Proteomic response to intracellular proteins of Monascus pilosus grown under phosphate-limited complex medium with different growth rates and pigment production

Monascus pigments are important colorings in food applications. Rice containing potassium phosphate and sodium nitrate was reported as a good pigment-producing medium for Monascus in previous studies. We found that the lack of potassium phosphate in this medium depressed red pigment production in cultivated Monascus pilosus. However, the influence of phosphate limitation on the biochemical metabolisms concerning culture growth and pigment production in Monascus remains unknown. Here, we used proteomic analysis by two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), tandem mass spectrometry (MS/MS), and database interrogation to separate and identify the proteins of M. pilosus grown between the lack of potassium phosphate and the control media. Phosphate limitation to this complex medium induced an up-regulation of aldehyde dehydrogenase and several glycolytic enzymes in Monascus relative to the control. In contrast, the metabolic enzymes such as glucosamine:fructose-6-phosphate aminotransferase and ADP-ribosylation factor 1 were up-regulated in the control.     

Proteome response of Monascus pilosus during rice starch limitation with suppression of monascorubramine production

For centuries, red mold rice has been made by fermentation of cooked rice with Monascus species. However, the influence of different carbon sources on the metabolism of Monascus cells remains unclear. We compared the proteome response of Monascus pilosus to replacement of the rice starch fraction with lactose during cultivation, using two-dimensional gel electrophoresis, matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry, and tandem mass spectrometry to identify the proteins expressed. The results showed that cell growth and monascorubramine pigment formation of M. pilosus were sensitive to rice starch limitation during cultivation. A total of 12 proteins were identified with statistically altered expression in the cells cultivated with lactose. These deregulated proteins were involved in glycolysis, TCA cycle, energy generation, protein folding, and peptide biosynthesis. The possible metabolic flux shifts induced by rice starch limitation were discussed. The results suggested that the suppression of monascorubramine formation could be related to the necessary energy-requiring adaptations executed in response to carbon depletion during rice starch limitation.     

Proteomic analysis of Caco-2 cells treated with monacolin K

Monascus species is an important traditional fermentation fungus used on food. Monacolin K (a secondary metabolite of Monascus, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase) in inhibition of mevalonate synthesis may result in reductions of isoprenoid prenylation in cells. Impairment of protein isoprenoid prenylation has been related to anticancer effect in cancer cells. As a functional food for Monascus, however, the molecular mechanisms responsible for the anti-proliferate effect of monacolin K in cancer cells are not clear. We used proteomic analysis by two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), tandem mass spectrometry (MS/MS), and database interrogation to separate and identify the proteins of Caco-2 cells treated with monacolin K. The results showed that monacolin K inhibited the proliferation of Caco-2 cells in a dose-dependent manner. The identified proteins in proteomic analysis included anti-oxidation enzymes related to reactive oxygen species stress, cytoskeleton proteins, glycolytic enzymes, and enzymes involved in mediating protein interactions. Furthermore, glutathione S-transferase P 1 and cytoskeleton-8, -18, and -19 revealed a down-regulation in a dose-dependent manner in exposure of Caco-2 cells to monacolin K.     

Cloning and characterization of monacolin K biosynthetic gene cluster from Monascus pilosus

Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus, and it has the same structure as lovastatin, which is mainly produced by Aspergillus terreus. In the present study, a bacterial artificial chromosome (BAC) clone, mps01, was screened from the BAC library constructed from Monascus pilosus BCRC38072 genomic DNA. The putative monacolin K biosynthetic gene cluster was found within a 42 kb region in the mps01 clone. The deduced amino acid sequences encoded by the nine genes designated as mokA- mokI, which share over 54% similarity with the lovastatin biosynthetic gene cluster in A. terreus, were assumed to be involved in monacolin K biosynthesis. A gene disruption construct designed to replace the central part of mokA, a polyketide synthase gene, in wild-type M. pilosus BCRC38072 with a hygromycin B resistance gene through homologous recombination, resulted in a mokA-disrupted strain. The disruptant did not produce monacolin K, indicating that mokA encoded the PKS responsible for monacolin K biosynthesis in M. pilosus BCRC38072.     

