Characterization of potassium-solubilizing fungi, Mortierella spp., isolated from a poplar plantation rhizosphere soil.

Potassium-solubilizing microorganisms are capable of secreting acidic chemicals that dissolve and release potassium from soil minerals, thus facilitating potassium uptake by plants. In this study, three potassium-dissolving filamentous fungi were isolated from the rhizosphere soil of a poplar plantation in Jiangsu Province, China. Phylogenetic analyses based on ITS, 18 S, and 28 S showed that these three isolates were most similar to Mortierella. These strains also possessed spherical or ellipsoidal spores, produced sporangia at the hyphal tip, and formed petal-like colonies on PDA media resembling those of Mortierella species. These findings, along with further phenotypic observations, suggest that these isolates were Mortierella species. In addition, the potassium-dissolution experiment showed that strain 2K4 had a relatively high potassium-solubilizing capacity among these isolated fungi. By investigating the influences of different nutrient conditions (carbon source, nitrogen source, and inorganic salt) and initial pH values on the potassium-dissolving ability, the optimal potassium-solubilization conditions of the isolate were determined. When potassium feldspar powder was used as an insoluble potassium source, isolate 2K4 exhibited a significantly better polysaccharide aggregation ability on the formed mycelium-potassium feldspar complex. The composition and content of organic acids secreted by strain 2K4 were further detected, and the potassium-dissolution mechanism of the Mortierella species and its growth promotion effect were discussed, using maize as an example.

Implications of carbon catabolite repression for Aspergillus-based cell factories: A review.

Carbon catabolite repression (CCR) is a global regulatory mechanism that allows organisms to preferentially utilize a preferred carbon source (usually glucose) by suppressing the expression of genes associated with the utilization of nonpreferred carbon sources. Aspergillus is a large genus of filamentous fungi, some species of which have been used as microbial cell factories for the production of organic acids, industrial enzymes, pharmaceuticals, and other fermented products due to their safety, substrate convenience, and well-established post-translational modifications. Many recent studies have verified that CCR-related genetic alterations can boost the yield of various carbohydrate-active enzymes (CAZymes), even under CCR conditions. Based on these findings, we emphasize that appropriate regulation of the CCR pathway, especially the expression of the key transcription factor CreA gene, has great potential for further expanding the application of Aspergillus cell factories to develop strains for industrial CAZymes production. Further, the genetically modified CCR strains (chassis hosts) can also be used for the production of other useful natural products and recombinant proteins, among others. We here review the regulatory mechanisms of CCR in Aspergillus and its direct application in enzyme production, as well as its potential application in organic acid and pharmaceutical production to illustrate the effects of CCR on Aspergillus cell factories.

A High-Quality Genome Sequence of the Penicillium oxalicum 5-18 Strain Isolated from a Poplar Plantation Provides Insights into Its Lignocellulose Degradation.

Studies on the degradation of plant cell wall polysaccharides by fungal extracellular enzymes have attracted recent attention from researchers. Xylan, abundant in hemicellulose, that play great role in connection between cellulose and lignin, has seen interest in its hydrolytic enzymatic complex. In this study, dozens of fungus species spanning genera were isolated from rotting leaves based on their ability to decompose xylan. Among these isolates, a strain with strong xylanase-producing ability was selected for further investigation by genome sequencing. Based on phylogenetic analysis of ITS (rDNA internal transcribed spacer) and LSU (Large subunit 28S rDNA) regions, the isolate was identified as Penicillium oxalicum. Morphological analysis also supported this finding. Xylanase activity of this isolated P. oxalicum 5-18 strain was recorded to be 30.83 U/mL using the 3,5-dinitro-salicylic acid (DNS) method. Further genome sequencing reveals that sequenced reads were assembled into a 30.78 Mb genome containing 10,074 predicted protein-encoding genes. In total, 439 carbohydrate-active enzymes (CAZymes) encoding genes were predicted, many of which were associated with cellulose, hemicellulose, pectin, chitin and starch degradation. Further analysis and comparison showed that the isolate P. oxalicum 5-18 contains a diverse set of CAZyme genes involved in degradation of plant cell wall components, particularly cellulose and hemicellulose. These findings provide us with valuable genetic information about the plant biomass-degrading enzyme system of P. oxalicum, facilitating a further exploration of the repertoire of industrially relevant lignocellulolytic enzymes of P. oxalicum 5-18.

