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The intracellular [ATP]/[ADP] ratio is crucial for Escherichia coli's cellular functions, impacting transport, phosphorylation, signaling, and stress responses. Overexpression of F1-ATPase genes in E. coli increases glucose consumption, lowers energy levels, and triggers transcriptional responses in central carbon metabolism genes, particularly glycolytic ones, enhancing carbon flux. In this contribution, we report the impact of the perturbation of the energetic level in a PTS- mutant of E. coli by modifying the [ATP]/[ADP] ratio by uncoupling the cytoplasmic activity of the F1 subunit of the ATP synthase. The disruption of [ATP]/[ADP] ratio in the evolved strain of E. coli PB12 (PTS-) was achieved by the expression of the atpAGD operon encoding the soluble portion of ATP synthase F1-ATPase (strain PB12AGD+). The analysis of the physiological and metabolic response of the PTS- strain to the ATP disruption was determined using RT-qPCR of 96 genes involved in glucose and acetate transport, glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and global regulators. The apt mutant exhibited reduced growth despite increased glucose transport due to decreased energy levels. It heightened stress response capabilities under glucose-induced energetic starvation, suggesting that the carbon flux from glycolysis is distributed toward the pentose phosphate and the Entner-Duodoroff pathway with the concomitant. Increase acetate transport, production, and utilization in response to the reduction in the [ATP]/[ADP] ratio. Upregulation of several genes encoding the TCA cycle and the glyoxylate shunt as several respiratory genes indicates increased respiratory capabilities, coupled possibly with increased availability of electron donor compounds from the TCA cycle, as this mutant increased respiratory capability by 240% more than in the PB12. The reduction in the intracellular concentration of cAMP in the atp mutant resulted in a reduced number of upregulated genes compared to PB12, suggesting that the mutant remains a robust genetic background despite the severe disruption in its energetic level.

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The culture of engineered Escherichia coli for shikimic acid (SA) production results in the synthesis of quinic acid (QA) and dehydroshikimic acid (DHS), reducing SA yield and impairing downstream processes. The synthesis of QA by quinate/shikimate dehydrogenase (YdiB, ydiB) has been previously proposed; however, the precise role for this enzyme in the production of QA in engineered strains of E. coli for SA production remains unclear. We report the effect of the inactivation or the overexpression of ydiB in E. coli strain PB12.SA22 on SA, QA, and DHS production in batch fermentor cultures. The results showed that the inactivation of ydiB resulted in a 75% decrease in the molar yield of QA and a 6.17% reduction in the yield of QA (mol/mol) relative to SA with respect to the parental strain. The overexpression of ydiB caused a 500% increase in the molar yield of QA and resulted in a 152% increase in QA (mol/mol) relative to SA, with a sharp decrease in SA production. Production of SA, QA, and DHS in parental and derivative ydiB strains suggests that the synthesis of QA results from the reduction of 3-dehydroquinate by YdiB before its conversion to DHS.

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BACKGROUND: Melanins comprise a chemically-diverse group of polymeric pigments whose function is related to protection against physical and chemical stress factors. These polymers have current and potential applications in the chemical, medical, electronics and materials industries. The biotechnological production of melanins offers the possibility of obtaining these pigments in pure form and relatively low cost. In this study, Escherichia coli strains were engineered to evaluate the production of melanin from supplemented catechol or from glycerol-derived catechol produced by an Escherichia coli strain generated by metabolic engineering. RESULTS: It was determined that an improved mutant version of the tyrosinase from Rhizobium etli (MutmelA), could employ catechol as a substrate to generate melanin. Strain E. coli W3110 expressing MutmelA was grown in bioreactor batch cultures with catechol supplemented in the medium. Under these conditions, 0.29 g/L of catechol melanin were produced. A strain with the capacity to synthesize catechol melanin from a simple carbon source was generated by integrating the gene MutmelA into the chromosome of E. coli W3110 trpD9923, that has been modified to produce catechol by the expression of genes encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, transketolase and anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. In batch cultures with this strain employing complex medium with 40 g/L glycerol as a carbon source, 1.21 g/L of catechol melanin were produced. The melanin was analysed by employing Fourier transform infrared spectroscopy, revealing the expected characteristics for a catechol-derived polymer. CONCLUSIONS: This constitutes the first report of an engineered E. coli strain and a fermentation process for producing a catechol melanin from a simple carbon source (glycerol) at gram level, opening the possibility of generating a large quantity of this polymer for its detailed characterization and the development of novel applications.

