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Effects of the principal nutrients on lovastatin production by monascus pilosus

Biosci. Biotechnol. Biochem., 70 (5), 1154–1159, 2006 Effects of the Principal Nutrients on Lovastatin Productionby Monascus pilosus Tsuyoshi MIYAKE,1;y Kumiko UCHITOMI,2 Ming-Yong ZHANG,1;3 Isato KONO,1Nobuyuki N OZAKI,1 Hiroyuki SAMMOTO,1 and Kenji INAGAKI 1Industrial Technology Center of Okayama Prefecture, Haga 5301, Okayama 701-1296, Japan2Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Tsushima 1-1-1, Okayama 700-8530, Japan3Japan Society for the Promotion of Science, Ichibancho 6, Chiyoda-ku, Tokyo 102-8471, Japan Received October 17, 2005; Accepted December 27, 2005 Lovastatin production is dependent on the substrates conditions are sufficient to allow high lovastatin pro- provided. We investigated how several carbon and duction: growth resulting in carbon limitation, and non- nitrogen sources in the medium affect lovastatin pro- glucose repression under nitrogen limitation. Informa- duction by Monascus pilosus. M. pilosus required a tion about the regulation of lovastatin biosynthesis suitable concentration of organic nitrogen peptone for should be useful for improving bioprocesses for high high lovastatin production. As sole carbon source with lovastatin production by A. terreus in the laboratory or peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high produc- It is known that lovastatin production by Monascus tion. Interestingly, glycerol combined with maltose species is also dependent on the culture conditions, enhanced lovastatin production, up to 444 mg/l in the because various bioprocess improvements have been most effective case. Moreover, an isolated mutant, in attempted to achieve higher production, including which glucose repression might be relieved, easily incubation processes, solid-state fermentation, and se- produced the highest level of lovastatin, 725 mg/l on lections of optimized strains and complex substrates glucose–glycerol–peptone medium. These observations provided.10–13) Little has been indicated about the indicate that lovastatin production by M. pilosus is regulation of lovastatin biosynthesis in this species, regulated by strict glucose repression and that an however. In order to improve lovastatin production, we appropriate release from this repression by optimizing focused on M. pilosus, since this species has long been medium composition and/or by a mutation(s) is re- popular for use in brewing foodstuffs in Japan. Although quired for high lovastatin production.
the lovastatin productivity of M. pilosus is thought stillto be relatively low, this species has the advantage of Monascus pilosus; lovastatin; production; lower risk of citrinin (a nephrotoxic agent) contamina- tion, unlike M. ruber and M. purpureus.14) But there hasbeen no study on the influence of principal nutrients Statins such as lovastatin are inhibitors of the enzyme such as carbon and nitrogen on lovastatin production by hydroxymethyglutaryl coenzyme A reductase, which catalyzes the rate-limiting step in cholesterol biosyn- In this study, we examined the effects of several thesis, resulting in lowered blood cholesterol.1,2) Lova- carbon and nitrogen sources on lovastatin production to statin can be produced by Penicillium species,3) Asper- develop a defined medium for high production, and isolated a regulatory mutant which would serve to polyketide pathway, which is responsible for synthesiz- develop such a level of production. We found that ing many secondary metabolites with complex chemical the type and dose of carbon and nitrogen sources had structures. The genes and the enzymes involved in a strong influence on lovastatin production by M. pilosus lovastatin biosynthesis have been identified and charac- with a variety of biomasses, indicating high potential terized in A. terreus,5,6) and it is becoming apparent that for such production. Based on these results, we also both carbon and nitrogen sources regulate lovastatin discuss the regulation of lovastatin biosynthesis in biosynthesis at the level of glucose repression and signaling of growth or substrate limitations.7,8) Thesestudies showed that two overlapping carbon source y To whom correspondence should be addressed. Tel: +81-86-286-9600; Fax: +81-86-286-9632; E-mail: tsuyoshi Lovastatin Production by Monascus pilosus after 14 d of growth at 25 C in PD medium. Onemutant, designated MK-1, which showed maximum Strains and growth conditions. Cultures of M. pilosus lovastatin productivity, 5-fold higher than that of the IFO4520 (wild type) and mutant strains were maintained wild type, was selected for further experiments. The on PDA (potato dextrose agar; BD, Franklin Lakes, NJ).
