Production of lovastatin by wild strains of Aspergillus terreus

A wild fungal strain of Aspergillus terreus, labeled as PM3, was isolated by using the Candida albicans bioassay and confirmed by 18S r DNA analyses. Lovastatin was produced by submerged and solid state fermentations. Of the 30 isolated fungal strains, 11 showed lovastatin production with Aspergillus terreus PM3 being the best with a yield of 240 mg/L at the 10th day of submerged fermentation. Carboxymethylcellulose had a stimulatory effect on lovastatin production. It restricted uncontrolled filamentous growth, induced pellet formation and, thereby, improved lovastatin yield. In solid state fermentation (SSF), of the agro wastes from five crops (bran of wheat and rice, husks of red gram and soybean, and green gram straw), wheat bran showed maximum lovastatin production (12.5 mg/g of dry substrate) at pH 7.1 and a temperature of 30 +/- 2 degrees C. Development of a lovastatin production process based on wheat bran as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Lovastatin biosynthesis in Aspergillus terreus: characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene.

BACKGROUND: Lovastatin, an HMG-CoA reductase inhibitor produced by the fungus Aspergillus terreus, is composed of two polyketide chains. One is a nonaketide that undergoes cyclization to a hexahydronaphthalene ring system and the other is a simple diketide, 2-methylbutyrate. Fungal polyketide synthase (PKS) systems are of great interest and their genetic manipulation should lead to novel compounds. RESULTS: An A. terreus mutant (BX102) was isolated that could not synthesize the nonaketide portion of lovastatin and was missing a approximately 250 kDa polypeptide normally present under conditions of lovastatin production. Other mutants produced lovastatin intermediates without the methylbutyryl sidechain and were missing a polypeptide of approximately 220 kDa. The PKS inhibitor cerulenin reacted covalently with both polypeptides. Antiserum raised against the approximately 250 kDa polypeptide was used to isolate the corresponding gene, which complemented the BX102 mutation. The gene encodes a polypeptide of 269 kDa containing catalytic domains typical of vertebrate fatty acid and fungal PKSs, plus two additional domains not previously seen in PKSs: a centrally located methyltransferase domain and a peptide synthetase elongation domain at the carboxyl terminus. CONCLUSIONS: The results show that the nonaketide and diketide portions of lovastatin are synthesized by separate large multifunctional PKSs. Elucidation of the primary structure of the PKS that forms the lovastatin nonaketide, as well as characterization of blocked mutants, provides new details of lovastatin biosynthesis.     

Putative Diels-Alder-Catalyzed Cyclization during the Biosynthesis of Lovastatin

A Diels-Alder cyclization proposed to occur during polyketide synthase assembly of the bicyclic core of lovastatin (1) (mevinolin) by Aspergillus terreus MF 4845 was examined via the synthesis of the N-acetylcysteamine (NAC) thioester of [2,11-(13)C(2)]-(E,E,E)-(R)-6-methyldodecatri-2,8,10-enoate (5a). In vitro Diels-Alder cyclization of the corresponding unlabeled NAC ester 5b, ethyl ester 18b, and acid 20b yielded two analogous diastereomers in each case, under either thermal or Lewis acid-catalyzed conditions. The reaction of thioester 5 proceeds readily at 22 degrees C in aqueous media. For 18b, one product is trans-fused ethyl (1R,2R,4aS, 6R,8aR)-1,2,4a,5,6,7,8,8a-octahydro-2,6-dimethylnaphthalene-1-carboxylate (30) (endo product), and the other is cis-fused ethyl (1R,2S,4aR,6R,8aR)-1,2,4a,5,6,7,8,8a-octahydro-2,6-dimethylnaphthalene-1-carboxylate (31) (exo product). Isomer 21 with stereochemistry analogous to 4a,5-dihydromonacolin L (2), a precursor of 1, was made by transformation of a tricyclic lactone, (1S,2S,4aR,6S,8S,8aS)-1-(ethoxycarbonyl)-1,2,4a,5,6,7,8,8a-octahydro-2-methyl-6,8-naphthalenecarbolactone (22) using reduction and Barton deoxygenation. Comparison of 21 with 30 and 31 confirmed the structural assignments and showed that the nonenzymatic 4 + 2 cyclizations of 5, 18, and 20 proceed via chairlike exo and endo transition states with the methyl substituent pseudoequatorial. The proposed biosynthetic Diels-Alder leading to lovastatin (1) would require an endo conformation with the methyl substituent pseudoaxial. Intact incorporation of the labeled hexaketide triene 5a into 1 was not achieved because of rapid degradation by A. terreus cells.     

