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

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 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 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

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

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

Impact of genotype of <i>E</i>. <i>coli</i> on butyric acid production using different thioesterases.

<p>Different <i>E</i>. <i>coli</i> strains were transformed with plasmid pQE-tesAT, pQE-tesBF and pQE-tesBT for expression of thioesterase TesAT (represented as AT), TesBF (represented as BF) and TesBT (represented as BT), respectively, and used for butyric acid production.</p