Important Filamentous Fungi Research Articles

CRISPR/Cas9-based genome engineering: A new breakthrough in the genetic manipulation of filamentous fungi.

Filamentous fungi have several industrial, environmental, and medical applications. However, they are rarely utilized owing to the limited availability of full-genome sequences and genetic manipulation tools. Since the recent discovery of the full-genome sequences for certain industrially important filamentous fungi, CRISPR/Cas9 technology has drawn attention for the efficient development of engineered strains of filamentous fungi. CRISPR/Cas9 genome editing has been successfully applied to diverse filamentous fungi. In this review, we briefly discuss the use of common genetic transformation techniques as well as CRISPR/Cas9-based systems in filamentous fungi. Furthermore, we describe potential limitations and challenges in the practical application of genome engineering of filamentous fungi. Finally, we provide suggestions and highlight future research prospects in the area.

Alternative transcription start sites of the enolase-encoding gene enoA are stringently used in glycolytic/gluconeogenic conditions in Aspergillus oryzae.

Gene expression using alternative transcription start sites (TSSs) is an important transcriptional regulatory mechanism for environmental responses in eukaryotes. Here, we identify two alternative TSSs in the enolase-encoding gene (enoA) in Aspergillus oryzae, an industrially important filamentous fungus. TSS use in enoA is strictly dependent on the difference in glycolytic and gluconeogenic carbon sources. Transcription from the upstream TSS (uTSS) or downstream TSS (dTSS) predominantly occurs under gluconeogenic or glycolytic conditions, respectively. In addition to enoA, most glycolytic genes involved in reversible reactions possess alternative TSSs. The fbaA gene, which encodes fructose-bisphosphate aldolase, also shows stringent alternative TSS selection, similar to enoA. Alignment of promoter sequences of enolase-encoding genes in Aspergillus predicted two conserved regions that contain a putative cis-element required for enoA transcription from each TSS. However, uTSS-mediated transcription of the acuN gene, an enoA ortholog in Aspergillus nidulans, is not strictly dependent on carbon source, unlike enoA. Furthermore, enoA transcript levels in glycolytic conditions are higher than in gluconeogenic conditions. Conversely, acuN is more highly transcribed in gluconeogenic conditions. This suggests that the stringent usage of alternative TSSs and higher transcription in glycolytic conditions in enoA may reflect that the A. oryzae evolutionary genetic background was domesticated by exclusive growth in starch-rich environments. These findings provide novel insights into the complexity and diversity of transcriptional regulation of glycolytic/gluconeogenic genes among Aspergilli.

A newly constructed Agrobacterium-mediated transformation system revealed the influence of nitrogen sources on the function of the LaeA regulator in Penicillium chrysogenum

Penicillium chrysogenum is not only an industrially important filamentous fungus for penicillin production, but it also represents as a promising cell factory for production of natural products. Development of efficient transformation systems with suitable selection markers is essential for genetic manipulations in P. chrysogenum. In this study, we have constructed a new and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system with two different selection markers conferring the resistance to nourseothricin and phleomycin for P. chrysogenum. Under the optimized conditions for co-cultivation at 22 °C for 60 h with acetosyringone concentration of 200 μM, the transformation efficiency of the ATMT system could reach 5009 ± 96 transformants per 106 spores. The obtained transformants could be exploited as the T-DNA insertion mutants for screening genes involved in morphogenesis and secondary metabolism. Especially, the constructed ATMT system was applied successfully to generate a knockout mutant of the laeA regulatory gene and relevant complementation strains in a wild strain of P. chrysogenum. Our results indicated that the LaeA regulator controls growth, sporulation, osmotic stress response and antibiotic production in P. chrysogenum, but its function is reliant on nitrogen sources. Furthermore, we showed that the laeA orthologous genes from the citrus postharvest pathogen P. digitatum and from the industrial fungus Aspergillus niger could recover the phenotypic defects in the P. chrysogenum laeA deletion mutant. Conclusively, this work provides a new ATMT system, which can be employed for T-DNA insertional mutagenesis, heterologous gene expression or for molecular inspections of potential genes related to secondary metabolism in P. chrysogenum.

The GATA-Type Transcriptional Factor Are1 Modulates the Expression of Extracellular Proteases and Cellulases in Trichoderma reesei.