M. pilosus mutant MK-1 was isolated from subcultures After growth on PDA for 10 d at 30 C, spores were containing cerulenin at 50 mg/l, and showed tolerance to harvested with a sterile solution (0.9% NaCl, 0.2% Tween 80), and approximately 107 spores were inocu-lated into 25 ml PD medium (potato dextrose broth; BD) and GGP medium (3% glucose, 7% glycerol, 3.8%peptone, 0.1% MgSO .
Effects of nitrogen sources on lovastatin production flasks. Liquid cultures were incubated at 25 C at In general, M. pilosus can produce moderate amounts 120 rpm for the indicated times. Carbon and nitrogen of lovastatin (approximately 130 mg/l) in glucose– sources in GGP medium were modified as indicated.
glycerol–peptone (GGP) medium after biomass forma- There was no large variation in initial pH of the media tion, though it requires a longer growth period than other used (approximately pH 6.5), nor in the final pH of lovastatin producing species such as A. terreus. First, peptone in GGP medium was replaced with severalnitrogen sources: nitrate is one of the standard inorganic Analytical methods. After growth in liquid culture, nitrogen sources for filamentous fungi, glutamate is one fermentation broth and cells were separated by filtration of simple organic nitrogen sources generally selected for (No. 5C; Advantec, Tokyo, Japan). Biomass was deter- lovastatin production of A. terreus and red pigment mined based on the dry-weight (dw) of the filtrated cells production of Monascus species,7,15) and tryptone is from cultures. Fermentation broth was assayed for another major complex organic nitrogen source for lovastatin production after filtration using membrane filamentous fungi. But growth of M. pilosus on nitrate filters (pore size, 0.45 mM). Lovastatin content in the and glutamate was very poor and no lovastatin was fermentation broth was determined by chromatograms detected, and tryptone did not contribute to lovastatin detected at 237 nm under established conditions, as production despite a well-formed biomass (data not described previously,14) and the peak of the statin was shown). Hence we redefined peptone as an organic identified by mass spectra. All data presented are the nitrogen source for lovastatin production in this study.
averages of results obtained from three independent We also examined the effect of peptone concentration in GGP medium on lovastatin production. As shown inTable 1, 3.8% peptone, the original concentration in Observation of morphological development. To ob- GGP medium, was re-established for high lovastatin serve the morphological development of mutant strains, production with suitable biomass. Lower concentrations plate cultures were grown for 7 d or more at 30 C. After of peptone led to production, but with unsuitable culturing, colonies, mycelia, and spores present on biomass levels. A higher concentration of peptone plates were observed directly under a light microscope.
strongly repressed production. We concluded thatM. pilosus requires a suitable concentration of organic Mutant isolation. Monascus species in general have a nitrogen peptone for high lovastatin production and teleomorphic life cycle, in which recessive phenotypes tend to be complemented and dominant phenotypes tendto be condensed through generations. We adopted a Effects of carbon sources on lovastatin production scheme using liquid culture combined with drug The effects of sole carbon sources combined with tolerance for positive selection of dominant mutations.
3.8% peptone on lovastatin production were determined.
In order to select spontaneous regulatory mutants in Because the secondary metabolism of filamentous fungi lovastatin production, cultivation and screening werecarried out in PD medium causing low or repressive Effect of Peptone Concentration on Lovastatin Production production. After treatment with N-methyl-N0-nitro-N- nitrosoguanidine (the survival rate was several percent),spores of M. pilosus IFO4520 were inoculated and cultivated at 30 C in PD medium containing cerulenin, an inhibitor of general polyketide synthases at 20– 100 mg/l, because cerulenin tolerance might mean an increase in polyketide synthases or the development of resistance to inhibition. After repeated subculturing at intervals of several days, we finally isolated several After cultures of M. pilosus IFO4520 were grown at 25 C for 21 d in a medium containing the indicated peptone concentrations with 3% glucose– cerulenin tolerant mutants. In the first screening, the 7% glycerol, the lovastatin contents in fermentation broth and biomass were mutants obtained were assayed for lovastatin production Effect of Sole Carbon Source on Lovastatin Production by Effect of Carbon Source Combinations on Lovastatin a After cultures of M. pilosus IFO4520 were grown at 25 C for 21 d in a medium containing the indicated combinations of carbon sources with 3.8% peptone, the lovastatin contents in fermentation broth and biomass were a After cultures of M. pilosus IFO4520 were grown at 25 C for 21 d in amedium containing the indicated carbon source concentrations with 3.8%peptone, the lovastatin contents in fermentation broth and biomass weredetermined.