Fermentative production of curdlan

Curdlan was produced by pure culture fermentation using Agrobacterium radiobacter NCIM 2443. Three different carbon sources (glucose, sucrose, maltose) were selected for study. Sucrose was found to be the most efficient. Utilization of sugar during the course of fermentation was studied, and the data were correlated to the production of curdlan. Curdlan mimics a secondary metabolite, in that its synthesis is associated with the poststationary growth phase of nitrogen-depleted batch culture. This was inferred from the results obtained from utilization of nitrogen. Regulation of pH at 6.1 +/- 0.3 resulted in an increased yield of curdlan from 2.48 to 4.8 g/L, and the corresponding increase in succinoglucan production was from 1.78 to 2.8 g/L. An attempt was made to increase curdlan production by the addition of the uridine nucleotides UMP and UDP-glucose to the fermentation broth. It was found that UDP-glucose at 0.8 microg/mL and UMP at 0.6 microg/mL served as precursors for curdlan and succinoglucan production when added after 18 h of nitrogen depletion in the fermentation broth.     

A novel butenoate derivative from Aspergillus niger

A novel butenoate derivative, methyl (Z)-4-{[(Z)-1-(hydroxymethyl)-2-phenyl-1-ethenyl] amino}-4-oxo-2-butenoate (1), was isolated from the fermentation broth of an Aspergillus niger strain Ta1. The structure (1) was elucidated by spectral analysis. Bioassays showed that compound 1 had a weak antioxidant activity.     

Enhanced Production of Spinosad in <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> by Genome Shuffling

Spinosad (spinosyns A and D) is a mixture of secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. In this study, we applied genome shuffling of S. spinosa to achieve a rapid improvement of spinosad production. Ten strains with subtle improvements in spinosad production were obtained from the populations generated by the mutation with nitrosoguanidine and ultraviolet irradiation, and then they were subjected for recursive protoplast fusion. After four rounds of genome shuffling, a high yielding strain, designated as S. spinosa 4-7, was successfully isolated. Its production reached 547 mg/L, which was increased by 200.55% and 436.27% in comparison with that of the highest parent strain and the original strain, respectively. The subculture experiments indicated that the high producer of S. spinosa 4-7 was stable. Spinosad fermentation experiments by S. spinosa 4-7 were carried out in a 5-L fermentor, and its production of spinosad reached 428 mg/L after 168 h of fermentation.     

Inulinase synthesis from a mesophilic culture in submerged cultivation

A newly isolated mesophilic bacterial strain from dahlia rhizosphere, identified as Staphylococcus sp. and designated as RRL-M-5, was evaluated for inulinase synthesis in submerged cultivation using different carbon sources individually or in combination with inulin as substrate. Inulin appeared as the most favorable substrate at a 0.5–1.0% concentration. Media pH influenced the enzyme synthesis by the bacterial strain, which showed an optimum pH at 7.0–7.5. Supplementation of fermentation medium with external nitrogen (organic and inorganic) showed a mixed impact on bacterial activity of enzyme synthesis. The addition of soybean meal and corn steep solid resulted in about an 11% increase in enzyme titers. Among inorganic nitrogen sources, ammonium sulfate was found to be the most suitable. Maximum enzyme activities (446 U/L) were obtained when fermentation was carried out at 30°C for 24 h with a medium containing 0.5% inulin as a sole carbon source and 0.5% soybean meal as the nitrogen source. Bacterial inulinase could be a good source for the hydrolysis of inulin for the production of d-fructose.     

Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3

Marine endosymbiotic fungi Aspergillus ustus (MSF3) which produce high yield of biosurfactant was isolated from the marine sponge Fasciospongia cavernosa collected from the peninsular coast of India. Maximum production of biosurfactant was obtained in Sabouraud dextrose broth. The optimized bioprocess conditions for the maximum production was pH 7.0, temperature 20 °C, salt concentration 3%, glucose and yeast extract as carbon source and nitrogen sources respectively. The response surface methodology based analysis of carbon and nitrogen ratio revealed that the carbon source can increase the biosurfactant yield. The biosurfactant produced by MSF3 was partially characterized as glycolipoprotein based on the estimation of macromolecules and TLC analysis. The partially purified biosurfactant showed broad spectrum of antimicrobial activity. The strain MSF3 can be used for the microbially enhanced oil recovery process.     

Development of minimal fermentation media supplementation for ethanol production using two Saccharomyces cerevisiae strains

Ethanol production by fermentation is strongly dependent on media composition. Specific nutrients, such as trace elements, vitamins and nitrogen will affect the physiological state and, consequently, the fermentation performance of the micro-organism employed. The purpose of this study has been to assess the highest ethanol production by a minimal medium, instead of the more complex nutrients supplementation used during alcoholic fermentation. All fermentation tests were carried out using a microwell plate reader to monitor the processes. Two Saccharomyces cerevisiae strains (NCYC 2826 and NCYC 3445) were tested using three nitrogen sources, supplied with different vitamin and salts. The results show that solutions made of urea phosphate, KCl, MgSO₄·7H₂O, Ca-panthothenate, biotin allowed an ethanol yield of 22.9 and 23.4 g/L for strain NCYC 2826 and NCYC 3445, respectively, representing 90 and 92% of the theoretical yield. All tests were carried out using glucose as common reference carbon source.     

