Staurosporine,a Prolyl Endopeptidase Inhibitor

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.     

红曲霉不同转化方法比较

为了研究红曲霉聚酮体途径,考察和比较了4种不同的转化方法以建立有效的红曲霉遗传转化系统。以潮霉素作为抗性筛选标记,pBC-Hygro作为转化载体,用基于原生质体的传统转化和电击转化、基于萌发孢子的电击转化以及REMI技术转化红曲霉。发现基于萌发孢子的电击转化由于转化率极低而不适于红曲霉转化。基于原生质体的传统转化和电击转化尽管每微克DNA分别能获得135个转化子和125个转化子,但因转化子稳定性差也适合红曲霉转化的转化。应用REMI技术,转化率提高约20倍,每微克DNA 2500个转化子,70%～75%的转化子的稳定,非常适合于红曲霉的转化。      

Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.     

Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.     

Identification of a cerulenin resistance gene from Monascus pilosus.

Detoxification is essential for the fungal growth in the drug stress environments, and the multidrug transporters play an important role in this process. Here a cerulenin transporter gene (MpMdt, AB206476) was identified from Monascus pilosus. MpMdt mRNA contains 1951 bp and encodes a protein of 559 amino acid residues with 11 trans-membrane domains; and there is no difference in the sequence of MpMdt mRNA between the wild type M. pilosus IFO4520 and its cerulenin resistant mutant MK-1. Up-expression of MpMdt renders the cerulenin resistance of the mutant MK-1. Over-expression of MpMdt could also increase the cerulenin tolerance in the transgenic M. pilosus IFO4520. These results suggested that MpMdt is able to efflux-transport the anti-fungal antibiotic cerulenin and increase the cerulenin resistance of M. pilosus.     

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.     

Development of monacolin K-enriched ganghwayakssuk (Artemisia princeps Pamp.) by fermentation with Monascus pilosus

Monacolin K-enriched ganghwayakssuk (Artemisia princeps Pamp.) was developed by fermentation with Monascus sp. Among the 15 Monascus spp. isolated previously from Monascus fermentation products, Monascus pilosus KMU108 produced 2,219 mg/kg of monacolin K during ganghwayakssuk fermentation with no detectable citrinin. The optimum concentrations of ganghwayakssuk and glucose determined from the response surface methodology (RSM) design were 2.2% and 3.8%, respectively. By applying these conditions, the monacolin K productivity was increased to 3,007 mg/kg after 15 days of fermentation. On the other hand, other characteristics such as the total content of flavonoids and phenolic compounds, and the antioxidant activity were relatively unchanged. Therefore, Monascusfermented ganghwayakssuk is an excellent biomaterial for the development of functional foods because of its high level of monacolin K, known to lower cholesterol levels.     

Dimerumic acid protected oxidative stress-induced cytotoxicity in isolated rat hepatocytes

Dimerumic acid (DMA) is contained in Monascus anka and Monascus pilosus fermented products. The purpose of this study was to evaluate the effect of DMA against salicylic acid (SA)- and tert-butylhydroperoxide (t-BHP)-induced oxidative stress and cytotoxicity in the liver, using rat liver microsomes and isolated rat hepatocytes. DMA was extracted from monascus-garlic-fermented extract using M. pilosus. In rat liver microsomes, 1 microM DMA decreased SA-induced lipid peroxidation but did not affect the production of the oxidative metabolite of SA via CYP. In isolated rat hepatocytes, 1 microM DMA decreased SA-induced lipid peroxidation and chemiluminescence (CL) generation and the intracellular glutathione-reduced form/oxidized form (GSH/GSSG) ratio in the presence of 1 microM DMA was higher than that without DMA; however, 100 microM DMA suppressed the leakage of lactate dehydrogenase (LDH). On the other hand, t-BHP-induced lipid peroxidation, CL generation, and LDH leakage were prevented by 100 microM DMA. Thus, DMA showed an antioxidative effect in hepatocytes and protected against hepatotoxicity by suppressing oxidative stress without affecting CYP enzymes.     

Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI)

The REMI method was used to introduce the plasmid pV2 harboring the hygromycin B phosphotransferase (hph) gene controlled by the Aspergillus nidulans trpC promoter and the trpC terminator into a taxol-producing endophytic fungus BT2. REMI transformation yielded stable transformants capable of continuing to grow on PDA medium containing 125 mug mL(-1) hygromycin B. The transformation efficiency was about 5-6 transformants mug(-1) plasmid DNA. The presence of hph gene in transformants was confirmed by PCR and Southern blot analyses. To the authors' knowledge, this is the first report on the transformation of taxol-producing endophytic fungi by the REMI technique. This study provides an effective approach for improving taxol production of endophytic fungi by the genetic engineering of taxol biosynthetic pathway genes in the future.     

红曲米和红腐乳中红曲菌种的分类与鉴定

对我省红腐乳及红曲米中红曲菌的资源、多样性进行了调查,收集、分离和纯化得到20株红曲菌的纯培养。根据李钟庆等人的红曲菌属分类检索表,通过菌落形态和显微特征进行观察常规袁型鉴定,初步确定为以下七个种：红色红曲菌（FA01、FA11）;橙色红曲菌（FA02、FA05、FA06、FA15、FA16、FA17）、紫色红曲菌（FA03、FA09、FA13、FA19、FA20）、丛毛红曲菌（FA04）、发白红曲菌（FA07、FA18）、旱生红曲菌（R如8、FA10、FA12）和血红红曲菌（FA14）。运用FTIR分析图谱构建了20株红曲菌株的系统发育图,探讨耵IR分析运用于红曲菌资源或其它真菌的多样性、亲缘关系和分类鉴定中的可行性。研究结果显示FTIR分析在一定程度上可区分不同的红曲菌种,可以作为红曲菌菌种和疑难菌株鉴别的辅助手段。     

Simultaneous enrichment of deglycosylated ginsenosides and monacolin K in red ginseng by fermentation with Monascus pilosus

To improve its bioavailability and pharmacological effects in humans, red ginseng was fermented with a newly isolated fungus, Monascus pilosus KMU103. Most of the ginsenosides were converted to deglycosylated ginsenocides, such as Rh(1), Rh(2), and Rg(3). The total amount of ginsenosides Rh(1), Rh(2), and Rg(3) was 838.7 mg/kg in the red ginseng, and increased to 4,117 mg/kg after 50 L fermentation in 13% red ginseng and 2% glucose. In addition, the Monascus-fermented red ginseng contained 3,089 mg/kg of monacolin K, one of the metabolites produced by Monascus known to reduce cholesterol in the blood. This newly developed Monascus-fermented red ginseng should result in improved health effects, not only by biotransforming gisenosides to deglycosylated ones but also by creating additional bioactive compounds.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants. Received: 6 March 1998 / Accepted: 25 May 1998     

Determination and kinetics of producing glucosamine using fungi

This work used three fungi, Rhizopus oligosorus BCRC 31996, Monascus pilosus BCRC31527, and Aspergillus sp. BCRC31742, to produce glucosamine by using submerged fermentation and flask cultures. The reaction of glucosamine with 1-naphthyl isothiocyanate as derivatizing agent was carried out in pyridine at 50 degrees C for 1 h. The derivative was accurately analyzed and quantified by using high performance liquid chromatography. The relative standard deviation of glucosamine determined between experimental and real values were less than 2%. The kinetic and strategy of producing glucosamine in a flask culture was investigated to achieve an optimum yield of glucosamine under different conditions including three kinds of fungi, medium, and pH values. The descending ability of producing glucosamine for the three fungi was Aspergillus sp. BCRC31742 > Monascus pilosus BCRC31527 > Rhizopus oligosorus BCRC 31996 under the conditions studied. The experimental result shows that the glucosamine concentration had an optimum value and was 3430 mg/L by using Aspergillus sp. BCRC31742 culture in glucose and peptone (GP) medium, the yield of which was the best amount using wild-type microorganisms in the past. The generation culture of fungi and the pH control played important roles in enhancing the yield of glucosamine. The specific growth rate of the microorganism and the biomass, content, yield, and productivity of glucosamine were calculated as well.     

