Simultaneous biosynthesis of (+)-geodin by a lovastatin-producing fungus Aspergillus terreus

The simultaneous biosynthesis of lovastatin (mevinolinic acid) and (+)-geodin by Aspergillus terreus ATCC 20542 with regard to the medium composition, i.e. the concentrations of carbon and nitrogen source, was described in this paper. A. terreus is a lovastatin producer but the formation of lovastatin was accompanied by the significant amounts of (+)-geodin, when the elevated concentration of carbon source (lactose) was still present in the medium in the idiophase and nitrogen source (yeast extract) was deficient. It was observed for runs, in which the higher (above 20 g l(-1)) initial lactose concentration was applied or when the nitrogen deficiency led to the decrease of biomass content in the system. In contrast to lovastatin, there was not optimum initial concentration of yeast extract, as its lowest tested initial concentration (2 g l(-1)) led to the highest (+)-geodin volumetric formation rates and final yield. What is more, even higher final (+)-geodin concentrations were achieved at elevated initial lactose concentration (40 g l(-1)) and in the lactose-fed fed-batch run. In the fed-batch run lovastatin concentration increased significantly too, as this metabolite formation is also carbon source dependent. Finally, (+)-geodin occurred to be a metabolite, whose formation, in contrast to lovastatin, is non-growth associated.     

Effects of the principal nutrients on lovastatin production by Monascus pilosus

Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.     

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.     

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 lovastatin production by solid state fermentation ofMonascus ruber

The purpose of this study was to optimize the solid state cultivation ofMonascus ruber on sterile rice. A single-level-multiple-factor and a single-factor-multiple-level experimental design were employed to determine the optimal medium constituents and to optimize carbon and nitrogen source concentrations for lovastatin production. Simultaneous quantitative analyses of the β-hydroxyacid form and β-hydroxylactone for of lovastatin were performed by the high performance liquid chromatography (HPLC) method with a UV photodiode-array (PDA) detector. The total lovastatin yield (4≈6 mg/g, average of five repeats) was achieved by adding soybean powder, glycerol, sodium nitrate, and acetic acid at optimal composition of the medium increased by almost 2 times the yield observed prior to optimization. The experimental results also indicated that the β-hydroxylactone form of lovastatin (LFL) and the β-hydroxyacid form of lovastatin (AFL) simultaneously existed in solid state cultures ofMonascus ruber, while the latter was the dominant form in the middle-late stage of continued fermentation. These results indicate that optimized culture conditions can be used for industrial production of lovastatin to obtain high yields.     

Biosynthesis of lovastatin and (+)-geodin by Aspergillus terreus in batch and fed-batch culture in the stirred tank bioreactor

The simultaneous formation of mevinolinic acid (lovastatin; antihypercholesterolemia drug) and (+)-geodin (by-product) by Aspergillus terreus ATCC 20542 in the batch and fed-batch cultivation in the stirred tank bioreactor was investigated and described in this paper. The main factors influencing the formation of these two secondary metabolites were the initial nitrogen concentration and the aeration rate of the medium. The experiments aimed at achieving as high as possible lovastatin titre accompanied by as low as possible (+)-geodin concentration. The application of lactose-fed discontinuous fed-batch process allowed increasing lovastatin formation, in comparison with the batch process. Nevertheless (+)-geodin titre increased too. But the control of pH at the levels of 7.6 and 7.8 was successfully applied to repress the formation of the by-product both in batch and fed-batch experiments. Additionally, apart from pH control, the supplementation of the medium with nicotinamide and calcium pantothenate was used to facilitate the formation of lovastatin. The simultaneous pH control and B-group vitamin supplementation allowed achieving the best results in the batch cultivation.     

Influence of medium design on lovastatin and mevastatin production byAspergillus terreus strains

In order to investigate the influence of medium design on lovastatin and mevastatin production byAspergillus terreus strains, several nitrogen complex sources, such as vegetal flour and peptones of different origin (animal and vegetal) were tested, together with the addition of methionine, an aminoacid that is directly involved in lovastatin biosynthetic pathway. Soybean peptone generally allowed the best lovastatin yields to be achieved (250–280 mg l^?1), particularly in the presence of soybean and peanut flours. For mevastatin, the best results (300–320 mg l^?1) were obtained at 7 days fermentation with modified base medium (CLD), and at 14 days with standard medium (STD), not being possible in this case to associate the best yield with a defined flour and/or peptone. The results show that lovastatin production is influenced by the presence of soybean peptone and by the addition of methionine; instead, the production of mevastatin appears more strictly strain-associated and not directly dependent on the complex ingredients employed.     