Trichoderma reesei is a biotechnologically important filamentous fungus with the remarkable ability to secrete large amounts of enzymes, whose production is strongly affected by both the carbon and nitrogen sources. While the carbon metabolism regulators are extensively studied, the regulation of enzyme production by the nitrogen metabolism regulators is still poorly understood. In this study, the GATA transcription factor Are1, which is an orthologue of the Aspergillus global nitrogen regulator AREA, was identified and characterized for its functions in regulation of both protease and cellulase production in T. reesei. Deletion of the are1 gene abolished the capability to secrete proteases, and complementation of the are1 gene rescued the ability to produce proteases. Quantitative RT-PCR analysis revealed that the transcripts of protease genes apw1 and apw2 were also significantly reduced in the Δare1 strain when grown in the medium with peptone as the nitrogen source. In addition, deletion of are1 resulted in decreased cellulase production in the presence of (NH4)2SO4. Consistent with the reduction of cellulase production, the transcription levels of the major cellulase genes, including cbh1, cbh2, egl1, and egl2, were dramatically decreased in Δare1. Sequence analysis showed that all promoter regions of the tested protease and cellulase genes contain the consensus GATA elements. However, the expression levels of the major cellulase transcription activator Xyr1 and the repressor Cre1 had no significant difference between Δare1 and the parental strain QM9414, indicating that the regulatory mechanism deserves further investigation. Taken together, these results demonstrate the important role of Are1 in the regulation of protease and cellulase production in T. reesei, although these processes depend on the kind of nitrogen sources. The findings in this study contribute to the understanding of the regulation network of carbon and nitrogen sources in filamentous fungi.

Structural Diversity and Bioactivities of Peptaibol Compounds From the Longibrachiatum Clade of the Filamentous Fungal Genus Trichoderma.

This study examined the structural diversity and bioactivity of peptaibol compounds produced by species from the phylogenetically separated Longibrachiatum Clade of the filamentous fungal genus Trichoderma, which contains several biotechnologically, agriculturally and clinically important species. HPLC-ESI-MS investigations of crude extracts from 17 species of the Longibrachiatum Clade (T. aethiopicum, T. andinense, T. capillare, T. citrinoviride, T. effusum, T. flagellatum, T. ghanense, T. konilangbra, T. longibrachiatum, T. novae-zelandiae, T. pinnatum, T. parareesei, T. pseudokoningii, T. reesei, T. saturnisporum, T. sinensis, and T. orientale) revealed several new and recurrent 20-residue peptaibols related to trichobrachins, paracelsins, suzukacillins, saturnisporins, trichoaureocins, trichocellins, longibrachins, hyporientalins, trichokonins, trilongins, metanicins, trichosporins, gliodeliquescins, alamethicins and hypophellins, as well as eight 19-residue sequences from a new subfamily of peptaibols named brevicelsins. Non-ribosomal peptide synthetase genes were mined from the available genome sequences of the Longibrachiatum Clade. Their annotation and product prediction were performed in silico and revealed full agreement in 11 out of 20 positions regarding the amino acids predicted based on the signature sequences and the detected amino acids incorporated. Molecular dynamics simulations were performed for structural characterization of four selected peptaibol sequences: paracelsins B, H and their 19-residue counterparts brevicelsins I and IV. Loss of position R6 in brevicelsins resulted in smaller helical structures with higher atomic fluctuation for every residue than the structures formed by paracelsins. We observed the formation of highly bent, almost hairpin-like, helical structures throughout the trajectory, along with linear conformation. Bioactivity tests were performed on the purified peptaibol extract of T. reesei on clinically and phytopathologically important filamentous fungi, mammalian cells, and Arabidopsis thaliana seedlings. Porcine kidney cells and boar spermatozoa proved to be sensitive to the purified peptaibol extract. Peptaibol concentrations ≥0.3 mg ml−1 deterred the growth of A. thaliana. However, negative effects to plants were not detected at concentrations below 0.1 mg ml−1, which could still inhibit plant pathogenic filamentous fungi, suggesting that those peptaibols reported here may have applications for plant protection.

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.

A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting

BackgroundIn the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. Trichoderma reesei is a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive.ResultsHerein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in T. reesei and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase cbh1 promoter in T. reesei, which allowed us to rapidly isolate cellulase hyperproducers from T. reesei progenies transformed with a dedicated transcriptional activator ace3 and from an atmospheric and room temperature plasma-created mutant T. reesei library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the T. reesei cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing hac1 and bip1) affecting regulatory stages beyond transcription. Using this method, T. reesei cellulase hyperproducers were also successfully isolated from an Agrobacterium-mediated random insertional mutant library.ConclusionsThe coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of T. reesei cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.

