Studies on mevinolin production by some fungi

The potentiality of 25 fungal species, belonging to fourteen genera isolated from Egyptian soils, to produce mevinolin, a hypocholesterolaemic agent, when grown on selected substrates was tested. Samples of culture filtrates were tested by thin layer chromatography and the positive results were further assessed by high pressure liquid chromatography analysis. It was found that nearly one-third of the fungi showed positive results for production of mevinolin. Aspergillus terreus was distinguished by its capacity to produce mevinolin when cultivated on selected media. Some factors influencing mevinolin production and the growth of A. terreus were also studied. The maximal mevinolin yields were achieved after 8 days incubation at 30 degrees C. An initial pH value of 5-6 was the optimum for growth of A. terreus and mevinolin production.     

Some nutritional factors influencing mevinolin production byAspergillus terreus strain

Aspergillus terreus produces the hypocholesterolemic compound mevinolin. Its growth and mevinolin production were affected by the composition of the culture medium. Both were at a maximum with glucose (6%) as the sole carbon source and in the presence of a mixture of yeast extract and sodium nitrate as nitrogen source. Influence of the concentration of some inorganic salts are also discussed.     

Optimization of fermentation medium by a modified method of genetic algorithms

Summary During the study of mevinolin biosynthesis by Aspergillus terreus ATCC 20542, 10 different medium components were selected for medium optimization. A new optimization method based on genetic algorithms and inductive learning was used for experimental design. For better efficiency the method was supported by a model, constructed with machine learning method, to predict the productivity. In four generations of fermentation experiments the productivity increased nearly three times.     

Aspergillus terreus and its toxic metabolites as a food contaminant in some Egyptian Bakery products and grains

A. terreus isolates isolated from some bakery products, corn and rice were found to be able to produce territrems. 90% of theA. terreus isolated from bakery products were able to produce territrem A, with a mean of 0.09 ppm, while 80% ofA. terreus isolates produce territrem B with a mean of 0.24 ppm. On the other hand 31.8% of the isolates ofA. terreus from corn were able to produce territrem A with a mean of 0.44 ppm. ConcerningA. terreus isolates from rice, 66.7% were found to produce territrem A, with a mean of 5.28 ppm, and 77.8% of the isolates produced territrem B with a mean of 1.79 ppm.     

High purity, high yield procedure for butyrolactone I production from Aspergillus terreus

Two experimental design methods, Plackett-Burman and Box-Behnken, were used to optimize media for the production of butyrolactone I, a chemical inhibitor of eukaryotic cyclin-dependent kinases, synthesized by Aspergillus terreus. The optimized medium produced as much as ten fold more butyrolactone I than the original medium. An isolation procedure is also described which generates highly purified butyrolactone I, free from other secondary metabolites produced by this strain of A. terreus. The results of this study provide the means to produce highly purified preparations of butyrolactone I.     

Capacity for biosurfactant production of environmental Pseudomonas and Vibrionaceae growing on carbohydrates

Summary Pseudomonas and Vibrionaceae strains with the capacity to produce biosurfactants when growing on sucrose were isolated from the environment by a simple screening procedure. Agargrown colonies were randomly selected; each colony was suspended in a water droplet on a microscope slide. The tested strain was regarded as positive if the droplet spread over the surface.1779 Pseudomonas and 660 Vibrionaceae isolates were tested; 1% and 0.8% of the isolates, respectively, were positive for biosurfactant production. No production was detected amongst the isolates of a control group of 538 Gram-positive and 1063 Gram-negative strains.Four biosurfactant producing strains were grown in fermenter cultures on a semisynthetic medium using sucrose as carbon and energy source. The terminal concentrations of biosurfactants were in the range of a factor 40 times the critical micelle dilution. One P. fluorescens strain was grown in a carbon limited chemostat (succinate). The biosurfactant production was successively decreasing until it stopped after less than ten generation times.     

Efficient Itaconic acid production via protein–protein scaffold introduction between GltA,AcnA, and CadA in recombinant Escherichia coli

Itaconic acid, which is a promising organic acid in synthetic polymers and some base-material production, has been produced by Aspergillus terreus fermentation at a high cost. The recombinant Escherichia coli that contained the cadA gene from A. terreus can produce itaconic acid but with low yield. By introducing the protein–protein scaffold between citrate synthesis, aconitase, and cis-aconitase decarboxylase, 5.7 g/L of itaconic acid was produced, which is 3.8-fold higher than that obtained with the strain without scaffold. The optimum pH and temperature for itaconic acid production were 8.5 and 30°C, respectively. When the competing metabolic network was inactivated by knock-out mutation, the itaconic acid concentration further increased, to 6.57 g/L.     