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Recombinant Escherichia coli strains for the production of valuable products are usually generated by transformation with plasmid expression vectors. However, in spite of their usefulness, common problems associated with plasmid use include segregrational and structural instability as well as undesired copy-number effects. A viable alternative to plasmid use is chromosomal gene integration. With the purpose of facilitating the process of stable strain generation, a novel chromosomal integration vector was developed and tested. We describe the construction and use of novel expression vector pLoxGentrc that contains the strong trc promoter (P(trc)), a multiple cloning site, the T1 and T2 rrnB terminator sequences, the lacI(q) gene and the aacC1 gene conferring gentamicin resistance flanked by two loxP sites. As a demonstration of utility, melanin-producing strains of E. coli were generated employing this vector. Melanin is a polymer synthesized by the enzyme tyrosinase using l-tyrosine as substrate. The melA gene encoding a tyrosinase from Rhizobium etli was ligated to pLoxGentrc to generate pLoxGentrcmelA. This plasmid was transformed into E. coli W3110 to generate a melanin-producing strain. A region from this plasmid including P(trc)melA, T1 and T2 rrnB and the aacC1 gene was amplified by PCR employing primers with 45 b regions of homology to the lacZ gene. The PCR product was electroporated into strain W3110 that expressed the λ-Red enzymes. From this experiment, strain W3110P(tr)(c)melA, was obtained having the melA gene inserted in the lacZ locus. Fermentor cultures with strain W3110/pLoxGentrcmelA grown in the presence and absence of gentamicin as well as W3110P(tr)(c)melA without antibiotic revealed that the latter displays high genetic stability as well as the highest melanin titer. Vector pLoxGentrc should be useful during strain generation processes, enabling direct comparison of plasmid and chromosome-based production systems.

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L-3,4-dihydroxyphenylalanine (L-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of L-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of L-tyrosine (L-Tyr), an L-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on L-Tyr production of PTS inactivation (PTS(-) gluc(+) phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of L-Tyr production (q( L-Tyr)), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q( L-Tyr) in the PTS(+) and the PTS(-) gluc(+) strains, respectively. An 8.6-fold increase in L-Tyr yield from glucose was observed in the PTS(-) gluc(+) tyrR (-) strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for L-Tyr production caused the synthesis of L-DOPA. One of such strains, having the PTS(-) gluc(+) tyrR (-) phenotype, displayed the best production parameters in minimal medium, with a specific rate of L-DOPA production of 13.6 mg/g/h, L-DOPA yield from glucose of 51.7 mg/g and a final L-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of L-DOPA in 50 h.

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We report a study to determine the role of pyruvate oxidase among Escherichia coli isogenic strains with active and inactive phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Strain PB11, displaying a specific growth rate (mu) in glucose minimal medium of 0.1 h(-1) is a ptsHI, crr operon deletion derivative of wild-type JM101 (displaying a mu of 0.70 h(-1)). Strain PB12 is a spontaneous mutant obtained from PB11 after selection for its capacity to grow on glucose with a mu of 0.40 h(-1). In minimal medium cultures supplemented with glucose plus acetate, strain JM101 displayed preferential consumption of glucose, whereas strains PB11 and PB12 did not display glucose catabolic repression of acetate consumption. Inactivation of poxB caused a severe reduction in growth rate in strain PB11 when grown in the fermentor with medium containing glucose or glucose plus acetate, whereas under the same conditions poxB(-)derivative strains of JM101 and PB12 were not affected. Relative transcript levels for 29 genes related to poxB transcriptional regulation and central metabolism were determined using RT-PCR. This analysis revealed 2-fold lower transcript levels for genes encoding subunits of the pyruvate dehydrogenase complex (Pdh) in strain PB11 and 4- to 6-fold higher transcript levels for poxB in strains PB11 and PB12, when compared to JM101. In addition, in the PTS(-) strains, upregulation of the poxB transcription factors rpoS, soxS and marA, was detected. The results presented here strongly suggest that AcCoA is mainly synthesized from acetate produced by pyruvate oxidase in strain PB11, whereas in strains JM101 and PB12, AcCoA is synthesized preferentially from pyruvate by Pdh.

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