The current status on secondary metabolites produced by plant pathogenic Colletotrichum species

The fungal genus Colletotrichum contains about 190 species, many of which are responsible for serious plant diseases including those of commercial crops. These species infect a wide range of crops in the tropical, sub-tropical and temperate regions of the world. The diseases caused by Colletotrichum species are known as “anthracnose diseases” and are characterized by sunken necrotic tissue in which masses of orange conidia are produced. A significant number of the metabolites of Colletotrichum species are known to contribute to their pathogenicity. These phytotoxic metabolites when applied to the leaves of their host plants, induced symptoms which were similar to those of the anthracnose caused by the fungus. These metabolites have been shown to play a significant role in the mechanism of infection and pathogenesis. This review deals with the structures and biological activity of the secondary metabolites which have been isolated from these economically important filamentous fungi. The literature up to July 2018 is reviewed and 160 references are cited.

BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae

Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. Although cell fusion is important for heterokaryon formation and sexual/parasexual reproduction required for cross breeding, knowledge on cell fusion and heterokaryon incompatibility in A. oryzae is limited because of low cell fusion frequency. Therefore, we aimed to develop a BiFC system to specifically visualise fused cells and facilitate the analysis of cell fusion in A. oryzae. The cell fusion ability and morphology of 15 A. oryzae strains were investigated using heterodimerising proteins LZA and LZB fused with split green fluorescence protein. Morphological investigation of fused cells revealed that cell fusion occurred mainly as conidial anastomosis during the early growth stage. Self-fusion abilities were detected in most industrial A. oryzae strains, but only a few strain pairs showed non-self fusion. Protoplast fusion assay demonstrated that almost all the pairs capable of non-self fusion were capable of heterokaryon formation and vice versa, thus providing the first evidence of heterokaryon incompatibility in A. oryzae. The BiFC system developed in this study provides an effective method in studying morphology of fused cells and heterokaryon incompatibility in the filamentous fungal species with low cell fusion efficiency.

A New Antifungal Antibiotic from Bacillus sp. KM5 Isolated from Rice Rhizospheric Soil

Bacterial strain, Bacillus sp. KM5 was recently isolated and characterized by strong antifungal activities against several agronomically important filamentous pathogenic fungi including Rhizoctonia solani Nees. This is a new study to characterize four prominent antibiotics synthesized by Bacillus sp. KM5 (termed M1, M2, M3, and M4), isolated from rice rhizospheric soil. Lipopeptide profiling and spectroscopic characterizations of antibiotics M1 and M2 identified them as Bacillomycin F and Bacillomycin D, respectively. Antibiotic M3 was recognized as another iturinic lipopeptide Bacillopeptin A. Finally, M4 emerged as a novel straight chain polyhydroxy polyether compound with a single double bond, indicating a non-polyene structure with no prior mention in the literature. Furthermore, in vitro bioassay of M4 showed maximum activity against Rhizoctonia solani among five plant pathogenic fungi. This research could help in developing new or improved antifungal formulations or novel strategies to prevent fungal spoilage.

Comprehensive evaluation of malt volatile compounds contaminated byFusarium graminearumduring malting

Fusarium graminearum is the most important filamentous fungi in the malting process, and greatly affects malting performance and malt quality. Analysis of the emitted volatiles from F. graminearum mycelium was used to evaluate the malt flavour contaminated by F. graminearum during malting based on solid-phase microextraction combined with gas chromatography–mass spectrometry. GC–MS analysis of different mycelium samples revealed a broad spectrum of volatile compounds including aldehydes, ketones, alcohols, organic acids and aromatics, which were highly similar to those of barley and malt. Statistical assessment of the data via principal component analysis demonstrated that the major contributors to the time-dependent dynamic changes in the volatiles were the volatile aldehyde, alcohol and ketone fractions in the contaminated malting process. The results showed that volatile concentration was modulated by the -metabolism of the green malt and F. graminearum. Volatile compounds from green malt were influenced by F. graminearum fungi contamination during malting, although some were probably generated by this fungus. Copyright © 2017 The Institute of Brewing & Distilling

In Vitro Activity of Calcium Channel Blockers in Combination with Conventional Antifungal Agents Against Clinically Important Filamentous Fungi