7 to 9 (Fig. 1). Even in the presence of glucose, which strongly represses production, the most suitable C/Nratio was close to from 7 to 9.
in general is commonly regulated by glucose repression, A mutant with increased lovastatin production three types of carbon sources of such repression were Lovastatin production by M. pilosus was shown to be tested for lovastatin production by M. pilosus: glucose strongly repressed in the presence of glucose. In order to as a repressive carbon source, maltose and fructose as relieve this repression spontaneously, we isolated a moderately repressive carbon sources, and glycerol and mutant called MK-1 that had 5-fold higher lovastatin lactose as non-repressive carbon sources. The effects of production in PD medium than the wild type (see all carbon sources tested on lovastatin production were ‘‘Materials and Methods’’). With respect to the morpho- dose-dependent up to 7%, positively or negatively.
logical characteristics of M. pilosus mutant MK-1, the Repressing carbon source glucose was shown to repress strain formed small red-appearing colonies and pro- lovastatin production strongly (Table 2). Moderately duced higher red pigments and more spores from more repressing carbon sources maltose and fructose led to branching mycelia on PDA in the short term than the high and intermediate production at a concentration of wild type (data not shown). Interestingly, there were 7% (Table 2). Non-repressing carbon source glycerol apparently higher differences in lovastatin production led to a slight but noticeable increase in lovastatin ability by the M. pilosus mutant MK-1 in the presence of production as compared to glucose, although lactose was glucose than in the parent, although lovastatin levels by not utilized for growth. Moreover, it is interesting that M. pilosus mutant MK-1 grown in the medium without combinations of carbon sources (maltose and glucose glucose tested in this study were also a little higher than combined with glycerol) resulted in enhanced lovastatin those of the wild type (data not shown). Especially, in production (Table 3). In this series of experiments, the GGP medium, M. pilosus mutant MK-1 produced the highest production, 444 mg/l, was achieved in the highest amount of lovastatin: up to 725 mg/l (Fig. 2).
presence of 1% maltose–7% glycerol. These results Additionally, biomass formation by M. pilosus mutant show that an appropriate selection of type and dose of MK-1 in tested cultures was relatively low as compared carbon sources, including combinations, to avoid strong with the wild type (data not shown). Based on these repression by glucose is required for higher lovastatin results, we propose that glucose repression and feedback inhibition of lovastatin production was substantiallyrelieved in M. pilosus mutant MK-1. Moreover, dif- Relationship of C/N ratio and lovastatin production ferences in the cell development of this mutant might The above results, shown in Tables 1 and 3, suggest hasten lovastatin production based on signals of growth.
that lovastatin production by M. pilosus requires amedium with a suitable C/N ratio. Hence, the effect of the peptone concentration (0.1–3.8%) in 1% maltose–7% glycerol medium was also determined, and the C/N In this study, the type and dose of carbon and nitrogen ratios for all conditions used in this study were sources strongly influenced lovastatin production by calculated and plotted against relative lovastatin pro- M. pilosus. M. pilosus produced very little lovastatin duction. The highest value under any one condition with grown on glucose, which commonly represses secondary one variable was set at 100%. It was discovered that a metabolite production. We believe that lovastatin bio- suitable C/N ratio for lovastatin production was from synthesis in M. pilosus is regulated by stricter glucose Lovastatin Production by Monascus pilosus Relationship between C/N Ratio and Lovastatin Production by M. pilosus IFO4520.
C/N ratios of the conditions used in this study were calculated and plotted against relative activities of lovastatin production, and the highest value at one variable was set at 100%.
indicates various conditions except in the presence of glucose; glucose–glycerol–peptone under which the peptone and glycerol concentrations were set and glucose was variable, and the glucose and glycerolconcentrations were set and the peptone concentration varied, respectively. The C-contents (% by dry weight) of glucose, maltose, fructose,glycerol, and lactose were 39.96, 39.96, 39.96, 39.08, and 39.96 respectively. Peptone contained 15.5% nitrogen and 45.95% carbon by dryweight. The N-content (% by dry weight) of NaNO3 is 16.47.
