Production of phleichrome by Cladosporium phlei as stimulated by diketopiperadines of Epichloe typhina

Epichloe typhina is an endophytic fungus, while Cladosporium phlei is a pathogenic fungus of the timothy plant (Phleum pretense L.). We found two activities in the culture filtrate of E. typhina: one stimulated the pathogenic fungus, C. phlei, to produce phleichrome and the other inhibited its growth. The active ingredients that stimulated the production of phleichrome and inhibited the growth of C. phlei were isolated and characterized. The isolated compounds were identified as cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), which were stimulatory compounds, and p-hydroxybenzaldehyde, which was the growth inhibitory compound, based on an analysis of their spectral data. Of the two stimulatory compounds, cyclo-(L-Pro-L-Phe) showed higher activity. However, when 500 microg of cyclo-(L-Pro-L-Phe) was spotted on the TLC plate for bio-autography, a growth inhibitory zone was identified in the central red region, which contained phleichrome. On the other hand, phleichrome showed antifungal activity against E. typhina in the light, so it is assumed that there might be antagonism between the endophytic fungus, E. typhina, and the pathogenic fungus, C. phlei.     

Regulation of secondary metabolite production in filamentous ascomycetes

Fungi are renowned for their ability to produce bioactive small molecules otherwise known as secondary metabolites. These molecules have attracted much attention due to both detrimental (e.g. toxins) and beneficial (e.g. pharmaceuticals) effects on human endeavors. Once the topic only of chemical and biochemical studies, secondary metabolism research has reached a sophisticated level in the realm of genetic regulation. This review covers the latest insights into the processes regulating secondary metabolite production in filamentous fungi.     

Inducing secondary metabolite production by the soil-dwelling fungus Aspergillus terreus through bacterial co-culture

The soil-dwelling fungus Aspergillus terreus was isolated from sediment collected from the lake of Wadi EI Natrun in Egypt. Co-cultivation of A. terreus with the bacteria Bacillus subtilis and Bacillus cereus on solid rice medium resulted in an up to 34-fold increase in the accumulation of constitutively present fungal natural products (4–15) compared to axenic cultures of A. terreus. The fungal products included two new butyrolactone derivatives, isobutyrolactone II (1) and 4-O-demethylisobutyrolactone II (2), together with the known N-(carboxymethyl)anthranilic acid (3) that were not present in axenic fungal controls and were only detected during co-cultivation with B. subtilis or with B. cereus. The structures of all compounds were unambiguously elucidated by 1D and 2D NMR spectroscopy, and by HRESIMS measurements, as well as by comparison with the literature. In a second set of experiments, A. terreus was co-cultured with Streptomyces lividans and with Streptomyces coelicolor. These co-cultivation experiments failed to induce fungal natural product accumulation in contrast to co-cultures with Bacillus sp. Compounds 5 and 14 showed weak inhibition of B. cereus with minimal inhibitory concentrations (MICs) of 64 μg/mL, whereas only 8 showed moderate cytotoxicity against the murine lymphoma (L5178Y) cell line with inhibition of 80% at a dose of 10 μg/mL.     

Efficient cellulase production by the filamentous fungus Acremonium cellulolyticus

Cellulase production was investigated in a culture of a strain of Acremonium cellulolyticus. The medium components were optimized for the improvement of cellulase production. The maximum production of cellulolytic enzymes was obtained in a medium containing (grams per liter) 50 Solka Floc, 5 (NH4)2SO4, 24 KH2PO4, 4.7 potassium tartrate hemihydrate, 1.2 MgSO4.7H2O, 1 Tween 80, 4 urea, 0.01 ZnSO4.7H2O, 0.01 MnSO4.6H2O, and 0.01 CuSO4.7H2O, with a pH of 4.0. In the flask culture, 15.5 filter paper units (FPU)/mL of maximum cellulase activity was obtained, 17.42 FPU/mL in a 7-L bioreactor, and 13.08 FPU/mL in a 50-L scale bioreactor for 4-8 d at 30 degrees C. Average production rates were 1.94 FPU/mL.d in flasks, 2.86 FPU/mL.d in the 7-L bioreactor, and 2.56 FPU/mL.d in the 50-L bioreactor. Cellulase production on a small scale was successfully reproduced in the 50-L pilot scale bioreactor. Saccharification activity from A. cellulolyticus was compared with cellulolytic enzymes produced by other strains. The A. cellulolyticus culture broth had a comparable saccharification yield in comparison with those of other Trichoderma enzymes (GC220 or Cellulosin T2) under the same total cellulase activity. Its saccharification yield (percent of released reducing sugar to used dried substrate) was 60%, and its glucose content was 83%.     

A sandwiched-culture technique for evaluation of heterologous protein production in a filamentous fungus

Aspergillus niger is known for its efficient excretion machinery. However, problems have often arisen in obtaining high amounts of heterologous proteins in the culture medium. Here we present a quick method using sandwiched colonies to evaluate transgenic strains for secretion of heterologous proteins. Expressing the ABH1 hydrophobin of Agaricus bisporus in A. niger, we showed that low production levels of the heterologous protein are probably due to extracellular proteolytic degradation of the protein.     