Phosphorus-Solubilizing Capacity of Mortierella Species Isolated from Rhizosphere Soil of a Poplar Plantation.

Phosphorus is one of the main nutrients necessary for plant growth and development. Phosphorus-dissolving microorganisms may convert insoluble phosphorus in soil into available phosphorus that plants can easily absorb and utilize. In this study, four phosphorus-solubilizing fungi (L3, L4, L5, and L12) were isolated from the rhizosphere soil of a poplar plantation in Dongtai, Jiangsu Province, China. Phylogenetic analysis based on the internal transcribed spacer (ITS) and large subunit (LSU) of the ribosomal DNA sequences showed that the ITS and 28S sequences of isolates were the most similar to those of Mortierella. Morphological observation showed that most colonies grew in concentric circles and produced spores under different culture conditions. These results and further microscopic observations showed that these isolated fungi belonged to the genus Mortierella. Pikovskaya (PKO) medium, in which tricalcium phosphate was the sole phosphorus source, was used to screen strain L4 with the best phosphorus-solubilizing effect for further study. When the carbon source was glucose, the nitrogen source was ammonium chloride, the pH was 5, and the available phosphorus content was the highest. By exploring the possible mechanism of phosphorus release by phosphorus-solubilizing fungi, it was found that strain L4 produces several organic acids, such as oxalic acid, lactic acid, acetic acid, succinic acid, tartaric acid, malic acid, and citric acid. At 24 h, the alkaline phosphatase and acid phosphatase activities reached 154.72 mol/(L·h) and 120.99 mol/(L·h), respectively.

Impacts of Biogas Slurry Fertilization on Arbuscular Mycorrhizal Fungal Communities in the Rhizospheric Soil of Poplar Plantations.

The majority of terrestrial plants are symbiotic with arbuscular mycorrhizal fungi (AMF). Plants supply carbohydrates to microbes, whereas AMF provide plants with water and other necessary nutrients-most typically, phosphorus. Understanding the response of the AMF community structure to biogas slurry (BS) fertilization is of great significance for sustainable forest management. This study aimed to look into the effects of BS fertilization at different concentrations on AMF community structures in rhizospheric soil in poplar plantations. We found that different fertilization concentrations dramatically affected the diversity of AMF in the rhizospheric soil of the poplar plantations, and the treatment with a high BS concentration showed the highest Shannon diversity of AMF and OTU richness (Chao1). Further analyses revealed that Glomerales, as the predominant order, accounted for 36.2-42.7% of the AMF communities, and the relative abundance of Glomerales exhibited negligible changes with different BS fertilization concentrations, whereas the order Paraglomerales increased significantly in both the low- and high-concentration treatments in comparison with the control. Furthermore, the addition of BS drastically enhanced the relative abundance of the dominant genera, Glomus and Paraglomus. The application of BS could also distinguish the AMF community composition in the rhizospheric soil well. An RDA analysis indicated that the dominant genus Glomus was significantly positively correlated with nitrate reductase activity, while Paraglomus showed a significant positive correlation with available P. Overall, the findings suggest that adding BS fertilizer to poplar plantations can elevate the diversity of AMF communities in rhizospheric soil and the relative abundance of some critical genera that affect plant nutrient uptake.

CRISPR/Cas9-Based Genome Editing and Its Application in Aspergillus Species.