Proteome changes in Caco-2 cells treated with Monascus-fermented red mold rice extract

Monascus-fermented red mold rice has been extensively used as a folk medicine for thousands of years. Monascus secondary metabolites, including monacolin K, monascorubrin, and ankaflavin, have been reported to have an antiproliferative effect on cancer cells. However, the cell machinery responsible for the antiproliferation of Monascus-fermented red mold rice treatment in cancer cells remains unclear. A proteomic approach using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry, and tandem mass spectrometry was used to identify proteins with modified expression in Caco-2 cells treated with Monascus-fermented red mold rice extract. A total of 20 proteins were identified with significantly altered expression (P < 0.05) in response to Monascus-fermented red mold rice extract treatment. The deregulated proteins that were identified included heat shock protein 70, protein kinase C epsilon type, clusterin-associated protein 1, and two tumor suppressors (N-chimaerin and calponin-2). Our results suggested the involvement of heat shock protein 70-mediated cytotoxicity in the Caco-2 cells treated with Monascus-fermented red mold rice extract.     

Enhanced anti-inflammatory activities of Monascus pilosus fermented products by addition of ginger to the medium

Hypercholesterolemia initiates the atherogenic process; however, chronic inflammation promotes atherogenesis. Monascus spp. fermented products are recognized for their anti-hypercholesterolemic effect, but their anti-inflammatory activity is not as significant as that of many plant-derived foods. To enhance the anti-inflammatory function of Monascus pilosus fermented products, ginger was added to the PDB medium at a ratio of 20% (v/v). The mycelia and broth were collected, freeze-dried, and extracted by ethanol for assays. Macrophage RAW264.7 was challenged with lipopolysaccharide (LPS) and coincubated with the extracts of fermented product cultured in ginger-supplemented medium (MPG) or extracts of fermented product cultured in regular PDB medium (MP) for 18 h. Human umbilical vein endothelial cell HUVEC was challenged with tumor necrosis factor (TNF)-α and coincubated with the extracts of either MPG or MP for 6 h. The results showed that MPG significantly (p<0.05) lowered the production of macrophage pro-inflammatory cytokines TNF-α, nitric oxide (NO), interleukin (IL)-1, IL-6, and prostaglandin E2 (PGE2) by 68.53%, 84.29%, 32.55%, 84.49%, and 69.49%, respectively; however, MP had no inhibitory effect. MPG significantly downregulated the expression of p-IκB, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) in macrophage by 42.16%, 50.87%, and 51.35%, respectively, while MP had no inhibition on COX-2 expression and only 16.64% and 19.22% downregulatory effect on iNOS and phosphorylated-IκB (p-IκB), respectively. Moreover, MPG significantly suppressed the expression of vessel cell adhesion molecule-1 (VCAM-1) and p-IκB in endothelial cell by 63.48% and 63.41%, respectively. LC/MS/MS analysis indicated that 6-gingerdiol was formed in the ginger-modified medium during fermentation. The results of this study will facilitate the development of Monascus spp. fermented products as antiatherosclerotic nutraceuticals.     

Two new Monascus metabolites with strong blue fluorescence isolated from red yeast rice

Red yeast rice obtained as cultures of Monascus AS3.4444 on rice was extracted and analyzed by high-performance liquid chromatography (HPLC). Two new Monascus metabolites with similar fluorescence spectra (lambda ex = 396 nm, lambda em = 460 nm) and UV absorption spectra (lambda max = 386 nm) were detected. They were isolated by rechromatography on a silica gel column and semipreparative HPLC, and two strong blue fluorescent compounds were obtained. Their structures were elucidated by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization tandem mass spectrometry (ESI-MS/MS), intensive ESI-MS, and nuclear magnetic resonance spectroscopy ( (1)H NMR, (13)C NMR, COSY, and HMBC) studies. High-resolution mass spectrometry indicated the molecular formulas C 21H 24O 5 and C 23H 28O 5. The two new compounds, named monasfluore A and monasfluore B, respectively, contain a alkyl side chain, gamma-lactone, and propenyl group, whereas the more lipophilic compound, monasfluore B, is a higher homologue of monasfluore A, with the more lipophilic octanoyl instead of the hexanoyl side chain.     