丝状真菌次级代谢产物生物合成的表观遗传调控

丝状真菌产生的次级代谢产物是新药的重要来源之一,其生物合成过程受到众多因素的调控。最近的研究表明,表观遗传对多种丝状真菌次级代谢产物的生物合成具有调控作用。DNA和组蛋白的甲基化与乙酰化修饰是目前所知的丝状真菌主要的表观遗传调控形式。通过过表达或缺失相关表观修饰基因和利用小分子表观遗传试剂改变丝状真菌染色体的修饰形式,不仅可以提高多种已知次级代谢产物产量,而且可以通过激活沉默的生物合成基因簇诱导丝状真菌产生新的未知代谢产物。丝状真菌表观遗传学正逐渐成为真菌菌株改良的新策略以及挖掘真菌次级代谢产物合成潜力的强有力手段。     

Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei

In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.     

Electrical phenotypes of calcium transport mutant strains of a filamentous fungus, Neurospora crassa

We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters-a mechanosensitive channel homolog (MscS), a Ca(2+)/H(+) exchange protein (cax), and Ca(2+)-ATPases (nca-1, nca-2, nca-3)-as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H(+)-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca(2+) levels, indicative of lesions in Ca(2+) homeostasis. However, the net Ca(2+) effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca(2+)-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca(2+) signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca(2+)] was elevated. Thus, although Ca(2+) homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654-661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H(+)-ATPase activity.     

Engineering the plant cell factory for secondary metabolite production

Plant secondary metabolism is very important for traits such as flower color, flavor of food, and resistance against pests and diseases. Moreover, it is the source of many fine chemicals such as drugs, dyes, flavors, and fragrances. It is thus of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a fine chemical, to produce less of a toxic compound, or even to make new compounds, Engineering of plant secondary metabolism is feasible nowadays, but it requires knowledge of the biosynthetic pathways involved. To increase secondary metabolite production different strategies can be followed, such as overcoming rate limiting steps, reducing flux through competitive pathways, reducing catabolism and overexpression of regulatory genes. For this purpose genes of plant origin can be overexpressed, but also microbial genes have been used successfully. Overexpression of plant genes in microorganisms is another approach, which might be of interest for bioconversion of readily available precursors into valuable fine chemicals. Several examples will be given to illustrate these various approaches. The constraints of metabolic engineering of the plant cell factory will also be discussed. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks. This revised version was published online in August 2006 with corrections to the Cover Date.     

cis-benzeneglycol production using a mutant Pseudomonas strain

The biotransformation of commodity aromatic chemicals into dihydroxy derivatives was studied. A strain isolated from the invironment, Pseudomonas JI104, used benzene, toluene, and other hydrocarbons as sole carbon and energy sources. We selected mutants unable to grow with benzene, and among these, screened for strains with deficient cis-benzenglycol dehydrogenase able to stably produce cis-benzeneglycol when another carbon source was co-metabolized.We exained the possibility of cis-benzeneglycol production by growing the mutant strain in the presence of benzene vapor. Ethanol was the carbon and energy source most adapted to the cis-benzeneglycol production phase, and lactate or propanol could also be used. Glucose inhibited the production of the metabolite.The growth rates were barely affected by the presence of benzene at a reduced partial pressure (less than 20% of saturation), showing that continuous culture is possible. In a batch process, 0.54g·1⁻¹ of a cell suspension produced 5.1 mmol·1⁻¹cis-benzeneglycol in 27 h, using ethanol as the energy source.     

Directed evolution of a filamentous fungus for thermotolerance

Background  

Filamentous fungi are the most widely used eukaryotic biocatalysts in industrial and chemical applications. Consequently, there is tremendous interest in methodology that can use the power of genetics to develop strains with improved performance. For example, Metarhizium anisopliae is a broad host range entomopathogenic fungus currently under intensive investigation as a biologically based alternative to chemical pesticides. However, it use is limited by the relatively low tolerance of this species to abiotic stresses such as heat, with most strains displaying little to no growth between 35–37°C. In this study, we used a newly developed automated continuous culture method called the Evolugator™, which takes advantage of a natural selection-adaptation strategy, to select for thermotolerant variants of M. anisopliae strain 2575 displaying robust growth at 37°C.     

Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.     

The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi

A secondary metabolite is a chemical compound produced by a limited number of fungal species in a genus, an order, or even phylum. A profile of secondary metabolites consists of all the different compounds a fungus can produce on a given substratum and includes toxins, antibiotics and other outward-directed compounds. Chemotaxonomy is traditionally restricted to comprise fatty acids, proteins, carbohydrates, or secondary metabolites, but has sometimes been defined so broadly that it also includes DNA sequences. It is not yet possible to use secondary metabolites in phylogeny, because of the inconsistent distribution throughout the fungal kingdom. However, this is the very quality that makes secondary metabolites so useful in classification and identification. Four groups of organisms are particularly good producers of secondary metabolites: plants, fungi, lichen fungi, and actinomycetes, whereas yeasts, protozoa, and animals are less efficient producers. Therefore, secondary metabolites have mostly been used in plant and fungal taxonomy, whereas chemotaxonomy has been neglected in bacteriology. Lichen chemotaxonomy has been based on few biosynthetic families (chemosyndromes), whereas filamentous fungi have been analysed for a wide array of terpenes, polyketides, non-ribosomal peptides, and combinations of these. Fungal chemotaxonomy based on secondary metabolites has been used successfully in large ascomycete genera such as Alternaria, Aspergillus, Fusarium, Hypoxylon, Penicillium, Stachybotrys, Xylaria and in few basidiomycete genera, but not in Zygomycota and Chytridiomycota.     

