High-yield strain of fusidic acid obtained by atmospheric and room temperature plasma mutagenesis and the transcriptional changes involved in improving its production in fungus Fusidium coccineum.

AIMS: To obtain the high-yield strain of fusidic acid, which is produced from fungus Fusidium coccineum and is the only fusidane-type antibiotic that has been used clinically, and confirm the changes in the transcription levels involved in increasing its production. METHODS AND RESULTS: By using the atmospheric and room temperature plasma mutagenesis technology, a high-yield mutant strain of fusidic acid-producing fungus F. coccineum was obtained. Using the genomic analysis of the original strain based on biosynthetic pathways of ergosterol and helvolic acid, we demonstrate that the pathway involved in the biosynthesis of 2,3-oxidosqualene from acetyl coenzyme A was shared by fusidic acid and ergosterol, and fusidic acid was finally synthesized by the catalysis of multiple cytochrome P450s and short-chain dehydrogenase/reductase from 2,3-oxidosqualene. Then, through the transcriptomic analysis of the original and mutagenized strain, it revealed that the proposed pathway from sucrose to fusidic acid was the most significantly up-regulated in the transcription levels of the mutant strain. CONCLUSIONS: The changes in the transcription levels of fusidic acid during its biosynthesis might result in high-yield of fusidic acid in the mutant strain. This is the first report on the whole biosynthetic pathway of fusidic acid in F. coccineum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study obtain the genetic basis for the biosynthesis of fusidic acid which could be beneficial for the molecular modifications of F. coccineum to further increase its yield by fermentation in future, and established the foundation to reveal the mechanism of the high-yield of the mutant strain.

Morphological, Pathogenic and Toxigenic Variability in the Rice Sheath Rot Pathogen Sarocladium Oryzae.

Sheath rot is an emerging rice disease that leads to considerable yield losses. The main causal agent is the fungus Sarocladium oryzae. This pathogen is known to produce the toxins cerulenin and helvolic acid, but their role in pathogenicity has not been clearly established. S. oryzea isolates from different rice-producing regions can be grouped into three phylogenetic lineages. When grown in vitro, isolates from these lineages differed in growth rate, colour and in the ability to form sectors. A diverse selection of isolates from Rwanda and Nigeria, representing these lineages, were used to further study their pathogenicity and toxin production. Liquid chromatography high-resolution mass spectrometry analysis was used to measure cerulenin and helvolic acid production in vitro and in planta. The three lineages clearly differed in pathogenicity on the japonica cultivar Kitaake. Isolates from the least pathogenic lineage produced the highest levels of cerulenin in vitro. Helvolic acid production was not correlated with the lineage. Sectorisation was observed in isolates from the two least pathogenic lineages and resulted in a loss of helvolic acid production. In planta, only the production of helvolic acid, but not of cerulenin, correlated strongly with disease severity. The most pathogenic isolates all belonged to one lineage. They were phenotypically stable, shown by the lack of sectorisation, and therefore maintained high helvolic acid production in planta.

Biosynthesis of helvolic acid and identification of an unusual C-4-demethylation process distinct from sterol biosynthesis.

Fusidane-type antibiotics represented by helvolic acid, fusidic acid and cephalosporin P1 are a class of bacteriostatic agents, which have drawn renewed attention because they have no cross-resistance to commonly used antibiotics. However, their biosynthesis is poorly understood. Here, we perform a stepwise introduction of the nine genes from the proposed gene cluster for helvolic acid into Aspergillus oryzae NSAR1, which enables us to isolate helvolic acid (~20 mg L-1) and its 21 derivatives. Anti-Staphylococcus aureus assay reveals that the antibacterial activity of three intermediates is even stronger than that of helvolic acid. Notably, we observe an unusual C-4 demethylation process mediated by a promiscuous short-chain dehydrogenase/reductase (HelC) and a cytochrome P450 enzyme (HelB1), which is distinct from the common sterol biosynthesis. These studies have set the stage for using biosynthetic approaches to expand chemical diversity of fusidane-type antibiotics.

De novo genome assembly and annotation of rice sheath rot fungus Sarocladium oryzae reveals genes involved in Helvolic acid and Cerulenin biosynthesis pathways.

