose Bengal treatment with A. niger TF05 were studied. A Plackett–Burman design (PBD) and a uniform design (UD) were used to optimize the decolorization conditions of A. niger TF05 and enhance its decolorization effect. The mechanism of Rose Bengal decolorization by A. niger TF05 was examined by analysing degradation products via UV–visible light spectroscopy, IR spectroscopy and GC-MS. The best decolorization effect was achieved in the single factor test with glucose and ammonium chloride as carbon and nitrogen sources, respectively. Mg2+ was an essential ion that could improve the mould ball state and adsorption efficiency if the spore concentration was maintained at 106 spores ml–1. The optimal decolorization conditions obtained using the PBD and UD methods were 11.5 g l−1 glucose, 6.5 g l−1 ammonium chloride, 0.4 g l−1 magnesium sulphate, pH 5.8, 28 °C, 140 r.p.m. rotational speed, 0.18 g l−1 dye concentration, 0.5 ml of inocula and 120 h decolorization time. Under these conditions, the maximum decolorization rate was 106%. Spectral analysis suggested that the absorption peak of the product changed clearly after decolorization; GC-MS analysis revealed that the intermediate product tetrachlorophthalic anhydride formed after decolorization. The combined use of the PBD and UD methods can optimize multi-factor experiments. A. niger TF05 decolorized Rose Bengal during intracellular enzymatic degradation after adsorption.