Despite the current therapeutic options, filamentous fungal infections are associated with high mortality rate especially in immunocompromised patients. In order to find a new potential therapeutic approach, the in vitro inhibitory effect of two antiarrhythmic agents, diltiazem and verapamil hydrochloride were tested against different clinical isolates of ascomycetous and mucoralean filamentous fungi. The in vitro combinations of these non-antifungal drugs with azole and polyene antifungal agents were also examined. Susceptibility tests were carried out using the broth microdilution method according to the instructions of the Clinical and Laboratory Standards Institute document M38-A2. Checkerboard microdilution assay was used to assess the interactions between antifungal and non-antifungal drugs. Compared to antifungal agents, diltiazem and verapamil hydrochloride exerted a relatively low antifungal activity with high minimal inhibitory concentration values (853–2731 μg/ml). Although in combination they could increase the antifungal activity of amphotericin B, itraconazole and voriconazole. Indifferent and synergistic interactions were registered in 33 and 17 cases, respectively. Antagonistic interactions were not revealed between the investigated compounds. However, the observed high MICs suggest that these agents could not be considered as alternative systemic antifungal agents.

Analysis of an acyl-CoA binding protein in Aspergillus oryzae that undergoes unconventional secretion

Acyl-CoA binding protein (ACBP) plays important roles in the metabolism of lipids in eukaryotic cells. In the industrially important filamentous fungus Aspergillus oryzae, although we have previously demonstrated that the A. oryzae ACBP (AoACBP) localizes to punctate structures and exhibits long-range motility, which is dependent on autophagy-related proteins, the physiological role of AoACBP remains elusive. Here, we describe identification and characterization of another ACBP from A. oryzae; we named this ACBP as AoAcb2 and accordingly renamed AoACBP as AoAcb1. The deduced amino acid sequence of AoAcb2 lacked a signal peptide. Phylogenetic analysis classified AoAcb2 into a clade that was same as the ACBP Acb1 of the model yeast Saccharomyces cerevisiae, but was different from that of AoAcb1. In contrast to punctate localization of AoAcb1, AoAcb2 was found to be dispersedly distributed in the cytoplasm, as was previously observed for the S. cerevisiae Acb1. Since we could not generate an Aoacb2 disruptant, we created an Aoacb2 conditional mutant that exhibited less growth under Aoacb2-repressed condition, suggesting that Aoacb2 is an essential gene for growth. Moreover, we observed that A. oryzae AoAcb2, but not A. oryzae AoAcb1, was secreted under carbon-starved condition, suggesting that AoAcb2 might be secreted via the unconventional protein secretion (UPS) pathway, just like S. cerevisiae Acb1. We also demonstrated that the unconventional secretion of AoAcb2 was dependent on the t-SNARE AoSso1, but was independent of the autophagy-related protein AoAtg1, suggesting that the unconventional secretion of AoAcb2, unlike that of S. cerevisiae Acb1, via the UPS pathway, is not regulated by the autophagy machinery. Thus, the filamentous fungus A. oryzae harbors two types of ACBPs, one of which appears to be essential for growth and undergoes unconventional secretion.

Nonsteroidal Anti-inflammatory Drugs (NSAIDS) Inhibit the Growth and Reproduction of Chaetomium globosum and Other Fungi Associated with Water-Damaged Buildings.

Indoor mold due to water damage causes serious human respiratory disorders, and the remediation to homes, schools, and businesses is a major expense. Prevention of mold infestation of building materials would reduce health problems and building remediation costs. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit yeasts and a limited number of filamentous fungi. The purpose of this research was to determine the possible inhibitory activity of nonsteroidal anti-inflammatory drugs (NSAIDs) on germination, fungal growth, and reproduction of Chaetomium globosum and other important filamentous fungi that occur in water-damaged buildings. Several NSAIDs were found to inhibit C. globosum germination, growth, and reproduction. The most effective NSAIDs inhibiting C. globosum were ibuprofen, diflunisal, and diclofenac. Fusarium oxysporum, Fusarium solani, Aspergillus niger, and Stachybotrys atra were also tested on the various media with similar results obtained. However, F. oxysporum and A. niger exhibited a higher level of resistance to aspirin and NaSAL when compared to the C. globosum isolates. The inhibition exhibited by NSAIDs was variable depending on growth media and stage of fungal development. These compounds have a great potential of inhibiting fungal growth on building materials such as gypsum board. Formulations of sprays or building materials with NSAID-like chemical treatments may hold promise in reducing mold in homes and buildings.