Phenolic acids in foods: an overview of analytical methodology

Phenolic acids are aromatic secondary plant metabolites, widely spread throughout the plant kingdom. Existing analytical methods for phenolic acids originated from interest in their biological roles as secondary metabolites and from their roles in food quality and their organoleptic properties. Recent interest in phenolic acids stems from their potential protective role, through ingestion of fruits and vegetables, against oxidative damage diseases (coronary heart disease, stroke, and cancers). High performance liquid chromatography (HPLC) as well as gas chromatography (GC) are the two separation techniques reviewed. Extraction from plant matrixes and cleavage reactions through hydrolysis (acidic, basic, and enzymatic) are discussed as are the derivatization reagents used in sample preparation for GC. Detection systems discussed include UV-Vis spectroscopy, mass spectrometry, electrochemical, and fluorometric detection. The most common tandem techniques are HPLC/UV and GC/MS, yet LC/MS is becoming more common. The masses and MS fragmentation patterns of phenolic acids are discussed and tabulated as are the UV absorption maxima.     

蛋白质定向进化及其在微生物代谢调控中的应用

蛋白质定向进化是非理性改造蛋白质的一种有效方法。利用蛋白质定向进化技术可以改变代谢流,扩展或构建新的代谢途径,弱化或消除不必要或有害的代谢途径,从而达到提高某种代谢产物产率或降解有害物质的目的。蛋白质定向进化技术在代谢调控中的应用有效拓宽了代谢工程的应用范围。本文介绍了主要的蛋白质定向进化技术如易错PCR技术、DNA改组技术、交错延伸技术和临时模板随机嵌合技术等,评述了蛋白质定向进化技术对微生物细胞代谢中的关键蛋白进行定向改造,从而改善其代谢能力,调控微生物代谢等的应用。     

Chromatographic methods for metabolite profiling of virus- and phytoplasma-infected plants of Echinacea purpurea

This study was focused on the effects of virus and phytoplasma infections on the production of Echinacea purpurea (L.) Moench secondary metabolites, such as caffeic acid derivatives, alkamides, and essential oil. The identification of caffeic acid derivatives and alkamides was carried out by means of high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-electrospray ionization-mass spectrometry (ESI-MS), and MS(2). Quantitative analysis of these compounds was carried out using HPLC-DAD. The results indicated that the presence of the two pathogens significantly decreases (P < 0.05) the content of cichoric acid, the main caffeic acid derivative. Regarding the main alkamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, a significant decrease (P < 0.05) in the content of this secondary metabolite was observed in virus-infected plants in comparison with healthy plants, while in the phytoplasma-infected sample the variation of this secondary metabolite was not appreciable. The % relative area of the E/Z isomers of this alkamide was also found to change in infected samples. The gas chromatography (GC) and GC-MS analysis of E. purpurea essential oil enabled the identification of 30 compounds. The main significant differences (P < 0.05) in the semiquantitative composition were observed for three components: limonene, cis-verbenol, and verbenone. The results indicate that the presence of virus and phytoplasma has an appreciable influence on the content of E. purpurea secondary metabolites, which is an important issue in defining the commercial quality, market value, and therapeutic efficacy of this herbal drug.     

New monascus metabolites with a pyridine structure in red fermented rice

Monascus purpureus produces several hitherto unknown compounds in addition to pigments and the mycotoxin citrinin. In red fermented rice (angkak, red koji) obtained as cultures of M. purpureus DSM1379 and DSM1603, we detected two compounds with identical UV absorption spectra and maxima at 306-307 nm. They were isolated by HPLC, and their structures were elucidated by intensive MS and NMR studies. Monascopyridine A (3) contains a gamma-lactone, propenyl group, hexanoyl side chain, and a pyridine ring, whereas the more lipophilic compound, monascopyridine B, is a higher homologue of monascopyridine A with the more lipophilic octanoyl instead of the hexanoyl side chain. This is the first report of Monascus metabolites with a pyridine ring.     