BACKGROUND: Sheath rot disease caused by Sarocladium oryzae is an emerging threat for rice cultivation at global level. However, limited information with respect to genomic resources and pathogenesis is a major setback to develop disease management strategies. Considering this fact, we sequenced the whole genome of highly virulent Sarocladium oryzae field isolate, Saro-13 with 82x sequence depth. RESULTS: The genome size of S. oryzae was 32.78 Mb with contig N50 18.07 Kb and 10526 protein coding genes. The functional annotation of protein coding genes revealed that S. oryzae genome has evolved with many expanded gene families of major super family, proteinases, zinc finger proteins, sugar transporters, dehydrogenases/reductases, cytochrome P450, WD domain G-beta repeat and FAD-binding proteins. Gene orthology analysis showed that around 79.80 % of S. oryzae genes were orthologous to other Ascomycetes fungi. The polyketide synthase dehydratase, ATP-binding cassette (ABC) transporters, amine oxidases, and aldehyde dehydrogenase family proteins were duplicated in larger proportion specifying the adaptive gene duplications to varying environmental conditions. Thirty-nine secondary metabolite gene clusters encoded for polyketide synthases, nonribosomal peptide synthase, and terpene cyclases. Protein homology based analysis indicated that nine putative candidate genes were found to be involved in helvolic acid biosynthesis pathway. The genes were arranged in cluster and structural organization of gene cluster was similar to helvolic acid biosynthesis cluster in Metarhizium anisophilae. Around 9.37 % of S. oryzae genes were identified as pathogenicity genes, which are experimentally proven in other phytopathogenic fungi and enlisted in pathogen-host interaction database. In addition, we also report 13212 simple sequences repeats (SSRs) which can be deployed in pathogen identification and population dynamic studies in near future. CONCLUSIONS: Large set of pathogenicity determinants and putative genes involved in helvolic acid and cerulenin biosynthesis will have broader implications with respect to Sarocladium disease biology. This is the first genome sequencing report globally and the genomic resources developed from this study will have wider impact worldwide to understand Rice-Sarocladium interaction.

A Critical Review of the Properties of Fusidic Acid and Analytical Methods for Its Determination.

Fusidic acid, an antibiotic produced from the Fusidium coccineum fungus, belongs to the class of steroids, but has no corticosteroid effects. It is indicated for the treatment of infections caused by methicillin-resistant Staphylococcus aureus strains. The aim of this study was to search for the properties of fusidic acid published so far in the literature, as well as the methods developed for its determination in biological samples and pharmaceutical formulations. From the findings, we can conclude that fusidic acid has been used for decades and is indicated for the treatment of serious infections caused by Gram-positive microorganisms to this day. Furthermore, it is a hypoallergenic agent, has low toxicity, shows low resistance, and has no cross-resistance with other clinically used antibiotics. The analytical method of high-performance liquid chromatography has been widely used for determining fusidic acid, since it can reduce the cost and time of analysis, making it more viable for routine quality control in the pharmaceutical industry.

RNA-seq reveals the pan-transcriptomic impact of attenuating the gliotoxin self-protection mechanism in Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus produces a number of secondary metabolites, one of which, gliotoxin, has been shown to exhibit anti-fungal activity. Thus, A. fumigatus must be able to protect itself against gliotoxin. Indeed one of the genes in the gliotoxin biosynthetic gene cluster in A. fumigatus, gliT, is required for self-protection against the toxin- however the global self-protection mechanism deployed is unclear. RNA-seq was employed to identify genes differentially regulated upon exposure to gliotoxin in A. fumigatus wild-type and A. fumigatus ∆gliT, a strain that is hypersensitive to gliotoxin. RESULTS: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated. Expression of 164 genes was differentially regulated (log2 fold change of 1.5) in A. fumigatus wild-type when exposed to gliotoxin, consisting of 101 genes with up-regulated expression and 63 genes with down-regulated expression. Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin. These consisted of 508 genes with up-regulated expression, and 1192 genes with down-regulated expression. Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism. Specifically, genes involved in gliotoxin biosynthesis, helvolic acid biosynthesis, siderophore-iron transport genes and also nitrogen metabolism genes and ribosome biogenesis genes underwent altered expression. It was confirmed that gliotoxin biosynthesis is induced upon exposure to exogenous gliotoxin, production of unrelated secondary metabolites is attenuated in A. fumigatus ∆gliT, while quantitative proteomic analysis confirmed disrupted translation in A. fumigatus ∆gliT challenged with exogenous gliotoxin. CONCLUSIONS: This study presents the first global investigation of the transcriptional response to exogenous gliotoxin in A. fumigatus wild-type and the hyper-sensitive strain, ∆gliT. Our data highlight the global and extensive affects of exogenous gliotoxin on a sensitive strain devoid of a self-protection mechanism and infer that GliT functionality is required for the optimal biosynthesis of selected secondary metabolites in A. fumigatus.

