30 research outputs found
Microwave-assisted extraction of anthocyanin from Chinese bayberry and its effects on anthocyanin stability
Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography
Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of at least 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins’ functions were obtained from their interactions with each of a panel of ligands
A broader role for AmyR in Aspergillus niger: regulation of the utilisation of d-glucose or d-galactose containing oligo- and polysaccharides
AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity assays, genes expression analysis and growth profiling on d-glucose- and d-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding α- and β-glucosidases, α- and β- galactosidases, as well as genes encoding α-amlyases and glucoamylases. In addition, we provide evidence that d-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed
Current state of genome-scale modeling in filamentous fungi
The group of filamentous fungi contains important species used in industrial biotechnology for acid, antibiotics and enzyme production. Their unique lifestyle turns these organisms into a valuable genetic reservoir of new natural products and biomass degrading enzymes that has not been used to full capacity. One of the major bottlenecks in the development of new strains into viable industrial hosts is the alteration of the metabolism towards optimal production. Genome-scale models promise a reduction in the time needed for metabolic engineering by predicting the most potent targets in silico before testing them in vivo. The increasing availability of high quality models and molecular biological tools for manipulating filamentous fungi renders the model-guided engineering of these fungal factories possible with comprehensive metabolic networks. A typical fungal model contains on average 1138 unique metabolic reactions and 1050 ORFs, making them a vast knowledge-base of fungal metabolism. In the present review we focus on the current state as well as potential future applications of genome-scale models in filamentous fungi
Aspergillus as a multi-purpose cell factory: current status and perspectives
Aspergilli have a long history in biotechnology as expression platforms for the production of food ingredients, pharmaceuticals and enzymes. The achievements made during the last years, however, have the potential to revolutionize Aspergillus biotechnology and to assure Aspergillus a dominant place among microbial cell factories. This mini-review will highlight most recent breakthroughs in fundamental and applied Aspergillus research with a focus on new molecular tools, techniques and products. New trends and concepts related to Aspergillus genomics and systems biology will be discussed as well as the challenges that have to be met to integrate omics data with metabolic engineering attempts
Systems biology methods and developments of filamentous fungi in relation to the production of food ingredients
Systems biology is rapidly evolving in many areas of biological sciences. In this chapter, we present a review of systems biology of filamentous fungi in relation to the production of enzymes, chemicals and food ingredients. We summarize the current status of systems biology through functional genomics (i.e. genomics, transcriptomics, proteomics, metabolomics) and bioinformatics of different food-related filamentous fungi. In addition, we present a number of case studies dealing with systems biology and functional genomics through the development of a genome-scale metabolic model of filamentous fungi, which serve as important cell factories in food biotechnology. © 2013 Woodhead Publishing Limited. All rights reserved
Increased Lipid Accumulation in Mucor circinelloides by Overexpression of Mitochondrial Citrate Transporter Genes
Mucor circinelloides has been commonly used as the model microbe to investigate lipid production as an oleaginous fungus. Mitochondrial citrate transporter can catalyze the translocation of the citrate, accumulated from TCA cycle, across the mitochondrial inner membrane. The extra-mitochondrial citrate is then cleaved by ATP-citrate lyase to oxaloacetate (OAA) and acetyl-CoA. Acetyl-CoA together with NADPH generated in cytosol is used for fatty acid biosynthesis. Thus, citrate transporters provide a link between TCA cycle in mitochondria and fatty acid biosynthesis in cytosol. However, the role of citrate transporters for lipid accumulation in oleaginous fungi is not clear. Two genes coding for citrate transporters, named citrate transporter (ct) and tricarboxylate transporter (tct) respectively, were present in the genome of oleaginous fungus M. circinelloides WJ11, a high lipid producing strain (36%, lipid/cell dry weight). As the mutant of strain CBS 277.49 (15%, lipid/cell dry weight) has been constructed and its genetic engineering tools are available for gene manipulation, so in this work, we investigated the role of citrate transporters in regulating lipid biosynthesis by overexpressing the