Aspergillus, a genus of filamentous fungi, is extensively distributed in nature and plays crucial roles in the decomposition of organic materials as an important environmental microorganism as well as in the traditional fermentation and food processing industries. Furthermore, due to their strong potential to secrete a large variety of hydrolytic enzymes and other natural products by manipulating gene expression and/or introducing new biosynthetic pathways, several Aspergillus species have been widely exploited as microbial cell factories. In recent years, with the development of next-generation genome sequencing technology and genetic engineering methods, the production and utilization of various homo-/heterologous-proteins and natural products in Aspergillus species have been well studied. As a newly developed genome editing technology, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been used to edit and modify genes in Aspergilli. So far, the CRISPR/Cas9-based approach has been widely employed to improve the efficiency of gene modification in the strain type Aspergillus nidulans and other industrially important and pathogenic Aspergillus species, including Aspergillus oryzae, Aspergillus niger, and Aspergillus fumigatus. This review highlights the current development of CRISPR/Cas9-based genome editing technology and its application in basic research and the production of recombination proteins and natural products in the Aspergillus species.

Advances in Genetic Engineering Technology and Its Application in the Industrial Fungus Aspergillus oryzae.

The filamentous fungus Aspergillus oryzae is an important strain in the traditional fermentation and food processing industries and is often used in the production of soy sauce, soybean paste, and liquor-making. In addition, A. oryzae has a strong capacity to secrete large amounts of hydrolytic enzymes; therefore, it has also been used in the enzyme industry as a cell factory for the production of numerous native and heterologous enzymes. However, the production and secretion of foreign proteins by A. oryzae are often limited by numerous bottlenecks that occur during transcription, translation, protein folding, translocation, degradation, transport, secretion, etc. The existence of these problems makes it difficult to achieve the desired target in the production of foreign proteins by A. oryzae. In recent years, with the decipherment of the whole genome sequence, basic research and genetic engineering technologies related to the production and utilization of A. oryzae have been well developed, such as the improvement of homologous recombination efficiency, application of selectable marker genes, development of large chromosome deletion technology, utilization of hyphal fusion techniques, and application of CRISPR/Cas9 genome editing systems. The development and establishment of these genetic engineering technologies provided a great deal of technical support for the industrial production and application of A. oryzae. This paper reviews the advances in basic research and genetic engineering technologies of the fermentation strain A. oryzae mentioned above to open up more effective ways and research space for the breeding of A. oryzae production strains in the future.

Identification and characterization of a DevR-interacting protein in Aspergillus oryzae.

The basic helix-loop-helix (bHLH) proteins belong to a superfamily of transcription factors. Recent research has shown that the bHLH transcription factor DevR is involved in both sexual and asexual development as well as conidial melanin production in Aspergillus species. Our previous research also found that DevR significantly influences polysaccharide metabolism in Aspergillus oryzae. In this study, to further explore the function of DevR, its interaction proteins were screened by a yeast two-hybrid assay. An A. oryzae cDNA library was transformed into the Y187 strain by using the SMART technique and the homologous recombination method, and then hybridized with a constructed DevR bait plasmid introducing strain to obtain positive clones. Through sequencing analysis, the potential interaction proteins of DevR were determined. Among them, an AO090701000363 gene-encoding protein (named DipA), which was predicted to be a basic leucine zipper (bZIP) transcription factor, was a possible candidate. Phenotypic analysis indicated that overexpression of the AodipA may significantly suppress growth of the strain. Additionally, although no obvious change in the growth rate was found, the deletion of AodipA resulted in thicker hyphae morphology relative to the control. Comparative proteomic analysis further indicated that DipA was potentially involved in the regulation of cell wall integrity, carbon utilization, acetate catabolic process and other biological processes. Partial similarity of the phenotype to that of DevR suggested a correlation between them and implied that the DipA has a function partially similar to that of DevR.

Studies of Cellulose and Starch Utilization and the Regulatory Mechanisms of Related Enzymes in Fungi.

Polysaccharides are biopolymers made up of a large number of monosaccharides joined together by glycosidic bonds. Polysaccharides are widely distributed in nature: Some, such as peptidoglycan and cellulose, are the components that make up the cell walls of bacteria and plants, and some, such as starch and glycogen, are used as carbohydrate storage in plants and animals. Fungi exist in a variety of natural environments and can exploit a wide range of carbon sources. They play a crucial role in the global carbon cycle because of their ability to break down plant biomass, which is composed primarily of cell wall polysaccharides, including cellulose, hemicellulose, and pectin. Fungi produce a variety of enzymes that in combination degrade cell wall polysaccharides into different monosaccharides. Starch, the main component of grain, is also a polysaccharide that can be broken down into monosaccharides by fungi. These monosaccharides can be used for energy or as precursors for the biosynthesis of biomolecules through a series of enzymatic reactions. Industrial fermentation by microbes has been widely used to produce traditional foods, beverages, and biofuels from starch and to a lesser extent plant biomass. This review focuses on the degradation and utilization of plant homopolysaccharides, cellulose and starch; summarizes the activities of the enzymes involved and the regulation of the induction of the enzymes in well-studied filamentous fungi.