D-amino acid oxidase,aromatic L-amino aminotransferase,and aromatic lactate dehydrogenase from several yeast species: Comparison of enzyme activities and enzyme specificities

Production of D-amino acid oxidase, L-aromatic aminotransferase and aromatic lactate dehydrogenase by several yeast species was examined. Of 16 strains tested, Trigonopsis variabilis and Rhodosporidium toruloides were found to be most suitable for D-amino acid oxidase production, T. variabilis and Brettanomyces anomalus for L-aromatic aminotransferase production, and Hansenula polymorpha, Cryptococcus terreus, and Candida maltosa for aromatic lactate dehydrogenase production. This selection is based on a high amount of enzyme activity as well as a broad enzyme specificity. The data will be reported here.     

Bioconversion of cellulose wastes to protein and sugar

A schematic representation of the variety of products which can be obtained by microbial conversion of cellulose is presented. Alkaline pre-treatment has been used after milling in all the experiments. Solka-floc or sugarcane bagasse was used as sources of cellulose. A cellulolytic strain of Aspergillus terreus (ATCC 30514) was cultivated in batch-, fed batch and continuous culture up to 7 liter stirred tank fermenter. The general growth characteristics were determined by growing on glucose. Results of experiments on the growth of A. terreus for production of biomass on Solka-floc or Sugarcane bagasse are given, also the ability of crude cellulases to produce sugar syrups by enzymatic hydrolysis of cellulose has been evaluated.     

Lovastatin and (+)‐geodin production by Aspergillus terreus from crude glycerol

The use of pure substrate represents a significant proportion of the cost of manufacturing a drug such as lovastatin. This study explores the production of lovastatin and (+)‐geodin by Aspergillus terreus ATCC 20542 using biodiesel‐derived crude glycerol (CG) as a feedstock. Shake flask experiments showed reduced lovastatin production and glycerol consumption in the presence of 10–50 g/L CG with respect to pure glycerol controls. At 50 g/L, lovastatin and (+)‐geodin production was significantly reduced by 82 and 73%, respectively. The lowest lovastatin inhibition was detected in 30 g/L of CG (48%), which was accompanied by a significant rise in (+)‐geodin production (338%). Further investigation was performed on three major impurities found in CG, namely methanol (MeOH), sodium chloride (NaCl), and fatty acids (oleic acid and palmitic acid (PA), soap). None was particularly inhibitory for lovastatin, except soap and PAs, which reduced its production by more than 50% at all concentrations tested. In contrast, (+)‐geodin was inhibited in the presence of MeOH and PA by up to 46 and 91%, respectively. These observations indicate that partial purification of CG would be potentially useful in improving production of lovastatin and (+)‐geodin by A. terreus.     

Lovastatin Biosynthesis by Aspergillus terreus in a Chemically Defined Medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Effects of iron level on the anatagonistic action of siderophores from non-pathogenicStaphylococcus spp

The production, detection and the effects of iron concentration on the siderophore production ofStaphylococcus strains used as meat starter cultures were studied. Non-pathogenicStaphylococcus strains produce extracellular low molecular weight compounds which exhibited positive reactivity when measured by a universal detection method for siderophores. The production of siderophores was very closely associated with the iron concentration in the medium, and very low additions considerably reduced siderophore production. Although the production of siderophores was highly iron-dependent, the antimicrobial activity of spent medium fromStaphylococcus cultures against selected yeasts and moulds remained considerable under high iron concentrations.     

Use of mutation strategies applied toAspergillus terreus ATCC 52430 to obtain mutants with improved cellulase productivity

Summary Mutation studies were carried out onAspergillus terreus ATCC 52430 to increase cellulase production. Optimal treatment conditions for increasing cellulase activity by UV mutagenesis were observed by exposure of spores to a UV germicidal lamp, wavelength 250 nm at a distance of 30 cm for 1 min. Nitrosoguanidine mutagenesis was optimal with respect to development of high cellulase mutants when spores were exposed to 500 ug/ml nitrosoguanidine for 1 h. Mutants were isolated on agar containing Walseth cellulose and the catabolite repressor, glycerol. Successive UV and nitrosoguanidine treatments resulted in isolation of strain UNG1-40 having 3.5, 4.6 and 3.3-fold increases in filter paper, β-glucosidase and carboxymethyl cellulase activity, respectively, compared toA. terreus ATCC 52430.     