[Microbial test-system for screening of inhibitors of sterol biosynthesis].

New effective economic microbial test-system for screening of microbial metabolites, that are inhibitors of sterol biosynthesis, is proposed. It is based on cultivation of fungal strain Acremonium fusidioides (former Fusidium coccineum), that produces fusidic acid (fusidin) that is antibiotic of steroid structure. Great similarity of fusidic acid biosynthesis in fungous strain and cholesterol biosynthesis in human organism, coincidence of their initial steps till squalene formation, allows use A. fusidioides as a model for estimation of microbial secondary metabolites that are potential inhibitors of sterol biosynthesis. Such inhibitors in A.fusidioides model are revealed as compounds that strongly reduce fusidin production without any visible influence on fungus growth. Mevalonate that is one of the crucial intermediates of sterol biosynthesis could be successfully applied for removal of inhibition induced by some microbial metabolites. A.fusidioides test-system can be easily mechanized because of miniaturization of microbiological procedures, cultivation in 6-, 24-, or 96-well plates and usage of automatic micropipettes and dispensers. The results of this model are well correlated with the ones obtained with human cells (Hep G2 test-system, offered earlier). A.fusidioides test-system can be applied at rather early stages of screening procedures and is quite effective for testing of crude extracts of producers' culture liquid.

Medium optimization for enhanced co-production of two bioactive metabolites in the same fermentation by a statistical approach.

This paper describes improved optimization method that combines the one-factor-at-a-time method (OFAT), Plackett-Burman design, and the response surface method (RSM), which were used to optimize the medium for the production of fumigaclavine C (FC) and helvolic acid (HA) from endophytic Aspergillus fumigatus CY018 simultaneously. The ideal carbon and nitrogen sources for the two compounds were assessed initially via the one-factor-at-a-time method. Three key cultivation factors (pH, phosphate, and inoculum size) were chosen based on the results of Plackett-Burman design, and subsequently optimized by the central composite design. The two metabolites were amply afforded when the cultivation was carried out with the inoculum size of 2.45% at pH 4.2 and 28°C for 19 days in the medium containing (g/l): mannitol 50, sodium succinate 5.4, NaNO3 2, MgSO4·7H2O 0.3, FeSO4·7H2O 0.01, and KH2PO4 0.67. The highest yields of FC and HA achieved herein were 17.26 and 16.88 mg/l. This work might be the first endeavor leading to the improved simultaneous production of two complex active metabolites with a single strain.

Cloning and functional analysis of the second geranylgeranyl diphosphate synthase gene influencing helvolic acid biosynthesis in Metarhizium anisopliae.

A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) gene was cloned from Metarhizium anisopliae NAFF635007. The ggs2 gene (1,239-bp open reading frame with no intron) encoded a protein of 412 amino acids, and the transcription occurred only after late log-phase during the growth. Gene disruption of ggs2, performed to clarify the function in M. anisopliae, resulted in decreased GGPP synthase activity together with a slight delay of sporulation. An high performance liquid chromatography (HPLC) comparison of compound profiles between the wild-type strain and the disruptant revealed that a compound was abolished by the ggs2 disruption. Purification and structural elucidation by 1H-NMR and mass spectrometry analyses revealed that the lost compound is helvolic acid. Furthermore, the pathogenicity assay against two species of insect larvae revealed that the ggs2-disruptant possessed much weaker toxicity than the wild-type strain. Based on these results, it was concluded that ggs2 encodes the GGPP synthase influencing the biosynthesis of secondary metabolites in various species, including helvolic acid in M. anisopliae. To the best of our knowledge, this is the first report to identify a GGPP synthase gene related to secondary metabolism in entomopathogenic fungi.

Protostadienol synthase from Aspergillus fumigatus: functional conversion into lanosterol synthase.

Oxidosqualene:protostadienol cyclase (OSPC) from the fungus Aspergillus fumigatus, catalyzes the cyclization of (3S)-2,3-oxidosqualene into protosta-17(20)Z,24-dien-3beta-ol which is the precursor of the steroidal antibiotic helvolic acid. To shed light on the structure-function relationship between OSPC and oxidosqualene:lanosterol cyclase (OSLC), we constructed an OSPC mutant in which the C-terminal residues (702)APPGGMR(708) were replaced with (702)NKSCAIS(708), as in human OSLC. As a result, the mutant no longer produced the protostadienol, but instead efficiently produced a 1:1 mixture of lanosterol and parkeol. This is the first report of the functional conversion of OSPC into OSLC, which resulted in a 14-fold decrease in the V(max)/K(M) value, whereas the binding affinity for the substrate did not change significantly. Homology modeling suggested that stabilization of the C-20 protosteryl cation by the active-site Phe701 through cation-pi interactions is important for the product outcome between protostadienol and lanosterol.

