Enhancing the production of cephalosporin C through modulating the autophagic process of Acremonium chrysogenum.

BACKGROUND: Autophagy is used for degradation of cellular components and nutrient recycling. Atg8 is one of the core proteins in autophagy and used as a marker for autophagic detection. However, the autophagy of filamentous fungi is poorly understood compared with that of Saccharomyces cerevisiae. Our previous study revealed that disruption of the autophagy related gene Acatg1 significantly enhanced cephalosporin C yield through reducing degradation of cephalosporin biosynthetic proteins in Acremonium chrysogenum, suggesting that modulation of autophagic process is one promising way to increase antibiotic production in A. chrysogenum. RESULTS: In this study, a S. cerevisiae ATG8 homologue gene Acatg8 was identified from A. chrysogenum. Acatg8 could complement the ATG8 mutation in S. cerevisiae, indicating that Acatg8 is a functional homologue of ATG8. Microscope observation demonstrated the fluorescently labeled AcAtg8 was localized in the cytoplasm and autophagosome of A. chrysogenum, and the expression of Acatg8 was induced by nutrient starvation. Gene disruption and genetic complementation revealed that Acatg8 is essential for autophagosome formation. Disruption of Acatg8 significantly reduced fungal conidiation and delayed conidial germination. Localization of GFP-AcAtg8 implied that autophagy is involved in the early phase of conidial germination. Similar to Acatg1, disruption of Acatg8 remarkably enhanced cephalosporin C yield. The cephalosporin C biosynthetic enzymes (isopenicillin N synthase PcbC and isopenicillin N epimerase CefD2) and peroxisomes were accumulated in the Acatg8 disruption mutant (∆Acatg8), which might be the main reasons for the enhancement of cephalosporin C production. However, the biomass of ΔAcatg8 decreased drastically at the late stage of fermentation, suggesting that autophagy is critical for A. chrysogenum cell survival under nutrition deprived condition. Disruption of Acatg8 also resulted in accumulation of mitochondria, which might produce more reactive oxygen species (ROS) which promotes fungal death. However, the premature death is unfavorable for cephalosporin C production. To solve this problem, a plasmid containing Acatg8 under control of the xylose/xylan-inducible promoter was introduced into ∆Acatg8. Conidiation and growth of the recombinant strain restored to the wild-type level in the medium supplemented with xylose, while the cephalosporin C production maintained at a high level even prolonged fermentation. CONCLUSIONS: Our results demonstrated inducible expression of Acatg8 and disruption of Acatg8 remarkably increased cephalosporin C production. This study provides a promising approach for yield improvement of cephalosporin C in A. chrysogenum.

RNAi knockdown of potent sugar sensor in cellulase-producing fungus Acremonium cellulolyticus.

A potent sugar sensor gene (g9105) was screened from the genomic data of the cellulase-producing fungus Acremonium cellulolyticus. The transcriptional level of g9105 in the SA49 transformant, which carried the knockdown RNA interference (RNAi) construct, was less than 10% compared with the parental YP-4 strain. The hairpin-type RNAi construct could be useful for this fungal gene knockdown. Changes in cellulase productivity and protein secretion between these two strains were not observed. The numbers of hyphal tips at subapical branching site were counted for the SA49 and YP-4 strains incubated with potato-dextrose medium at 30 °C for 4 days. The hyphal branching ratio of the SA49 strain was higher than that of the YP-4 strain. The present results suggest that the potent sugar sensor gene in A. cellulolyticus could be related with hyphal branch formation.

Maxillary sinus fungal infection by Acremonium.

OBJECTIVE: To illustrate by this case report that Acremonium must now be considered as a differential diagnosis in cases of maxillary fungus balls. CASE REPORT: Seventy-seven-year-old woman consulted for persistent pain of the right maxillary sinus, with rhinorrhea and nocturnal coughing. Computed tomography (CT) of the sinuses showed a heterogeneous opacification of the right maxillary sinus with well-defined hyperdense foci suggesting aspergillosis. She underwent a middle meatus antrectomy by an endonasal approach. Six months after the surgery, her symptoms were gone and had not recurred. Mycological examination found Acremonium. DISCUSSION/CONCLUSION: Acremonium is a genus of saprobic fungi that rarely cause disease in humans. Infection with Acremonium has recently been described in immunocompromised patients. We describe the first case of fungal maxillary sinusitis caused by Acremonium in an immunocompetent person. Clinically and radiologically, the initial diagnosis was aspergillosis. Acremonium must be considered together with aspergillosis in all situations of fungus ball chronic sinusitis.

[Structure peculiarities of cell walls of Acremonium chrysogenum--an autotroph of cephalosporin C].

