Project description:Investigation of whole genome gene expression level changes in a E. coli fatty acid overproducing strain with or without heterologous expression of the M. luteus FabH. The strain expressing M. luteus FabH produces more methyl ketones. This study will be further described in Goh, E.B., E.E.K. Baidoo, J.D. Keasling, and H.R. Beller. Engineering of bacterial methyl ketone synthesis for biofuels. A 11 microarray study using total RNA recovered from six separate control cultures of Escherichia coli K-12 DH1 fatty acid overproducing strain with empty vector and five separate cultures of test strain, Escherichia coli K-12 DH1 fatty acid overproducing strain with vector overexpressing M. luteus FabH. Each chip measures the expression level of 4,254 genes from Escherichia coli K-12 with eight 60-mer probe pairs (PM/MM) per gene, with 2-fold technical redundancy.
Project description:Exogenous fatty acids are directly incorporated into bacterial membranes, heavily influencing bacterial ecology and antibiotic susceptibility. We use liquid chromatography/mass spectroscopy to characterize how exogenous fatty acids impact the Escherichia coli fatty acid synthesis pathway. We quantify acyl-ACP, phospholipids and phospholipid synthesis intermediates, and key proteins using a targeted protcol.
Project description:Comparative analysis of changes in gene and protein expression and fatty acid profiles between Escherichia coli K-12 MG1655 ΔfadD ΔaraBAD expressing an acyl-acyl carrier protein thioesterase from Umbellularia californica (BTE) or a non-functional mutant thioesterase (BTE-H204A) to determine the functional basis for losses in cell viability, membrane integrity, or other stresses and metabolic perturbations that may be present. New hypotheses obtained from the study will assist in metabolic engineering efforts of improved strains exhibiting higher fatty acid yields and productivities.
Project description:Carboxylic acids are an attractive biorenewable chemical. Enormous progress has been made in engineering microbes for production of these compounds though titers remain lower than desired. Here we used transcriptome analysis of Escherichia coli during exogenous challenge with octanoic acid (C8) at pH 7.0. This analysis suggests that C8 challenge causes intracellular acidification and membrane damage. Escherichia coli MG1655 was grown to midlog (OD550 ~0.8) with or without 10 mM octanoic acid (pH=7.0) and the RNA was harvested and prepared for Affymetrix Microarray analysis.
Project description:Comparative analysis of changes in gene and protein expression and fatty acid profiles between Escherichia coli K-12 MG1655 ΔfadD ΔaraBAD expressing an acyl-acyl carrier protein thioesterase from Umbellularia californica (BTE) or a non-functional mutant thioesterase (BTE-H204A) to determine the functional basis for losses in cell viability, membrane integrity, or other stresses and metabolic perturbations that may be present. New hypotheses obtained from the study will assist in metabolic engineering efforts of improved strains exhibiting higher fatty acid yields and productivities. Cultures of fatty acid overproducing (BTE-expressing) and negative control (non-functional BTE-H204A-expressing) strains of Escherichia coli K-12 MG1655 delta-fadD delta-araBAD (deficient in beta-oxidation and L-arabinose catabolism) were sampled under two different sets of media/induction/antibiotic conditions. These conditions were shake flasks at 37C, 250 rpm shaking, in EZ rich defined medium supplemented with 0.2% glucose and 0.01 mM biotin (EZglu), and in fermentors at 37C with controlled air sparging, agitation, and pH in EZ rich defined medium supplemented with 0.4% glycerol and 0.01 mM biotin (EZgly). Two strains were analyzed in the EZglu experiment, the background strain harboring either pTrc99A-BTE (fatty acid overproducing) or pTrc99A-BTE-H204A (control phenotype). Three strains were analyzed in the EZgly experiment, with the background strain haboring either pBAD35-BTE and pBAD33 (fatty acid overproducing), pBAD35-BTE and pBAD33-ACC (fatty acid overproducing, ACC are the 4 subunits of E. coli K-12 acetyl-CoA carboxylase expressed as an artificial operon accDABC in plasmid pBAD33), and pBAD35-BTE-H204A and pBAD33 (control phenotype). RNA was extracted from harvested cell pellets from biological triplicates (EZglu) or biological duplicates (EZgly) of each strain at three different sampling times as defined in each sample description. Due to a hybridization or scanning problem, biological duplicates rather than triplicates were analyzed at the mid-stationary phase sampling point in the EZglu experiment for the control strain harboring pTrc99A-BTE-H204A. Multiple technical replicates at either the hybridization or sample level were analyzed from the biological duplicates of the fermentor experiment. The form of technical replicate (sample or hybridization) is specified in each EZgly sample description.