The low affinity glucose transporter HxtB is also involved in glucose signalling and metabolism in Aspergillus nidulans

One of the drawbacks during second-generation biofuel production from plant lignocellulosic biomass is the accumulation of glucose, the preferred carbon source of microorganisms, which causes the repression of hydrolytic enzyme secretion by industrially relevant filamentous fungi. Glucose sensing, subsequent transport and cellular signalling pathways have been barely elucidated in these organisms. This study therefore characterized the transcriptional response of the filamentous fungus Aspergillus nidulans to the presence of high and low glucose concentrations under continuous chemostat cultivation with the aim to identify novel factors involved in glucose sensing and signalling. Several transcription factor- and transporter-encoding genes were identified as being differentially regulated, including the previously characterized glucose and xylose transporter HxtB. HxtB was confirmed to be a low affinity glucose transporter, localizing to the plasma membrane under low- and high-glucose conditions. Furthermore, HxtB was shown to be involved in conidiation-related processes and may play a role in downstream glucose signalling. A gene predicted to encode the protein kinase PskA was also identified as being important for glucose metabolism. This study identified several proteins with predicted roles in glucose metabolic processes and provides a foundation for further investigation into the response of biotechnologically important filamentous fungi to glucose.

The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae.

Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.

Signaling pathways for stress responses and adaptation in Aspergillus species: stress biology in the post-genomic era.

Aspergillus species are among the most important filamentous fungi in terms of industrial use and because of their pathogenic or toxin-producing features. The genomes of several Aspergillus species have become publicly available in this decade, and genomic analyses have contributed to an integrated understanding of fungal biology. Stress responses and adaptation mechanisms have been intensively investigated using the accessible genome infrastructure. Mitogen-activated protein kinase (MAPK) cascades have been highlighted as being fundamentally important in fungal adaptation to a wide range of stress conditions. Reverse genetics analyses have uncovered the roles of MAPK pathways in osmotic stress, cell wall stress, development, secondary metabolite production, and conidia stress resistance. This review summarizes the current knowledge on the stress biology of Aspergillus species, illuminating what we have learned from the genomic data in this "post-genomic era."

Cell wall structure and biogenesis in Aspergillus species.

Aspergillus species are among the most important filamentous fungi from the viewpoints of industry, pathogenesis, and mycotoxin production. Fungal cells are exposed to a variety of environmental stimuli, including changes in osmolality, temperature, and pH, which create stresses that primarily act on fungal cell walls. In addition, fungal cell walls are the first interactions with host cells in either human or plants. Thus, understanding cell wall structure and the mechanism of their biogenesis is important for the industrial, medical, and agricultural fields. Here, we provide a systematic review of fungal cell wall structure and recent findings regarding the cell wall integrity signaling pathways in aspergilli. This accumulated knowledge will be useful for understanding and improving the use of industrial aspergilli fermentation processes as well as treatments for some fungal infections.

Comparative proteomic analysis reveals intracellular targets for bacillomycin L to induce Rhizoctonia solani Kühn hyphal cell death

Bacillomycin L, a natural iturinic lipopeptide produced by Bacillus amyloliquefaciens, is characterized by strong antifungal activity against a variety of agronomically important filamentous fungi including Rhizoctonia solani Kühn. To further understand its antifungal actions, proteomes were comparatively studied within R. solani hyphal cells treated with or without bacillomycin L. The results show that 39 proteins were alternatively expressed within cells in response to this lipopeptide, which are involved in stress response, carbohydrate, amino acid and nucleotide metabolism, cellular component organization, calcium homeostasis, protein degradation, RNA processing, gene transcription, and others, suggesting that, in addition to inducing cell membrane permeabilization, iturin exhibits antibiotic activities by targeting intracellular molecules. Based on these results, a model of action of bacillomycin L against R. solani hyphal cells was proposed. Our study provides new insight into the antibiotic mechanisms of iturins.

English

L-Lactic acid is a promising three-carbon building-block chemical, widely used in the food, pharmaceutical, leather and textile industries and Rhizopus oryzae is an important filamentous fungus for the production of L-lactic acid with high optical purity. This study investigated the medium compositions for the maximum biomass cultivation of R. oryzae L-lactic acid fermentation, and optimized the operation parameters for semi-continuous repeated fermentation in a stirred tank fermentor using response surface method (RSM) analysis. The results indicated that a higher biomass cultivation of 3.750±0.05 g/L was achieved when the medium was composed of 12% (w/v) glucose, 0.4% (w/v) ammonium sulfate and 0.045% (w/v) monopotassium phosphate. The optimal fermentation conditions for the initial batch were as follows: the aeration was 0.75 L/(L·min), inoculation of germs was 11% and agitation speed was 560 rpm. The fermentative intensity of the initial batch and the sequentially repeated batches with self-immobilized pelletized R. oryzae were 2.162 g/(L·h) and 3.704 g/(L·h), respectively.   Key words: Self-immobilized, Rhizopus oryzae, pellet, lactic acid, response surface method (RSM).