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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., (© 2019 Wiley Periodicals, Inc.)

Lactate dehydrogenase is an enzyme of glycolytic pathway which catalyzes the interconversion of pyruvate and lactate. The present study describes cDNA cloning, E. coli expression and characterization of lactate dehydrogenase B (LDH-B) from the heart ventricles of river buffalo (Bubalus bubalis). Total RNA was isolated from the heart tissue, a 1005bp cDNA encoding complete polypeptide chain of 334 amino acids was generated by reverse transcriptase reaction and analyzed for nucleotide sequence. The consensus sequence obtained from both strands has shown 84% to 98% homology with that of different mammalian species. The attributed gene was cloned, expressed in BL21 (DE3) RIPL Codon Plus strain of E. coli using pET21a (+) plasmid. The purified recombinant enzyme displayed a KM value of 50 µM for pyruvate, an optimum activity at 35°C and pH 7.0. The enzyme was found as a homotetramer of 140 kDa on FPLC based gel-filtration column. Molecular weight of a subunit of enzyme as determined by mass spectrometric analysis was 36530.21 Da. The present study describes the first ever report about the cDNA sequence and characteristics of recombinant LDH-B from River buffalo.