Effect of bongkrekic acid on growth and metabolism of filamentous fungi

The antifungal activity of bongkrekic acid against 17 tested molds was determined. Bongkrekic acid prevented spore germination and mycelial proliferation of Aspergillus niger, Rhizopus oryzae and Penicillium italicum. The action of bongkrekic acid was fungicidal. Under these conditions, the incorporation of ¹⁴C-leucine and ¹⁴C-uracil into the perchloric acid insoluble material of germinating A. niger conidia was significantly reduced by bongkrekic acid. Respiratory activity of resting spores was not affected by bongkrekic acid. Respiratory activity of germinated spores was inhibited by bongkrekic acid to the extent of 30 to 60% of controls for A. niger, R. oryzae and P. italicum. It has been concluded that operation of adenine nucleotide translocation in mitochondria of tested fungi is obligatory both for normal spore germination and fungal growth.     

Modeling the exponential growth of filamentous fungi during batch cultivation

In certain conditions, filamentous fungi are observed to grow exponentially during batch submerged growth. It is shown for three cases, with simple mechanistic models, that an exponential growth assumption is reasonable. The basis of these models is the identification of a growth unit, and a mechanism for its doubling with a constant generation time. The importance of the variation of morphological properties within populations is demonstrated by the comparison of computer simulations of simplified models using average values and either experimental data or computer simulations of detailed stochastic models. Copyright 1998 John Wiley & Sons, Inc.     

Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis

he presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).     

Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis

The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).     

Chain growth rate of -galactosidase during exponential growth and amino acid starvation

S_d phage were incubated in 1 m-O-methylhydroxylamine. At various time-intervals, samples of modified phage were isolated and disrupted either by heating or by treatment with detergent. Changes in viscosity and buoyant density of disrupted preparations took place in the course of modification. Three transient synchronous drops in viscosity and buoyant density levels were observed with minima at five minutes, one and three hours of modification. The specific viscosity of the preparations at minima was 10 to 20% that of the disrupted unmodified phage.Properties of the phage preparation isolated during the third period of decreased viscosity were studied in more detail. This preparation, subjected to thermal disruption, gives a single DNA-containing band in Cs₂SO₄ gradient centrifugation corresponding to a buoyant density of 1.37 g/cm³ (cf. 1.39, 1.29 and 1.43 g/cm³ for whole phage, phage ghosts and native phage DNA, respectively).The band contains practically all the ³⁵S label that was present in the starting phage, suggesting that it corresponds to a complex of phage DNA with protein. Electron microscopy revealed complexes as thick strands of 50 to 300 Å diameter bonded to globular particles of varying size.In four hours of modification, the viscosity and buoyant density of disrupted phage returned to values characteristic of unmodified preparations. The DNA band contained no ³⁵S label. Electron microscopy of the substance of this band revealed fibres of 20 Å diameter.A possible explanation of the results is based on the assumption of pre-existing non-covalent interaction of C₍₄₎—NH₂ moieties of cytidine residues with nucleophilic groupings of coating protein within the virion. It is assumed that it is this interaction that holds DNA in “non-native” conformation within intact phage particles and thus explains its peculiar properties discovered earlier. In the present case, the interaction determines the formation of DNA-protein crosslinks under O-methylhydroxylamine treatment via the earlier postulated intermediate product of cytosine modification. Restoration of “normal” physical properties of disrupted phage after more prolonged modification is explained by cleavage of the DNA-protein cross-links due to reaction of the postulated intermediate with O-methylhydroxylamine affording N₍₄₎-methoxy-6-methoxy-amino-5,6-dihydrocytidine residues.     

l-leucine aminopeptidase production by filamentous Aspergillus fungi

AIMS: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. METHODS AND RESULTS: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2.61, 2.59 and 1.30 IU ml(-1) for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11.39, 17.40 and 13.02 IU g(-1) dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65-75 degrees C at pH 8.0-9.0 for all three fungi. Metal ions, Co(2+) and Fe(2+) in 2 mmol l(-1) concentration apparently enhanced the relative enzyme activity and heat stability. CONCLUSIONS: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Biodiesel fuel production from lipids of filamentous fungi

The main stages in the production of biodiesel fuel from lipids of filamentous fungi belonging to the order Mucorales are described. Fungi of the family Cunninghamellaceae have been screened; the lipogenic activity of the examined strains has been assessed; and a producer generating up to 50% of lipids, represented by triacylglycerols, has been found. The substitution effect of a source of carbon and nitrogen with less expensive components (in particular, various industrial wastes) has been studied, as well as their influence on the quantity and major characteristics of the final product. An ecologically friendly method for extracting lipids from fungal mycelia, utilizing supercritical technologies, has been used. A correlation between the lipid content in the spore inoculum and the maximal lipid content in biomass has been discovered; this correlation is proposed for optimizing the biotechnology and increasing the yield of final products.     

Comparative study of acid and alkaline phosphatase during the autolysis of filamentous fungi

Acid and alkaline phosphatase activities in culture liquid and mycelial extract during autolysis were studied in seven fungi of the general Ascomycotina, Basidiomycotina and Zygomycotina. High activities of extracellular and mycelial extract acid phosphatase and lower activities of alkaline phosphatase were found in Ascomycotina, and acid phosphatase was present in Basidiomycotina. In Zygomycotina only mycelial extract alkaline phosphatase activity was detected. A correlation between degree of autolysis, pH and acid phosphatase activity was demonstrated.     

Laccase production at reactor scale by filamentous fungi

Laccases have received much attention from researchers during the past decades due to their broad substrate specificity and to the fact that they use molecular oxygen as the final electron acceptor instead of hydrogen peroxide as used by peroxidases. This makes laccases highly interesting for a wide variety of processes, such as textile dye decolouration, pulp bleaching, effluent detoxification, biosensors and bioremediation.