citrate transporters of M. circinelloides WJ11 in CBS 277.49. Results: Our results showed that overexpression of ct and tct led to increased lipid accumulation by 44% (from 13.0% to 18.8%, w/w, CDW) and 68% (from 13.0% to 21.8%, w/w, CDW), respectively. Moreover, extracellular citrate concentration in ct-overexpressing strains (4.91 mM) and tct-overexpressing (3.25 mM) were significantly decreased by 20% and 47% respectively compared to the control (6.09 mM). Furthermore, overexpression of the citrate transporter genes activated the downstream steps in lipid biosynthesis, such as ATP citrate lyase (acl gene) and fatty acid synthases (fas1 and fas2 genes), indicating a greater flux of carbon went into fatty acid biosynthesis. Conclusions: This is the first report showing that citrate transporters involved in lipid accumulation in M. circinelloides. Both citrate transporter and tricarboxylate transporter could transport mitochondrial citrate to cytoplasm, which could provide more citrate to be cleaved by increased ACL to provide more acetyl-CoA and NADPH for increased FAS to synthesize fatty acids, thus, play a vital role in lipid biosynthesis in oleaginous fungus M. circinelloides
Molecular mechanism of Forkhead box M1 inhibition by thiostrepton in breast cancer cells
Breast cancer is the most common type of malignancies in women worldwide, and genotoxic chemotherapeutic drugs are effective by causing DNA damage in cancer cells. However, >90% of patients with metastatic cancer are resistant to chemotherapy. The Forkhead box M1 (FOXM1) transcription factor plays a pivotal role in the resistance of breast cancer cells to chemotherapy by promoting DNA damage repair following genotoxic drug treatment. The aim of the present study was to investigate the inhibition of the FOXM1 protein by thiostrepton, a natural antibiotic produced by the Streptomyces species. Experimental studies were designed to examine the effectiveness of thiostrepton in downregulating FOXM1 mRNA expression and activity, leading to senescence and apoptosis of breast cancer cells. The cytotoxicity of thiostrepton in breast cancer was determined using cell viability assay. Additionally, thiostrepton treatment decreased the mRNA expression of cyclin B1 (CCNB1), a downstream target of FOXM1. The present results indicated that thiostrepton inhibited FOXM1 mRNA expression and its effect on CCNB1. Molecular dynamic simulations were performed to study the interactions between FOXM1-DNA and thiostrepton after molecular docking. The results revealed that the possible mechanism underlying the inhibitory effect of thiostrepton on FOXM1 function was by forming a tight complex with the DNA and FOXM1 via its binding domain. Collectively, these results indicated that thiostrepton is a specific and direct inhibitor of the FOXM1 protein in breast cancer. The findings of the present study may lead to the development of novel therapeutic strategies for breast cancer and help overcome resistance to conventional chemotherapeutic drugs
Increased fatty acid unsaturation and production of arachidonic acid by homologous over-expression of the mitochondrial malic enzyme in Mortierella alpina
Unravelling evolutionary strategies of yeast for improving galactose utilization through integrated systems level analysis
Identification of the underlying molecular mechanisms for a derived phenotype by adaptive evolution is difficult. Here, we performed a systems-level inquiry into the metabolic changes occurring in the yeast Saccharomyces cerevisiae as a result of its adaptive evolution to increase its specific growth rate on galactose and related these changes to the acquired phenotypic properties. Three evolved mutants (62A, 62B, and 62C) with higher specific growth rates and faster specific galactose uptake were isolated. The evolved mutants were compared with a reference strain and two engineered strains, SO16 and PGM2, which also showed higher galactose uptake rate in previous studies. The profile of intermediates in galactose metabolism was similar in evolved and engineered mutants, whereas reserve carbohydrates metabolism was specifically elevated in the evolved mutants and one evolved strain showed changes in ergosterol biosynthesis. Mutations were identified in proteins involved in the global carbon sensing Ras/PKA pathway, which is known to regulate the reserve carbohydrates metabolism. We evaluated one of the identified mutations, RAS2Tyr112, and this mutation resulted in an increased specific growth rate on galactose. These results show that adaptive evolution results in the utilization of unpredicted routes to accommodate increased galactose flux in contrast to rationally engineered strains. Our study demonstrates that adaptive evolution represents a valuable alternative to rational design in bioengineering of improved strains and, that through systems biology, it is possible to identify mutations in evolved strain that can serve as unforeseen metabolic engineering targets for improving microbial strains for production of biofuels and chemicals