Enhancement of Lovastatin Production by Supplementing Polyketide Antibiotics to the Submerged Culture of <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Feedback inhibition existed in lovastatin biosynthesis from Aspergillus terreus. Exogenous lovastatin and other different polyketide antibiotics biosynthesized by polyketide synthase were supplemented to the cultures of A. terreus to investigate their influences on lovastatin production. Supplementing exogenous lovastatin of 100 mg l⁻¹ at the early stage of fermentation and the fast stage of its biosynthesis resulted in decreases of 76.4% and 20% in final lovastatin production, respectively. However, the fungal cell growth was not affected; the growing cycle was only prolonged in the submerged cultivation. Separate supplementation of the five kinds of polyketide antibiotics such as tylosin, erythromycin, tetracycline, daunorobin, and rifamycin to the cultures resulted in increases of about 20~25% in the final lovastatin production. Especially, supplementing tylosin of 50 mg l⁻¹ at the beginning of lovastatin biosynthesis led to the final lovastatin production of 952.7 ± 24.3 mg l⁻¹, which was improved by 42% and 22% compared with that produced in the control and the original culture, respectively. These results are helpful to understand the regulations on lovastatin biosynthesis and improve the final desired metabolite contents in many antibiotics production.     

Adding Value to the Oil Cake as a Waste from Oil Processing Industry: Production of Lipase and Protease by <Emphasis Type="Italic">Candida utilis</Emphasis> in Solid State Fermentation

Olive oil cake is a by-product from the olive oil processing industry and can be used for the lipase and protease production by Candida utilis in solid state fermentation. Different carbon and nitrogen sources were evaluated, and the results showed that the supplementation of the substrate with maltose and starch as carbon sources and yeast extract as a nitrogen source significantly increased the lipase production. The best results were obtained with maltose, whereas rather low lipase and protease activities were found with glucose and oleic acid. Response surface methodology and a five-level–three-factor central composite rotatable design were used to evaluate the effects of the initial moisture content, inoculum size and fermentation time on both lipase and protease activity levels. A lipase activity value of ≈25 U g^-1 and a protease activity value of 110 U g^-1 were obtained under the optimized fermentation conditions. An alkaline treatment of the substrate appeared to be efficient, leading to increases of 39% and 133% in the lipase and protease production, respectively. The results showed that the olive cake could be a good source for enzyme production by solid state fermentation.     

Development of Industrial Brewing Yeast with Low Acetaldehyde Production and Improved Flavor Stability

Higher acetaldehyde concentration in beer is one of the main concerns of current beer industry in China. Acetaldehyde is always synthesized during beer brewing by the metabolism of yeast. Here, using ethanol as the sole carbon source and 4-methylpyrazole as the selection marker, we constructed a new mutant strain with lower acetaldehyde production and improved ethanol tolerance via traditional mutagenesis strategy. European Brewery Convention tube fermentation tests comparing the fermentation broths of mutant strain and industrial brewing strain showed that the acetaldehyde concentration of mutant strain was 81.67 % lower, whereas its resistant staling value was 1.0-fold higher. Owing to the mutation, the alcohol dehydrogenase activity of the mutant strain decreased to about 30 % of the wild-type strain. In the meantime, the fermentation performance of the newly screened strain has little difference compared with the wild-type strain, and there are no safety problems regarding the industrial usage of the mutant strain. Therefore, we suggest that the newly screened strain could be directly applied to brewing industry.     

高效液相色谱法分离测定洛伐他汀及其开环酸

改进了洛伐他汀的分析方法，以甲醇：水＝80：20为流动相，采用紫外检测器检测，使用C4色谱柱定性、定量分析了洛伐他汀及洛伐他汀羟基酸，并得出了相关方程．实验证明，采用新的色谱条件可在有效地分析洛伐他汀及其开环酸的基础上，节约分析成本．     

Red Yeast Rice Fermentation by Selected Monascus sp. with Deep-Red Color, Lovastatin Production but No Citrinin, and Effect of Temperature-Shift Cultivation on Lovastatin Production

Monascus pilosus NBRC4520 was selected for functional fermented food inoculation for its high lovastatin and low citrinin production with a deep-red color. For koji (mold rice) with high lovastatin production, separation of the growth phase and lovastatin production phase by shifting the temperature from 30 to 23 °C increased lovastatin production by nearly 20 times compared to temperature-constant cultivation. In addition, citrinin was not produced even in the lovastatin production phase, although the pigment was increased. With temperature-shift cultivation, 225 μg lovastatin/g dry koji was produced in 14 days without citrinin.     

