Time profiles of cellobiose fermentation by the engineered strain.

<p>The engineered <i>E</i>. <i>coli</i> strain SSY12 bearing the plasmid pPgap-OsmY-Gluc1C was grown in minimal medium (A) or complex medium (B) containing cellobiose under a microaerobic condition, and the metabolites and cell growth were monitored throughout the cultivation period.</p

Expression of β-galactosidase via its native and heterologous promoter in plasmid based system.

<p>Cells were grown aerobically and anaerobically, harvested and used to monitor β-galactosidase activity. The data are presented as the average and standard deviation of two independent experiments.</p

Time profiles of hydrolysate fermentation by the engineered strain.

<p>The engineered <i>E</i>. <i>coli</i> strain SSY12 bearing the plasmid pPgap-OsmY-Gluc1C was grown in LB+biomass hydrolysate medium under a microaerobic condition and the metabolites were monitored throughout the cultivation period. The data are presented as an average and standard deviation of two bioreactor batches.</p

Expression of cellulases under the constitutive <i>gapA</i> promoter and the inducible T7 promoter.

<p>Cells were grown aerobically for 16 hr, harvested and used to monitor the endoglucanase (A) and β-glucosidase (B) and activity in both the extracellular and intracellular fractions. The data are presented as the average and standard deviation of two independent experiments.</p

Time profiles of anaerobic cellulase expression under the constitutive <i>gapA</i> promoter and the inducible T7 promoter.

<p>Cells were grown anaerobically and used to monitor the (A) endoglucanase and (B) β-glucosidase activity in both the extracellular and intracellular fractions. The data are presented as the average and standard deviation of two independent experiments.</p

Expression of β-galactosidase via its native and heterologous promoter in genome integration based system.

<p>Cells were grown (A) aerobically and (B) anaerobically, harvested and used to monitor β-galactosidase activity. The data are presented as the average and standard deviation of two independent experiments.</p

Presentation1_Secretome analysis of breast cancer cells to identify potential target proteins of Ipomoea turpethum extract-loaded nanoparticles in the tumor microenvironment.pdf

Background: Breast cancer is the leading cause of frequent malignancy and morbidity among women across the globe, with an increment of 0.5% incidences every year. The deleterious effects of traditional treatment on off-target surrounding cells make it difficult to win the battle against breast cancer. Hence, an advancement in the therapeutic approach is crucial. Nanotechnology is one of the emerging methods for precise, targeted, and efficient drug delivery in cells. The previous study has demonstrated the cytotoxic effect of Ipomoea turpethum extract on breast cancer cells delivered via NIPAAM-VP-AA nanoparticles (NVA-IT). Manipulating the tumor microenvironment (TME) to inhibit cancer progression, invasion, and metastasis seems to be very insightful for researchers these days. With the help of secretome analysis of breast cancer cells after treatment with NVA-IT, we have tried to find out the possible TME manipulation achieved to favor a better prognosis of the disease.Method: MCF-7 and MDA MB-231 cells were treated with the IC50 value of NVA-IT, and the medium was separated from the cells after 24 h of the treatment. Nano LCMS/MS analysis was performed to identify the secretory proteins in the media. Further bioinformatics tools like GENT2, GSCA, GeneCodis 4, and STRING were used to identify the key proteins and their interactions.Result: From the nano LCMS/MS analysis, 70 differentially expressed secretory proteins in MCF-7 and 191 in MDA MB-231 were identified in the cell’s media. Fifteen key target proteins were filtered using bioinformatics analysis, and the interaction of proteins involved in vesicular trafficking, cell cycle checkpoints, and oxidative stress-related proteins was prominent.Conclusion: This study concluded that I. turpethum extract-loaded NIPAAM-VP-AA nanoparticles alter the secretory proteins constituting the TME to cease cancer cell growth and metastasis.</p

Engineered Production of Short Chain Fatty Acid in <i>Escherichia coli</i> Using Fatty Acid Synthesis Pathway

<div><p>Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from <i>Anaerococcus tetradius</i>, TesBF from <i>Bryantella formatexigens</i> and TesBT from <i>Bacteroides thetaiotaomicron</i>, for production of SCFAs in <i>Escherichia coli</i> utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. <i>E</i>. <i>coli</i> strain expressing <i>tesBT</i> gene yielded maximum butyric acid titer at 1.46 g L^-1, followed by <i>tesBF</i> at 0.85 g L^-1 and <i>tesAT</i> at 0.12 g L^-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the <i>fadD</i> and <i>fadE</i> that initiates the fatty acid degradation cycle and overexpression of <i>fadR</i> that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. <i>In vitro</i> enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L^-1 butyric acid and 17.5 g L^-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in <i>E</i>. <i>coli</i> through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.</p></div

Effect of oxygen availability on production of butyric acid.

<p>(A) <i>E</i>. <i>coli</i> MG1655 transformed with pZA-tesBT plasmid was grown in 100 ml flask containing different volume of culture medium to vary oxygen availability and checked for butyric acid production. (B) <i>E</i>. <i>coli</i> MG1655 transformed with pZA-tesBT plasmid was grown in the bioreactor containing 350 ml culture medium with different oxygen saturation level and checked for butyric acid production.</p

Impact of cerulenin on thioesterase mediated butyric acid production.

<p><i>E</i>. <i>coli</i> MG1655 transformed with either (A) pQE-tesBF for expression on thioesterase TesBF or (B) pZA-tesBT for expression of thioesterase TesBT were grown in presence of different concentration of cerulenin and extracellular metabolites were analyzed using HPLC.</p