The significance of peroxisomes in secondary metabolite biosynthesis in filamentous fungi

Peroxisomes are ubiquitous organelles characterized by a protein-rich matrix surrounded by a single membrane. In filamentous fungi, peroxisomes are crucial for the primary metabolism of several unusual carbon sources used for growth (e.g. fatty acids), but increasing evidence is presented that emphasize the crucial role of these organelles in the formation of a variety of secondary metabolites. In filamentous fungi, peroxisomes also play a role in development and differentiation whereas specialized peroxisomes, the Woronin bodies, play a structural role in plugging septal pores. The biogenesis of peroxisomes in filamentous fungi involves the function of conserved PEX genes, as well as genes that are unique for these organisms. Peroxisomes are also subject to autophagic degradation, a process that involves ATG genes. The interplay between organelle biogenesis and degradation may serve a quality control function, thereby allowing a continuous rejuvenation of the organelle population in the cells.     

Solid-state fermentation: a promising microbial technology for secondary metabolite production

Solid state (substrate) fermentation (SSF) has been used successfully for the production of enzymes and secondary metabolites. These products are associated with the stationary phase of microbial growth and are produced on an industrial scale for use in agriculture and the treatment of disease. Many of these secondary metabolites are still produced by submerged liquid fermentations (SmF) even though production by this method has been shown to be less efficient than SSF. As large-scale production increases further, so do the costs and energy demands. SSF has been shown to produce a more stable product, requiring less energy, in smaller fermenters, with easier downstream processing measures. In this article we review an important area of biotechnology, since the recent evidence indicates that bacteria and fungi, growing under SSF conditions, are more than capable of supplying the growing global demand for secondary metabolites.     

Enhanced enzyme production by the cellulolytic fungus Penicillium pinophilum,mutant strain NTG III/6

Production of extracellular cellulase, xylanase and β-glucosidase by a mutant strain of Penicillium pinophilum (NTG III/6) increased dramatically on raising the temperature of incubation in a fermenter from 28 to 35°C. The effect of incubation temperature on enzyme production by the wild type strain (IMI 87160iii) was insignificant in comparison. Enzyme production was best when the pH of the culture medium was allowed to fluctuate naturally between 3.5 and 5.0 when barley straw or Solka Floc was the carbon source.     

NAD+/NADH homeostasis affects metabolic adaptation to hypoxia and secondary metabolite production in filamentous fungi*

Filamentous fungi are used to produce fermented foods, organic acids, beneficial secondary metabolites and various enzymes. During such processes, these fungi balance cellular NAD⁺:NADH ratios to adapt to environmental redox stimuli. Cellular NAD(H) status in fungal cells is a trigger of changes in metabolic pathways including those of glycolysis, fermentation, and the production of organic acids, amino acids and secondary metabolites. Under hypoxic conditions, high NADH:NAD⁺ ratios lead to the inactivation of various dehydrogenases, and the metabolic flow involving NAD⁺ is down-regulated compared with normoxic conditions. This review provides an overview of the metabolic mechanisms of filamentous fungi under hypoxic conditions that alter the cellular NADH:NAD⁺ balance. We also discuss the relationship between the intracellular redox balance (NAD/NADH ratio) and the production of beneficial secondary metabolites that arise from repressing the HDAC activity of sirtuin A via Nudix hydrolase A (NdxA)-dependent NAD⁺ degradation.     

Polysaccharide production by a reduced pigmentation mutant of the fungus Aureobasidium pullulans

Abstract A reduced pigmentation mutant was isolated from Aureobasidium pullulans ATCC 42023 by chemical mutagenesis and was subsequently characterized. The pigment melanin was present not only in A. pullulans cells but also contaminated the elaborated polysaccharide and thus, was measured in both fractions. Cellular and polysaccharide melanin levels of the mutant strain were at least 11-fold and 18-fold reduced, respectivelu, compared toits parent strain after 7 days of growth at 30°C whether sucrose or glucose served as the carbon source in the culture medium. Polysaccharide and cell dry weight levels of the mutant were very similar to those observed for the parent after growth on sucrose or glucose as the source of carbon over a period of 7 days at 30°C. The pullulan content of the polysaccharide produced by the parent or mutant strain was lower for sucrose-grown cells than for glucose-grown cells. It was also noted that the pullulan content of the polysaccharide elaborated by the mutant strain was slightly higher than that of the polysaccharide produced by the parent strain after growth on either sucrose or glucose.