Natividad Cabrera-Valladares

University of Queensland , Brisbane, Queensland, Australia

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Publications (6)13.1 Total impact

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    ABSTRACT: The glycolytic intermediate phosphoenolpyruvate (PEP) is a precursor of several cellular components, including various aromatic compounds. Modifications to the PEP node such as PEP:sugar phosphotransferase system (PTS) or pyruvate kinase inactivation have been shown to have a positive effect on aromatics production capacity in Escherichia coli and Bacillus subtilis. In this study, pyruvate kinase and PTS-deficient B. subtilis strains were employed for the construction of derivatives lacking shikimate kinase activity that accumulate two industrially valuable chemicals, the intermediates of the common aromatic pathway, shikimic and dehydroshikimic acids. The pyruvate kinase-deficient strain (CLC6-PYKA) showed the best production parameters under resting-cell conditions. Compared to the PTS-deficient strain, the shikimic and dehydroshikimic acids specific production rates for CLC6-PYKA were 1.8- and 1.7-fold higher, respectively. A batch fermentor culture using complex media supplemented with 83 g/l of glucose was developed with strain CLC6-PYKA, where final titers of 4.67 g/l (shikimic acid) and 6.2 g/l (dehydroshikimic acid) were produced after 42 h. © 2013 S. Karger AG, Basel.
    Journal of Molecular Microbiology and Biotechnology 10/2013; 24(1):37-45. · 1.95 Impact Factor
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    ABSTRACT: The phosphoenolpyruvate (PEP) node is an important carbon distribution point in the central metabolic networks; therefore, its modification is a common strategy employed for developing microbial production strains. In this study, mutants of Bacillus subtilis 168 were generated with deletions of pykA (which encodes pyruvate kinase), ptsG (which encodes the glucose-specific IICBA(Glc) component) or the ptsGHI operon [which encodes IICBA(Glc), HPr protein and enzyme I from the PEP:sugar phosphotransferase system (PTS)]. These modifications caused a reduction in the initial rate of [(14)C]-glucose import, corresponding to 10.99, 2.83 and 0.50% of that found in B. subtilis 168 for strains with inactive pykA, ptsG or ptsGHI genes, respectively. Characterization of derivative strains lacking 3-dehydroquinate synthase activity showed that inactivation of pykA leads to an 8-fold increase in carbon flow to the common aromatic pathway. Quantitative real-time PCR analyses of 76 genes from several functional classes revealed a carbon starvation transcriptional pattern that includes a partial gluconeogenic response and overexpression of genes encoding non-PTS glucose importers in the strains lacking functional pykA, ptsG or ptsGHI genes. A transcriptional response consistent with pyruvate limitation was also detected, which includes upregulation of genes encoding malic enzymes that generate pyruvate from malate.
    Journal of Molecular Microbiology and Biotechnology 07/2012; 22(3):177-97. · 1.95 Impact Factor
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    ABSTRACT: Anthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products. Chemical synthesis of anthranilate is an unsustainable process since it implies the use of nonrenewable benzene and the generation of toxic by-products. In Escherichia coli anthranilate is synthesized from chorismate by anthranilate synthase (TrpED) and then converted to phosphoribosyl anthranilate by anthranilate phosphoribosyl transferase to continue the tryptophan biosynthetic pathway. With the purpose of generating a microbial strain for anthranilate production from glucose, E. coli W3110 trpD9923, a mutant in the trpD gene that displays low anthranilate producing capacity, was characterized and modified using metabolic engineering strategies. Sequencing of the trpED genes from E. coli W3110 trpD9923 revealed a nonsense mutation in the trpD gene, causing the loss of anthranilate phosphoribosyl transferase activity, but maintaining anthranilate synthase activity, thus causing anthranilate accumulation. The effects of expressing genes encoding a feedback inhibition resistant version of the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (aroGfbr), transketolase (tktA), glucokinase (glk) and galactose permease (galP), as well as phosphoenolpyruvate:sugar phosphotransferase system (PTS) inactivation on anthranilate production capacity, were evaluated. In shake flask experiments with minimal medium, strains W3110 trpD9923 PTS- and W3110 trpD9923/pJLBaroGfbrtktA displayed the best production parameters, accumulating 0.70-0.75 g/L of anthranilate, with glucose-yields corresponding to 28-46% of the theoretical maximum. To study the effects of extending the growth phase on anthranilate production a fed-batch fermentation process was developed using complex medium, where strain W3110 trpD9923/pJLBaroGfbrtktA produced 14 g/L of anthranilate in 34 hours. This work constitutes the first example of a microbial system for the environmentally-compatible synthesis of anthranilate generated by metabolic engineering. The results presented here, including the characterization of mutation in the trpD gene from strain W3110 trpD9923 and the development of a fermentation strategy, establish a step forward towards the future improvement of a sustainable process for anthranilate production. In addition, the present work provides very useful data regarding the positive and negative consequences of the evaluated metabolic engineering strategies.