Alterations of cell walls of Acremonium chrysogenum occurring at intensive synthesis of cephalosporin C has been studied. It is shown, using electron microscopy, that the cell wall of the cells ofATCC 11550 strain ("wild" type) became looser and thicker during growth. The cell wall of the cells of strain 26/8 (hyperautotroph of cephalosporin C) considerably degraded by the end of the stationary phase. Biochemical analysis has shown that these alterations entailed decrease of the proteins' content covalently or noncovalently linked with the polysaccharides of cell walls of both strains. An increase of sensitivity of cell walls of the strain-superproducer to an activity of lytic enzymes of chitinase, laminarinase, proteinase K, and lyticase preparation has been observed during the growth, but this increase has not been found in the case of "wild" type strain. The obtained results evidence to the structure failure of the cell wall of A. chrysogenum entailing the intensive creation of antibiotic.

Fatty acids reduce the tensile strength of fungal hyphae during cephalosporin C production in Acremonium chrysogenum.

Fragmentation rate constants, which can be used to estimate the tensile strength of fungal hyphae, were used to elucidate relationships between morphological changes and addition of fatty acids during cephalosporin C production in Acremonium chrysogenum M35. The number of arthrospores increased gradually during fermentation, and, in particular, was higher in the presence of rice oil, oleic acid or linoleic acid than in their absence. Because supplementation of rice oil or fatty acids increased cephalosporin C, we concluded that differentiation to arthrospores is related to cephalosporin C production. To estimate the relative tensile strengths of fungal hyphae, fragmentation rate constants (k (frag)) were measured. When rice oil, oleic acid, or linoleic acid were added into medium, fragmentation rate constants were higher than for the control, and hyphal tensile strengths reduced. The relative tensile strength of fungal hyphae, however was not constant presumably due to differences in physiological state.

Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum.

Using bimolecular fluorescence complementation assays, we were able to demonstrate protein-protein interaction of the transcription factors AcFKH1 and CPCR1 in living cells from the filamentous fungus Acremonium chrysogenum. This was accomplished by splitting the gene for the enhanced yellow fluorescent protein (EYFP) into two parts encoding the N- and C-terminus. Both fragments were fused to different gene derivatives of the fungal transcription factors. The recombinant plasmids were used to generate transgenic fungal strains for subsequent confocal laser microscopy. Only when the full-length transcription factors were fused to EYFP fragments yellow fluorescence was observed due to the bimolecular complementation of both chimeric proteins. The nuclear localization of the protein-protein interaction was verified by staining fungal cells with the nucleic acid dye TOTO-3.

Analysis of the relationship between growth, cephalosporin C production, and fragmentation in Acremonium chrysogenum.

Mycelial fragmentation in submerged cultures of the cephalosporin C (CPC) producing fungus Acremonium chrysogenum was characterized by image analysis. In both fed-batch and chemostat cultures, the proportion of mycelial clumps seemed to be the most sensitive morphological indicator of fragmentation. In a fed-batch fermentation culture, this declined from roughly 60% at inoculation to less than 10% after 43 h. Subsequent additions of glucose resulted in a sharp increase back to near the initial value, an increase that reversed itself a few hours after glucose exhaustion. Meanwhile CPC production continued to decline steadily. On the other hand, the addition of soybean oil enhanced CPC production, but had no significant effect on the morphology. Although it may sometimes appear that morphology and productivity are related in batch or fed-batch cultures, this study suggests that this is because both respond simultaneously to more fundamental physiological changes, dependent on the availability of carbon. In circumstances, such as supplementary carbon source addition, the relationship is lost. Chemostat cultures supported this belief, as CPC-production rates were hardly affected by the specific growth rate, but the morphology showed significant differences, i.e., lower dilution rates resulted in a lower proportion of clumps and in smaller clumps.

Glucosylation of the peptide leucinostatin A, produced by an endophytic fungus of European yew, may protect the host from leucinostatin toxicity.