Antimicrobial activity of Monascus pilosus IFO 4520 against contaminant of Koji.

Antimicrobial activity of Monascus pilosus IFO 4520 was examined to prevent contamination during beni-koji making in the open air. The antibacterial effect of the beni-koji prepared with this strain occured with 30 mg/ml of beni-koji extract in combination with 0.5% lactic acid against two contaminants of koji, Micrococcus varians and Bacillus subtilis. There were two compounds, antibacterial and antiyeast substances, in the beni-koji extract. These results suggest a possibility of inhibiting the growths of contaminants during beni-koji making using beni-koji extract and lactic acid.     

Enhanced frequency of Beauveria bassiana blastospore transformation by restriction enzyme-mediated integration and electroporation

The techniques of restriction enzyme-mediated integration (REMI) and electroporation (EP) were applied for the first time to improving the blastospore transformation of fungal biocontrol agent Beauveria bassiana for higher frequency. The blastospores from < or =24 h incubation in glucose-mineral medium after shaking conidia for 48 h in Subouraud dextrose broth were found most competent for integrating 1 microg plasmid DNA vectoring the phosphinothricin (PPT) resistance gene bar in 360 microL reaction system containing 100 U HindIII or XbaI. Such blastospores were also most suitable for EP transformation at the optimized field strength of 10 kV cm(-1). The optimized REMI and EP generated averagely 39 and 53 transformants microg(-1) plasmid DNA whereas polyethylene glycol (PEG) integration yielded only 22. All transformants grew well on Czapek's agar containing 400 microg PPT mL(-1) after three rounds of cultivation on the same agar excluding PPT but their parental strain showed no resistance. The target gene inserted into the genomes of 10 transformants randomly taken from REMI or EP transformation was consistently detected by both PCR and Southern blotting. Compared to the PEG integration, REMI and EP enhanced the frequency of the blastospore transformation by 73 and 137%, respectively.     

Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus, Coniothyrium minitans

Restriction enzyme mediated integration (REMI) and Agrobacterium-mediated transformation (ATMT) were used to transform protoplasts or germinated conidia of the mycoparasite Coniothyrium minitans to hygromycin resistance. Using REMI, up to 32 transformants mug DNA(-1) were obtained, while 37.8 transformants 5 x 10(5) germlings(-1) were obtained using ATMT. Single-copy integrations occurred in 8% and 40% of REMI and ATMT transformants, respectively. A novel microtitre plate-based test was developed to expedite screening of 4000 REMI and ATMT C. minitans transformants. Nine pathogenicity mutants that displayed reduced or no pathogenicity on sclerotia of Sclerotinia sclerotiorum were identified.     

Monascuspiloin enhances the radiation sensitivity of human prostate cancer cells by stimulating endoplasmic reticulum stress and inducing autophagy

Prostate cancer is a very common cancer among males. Traditional treatments for prostate cancer have limited efficacy; therefore, new therapeutic strategies and/or new adjuvant drugs must be explored. Red yeast rice (RYR) is a traditional food spice made in Asia by fermenting white rice with Monascus purpureus Went yeast. Accumulating evidence indicates that RYR has antitumor activity. In this study, PC-3 cells (human prostate cancer cells) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with monascuspiloin (MP, a yellow pigment isolated from Monascus pilosus M93-fermented rice) and to determine the underlying mechanisms of these effects in vitro and in vivo. We found that IR combined with MP showed increased therapeutic efficacy when compared with either treatment alone in PC-3 cells. In addition, the combined treatment enhanced DNA damage and endoplasmic reticulum (ER) stress. The combined treatment induced primarily autophagy in PC-3 cells, and the cell death that was induced by the combined treatment was chiefly the result of inhibition of the Akt/mTOR signaling pathways. In an in vivo study, the combination treatment showed greater anti-tumor growth effects. These novel findings suggest that the combined treatment could be a potential therapeutic strategy for prostate cancer.     

Deletions in the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi by Restriction Enzyme-Mediated Integration and Conventional Transformation-Mediated Mutagenesis

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA₁₂-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.