Upstream and Downstream Processing of Lovastatin by Aspergillus terreus

The present study describes the enhanced production and purification of lovastatin by Aspergillus terreus in submerged batch fermentation. The enhancement of lovastatin production from A. terreus was attempted by random mutagenesis using ultraviolet radiations and nitrous acid. UV mutants exhibited increased efficiency for lovastatin production as compared with nitrous acid mutants. Among all the mutants developed, A. terreus UV-4 was found to be the hyper producer of lovastatin. This mutant gave 3.5-fold higher lovastatin production than the wild culture of A. terreus NRRL 265. Various cultural conditions were also optimized for hyper-producing mutant strain. 5 % glucose as carbon source, 1.5 % corn steep liquor as nitrogen source, initial pH value of 6, 120 h of incubation period, and 28 °C of incubation temperature were found as best parameters for higher lovastatin production in shake flasks. Production of lovastatin by wild and mutant strains of A. terreus was also scaled up to laboratory scale fermentor. The fermentation process was conducted at 28 °C, 200 rpm agitation, and 1vvm air flow rate without pH control. After the optimization of cultural conditions in 250 ml Erlenmeyer flasks and scaling up to laboratory scale fermentor, the mutant A. terreus UV-4 gave eightfold higher lovastatin production (3249.95 μg/ml) than its production by wild strain in shake flasks. Purification of lovastatin was carried out by solvent extraction method which yielded 977.1 mg/l of lovastatin with 98.99 % chromatographic purity and 26.76 % recovery. The crystal structure of lovastatin was determined using X-ray diffraction analysis which is first ever reported.     

Production of lovastatin by a wild strain of Aspergillus terreus

Of 68 Aspergillus terreus, three produced lovastatin with equivalent or better yield than strain ATCC 20542 originally described for lovastatin production. Medium optimization experiments with the best isolate (TUB F-514) indicated that lactose, rapeseed meal and KNO3 were the best carbon, organic nitrogen and inorganic nitrogen sources, respectively. In shake-flasks with optimized medium containing 4 % (w/v) lactose, 400 g lovastatin/ml was produced, with a yield of 10 mg/g lactose. In solid substrate fermentation on extracted sweet sorghum pulp supplemented with cheese whey 1500 g lovastatin/g dry weight was produced with a yield of 37.5 mg/g lactose. © Rapid Science Ltd. 1998     

Lovastatin biosynthesis depends on the carbon–nitrogen proportion: Model development and controller design

Lovastatin biosynthesis depends on the relative concentrations of dissolved oxygen and the carbon and nitrogen resources. An elucidation of the underlying relationship would facilitate the derivation of a controller for the improvement of lovastatin yield in bioprocesses. To achieve this goal, batch submerged cultivation experiments of lovastatin production by Aspergillus flavipus BICC 5174, using both lactose and glucose as carbon sources, were performed in a 7‐L bioreactor and the data used to determine how the relative concentrations of lactose, glucose, glutamine, and oxygen affected lovastatin yield. A model was developed based on these results and its prediction was validated using an independent set of batch data obtained from a 15‐L bioreactor using five statistical measures, including the Willmott index of agreement. A non‐linear controller was designed considering that dissolved oxygen and lactose concentrations could be measured online, and using the lactose feed rate and airflow rate as process inputs. Simulation experiments were performed to demonstrate that a practical implementation of the non‐linear controller would result in satisfactory outcomes. This is the first model that correlates lovastatin biosynthesis to carbon–nitrogen proportion and possesses a structure suitable for implementing a strategy for controlling lovastatin production.     

Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.     

Bacillus subtilis var. amyloliquefaciens biosynthesis of extracellular protease possessing coagulase activity and formed under conditions of limiting the nitrogen sources in the medium

An exocellular protease having the activity of coagulase was synthesized by Bacillus subtilis var. amyloliquefaciens when the growth medium contained no nitrogen sources. The removal of a nitrogen source from the medium was found to induce the synthesis of exoproteases by washed bacterial cells. Protein as a sole source of nitrogen in the medium inhibited rather than induced the biosynthesis of proteases possessing the activity of coagulase by the cells. The production of exoproteases with the coagulase activity was inhibited when a mixture of amino acids was used as a sole nitrogen source.     

