Theoretical and experimental investigation of chaperone effects on soluble recombinant proteins in Escherichia coli: effect of free DnaK level on temperature-induced recombinant streptokinase production

Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK–DnaJ–GrpE and GroEL–GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a σ32 mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in principle extended to bigger genetic networks as well as other recombinant proteins and expression systems

INTERGENUS PROTOPLAST FUSION STUDIES BETWEEN MARINE STREPTOMYCES SP. ANS4 AND ESCHERICHIA COLI

Objective: To study the intergenus protoplast fusion bioactive marine Streptomyces sp ANS4 with the Escherichia coli for the strain improvement. Methods: Protoplast of both the organisms was prepared by lysozyme treatment and fused using polyethylene glycol (PEG). Fusant colonies were regenerated using regeneration medium where all the regenerated colonies are powdery in consistency. Microscopic, cultural, physiological, enzymatic and antibiotic susceptibility characteristics of one fusant culture and both the parent cultures are studied. Growth curve and bioactive metabolite production by both the parent and fusant cultures was also studied. Results: oval shaped protoplasts of both the parent cultures are clearly seen under the phase contrast microscope. All the colonies developed after protoplast fusion on regeneration medium looked like one of the parent culture Streptomyces sp ANS4. One among the 10 antagonistic colonies selected from the regeneration medium was expressed the characteristics of either or both of the parental cultures and certain new characteristics. The results of growth curve study showed that the fusant culture possess 10 hours reduction in attaining stationary phase compared to parent Streptomyces sp ANS4. The antibiotic activity of fusant culture was also higher than the parent cultures. Conclusion: The intergenus protoplast fusion between the Streptomyces sp ANS4 and the E.coli strain results in the reduction in time for secondary metabolite production and also increase in antimicrobial activity. This suggested that the potential bioactive compounds may be obtained in increased quantity from the fusant culture with lesser fermentation period

Reconstruction and analysis of a genome-scale metabolic model for Scheffersomyces stipitis

<p>Abstract</p> <p>Background</p> <p>Fermentation of xylose, the major component in hemicellulose, is essential for economic conversion of lignocellulosic biomass to fuels and chemicals. The yeast <it>Scheffersomyces stipitis </it>(formerly known as <it>Pichia stipitis</it>) has the highest known native capacity for xylose fermentation and possesses several genes for lignocellulose bioconversion in its genome. Understanding the metabolism of this yeast at a global scale, by reconstructing the genome scale metabolic model, is essential for manipulating its metabolic capabilities and for successful transfer of its capabilities to other industrial microbes.</p> <p>Results</p> <p>We present a genome-scale metabolic model for <it>Scheffersomyces stipitis</it>, a native xylose utilizing yeast. The model was reconstructed based on genome sequence annotation, detailed experimental investigation and known yeast physiology. Macromolecular composition of <it>Scheffersomyces stipitis </it>biomass was estimated experimentally and its ability to grow on different carbon, nitrogen, sulphur and phosphorus sources was determined by phenotype microarrays. The compartmentalized model, developed based on an iterative procedure, accounted for 814 genes, 1371 reactions, and 971 metabolites. In silico computed growth rates were compared with high-throughput phenotyping data and the model could predict the qualitative outcomes in 74% of substrates investigated. Model simulations were used to identify the biosynthetic requirements for anaerobic growth of <it>Scheffersomyces stipitis </it>on glucose and the results were validated with published literature. The bottlenecks in <it>Scheffersomyces stipitis </it>metabolic network for xylose uptake and nucleotide cofactor recycling were identified by in silico flux variability analysis. The scope of the model in enhancing the mechanistic understanding of microbial metabolism is demonstrated by identifying a mechanism for mitochondrial respiration and oxidative phosphorylation.</p> <p>Conclusion</p> <p>The genome-scale metabolic model developed for <it>Scheffersomyces stipitis </it>successfully predicted substrate utilization and anaerobic growth requirements. Useful insights were drawn on xylose metabolism, cofactor recycling and mechanism of mitochondrial respiration from model simulations. These insights can be applied for efficient xylose utilization and cofactor recycling in other industrial microorganisms. The developed model forms a basis for rational analysis and design of <it>Scheffersomyces stipitis </it>metabolic network for the production of fuels and chemicals from lignocellulosic biomass.</p