    Microbial Cell Factories 05/2009; 8:19. · 3.31 Impact Factor
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    ABSTRACT: Pseudomonas aeruginosa produces the biosurfactants rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs). In this study, we report the production of one family of rhamnolipids, specifically the monorhamnolipids, and of HAAs in a recombinant Escherichia coli strain expressing P. aeruginosa rhlAB operon. We found that the availability in E. coli of dTDP-L: -rhamnose, a substrate of RhlB, restricts the production of monorhamnolipids in E. coli. We present evidence showing that HAAs and the fatty acid dimer moiety of rhamnolipids are the product of RhlA enzymatic activity. Furthermore, we found that in the recombinant E. coli, these compounds have the same chain length of the fatty acid dimer moiety as those produced by P. aeruginosa. These data suggest that it is RhlAB specificity, and not the hydroxyfatty acid relative abundance in the bacterium, that determines the profile of the fatty acid moiety of rhamnolipids and HAAs. The rhamnolipids level produced in recombinant E. coli expressing rhlAB is lower than the P. aeruginosa level and much higher than those reported by others in E. coli, showing that this metabolic engineering strategy lead to an increased rhamnolipids production in this heterologous host.
    Applied Microbiology and Biotechnology 12/2006; 73(1):187-94. · 3.69 Impact Factor
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    ABSTRACT: A parametric study was conducted to define optimum conditions to achieve high yields in the conversion of tyrosine to eumelanin (EuMel) using recombinant Escherichia coli. Escherichia coli W3110 (pTrcMutmelA) expressing the tyrosinase coding gene from Rhizobium etli and glucose-mineral media were used to transform tyrosine into EuMel. Batch aerobic fermentor cultures were performed to study the effect of temperature, pH and inducer concentration (isopropyl-D-thio-galactopyranoside) on melanin production. Under optimum conditions, 0.1 mmol l(-1) of isopropyl-D-thio-galactopyranoside, temperature of 30 degrees C, and changing pH from 7.0 to 7.5 during the production phase, a 100% conversion of tyrosine into EuMel is obtained. Furthermore, tyrosine feeding allowed us to obtain the highest level (6 g l(-1)) of EuMel produced by recombinant E. coli reported until now. The most important factors affecting melanin formation and hence influencing the rate and efficiency in the conversion of tyrosine into EuMel in this system, are the temperature and pH. Maximum theoretical yield was obtained using a simple culture process and mineral media to convert tyrosine (a medium value compound) into melanin, a high value compound. The process reported here avoids the use of purified tyrosinase, expensive chemical methods or the cumbersome extraction of this polymer from animal or plant tissues.
    Journal of Applied Microbiology 12/2006; 101(5):1002-8. · 2.20 Impact Factor
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    ABSTRACT: The gene melA from the nitrogen-fixing bacterium Rhizobium etli CFN42 was amplified using PCR, cloned in the expression vector pTtrc99A to obtain plasmid pTrcmelA, and transformed into E. coli strain W3110. The resulting recombinant strain W3110/pTrcmelA synthesized a dark pigment when growing in solid or liquid media containing l-tyrosine and copper. This pigment was identified as melanin by comparing it with analytical grade melanin using a spectrophotometric assay. Melanin was synthesized when recombinant E. coli cells were incubated at 30 °C; however, at 37 °C significantly less polymer was produced. The recombinant tyrosinase expressed intracellularly in E. coli was purified 40-fold with a 25% yield from a cell extract by ammonium sulfate precipitation and ion exchange chromatography. With the partially purified tyrosinase, the Km for l-dopa and l-tyrosine were determined as 2.44 and 0.19 mM, respectively. Temperature and pH for maximum activity were 50 °C and 6.5–7.5, respectively. Activation energy for thermal inactivation (50.77 kJ/mol; using l-dopa as substrate at pH 7) and half-life values indicate a higher thermal stability of R. etli tyrosinase in comparison with mushroom tyrosinase. Interestingly, for a bacterial tyrosinase, MelA showed an unusually higher activity for l-tyrosine than for l-dopa.
    Enzyme and Microbial Technology. 01/2006;

Publication Stats

72 Citations
13.10 Total Impact Points


  • 2013
    • University of Queensland 
      • Australian Institute for Bioengineering and Nanotechnology
      Brisbane, Queensland, Australia
  • 2009–2012
    • Universidad Nacional Autónoma de México
      • Institute of Biotechnology
      Mexico City, The Federal District, Mexico
    • Universidad Autónoma del Estado de Morelos
      • Centre of Biotechnological Research (CEIB)
      Cuernavaca, Morelos, Mexico