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Overproduction of Native and Click-able Colanic Acid Slime from Engineered Escherichia coli

ABSTRACT: The fundamental biology

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PROVIDER: S-EPMC9976346 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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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.

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Overproduction of Native and Click-able Colanic Acid Slime from Engineered Escherichia coli

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