Biosynthesis of steroidal antibiotic fusidanes: functional analysis of oxidosqualene cyclase and subsequent tailoring enzymes from Aspergillus fumigatus.

Three putative oxidosqualene cyclase (OSC) genes exist in the genome of the fungus Aspergillus fumigatus that produces a steroidal antibiotic, helvolic acid. One of these genes, Afu4g14770, designated AfuOSC3, is clustered with genes of cytochrome P450 monooxygenases (P450s), a short-chain dehydrogenase/reductase (SDR), and acyltransferases, which presumably function in triterpene tailoring steps, suggesting that this gene cluster codes for helvolic acid biosynthesis. AfuOSC3 was PCR amplified from A. fumigatus IFO8866 genomic DNA and expressed in yeast. The yeast transformant accumulated protosta-17(20)Z,24-dien-3beta-ol, an established precursor for helvolic acid. Its structural isomer, (20R)-protosta-13(17),24-dien-3beta-ol, was also isolated from the transformed yeast. To further identify the function of triterpene tailoring enzymes, four P450 genes (CYP5081A1-D1) and a SDR gene (AfuSDR1) in the cluster were each coexpressed with AfuOSC3 in yeast. As a result, coexpression of AfuSDR1 gave a 3-keto derivative of protostadienol. On the other hand, coexpression with CYP5081A1 gave protosta-17(20)Z,24-diene-3beta,29-diol and protosta-17(20)Z,24-dien-3beta-ol-29-oic acid. These metabolites are in well accord with the oxidative modification involved in helvolic acid biosynthesis. AfuSDR1 and CYP5081A1 presumably function together to catalyze demethylation of C-29 methyl group. These results provided a firm ground for identification of the present gene cluster to be involved in helvolic acid biosynthesis.

Molecular cloning, expression, and site-directed mutations of oxidosqualene cyclase from Cephalosporium caerulens.

A cDNA for oxidosqualene:lanosterol cyclase (OSLC) was cloned and sequenced from the fungus Cephalosporium caerulens, that produces a steroidal antibiotic, helvolic acid. A 2280 bp open reading frame encoded an M(r) 87078 protein with 760 amino acids. The cDNA was functionally expressed in the OSLC-deficient mutant GIL77 strain of Saccharomyces cerevisiae. A truncated recombinant enzyme (Delta49N) starting from the second methionine (M50) residue was completely inactive, suggesting that ca. 30 additional hydrophilic amino acid residues at the N-terminal are essential for the folding of the enzyme. Furthermore, the active site residues, H234 and D456 (numbering in S. cerevisiae OSLC), were chosen for site-directed mutagenesis experiments; H234E, H234Y, H234F, D456E, D456N, and D456H mutants were inactive, while H234W and H234K mutants retained lanosterol-forming activity.

[Screening of drugs inhibiting cholesterol synthesis]. / Poisk preparatov, ingibiruiushchix sintez xolesterina

The biosynthesis of fusidin, an antibiotic of steroid structure, was used as a model of steroid synthesis. Screening of compounds modifying the fusidin synthesis included 80 strains of mycelial fungi. A high frequency of such fungal cultures was observed. 9 cultures forming compounds which inhibited the synthesis of cholesterol in the cell culture of human hepatocytes were screened. A method for biological estimation of the activity of cholesterol synthesis inhibitors was developed on rabbits with high blood levels of cholesterol. It was shown that the cholesterol synthesis inhibitors, isolated as a result of the specific screening were not toxic and markedly lowered the cholesterol blood levels.

[Fatty acid composition of Fusidium coccineum lipids]. / Sostav zhirnykh kislot lipidov Fusidium coccineum.

The fatty acid composition of the lipids synthesized de novo or during growth of the organism producing fusidic acid, Fusidium coccineum on complex media was studied. The content of the total extractable lipids in the mycelium did not exceed 10-11 per cent ty the biomass dry weight. Unsaturated fatty acids with 18 carbon atoms in the chain, mainly oleinic and linoleic acids and saturated fatty acids such as palmitic and stearic acids predominated. The ratio of the main fatty acids and the content of the minor acids were subject to strains and conditions of their cultivation. However, linoleic and oleinic acids predominated in all the lipids except the lipids from submerged cultures growing in the form of unusually large clots. Such lipids contained up to 60 per cent of palmitic acid.