Oxidative in vitro metabolism of the soy phytoestrogens daidzein and genistein

The oxidative metabolism of the major soy isoflavones daidzein and genistein was investigated using liver microsomes from Aroclor-treated male Wistar rats. Both daidzein and genistein were extensively metabolized and are therefore excellent substrates for cytochrome P450 enzymes. The identity of the metabolites was elucidated using high-performance liquid chromatography (HPLC) with diode array detection, gas chromatography-mass spectrometry (GC/MS), and HPLC/atmospheric pressure ionization electrospray mass spectrometry (API-ES MS) as well as reference substances. Daidzein was converted to nine metabolites, comprising four monohydroxylated, four dihydroxylated, and one trihydroxylated metabolite. Genistein was metabolized to four monohydroxylated and two dihydroxylated products. With both isoflavones the additional hydroxy groups are exclusively introduced into the ortho positions of existing phenolic hydroxy groups. The major metabolites of daidzein were identified as 6,7,4'-trihydroxyisoflavone, 6,7,3',4'-tetrahydroxyisoflavone, 7,8, 4'-trihydroxyisoflavone, and 5,6,7,4'-tetrahydroxyisoflavone. The main microsomal metabolites of genistein were 5,6,7, 4'-tetrahydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone. Furthermore, the GC/MS and HPLC/API-ES MS analysis support the conclusion that one monohydroxylated metabolite of daidzein and genistein is hydroxylated at the aliphatic position C-2 of the C-ring. The UV-vis, GC/MS, and HPLC/MS data of all detected metabolites as well as the derived chemical structure of the metabolites are presented. Most metabolites are reported in this paper for the first time. On the basis of these findings it is suggested that hydroxylation reactions may also play an important role in the in vivo metabolism of the soy isoflavones daidzein and genistein.     

A novel formulation approach for preparation of nanoparticulate red mold rice

Monascus has been used for thousands of years. In China, Monascus has been widely used as a natural food-coloring agent for many kinds of foods. The metabolites of Monascus species, specifically, monacolin K, gamma-aminobutyric acid, and dimerumic acid, have been proven to have cholesterol-lowering, blood pressure-lowering, and antioxidant effects. Nowadays, the public has recognized the importance of Monascus products for its many health benefits. The focus of this study is to explore the effects of nanoparticulate dispersion of red mold rice (RMR) after wet-milling technology treatment. An RMR nanoparticulate formulation was reproducibly obtained after milling in the presence of dispersing agent and water. Furthermore, the physical and chemical properties of these RMR particles were studied using electron microscopy, laser light scattering, pH meter, high-performance liquid chromatography (HPLC), and photometry. The results demonstrate that RMR (mean size = 20.15 microm), processed with wet-milling technology, forms an aqueous-based nanoparticle dispersion (mean particle size of less than 0.41 microm). In addition, HPLC analyses, performed on the secondary metabolites, demonstrated that monacolin K was reduced to 50-92% of its base level and citrinin was reduced to 48-74% of its base level. When testing for the levels of pH, the processed RMR had increased from a pH level of 4.47-4.82 to 5.56-6.4; also, pigment analysis showed that yellow and red pigments were reduced to 36 and 39% of its base level after the wet-milling process. Partial agglomeration has been observed in RMR dispersion when stored in refrigeration after 2 months. RMR can be formulated as a nanoparticulate dispersion without compromising its stability, but its secondary metabolite extraction rate was changed. Further experimentation will be needed to verify safety and functionality evaluations.     

Improving the ratio of monacolin K to citrinin production of Monascus purpureus NTU 568 under dioscorea medium through the mediation of pH value and ethanol addition

Using dioscorea root as substrate of Monascus species was found to stimulate monacolin K (cholesterol-lowering agent) formation in our previous study, but the mycotoxin-citrinin has never been studied. This study used dioscorea root as the liquid medium to culture Monascus purpureus NTU 568 using a 6.6 L jar fermentor. Culture pH value, dioscorea concentration, and ethanol concentration were used as the factors of response surface methodology (RSM) to investigate the optimal culture condition for high monacolin K production and low citrinin formation. Monacolin K and citrinin formation of M. purpureus NTU 568 under submerged dioscorea medium were respectively found to be significantly formed by 148% and 147%, as compared to that under submerged rice medium. The reason is due to the pH value (3.5) of dioscorea medium involved in the formation of Monascus cell amount and secondary metabolite. RSM results further indicated that lowering the pH value to 2.5 would result in high monacolin K and citrinin concentrations as well as high biomass in fixed dioscorea amount, implying that pH value may stimulate the formation of monacolin K and citrinin through increasing Monascus cell amount. Lowering dioscorea and ethanol concentration was able to increase the ratio of monacolin K level to citrinin level. The optimal culture condition (pH 5.7, 1% dioscorea concentration, and 0.5% ethanol concentration) would increase monacolin K levels to 27.9 mg/g (by 47%) and decrease citrinin level to 2.15 microg/g (by 54%), as compared to control conditions (pH 3.5, 5% dioscorea, and ethanol free).     