on earlier glucose consumption and a relatively large amount of glycerol, a non-repressing carbon, can remain in the limited condition of carbon assimilation after growth. Glycerol in combination with maltose enhanced further induction of lovastatin production. In this case, the growth must depend mainly on rapidly metabolized glycerol, and thus lower levels of maltose can more closely mimic limitation of carbon assimilation duringgrowth, which explains how 1% maltose–7% glycerol Lovastatin Productivity of M. pilosus Mutant MK-1 with was most effective condition. A similar benefit of high lovastatin production by a combination of a rapidly and After culturing M. pilosus IFO4520 ( ) and M. pilosus mutant a slowly metabolized carbon was also found in Asper- MK-1 ( ) at 25 C in GGP medium (3% glucose, 7% glycerol, 3.8%peptone), the lovastatin levels in the fermentation broth were gillus.7) The nitrogen source is another significant limiting factor influencing the regulation of lovastatinbiosynthesis via growth. Among several nitrogen sour-ces tested, peptone was better for lovastatin production repression than in A. terreus and other Monascus species by M. pilosus; this is one reason that the growth of because they can relatively easily produce more lova- M. pilosus was largely dependent on the type of nitrogen statin grown even on glucose.7) Glucose repression in source. Moreover, a suitable concentration of peptone Aspergillus is mediated mainly by CreA as a repressor to was important for high production. Low biomass by expression of genes involved in the utilization of lower peptone concentration resulted in the reduction of alternative carbons, and CreA might also be involved total production. In the case of higher peptone concen- in lovastatin biosynthesis.7) Maltose and fructose do not tration, non-limitation of nitrogen and consumption of activate CreA strongly, and glycerol does not at all. The carbon sources might repress lovastatin biosynthesis and onset of lovastatin production by M. pilosus in these reduce the statin’s production, respectively. The stricter carbon sources can be explained by the mediation of regulation by carbon and nitrogen of lovastatin biosyn- CreA, which M. pilosus might also have. Maltose led to thesis by M. pilosus well reflected the suitable C/N ratio higher induction of lovastatin production than did calculated in this study, from 7 to 9, very narrow, and fructose or glycerol. Limitation of carbon assimilation lower than other lovastatin producing species.
for growth is required for initiation of secondary From the above, M. pilosus has mechanisms for metabolite production,7) which explains this phenom- regulating lovastatin biosynthesis similar to those of enon. Maltose is slowly metabolized, and this condition A. terreus, such as those at the level of glucose can mimic limitation of carbon assimilation during repression and via signals of growth or substrate growth, while fructose and glycerol are rapidly metab- limitations.7,8) The details, however, are a little different olized and a limited condition of carbon assimilation and control is tighter. In GGP medium, lovastatin must arise after growth. Interestingly, glycerol in production by M. pilosus appears to be initiated after combination with glucose appears to relax strict glucose consumption of glucose and nitrogen for growth, and is repression by glucose only, because growth must depend almost saturated at approximately 130 mg/l after 21 d. A similar effect of feedback inhibition has been reported in A. terreus.16) This productivity was elevated 3-fold, upto 444 mg/l, the highest level reported in liquid culture This study was supported in part by grants from the of wild strains,9) by appropriate selection of carbon and Ministry of Education, Culture, Sports, Science and nitrogen sources with a suitably designed C/N ratio.
Technology of Japan and the Japan Society for the This highest achieved productivity also indicates the high potential for lovastatin production by M. pilosus,thought so far to have a relatively low ability. Moreover, it should be emphasized that this productivity understrict glucose repression can be greatly elevated, to Albert, A. W., Chen, J., Kuron, G., Hunt, V., Huff, J., 725 mg/l, by a mutation(s) in M. pilosus mutant MK-1.