Isolation,Identification and Optimization of a New Extracellular Lipase Producing Strain of <Emphasis Type="Italic">Rhizopus</Emphasis> sp.

A lipolytic mesophilic fungus which produces lipase extracellularly was isolated from soil. Based on ITS1-5.8S–ITS4 region sequences of ribosomal RNA, it was concluded that the isolate JK-1 belongs to genus Rhizopus and clades with Rhizopus oryzae. The present paper reports the screening, isolation, identification, and optimization of fermentation conditions for the production of lipase (EC 3.1.1.3). Culture conditions were optimized, and the highest lipase production was observed in basal medium with corn steep liquor as nitrogen source and glucose as carbon source. Maximum lipase production was observed at 72 h, which is about 870 U/ml. Optimization of fermentation conditions resulted in 16-fold enhancement in enzyme production.     

Butyrolactones from Aspergillus terreus

In the process development of lovastatin using Aspergillus terreus DRCC 152 in solid state fermentation, we have isolated a new butyrolactone-IV (3) along with the previously reported butyrolactone-I (1) and butyrolactone-II (2) produced under submerged conditions. The structure of compound 3 has been characterized as 3-hydroxy-5-[2-(1-hydroxy-1-methylethyl)-2(R)-2,3-dihydro-benzo[b]furan- 5 ylmethyl]-4-(4-hydroxyphenyl)-5-methoxycarbonyl-(5R)-2,5-dihydro-2 -furanone on the basis of spectroscopic studies. The absolute stereochemistry has been determined by single crystal X-ray diffraction studies. The cytotoxic and antibacterial activities of these compounds were determined.     

Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis

The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0−3.8×10⁵ Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index =1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0–5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22°C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.     

Production of Oils from Acetic Acid by the Oleaginous Yeast Cryptococcus curvatus

The feasibility of the conversion of acetic acid, a metabolite commonly obtained during anaerobic fermentation processes, into oils using the yeast Cryptococcus curvatus was reported. This microorganism exhibited very slow growth rates on acetate as carbon source, which led to design a two-stage cultivation process. The first consisted of cell growth on glucose as carbon source until its complete exhaustion. The second step involved the use of acetate as carbon source under nitrogen limitation in order to induce lipid accumulation. A typical experiment performed in a bioreactor involved a preliminary yeast growth with a glucose initial concentration of 15?g/L glucose. Further additions of acetate and nitrogen source allowed a final lipid accumulation up to 50% (w/w). These promising results demonstrated the suitability of the technique proposed.     

Production of Keratinase by Free and Immobilized Cells of <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Strain PPKS-2: Partial Characterization and Its Application in Feather Degradation and Dehairing of the Goat Skin

An extremely alkaliphilic bacterial strain, Bacillus sp. PPKS-2, was isolated from rice mill effluents and screened for the production of extracellular keratinase. The maximum production of keratinase occurred after 48 h in shaking culture at pH 11.0 and 37 °C in a medium containing 0.5% soybean flour. The strain grew and produced alkaline keratinase using chicken feather and horn meal as the sole source of carbon and nitrogen. An addition of 0.1% soybean flour or feather hydrolysate and sodium sulfite to feather medium increased the production and complete solubilization of feather took place within 5 days under solid-state fermentation conditions. The partially purified enzyme displayed maximum activity at pH 11.0 and 60 °C in a broad range of NaCl, 0–16%, and was not inhibited by sodium dodecyl sulfate (10%), ethylenediaminetetraacetic acid (10 mM), H₂O₂ (15%), and other commercial detergents. Immobilization of the whole cells proved to be useful for continuous production of keratinase and feather degradation. The enzyme was effectively used to remove hair from goat hide. The strain PPKS-2 can be effectively used for solid waste management of poultry feather in submerged as well as solid-state fermentation.     

Rheological Analysis of Biopolymer Produced by Aureobasidium Pullulans in Different Sources of Nitrogen

Summary: In this study, the rheological behaviors of media fermented by two Aureobasidium pullulans strains (IOC 3467 and IOC 3011) were evaluated in different nutritional conditions. The media consisted of crystal sugar (sucrose), as the main carbon source, and different nitrogen sources (ammonium sulfate, sodium nitrate, ammonium nitrate, urea or residual brewery yeast - RBY). Viscosity measurements were performed on cell-free supernatants, from 48-hour fermentation assays, at 25 ^°C. Shear rates ranged between 0.1000 and 500 s⁻¹. All samples showed pseudoplastic behavior. Nevertheless, the viscosimetric profile of each one varied according to the nitrogen source, its concentration and the strain used. The maximum viscosity of 0.06 Pa.s. was achieved at 15.6 s⁻¹ for IOC 3011 strain grown on RBY as nitrogen source.