[Morphofunctional characteristics of the development of strains of Fusidium coccineum differing in antibiotic activity in stab cultivation]. / Morfofunktsional'nye osobennosti razvitiia raznykh po antibioticheskoi aktivnosti shtammov Fusidium coccineum pri glubinnom kul'tivirovanii.

The object of the work was to study the morphological and functional characteristics of Fusidium coccineum strains producing fusidic acid and differing in the antibiotic activity. The high metabolic activity of the culture is accompanied by the following morphological characteristics: the cells are rich in ribosomes and mitochondria, they have early vacuolisation, are filled with lipid granules, and then the mycelium undergoes autolysis. As strains with a high activity grow, the structure of the cells changes, the number of ribosomes and mitochondria falls down, and the latter are destroyed. For a long time, the cells contain electron-dense granular structures limited with the membrane and capable of transformation into lipid granules and membranous structures. As was shown by cytochemical studies, the structures have not merely proteins and lipids, but also phosphorus compounds. Their functional role in the fungal metabolism is discussed. As soon as super-synthesis of fusidic acid commences, the cells of the highly active strains are filled with lipid granules associated possibly with the steroid antibiotic. These formations are released from the cell during local lysis of the cell wall and the cytoplasmic membrane.

[Metabolic characteristics of a nystatin-resistant strain of a fungal producer of fusidic acid]. / Osobennosti metabolizma nistatinorezistentnogo shtamma griba-produtsenta fuzidievoi kisloty.

The physiological and morphofunctional properties of the polyene-sensitive and resistant mutants of fungus producing fusidic acid were studied comparatively. The highly active nystatin resistant mutant was characterized by the decreased growth rates, lowered sporulation levels, retarded synthesis of nucleic acids and protein, lower respiration activity and higher mycelium lipid contents. The ultrastructural investigation of the polyene-resistant strain revealed the presence of lower numbers of ribosomes, membrane structures and mitochondria. Destructive changes in mitochondria and lipid masses in the hyphal cytoplasm were also observed.

[Ultrastructure of the spores of fungal strains, producers of fusidic acid, varying in antibiotic activity]. / Ul'trastruktura spor raznykh po antibioticheskoi aktivnosti shtammov griba produtsenta fuzidievoi kisloty.

The nature of the fine structure of the spores of fusidic acid-producing organism changed with an increase in the antibiotic activity of its strains. Impairments in the structure of the cell coating were observed. The surface layers of the spore wall became labile, they were capable of separating, destructive impairment in the electron solid structures were detected in the spore cytoplasm, such impairments resulted in autolysis of extended areas. The lability of the surface layers of the spore wall promoted contacts between the spore cell coatings. The increased adhesive properties of the spore coatings resulted in formation of the spore "heads" typical of sporulation in highly active strains. The latter were characterized by a marked increase in the number of the spores filled with lipids and devoid of the main cell organelles and having a changed structure of the spore wall. This explained the marked decrease in the viability of the conidia of the highly active strains of the organism producing fusidic acid.

[Possible role of the respiratory system of Fusidium coccineum in regulating fusidic acid biosynthesis]. / Vozmozhnaia rol' dykhatel'noi sistemy Fusidium cocineum v reguliatsii biosinteza fuzidievoi kisloty.

The respiration system was studied in three strains of the fungus Fusidium coccineum differing in their capability to synthesize fusidic acid. In all of the three strains, the system of oxidative phoshorylation predominated in supplying the cells with energy. In the strains with low and zero activities, the terminal oxidation of reduced equivalents occurred mainly via the respiration chain with cytochrome oxidase as a terminal component. In the strain with a high activity, there was an alternative cyanide resistant pathway, along with the classical cytochrome chain, and the complete switching to the alternative pathway coincided with the period of the antibiotic maximal accumulation. The induction of the alternative pathway in the strain with a high activity did not involve inhibition of the cytochrome region of the respiration chain. It was shown for the first time that the antibiotic synthesis and the character of cell differentiation can be changed by modifying the pathways of oxidation with specific inhibitors such as chloramphenicol and salicyl hydroxamate. Apparently, there is some general mechanism involved in regulating the production of the antibiotic, cell differentiation, and switching to the alternative oxidative pathway.