Biotechnological production and applications of statins

Statins are a group of extremely successful drugs that lower cholesterol levels in blood; decreasing the risk of heath attack or stroke. In recent years, statins have also been reported to have other biological activities and numerous potential therapeutic uses. Natural statins are lovastatin and compactin, while pravastatin is derived from the latter by biotransformation. Simvastatin, the second leading statin in the market, is a lovastatin semisynthetic derivative. Lovastatin is mainly produced by Aspergillus terreus strains, and compactin by Penicillium citrinum. Lovastatin and compactin are produced industrially by liquid submerged fermentation, but can also be produced by the emerging technology of solid-state fermentation, that displays some advantages. Advances in the biochemistry and genetics of lovastatin have allowed the development of new methods for the production of simvastatin. This lovastatin derivative can be efficiently synthesized from monacolin J (lovastatin without the side chain) by a process that uses the Aspergillus terreus enzyme acyltransferase LovD. In a different approach, A. terreus was engineered, using combinational biosynthesis on gene lovF, so that the resulting hybrid polyketide synthase is able to in vivo synthesize 2,2-dimethylbutyrate (the side chain of simvastatin). The resulting transformant strains can produce simvastatin (instead of lovastatin) by direct fermentation.     

Itaconic Acid Production by Filamentous Fungi in Starch-Rich Industrial Residues

Several fungi and starch-rich industrial residues were screened for itaconic acid (IA) production. Out of 15 strains, only three fungal strains were found to produce IA, which was confirmed by HPLC and GC–MS analysis. These strains were identified as Aspergillus terreus strains C1 and C2, and Ustilago maydis strain C3 by sequencing of 18S rRNA gene and internal transcribed spacer regions. Cis-aconitate decarboxylase (cad) gene, which encodes a key enzyme in IA production in A. terreus, was characterized from strains C1 and C2. C1 and C2 cad gene sequences showed about 96% similarity to the only available GenBank sequence of A. terreus cad gene. 3-D structure and cis-aconitic acid binding pocket of Cad enzyme were predicted by structural modeling. Rice, corn and potato starch wastes were screened for IA production. These materials were enzymatically hydrolyzed under experimentally optimized conditions resulting in the highest glucose production of 230 mg/mL from 20% potato waste. On comparing the production potential of selected strains with different wastes, the best IA production was achieved with strain C1 (255.7 mg/L) using potato waste. Elemental composition as well as batch-to-batch variation in waste substrates were analyzed. The difference in IA production from two different batches of potato waste was found to inversely correlate with their phosphorus content, which indicated that A. terreus produced IA under phosphate limiting condition. The potato waste hydrolysate was deionized to remove inhibitory ions like phosphate, resulting in improved IA production of 4.1 g/L by C1 strain, which is commercially competitive.     

Acceleration of fungal spore production by embedding a hydrophobic polymer net in a nutrient agar plate

A simple and novel procedure for the acceleration of fungal spore production was developed. A net of hydrophobic polymer such as polypropylene (PP) and polytetrafluoroethylene (PTFE) was embedded in a nutrient agar plate, and effect of the polymer net on spore production by 6 fungal strains, such as Aspergillus terreus, Penicillium multicolor, and Trichoderma virens were estimated. The effect of hydrophobic polymer net was insufficient in a liquid-surface immobilization (LSI) system with fungal cells immobilized on a ballooned microsphere layer formed on a liquid medium surface. On the other hand, the embedding of a PTFE net in an agar plate remarkably enhanced the spore production in all 6 strains tested to produce 2.0–8.5 × 10⁷ spores/cm²-agar plate surface. Especially, the spore production by A. terreus ATCC 20542 in the presence of a PTFE net was 7.7 times as much than that in no net. Positive correlations between the hydrophobicity of net and the spore production were observed in all 6 strains (R², 0.653–0.999).     

Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.     

Extracellular proteinase spectrum ofAspergillus terreus grown on various substrates

The level of proteinase activity and the ratio of proteinases I and II, secreted byAspergillus terreus, a cellulase producer, was followed during its growth on media containing various carbon sources. Correlation was found between the level of proteinase secretion and the rate of change of the cellulase complex spectrum. The extracellular proteolytic system ofA. terreus was presented mainly by proteinase II (metalloproteinase) during cultivation under conditions favoring fast accumulation of low-molar mass cellulases. The results indicate that proteinase II could be responsible for the limited proteolysis of high-molar mass cellulases ofA. terreus into smaller enzymes of the cellulolytic complex, thus changing their substrate specificity.