BACKGROUND: Yew species (Taxus spp.) throughout the world are hosts to hundreds, or perhaps thousands, of endophytic organisms. Most commonly, these organisms are fungi, living in a commensal or a symbiotic relationship with their host plant, so the plants exhibit little or no outward evidence that they are supporting these microorganisms. Little is known about any of the biochemical mechanisms that mediate the interactions between the yew host and its associated microbes. We feel that such information may not only contribute to our understanding of endophyte-tree biology, but also may provide novel pharmaceutical leads, because some of the compounds produced by these endophytes have demonstrated pharmacological activities. RESULTS: Acremonium sp. was isolated as an endophytic fungus of the European yew, Taxus baccata. Entry of Acremonium sp. into the plant may proceed via invasion of natural openings such as stomata. The relationship between Acremonium sp. and T. baccata may be a symbiotic one, because no symptoms are seen when Taxus media p.v. Hicksii is inoculated with this fungus. In culture, the fungus makes leucinostatin A, a peptide with phytotoxic, anticancer and antifungal properties. Although this peptide causes necrotic symptoms in many non-host plants and other cell types, it causes no visible symptoms in the host plant. T. baccata and several other plants have a UDP glucose; leucinostatin A glucosyl transferase that catalyzes the production of leucinostatin A beta di-O-glucoside from leucinostatin A. This glucoside, also made by the fungus, has a lower bioactivity against plants, fungi and a breast cancer cell line, BT-20, than leucinostatin A. CONCLUSIONS: Leucinostatin A may be one of several potentially toxic peptides produced by Acremonium sp. that contribute to the defense of the host, thereby preserving the fungus' own biological niche. The host plant is relatively immune to leucinostatin A because it has an enzyme which transfers two glycosyl residues to leucinostatin A, markedly reducing the peptide's bioactivity. Our results suggest that glucosylation reactions may play a more general role in plant defenses, especially against toxin-mediated disease development.

Coagulation autolysis in microorganisms and its relation to coagulase production.

The phenomenon of coagulation autolysis was observed in two model microorganisms, i.e., a bacterial culture and an imperfect fungus. It was characterized by impairment of the cell membranes, followed by condensation and dehydration of the cytoplasm and long-term preservation of the cells in the form of coagulated cytoplasm. In this respect, it was similar to coagulation necrosis of human tissues. The autolysis in the microorganisms was accompanied by increase of their coagulase activity, the substrate specificity of the enzyme rather broad. The coagulase activity of the microorganisms was detected during the culture period between the lag-phase and the exponential growth phase, i.e., the phase of their active growth. It served as a signal to induce biosynthesis of peptidohydrolase and cleavage of proteins. We believe that the phenomenon of coagulation autolysis in these microorganisms is rather typical and can be considered as an adaptative reaction, inducing a cascade of events from synthesis of coagulase to overproduction of peptidohydrolases with proteolytic activity.

[Isolation and characteristics of mitochondrial DNA from Acremonium chrysogenum]. / Vydelenie i kharakteristika mitokhondrial'noí DNK iz Acremonium chrysogenum.

Circular mDNAs 26.85 and 26.94 kb in length were isolated from two isogenic strains of A. chrysogenum producing cephalosporin C. The strains differed in antibiotic production capacity. Restriction analysis of the mDNAs was performed with using 6 endonucleases. Comparison of the restriction data revealed identity of mDNAs. A restriction map of the mDNAs was constructed. It is useful as a basis for further studies with molecular cloning.

[The protoplasts of Acremonium chrysogenum. Biochemical and morphological studies]. / Protoplasty Acremonium chrysogenum. Biokhimicheskie i morfologicheskie issledovaniia.

A procedure for preparing stable A. chrysogenum protoplasts capable of 60 per cent regeneration was developed. Two morphogenetic types of the regeneration were detected. The variants isolated after the protoplast regeneration were characterized by wide ranges of morphological variation. Capacity for the antibiotic production varied from 60 to 160 per cent of the activity of the starting strain. A procedure for isolating functionally active mitochondria from protoplasts of A. chrysogenum was also developed. Their main bioenergetic parameters were studied. In the respiratory chain of the A. chrysogenum mitochondria there were detected three conjugation sites of oxidative phosphorylation.

Differentition of mutants of Cephalosporium acremonium in complex medium: the formation of unicellular arthrospores and their germination.

Differentiation of swollen hyphal fragments to unicellular arthrospores accompanied the synthesis of cephalosporin C by a series of Cephalosporium acremonium mutants during propagation in a complex medium. The complex medium supported significantly higher synthesis than the defined medium used in previous studies of differentiation in C. acremonium. The mutants differed in their ability to form unicellular arthrospores and to synthesize cephalosporin C, but a one-to-one correspondence between the two properties was not observed. An inverse relation was observed between the growth rates of the mutants and their ability to synthesize cephalosporin C: each mutant produced more antibiotic but grew more slowly than its parent strain. Germination of the unicellular arthrospores occurred in complex medium but differed significantly from the germination of conidia in seed medium. The unicellular arthrospores were examined by electron microscopy and compared with swollen hyphal fragments and slender hyphal filaments. The unicellular arthrospores had a thicker cell wall, rougher cell surface, and had one or more small identations in their surface. The internal structure of the unicellular arthrospore resembled those of the swollen hyphal fragment and slender hyphal filament. Filaments had lower concentrations of lipid-containing vacuoles which were prevalent in both the swollen hyphal fragments and the unicellular arthrospores.