ANTIFUNGAL ACTIVITY OF ACTINOBACTERIA WITH A POTENTIAL TO INHIBIT RICE BLAST FUNGUS MAGNAPORTHE ORYZAE (ANAMORPH PYRICULARIA ORYZAE)

Objective: The aims of the present study were to screen the actinobacteria with high potential ability to produce secondary metabolites that have inhibitory activity against plant pathogenic fungi, Magnaporthe oryzae. Production of secondary metabolites was analysis by thin-layer chromatography and bioautography assay. Methods: Screening and selection of potential Streptomyces sp. morphological, cultural, physiological, and biochemical characterization of the screened isolate was carried out. Antifungal compound was confirmed by bioautography assay. Results: Bioautography method use in this study was found to be antifungal fraction from the crude extract. Antifungal secondary metabolites can be readily located on the plates by observing clear zones where active compounds inhibit fungal growth. Conclusion: The bioautography assay shows that this isolates can produce antifungal compound. Therefore, this isolate proves to be a promising microbe which can be further studied for its applications a biocontrol agent against rice blast fungi

IN SILICO ANALYSIS FOR THE PRODUCTION OF HIGHER CARBON ALCOHOLS USING SACCHAROMYCES CEREVISIAE

Technology for the production of alternative fuels is receiving increased attention owing to concerns on global energy and environmental problems. Using higher carbon alcohols as gasoline substitutes has several advantages compared to ethanol, the first generation biofuel. Higher carbon alcohols also have other applications as flavor/aroma compounds and as building blocks for several other products. Two different pathways for the production of higher carbon alcohols have been recently reported. This work looks at evaluating the different pathways for higher carbon alcohol production and identification of metabolic bottlenecks for their production using Saccharomyces cerevisiae . Quantitative characterization of the metabolic pathways of Saccharomyces cerevisiae is essential for understanding the metabolic behavior of the microorganism. Several mathematical modeling frameworks have been developed to describe and analyze the metabolic behavior of an organism. Stoichiometric modeling is one such approach which relies on mass balances over intracellular metabolites and the assumption of pseudo-steady-state conditions to determine intracellular metabolic fluxes. The development of stoichiometric models (metabolic models) and analysis of intracellular metabolic fluxes have several applications in metabolic engineering and strain improvement. The production of higher carbon alcohols (such as 1-butanol, isobutanol, isopropanol) was analyzed by introducing the pathways into the genome scale metabolic model of Saccharomyces cerevisiae . The yield of higher carbon alcohols obtained from the fermentative and non-fermentative pathways was calculated and compared with maximum theoretical yield. The effect of different industrially relevant carbon sources on the production of higher carbon alcohols was also analyzed. Constraint based analysis was carried out on the genome scale metabolic model to obtain the intracellular metabolic flux distribution during the production of these alcohols. Detailed analysis of the metabolic flux distribution was carried out based on the shadow prices and reduced costs obtained from metabolic flux analysis. The metabolic bottlenecks for the production of higher carbon alcohols and the rate limiting steps in the metabolism were identified based on these analyses. Strategies for enhancing the yield of higher carbon alcohols will be proposed based on these analyses

Screening, production, and characterization of biologically active secondary metabolite(s) from marine Streptomyces sp. PA9 for antimicrobial, antioxidant, and mosquito larvicidal activity

1319-1326Bioprospecting of actinobacteria from understudied ecosystems is a promising source for extracting novel bioactive metabolites. A study was undertaken to characterize and analyze the bio-efficacy of actinobacterial extract for antimicrobial, larvicidal, and antioxidant activities. Seven morphologically different actinobacterial cultures isolated from mangrove rhizosphere sediment near Parangipettai, South India, were tested for antimicrobial activity. Bioactive metabolites from one potential strain PA9 were produced by submerged fermentation. The selected Streptomyces sp. PA9 was subjected to the production of crude extract for antimicrobial, larvicidal, and antioxidant activity. The actinobacterial compound was characterized by Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC), and gas chromatography–mass spectrometry (GC–MS). The PA9 actinobacterial crude extract showed best antimicrobial activity against clinical bacteria, Salmonella typhi (21.6 ± 0.88 mm) and fungi, Candida albicans (26.6 ± 0.88 mm). The PA9 extract showed significant larvicidal activity against Culex quinquefasciatus with LC50=173.21 µg/ml and r2=0.841. The PA9 extract also exhibited antioxidant activity from DPPH (72%) and nitric oxide free radicals (85%). The characterization of the PA9 extract by FTIR analysis showed the presence of possible functional groups. Active compounds were isolated by HPLC and GC–MS with major and minor peaks observed on the basis of retention time. The bio-efficacy of PA9 has warranted further studies to develop a baseline for the drug development