Decrease of cholesterol in mouse melanoma causes secretion of lysosomal enzymes

We examined the change in the subcellular distribution of a lysosomal enzyme, beta-glucuronidase (beta-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of beta-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and beta-G activity in the cells, beta-G activity in the medium increased. Also, the ratio of beta-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat.     

Formation of secreted acid phosphatase during the growth of Saccharomyces cerevisiae yeasts on different sources of carbon and nitrogen nutrition

The effect of certain components in the growth medium on the secretion of acid phosphatase was studied with Saccharomyces cerevisiae. The presence of phosphate at a concentration of 10 mM in the medium inhibited the formation of repressible forms of this enzyme. The synthesis of the secreted enzyme depended on the sources of carbon and nitrogen nutrition. The enzyme yield was highest in a medium with sucrose as a carbon source and ammonium chloride as a nitrogen source. The secretion of acid phosphatase is stimulated by an increase in the sugar content and a deficiency of the nitrogen source in the medium.     

Lovastatin and (+)‐geodin formation by Aspergillus terreus ATCC 20542 in a batch culture with the simultaneous use of lactose and glycerol as carbon sources

The influence of initial glycerol and lactose concentrations on lovastatin and (+)‐geodin formation in batch cultures of Aspergillus terreus ATCC 20542 was presented. At first the experiments comprised lovastatin biosynthesis on glycerol as the sole carbon source. Lovastatin titers below 40 mg/L were found under these conditions and they were lower than previously obtained results when lactose was used as the sole carbon source. However, the application of the mixture of glycerol and lactose allowed in achieving higher lovastatin concentration in the broth. It even exceeded 122 mg/L when 10 g lactose and 15 g glycerol per liter were used. The calculated lovastatin volumetric and specific formation rates on glycerol or lactose and on the mixture of these two showed that lovastatin was faster produced on lactose than on glycerol. In the trophophase, the maximum volumetric lovastatin formation rate on lactose was up to four times higher than on glycerol and so was the lovastatin specific formation rate. Similar relations for the accompanying (+)‐geodin biosynthesis were also studied. When the mixture of lactose and glycerol was used, the transformation of (+)‐geodin to other polyketide metabolites also took place.     

不同氮源对悬浮培养玫瑰茄细胞的生长及硝态氮同化指征的影响

本文通过改变培养基中的氮源组成,来研究氮源的变化对悬浮培养玫瑰茄细胞的生长及硝态氮同化指征（活体内硝酸还原酶活力）的影响。实验表明,仅在含ＮＯ－３的培养基中能检测到硝酸还原酶活力,而仅含ＮＨ＋４的配方不能检测到该酶活力,表明硝酸还原酶是底物诱导酶。还探讨了培养基中不同的氮源组成对细胞生物量及ｐＨ的影响。     

S5 Lipase: an organic solvent tolerant enzyme

In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.     

Use of response surface methodology to optimize culture medium for production of lovastatin by Monascus ruber

Response surface methodology (RSM) was employed to study the effect of culture medium on the production of lovastatin in mixed solid-liquid state (or submerged) cultures by Monascus ruber. The maximal lovastatin yield (131 mg/L, average of three repeats) appeared at the region where the respective concentrations of rice powder, peptone, glycerin, and glucose were around 34.4 g/L, 10.8 g/L, 26.4 ml/L, and 129.2 g/L, respectively. The optimized medium resulted in a significant increase of lovastatin yield, as compared with that obtained by the fermentation of many other M. ruber species.     

Preparation and testing of an autolysate of fish viscera as growth substrate for bacteria.

The aqueous soluble phase of acidified and autolyzed fish viscera was used as the nitrogen source in a growth medium for bacteria. The bacteria tested grew faster and produced higher yields of cell mass on this growth medium than on corresponding media with standard tryptone preparations as the nitrogen source.     

Preparation and testing of an autolysate of fish viscera as growth substrate for bacteria

The aqueous soluble phase of acidified and autolyzed fish viscera was used as the nitrogen source in a growth medium for bacteria. The bacteria tested grew faster and produced higher yields of cell mass on this growth medium than on corresponding media with standard tryptone preparations as the nitrogen source.