Hoffman, C., Rothrock, J., Lopez, M., Josua, H., Harris, The lovastatin levels of this mutant grown in a medium E., Patchett, A., Monaghan, R., Currie, S., Stapley, E., containing glucose were especially higher from those of Albers-Schonberg, G., Hensens, O., Hirshfield, J., the wild type, strongly implying that glucose repression Hoogsteen, K., Liesch, J., and Springer, J., Mevinolin: can be relieved in this mutant. Taken together, we a highly potent competitive inhibitor of hydroxymethyl- propose that an appropriate release from strict glucose glutaryl-coenzyme A reductase and a cholesterol-low- repression by optimizing medium compositions and/or ering agent. Proc. Natl. Acad. Sci. USA, 77, 3957–3961 by a mutation(s) is required for high lovastatin produc- Albert, A. W., Lovastatin and simvastatin-inhibitors of HMG CoA reductase and cholesterol-biosynthesis. Car- M. pilosus mutant MK-1 might also have a muta- tion(s) in cell development with respect to morpholog- Endo, A., Kuroda, M., and Tsujita, Y., ML-236A, ML- ical characteristics, and have the ability to produce 236B, and ML-236C, new inhibitors of cholesterogen- higher levels of red pigments, which are also polyke- esis produced by Penicillium citrinium. J. Antibiotics tides. These morphological and metabolic characteristics of MK-1 suggest that cell development has a significant Endo, A., Monacolin K, a new hypocholesterolemic relationship with secondary metabolism (including agent produced by a Monascus species. J. Antibiotics polyketide synthesis) in M. pilosus, as in the well- described fungus A. nidulans.17) We expect that dif- Hendrickson, L., Davis, C. R., Roach, C., Nguyen, D. K., ferences in signal transduction during cell development Aldrich, T., McAda, P. C., and Reeves, C. D., Lovastatinbiosynthesis in Aspergillus terreus: characterization of in M. pilosus mutant MK-1 can result in derepression or blocked mutants, enzyme activities and a multifunctional activation of polyketide synthesis under glucose repres- polyketide synthase gene. Chem. Biol., 6, 429–439 sion via some factor operating upstream of this syn- thesis. Recently, LaeA, a global transcriptional regulator Kennedy, J., Auclair, K., Kendrew, S. G., Park, C., of secondary metabolism in Aspergillus species, was Vederas, J. C., and Hutchinson, C. R., Modulation of identified and characterized.18) LaeA has features of a polyketide synthase activity by accessory proteins during nuclear protein methyltransferase, is localized in the lovastatin biosynthesis. Science, 284, 1368–1372 (1999).
nucleus, and has been shown to up-regulate the Hajjaj, H., Niederberger, P., and Duboc, P., Lovastatin expression of gene clusters for various secondary biosynthesis by Aspergillus terreus in a chemically metabolites, including sterigmatocystin synthesis, pen- defined medium. Appl. Environ. Microbiol., 67, 2596– icillin synthesis, hyphal pigment production, and lova- Casas-Lopez, J. L., Sanchez-Perez, J. A., Fernandez- statin synthesis (heterologous expression). LaeA expres- Sevilla, J. M., Acien-Fernandez, F. G., Molina-Grima, sion is negatively regulated by two signal transduction E., and Chisti, Y., Production of lavastatin by Aspergil- elements, protein kinase A and Ras A, these are lus terreus: effects of C:N ratio and the principal involved in cell development and also negatively nutrients on growth and metabolite production. Enzyme regulate sporulation. Interestingly, delta-laeA mutants Microb. Technol., 33, 270–277 (2003).
showed little difference in spore production as compared Casas-Lopez, J. L., Sanchez-Perez, J. A., Fernandez- with wild type controls, suggesting a role for LaeA in Sevilla, J. M., Acien-Fernandez, F. G., Molina-Grima, the regulation of metabolic gene clusters. It should also E., and Chisti, Y., Fermentation optimization for the be mentioned that LaeA homologs appear to be present production of lovastatin by Aspergillus terreus: use of only in the genomes of filamentous fungi. Hence, it will response surface methodology. J. Chem. Technol. Bio- be of interest to identify the LaeA homolog in Wang, U. L., Houng, J. Y., Chang, H. S., Roger-Chien, M. pilosus. Additionally, in M. pilosus, the role of a H., and Hsu, W. H., Selection of drug-resistant mutant of protein kinase(s) in cell development must be elucidated Monascus pilosus for enhanced monacolin K production.
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Chang, Y. N., Lin, Y. C., Lee, C. C., Liu, B. L., and Experiments to investigate these issues have been Tzeng, Y. M., Effect of rice-glycerol complex medium described elsewhere (Zhang and Miyake, submitted; on the production of lovastatin by Monascus ruber. Folia Lovastatin Production by Monascus pilosus Su, Y. C., Wang, J. J., Lin, T. T., and Pan, T. M., R., Production of Monascus biopigments: an overview.
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