The successful application of laccases to the above-mentioned processes requires the production of large quantities of enzyme at low cost. Filamentous fungi are able to produce laccases in high amounts, however, an efficient production system at bioreactor scale is still lacking. This is mainly due to the fact that laccase production by wild-type strains of filamentous fungi is linked to secondary metabolism, which implies that the following drawbacks must be overcome: uncontrolled fungal growth, the formation of polysaccharides around mycelia and the secretion of certain compounds (i.e. proteases) that inactivate laccases. This review summarizes the current status of laccase production by wild-type strains of filamentous fungi at the bioreactor scale.     

Lignocellulolytic enzyme production on pretreated poplar wood by filamentous fungi

Gliocladium sp. TUB F-498, a wild strain of a lignocellulolytic fungus with a fast growth rate and enzyme production rate was selected as a potential in situ enzyme source for the bioprocessing of pretreated poplar wood (PPW) to ethanol in a simultaneous saccharification and fermentation process. TUB F-498 produced 77 filter paper units of cellulase activity and 246 IU of -glucosidase activity per g dry weight of substrate utilized, as compared with the highly successful mutant reference strain Trichoderma reesei Rut-C30, which produced 100 filter paper units and 92 IU per g dry weight substrate in a stirred-tank fermentation. TUB F-498 also produced more xylanase and endoglucanase activity than Rut-C30 on PPW in shake-flask fermentations.     

Coordinated process of polarized growth in filamentous fungi

Filamentous fungi are extremely polarized organisms, exhibiting continuous growth at their hyphal tips. The hyphal form is related to their pathogenicity in animals and plants, and their high secretion ability for biotechnology. Polarized growth requires a sequential supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeleton. Therefore, the arrangement of the cytoskeleton is a crucial step to establish and maintain the cell polarity. This review summarizes recent findings unraveling the mechanism of polarized growth with special emphasis on the role of actin and microtubule cytoskeleton and polarity marker proteins. Rapid insertions of membranes via highly active exocytosis at hyphal tips could quickly dilute the accumulated polarity marker proteins. Recent findings by a super-resolution microscopy indicate that filamentous fungal cells maintain their polarity at the tips by repeating transient assembly and disassembly of polarity sites.     

丝状真菌中纤维素酶与半纤维素酶的合成调控简

综述了丝状真菌合成纤维素酶和半纤维素酶的相关调控研究进展。对最近研究文章分析发现：细胞外信号分子（C源、光信号）,转录因子以及染色质重建等对丝状真菌合成调控纤维素酶及半纤维素酶有重要影响。同时解析丝状真菌合成纤维素酶和半纤维素酶调控网络,以期为利用基因工程改造纤维素酶和半纤维素酶生产工业菌株提供理论指导。     

Decrease of polygalacturonic acid synthase during xylem differentiation in sycamore

The activity of a polygalacturonic acid synthase, a membrane bound enzyme, was measured in particulate preparations obtained from homogenates of cambial cells, differentiating xylem cells and differentiated xylem cells isolated from actively growing trees of sycamore (Acer pseudoplatanus). The specific acitivity of the enzymic system fell at least 6–10-fold as cells differentiated from the vascular cambium to xylem. The percentage of radioactivity incorporated as galacturonic acid was 88% for cambium, 69% for differentiating xylem and 57% for differentiated xylem. The remainder of the radioactivity was in glucuronic acid. UDP-d-galacturonic acid-4-epimerase (EC 5.1.3.6) in the membrane preparation, therefore, probably enabled incorporation of radioactivity in the form of glucuronic acid by other synthases into polymers which are characteristic of secondary walls. Nevertheless, a considerable decrease of polygalacturonic acid synthase activity occurred which was correlated with the cessation of pectin deposition. This contrasts with the marked increase observed in xylan synthase activity as the cells differentiate.     

Approaches for refining heterologous protein production in filamentous fungi

Fungi combine the advantages of a microbial system such as a simple fermentability with the capability of secreting proteins that are modified according to a general eukaryotic scheme. Filamentous fungi such as Aspergillus niger efficiently secrete genuine proteins but the secretion of recombinant proteins turned out be a difficult task. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production of heterologous proteins. To fully exploit the potential of filamentous fungi, understanding of the molecular genetics, their physiology, and the glycosylation metabolism has to be investigated and clarified in more detail. This review summarizes recent developments in heterologous protein production by filamentous fungi and also generalizes the possibilities of improving the protein production by various genetic and bioprocessing approaches, thereby easing recognition of filamentous fungi as a relevant and reliable expression platform.     

Microtiter plate‐based cultivation to investigate the growth of filamentous fungi

Microscale bioprocessing techniques are rapidly emerging as a means to increase the speed of bioprocess design and to reduce material consumption. However, there is still a lack of suitable parallelized techniques to investigate the industrially important group of filamentous bacteria and fungi. Cultivation of filamentous organisms in shake flasks is still the favored technique for comparing and optimizing cultivation conditions of production strains at mL‐scale. In this paper, the application of a microtiter plate‐based cultivation system in combination with the filamentous fungus Aspergillus niger was investigated. A protocol for reproducible cultivation was developed and evaluated. Productivity of A. niger concerning the rose‐like aroma compound 2‐phenylethanol showed low standard deviations while regular and consistent morphologies appeared in the parallelized system. Furthermore, the effect of addition of microparticles on the morphology was investigated. The results can be used to accelerate the process development with A. niger and other filamentous organisms.