Physiological response and protein expression under acid stress of Escherichia coli O157:H7 TWC01 isolated from Taiwan

Escherichia coli O157:H7 has an unusually high resistance to acidic environments. Some research has revealed that acid-adapted cells, by exposure to moderately acidic conditions, are more resistant to a subsequent strong acidic challenge or other stress. This study was conducted to understand the protein expression regulation of acid tolerance response (ATR) of a local isolated E. coli O157:H7 TWC01 (TWC01) induced by an acidic environment. TWC01 cells were acid adapted by using hydrochloric acid (HCl) or lactic acid as acidifier to induce ATR. The total proteins of adapted cells were extracted for proteomic analysis and protein identification by matrix-assisted laser desorption ionization quadrupole time-of-flight tandem mass spectrometry (MALDI-Q-TOF MS/MS). Furthermore, the effects of acid adaptation on shiga-like toxin (stx) secretion were examined. Results revealed that acid adaptation depressed stx production of E. coli O157:H7 TWC01 during adaptation and did not improve post-stress toxin production. Image analysis of the gel indicated that numerous proteins were up-regulated and that lactic acid had a greater effect than HCl did (percentages of up-regulated proteins were 57.64 and 35.47%, respectively). Analysis of proteins by mass spectrometry revealed that most of the up-regulated proteins were metabolism-related, including phosphoglycerate kinase (PGK), glutamate decarboxylases alpha and beta (GadA, GadB), adenine phosphoribosyltransferase (APRT), and dihydrodipicolinate synthase (DHDPS). Others were related to translation (e.g., elongation factor Tu, elongation factor G), protein folding (e.g., alkyl hydroperoxide reductase), and membrane proteins (e.g., ompA precursor and ompR). The variation of protein expression showed that acid resistance was induced in TWC01 and was primarily manifested via expression of up-regulated proteins that contribute to increased energy conservation and polypeptide synthesis.     

Investigation of the metabolism of ergot alkaloids in cell culture by fourier transformation mass spectrometry

Ergot alkaloids are known toxic secondary metabolites of the fungus Claviceps purpurea occurring in various grains, especially rye products. The liver is responsible for converting the ergot alkaloids into metabolites; however, the toxic impact of these end products of metabolism is still unknown. The aim of this study was to analyze the metabolism of ergot alkaloids in colon and liver cell lines (HT-29, HepG2), as well as in human primary renal cells (RPTEC). It was shown that cells in vitro are able to metabolize ergot alkaloids, forming a variety of metabolic compounds. Significant differences between the used cell types could be identified, and a suitable model system was established using HT-29 cells, performing an intensive metabolism to hydroxylated metabolites. The formed substances were analyzed by coupling of high-performance liquid chromatography with fluorescence detection and Fourier transformation mass spectrometry (HPLC-FLD-FTMS) as a powerful tool to identify known and unknown metabolites.     

棉铃虫成虫野生型与体色突变体的蛋白组成比较

为进一步了解棉铃虫体色突变体的成色和调控机理,利用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术、基质辅助激光解吸电离串联飞行时间质谱技术(MALDI-TOF-TOF/MS)和生物信息学技术,对棉铃虫显性、隐性体色突变体与野生型成虫的蛋白质组成进行了比较分析。研究发现:显性体色突变体与野生型之间差异不明显,而隐性体色突变体和野生型之间存在一些明显差异,选取其中比较明显的3 条差异条带进行质谱分析,发现一些与能量代谢、细胞骨架相关的蛋白。结果表明,这2类蛋白可能与棉铃虫体色突变性状的发生和调控相关。