OPTIMIZATION OF MEDIUM COMPONENTS FOR ANTIBACTERIAL METABOLITE PRODUCTION FROM MARINE STREPTOMYCES SP. PUA2 USING RESPONSE SURFACE METHODOLOGY

Objective: The present study is an attempt to optimize the fermentation conditions for the antibacterial compound production from a newly isolated marine Streptomyces strain PUA2 by adopting response surface methodology as the statistical tool. Methods: Prior to using the Response Surface Methodology, Plackett Burmann (PB) design was used to explore the effect of variables on the antibacterial compound production. In PB method, high and low values were assigned for the eight variables viz., glucose, glycerol, soybean meal, manganese chloride, calcium carbonate, peptone and pH. Calcium carbonate and peptone were used as dummy variables. Based on the results of combined effects glycerol, soybean meal, manganese chloride and pH were investigated by 24 full-factorial central composite design. Results: The results of PB method showed the significant effect of glycerol, soybean meal, manganese chloride and pH on the antibacterial compound production. The results of ANOVA and regression of second order model showed that the linear effects of glycerol and manganese chloride and cross products effects of manganese chloride and pH were more significant. All the critical variables having greatest effect on the production of antibacterial compound from marine Streptomyces species PUA2. Optimization of process parameters resulted in increase in antibacterial activity from 7 mm to 14 mm. Conclusion: The factors optimized in the present study were useful for the increased production of antibacterial metabolite from Streptomyces sp PUA2. The result confirms the feasibility of medium optimization to improve antibiotic production

Quantitative Imaging Features Predict Response of Immunotherapy in Non-Small Cell Lung Cancer Patients

[No Abstract Available

Bioactive potential of selected actinobacterial strains against Mycobacterium tuberculosis and other clinical pathogens

1307-1311Marine actinobacteria produces diverse array of metabolites with novel chemical structures with potential bioactivities. Exploring the understudied ecosystems may increase the chance of getting novel actinobacteria and new metabolites.The present study explores the bioactive potential of actinobacteria isolated from the marine ecosystem of Andaman and Nicobar Islands, Bay of Bengal, against Mycobacterium tuberculosis and other clinical pathogens. The crude extracts from 15 marine actinobacterial strains were produced through agar surface fermentation using YEME agar and extracted using ethyl acetate. The crude extracts were tested against the standard strain M. tuberculosis H37Rv, clinical drug sensitive M. tuberculosis, and MDR M. tuberculosis strains by luciferase reporter phage (LRP) assay at 500 µg/ml concentration. The anti-microbial activity against other clinical pathogens, namely, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi, Klebsiellapneumoniae, Pseudomonas aeruginosa, Candida albicans, and Cryptococcusneoformans and non-tubercular mycobacteria, M. smegmatis was studied by agar plug method. Among the 15 extracts that were tested for anti-tubercular activity, the crude ethyl acetate extract of the 14 actinobacterial strains showed anti-tubercular activity against at least one of the three M. tuberculosis strains. Exceptionally, the ethyl acetate extract of strain SACC 168 inhibited all three M. tuberculosis strains tested. In anti-microbial screening, the crude extracts of eight strains showed anti-microbial activity including six strains, which were active against the non-tuberculous mycobacteria. Further purification and characterization of the active molecule from the potential extracts will pave way for the potential natural product candidate for tuberculosis and other microbial infections

Tuberculosis, A Great Masquerader: A Case Report of Disseminated Multifocal Skeletal TB and Tracheoesophageal Fistulae Mimicking Metastatic Cancer

Disseminated tuberculosis (TB) can mimic metastatic disease because of its multi-organ involvement (including bones), which can make the diagnosis much more complicated. Tracheoesophageal fistula is a very uncommon manifestation of TB, as is multifocal skeletal TB. There are reports of TB presenting either as multifocal skeletal TB or as tracheoesophageal fistulae, but we could not find any case reports describing both of these entities in a single patient and essentially mimicking a metastatic oesophageal neoplasm. However, we here describe one such case, which was managed medically