Project description:The aim of the study is to establish the existence of a relationship between the dietary intake of polyunsaturated fatty acids (PUFA) and the risk of colorectal cancer in humans, using 2 reliable and complementary biomarkers: the fatty acid-composition of lipids of the abdominal subcutaneous adipose tissue and the fatty acid composition of erythrocyte phospholipids.
Project description:Carboxylic acids are an attractive biorenewable chemical. Enormous progress has been made in engineering microbes for production of these compounds though titers remain lower than desired. Here we used transcriptome analysis of Escherichia coli during exogenous challenge with octanoic acid (C8) at pH 7.0. This analysis suggests that C8 challenge causes intracellular acidification and membrane damage.
Project description:Biosynthesis of carboxylic acids and fatty acids from renewable biomass is a key issue in biorefinery. However, their productivities are often limited due to various toxic effects of the products on the host organisms. Here, we have investigated the factors that influence tolerance of Escherichia coli to long chain carboxylic acid (e.g., n-heptanoic acid)-induced stress by using transcriptome analysis. The metabolic and genomic responses of E. coli BL21 and MG1655 strains with n-heptanoic acid indicated that acid stress is one of the major stresses, which might be generated by n-heptanoic acid in addition to the presumed solvent-like stress.
Project description:Investigation of whole genome gene expression level changes in a E. coli fatty acid overproducing strain with or without heterologous expression of the M. luteus FabH. The strain expressing M. luteus FabH produces more methyl ketones. This study will be further described in Goh, E.B., E.E.K. Baidoo, J.D. Keasling, and H.R. Beller. Engineering of bacterial methyl ketone synthesis for biofuels.
Project description:For over 30 years, serine hydroxamate has been used to chemically stimulate a stringent response in Escherichia coli and other bacteria. These studies have elucidated numerous characteristics of the classical stringent response beyond the simple cellular response to an amino acid shortage, including phospholipid synthesis and protease up-regulation. In this study, the effects of a serine hydroxamate addition on high cell density recombinant E. coli were examined and compared to the effects of recombinant protein production to determine overlaps, as recombinant protein production stress has often been attributed to amino acid shortages. Both the transcriptome and growth characteristics were evaluated and compared. The serine hydroxamate addition profoundly decreased the culture growth rate, whereas, recombinant protein production did not. Conversely, the transcriptome profile of the recombinant E. coli cultures were relatively unaffected by the serine hydroxamate addition, yet recombinant protein production dramatically changed the transcriptome profile. A subset of the classical stringent response genes were effected by the serine hydroxamate addition, whereas, recombinant protein production regulated numerous classical stringent response genes; however, not all. The genes that were regulated by the serine hydroxamate addition include numerous fatty acid synthesis genes, in agreement with altered phospholipids synthesis reports. These results indicate that recombinant protein production and the stringent response have many overlapping responses; however, are far from identical. It was hypothesized that recombinant protein production leads to a stringent response due to the high amino acid synthesis demands related to recombinant protein synthesis. A comparison of the transcriptomes during recombinant protein production and a chemical imposed stringent response would assist with determining what portion of the “metabolic burden” associated with recombinant protein production is due to amino acid shortages. In this study, the transcriptome profiles of recombinant E. coli were examined and compared for the three culture conditions: 1) Normal growth, no external stress; 2) L-serine hydroxamate addition (to mediate a stringent response); and 3) IPTG-induction to produce the recombinant protein chloramphenicol acetyltransferase (CAT). The transcriptome profiles from these three conditions were analyzed using Affymetrix Anti-sense E. coli GeneChip® microarrays.