Research progress on the basic helix-loop-helix transcription factors of Aspergillus species.

Basic helix-loop-helix (bHLH) proteins belong to a superfamily of transcription factors, and they are widely distributed in eukaryotic organisms. Members of the bHLH protein family can form homodimers or heterodimers with themselves or other family members, and they often play bifunctional roles as activators and repressors to uniquely regulate the transcription of downstream target genes. The bHLH transcription factors are usually involved in developmental processes, including cellular proliferation and differentiation. Therefore, these transcription factors often play crucial roles in regulating growth, development, and differentiation in eukaryotes. Aspergillus species fungi are widely distributed in the environment, and they play important roles not only in the decomposition of organic matter as an important environmental microorganism but also in the fermentation and the food processing industry. Furthermore, some pathogenic fungi, such as Aspergillus flavus and Aspergillus fumigatus, affect the environment and human health in important ways. Recent research has shown that some Aspergillus bHLH proteins are significantly involved in the regulation of asexual and sexual reproduction, secondary metabolite production, carbohydrate metabolism, conidial and sclerotial production, among other processes. Here, we review the regulatory mechanisms and biological functions of the bHLH transcription factors of the Aspergillus genus to provide a theoretical reference for further study on the growth and development of Aspergillus and the functions of bHLHs.

The Basic-Region Helix-Loop-Helix Transcription Factor DevR Significantly Affects Polysaccharide Metabolism in Aspergillus oryzae.

Basic-region helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that are often involved in the control of growth and differentiation. Recently, it was reported that the bHLH transcription factor DevR is involved in both asexual and sexual development in Aspergillus nidulans and regulates the conidial melanin production in Aspergillus fumigatus In this study, we identified and characterized an Aspergillus oryzae gene that showed high similarity with devR of A. nidulans and A. fumigatus (AodevR). In the AodevR-disrupted strain, growth was delayed and the number of conidia was decreased on Czapek-Dox (CD) minimal agar plates, but the conidiation was partially recovered by adding 0.6 M KCl. Simultaneously, the overexpression of AodevR was induced and resulted in extremely poor growth when the carbon source changed from glucose to polysaccharide (dextrin) in the CD agar plate. Scanning electron microscopy (SEM) indicated that the overexpression of AodevR resulted in extremely thin aberrant hyphal morphology. Conversely, the deletion of AodevR resulted in thicker hyphae and in more resistance to Congo red relative to the control strain. Quantitative reverse transcriptase PCR (RT-PCR) further indicated that AoDevR significantly affects chitin and starch metabolism, and importantly, the overexpression of AodevR inhibited the expression of genes related to starch degradation. A yeast one-hybrid assay suggested that the DevR protein possibly interacted with the promoter of amyR, which encodes a transcription factor involved in amylase production. Importantly, AoDevR is involved in polysaccharide metabolism and affects the growth of the A. oryzae strain.IMPORTANCEAspergillus oryzae is an industrially important filamentous fungus; therefore, a clear understanding of its polysaccharide metabolism and utilization is very important for its industrial utilization. In this study, we revealed that the basic-region helix-loop-helix (bHLH) transcription factor AoDevR is importantly involved in chitin and starch metabolism in A. oryzae The overexpression of AodevR strongly suppressed the expression of amylase-related genes. The results of a yeast one-hybrid assay suggested that the DevR protein potentially interacts with the promoter of amyR, which encodes a transcription factor involved in amylase production and starch utilization. This study provides new insight for further revealing the regulation mechanism of amylase production in A. oryzae.