LC‐MS/MS‐based metabolic profiling of Escherichia coli under heterologous gene expression stress

Abstract

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin‐resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)‐induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography‐mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN‐based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA‐CoA, p‐aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l‐carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.

Untargeted Metabolomics Identifies Key Metabolic Pathways Altered by Thymoquinone in Leukemic Cancer Cells

“…Further, dry extracts were reconstituted in 100 μL of acetonitrile: water (1:1, v/v ), vortexed for 10 min and spin for 15 min at 13,000 rpm at 4 °C to remove insoluble debris. Finally, the resultant supernatants were taken for LC-MS/MS run [ 9 , 10 ].…”

“…The mobile phase consisted of 0.1% Formic acid and 99.9% ACN formic acid (0.1%, v/v ), using a linear gradient programme where the component of solution was changed from 5% B to 100% B over 90 min, at a constant flow rate of 0.2 mL/min (95% A from 5% to 30% over 72 min, 30% to 100% over 10 min, and kept at 100% for 5 min at a flow rate of 0.25 mL/min). The column temperature was maintained at 30 °C [ 9 , 10 ].…”

Integration of Transcriptome and Metabolome Provides Unique Insights to Pathways Associated With Obese Breast Cancer Patients

“…Each sample (10 µL) was injected individually into Hypersail gold high-performance liquid chromatography (HPLC) column (150 × 4.6 mm, 5 µm) with a flow rate of 0.250 ml/min and mobile phase A 0.1% formic acid in 99.9% acetonitrile formic acid (0.1%, volume/volume) and mobile phase B is 0.1% formic acid in MilliQ. Mass Spec parameter performed as an earlier report (34). Raw data processed using the online XCMS database (35).…”

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“…This is especially true for over-expressing systems, where the advent of strong promoters like the T7 or pBAD promoter, has ensured that the bottleneck in protein synthesis is no longer at the transcriptional step. The post induction transcriptomic, proteomic as well as metabolomic profiles of E. coli expressing different proteins has been studied in detail to understand the exact nature of the CSR and its effect on protein synthesis rates [2,[7][8][9][10][11][12]. Strategies to alleviate the deleterious effects of the CSR have been proposed which essentially involve the upregulation of critical genes involved in substrate uptake, ATP synthesis or energy metabolism [13][14][15][16].…”