Editorial: Genomic strategies for efficient microbial cell factories

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The effect of heating rate on Escherichia coli metabolism, physiological stress, transcriptional response, and production of temperature-induced recombinant protein: a scale-down study

At the laboratory scale, sudden step increases from 30 to 42\ub0C can be readily accomplished when expressing heterologous proteins in heat-inducible systems. However, for large scale-cultures only slow ramp-type increases in temperature are possible due to heat transfer limitations, where the heating rate decreases as the scale increases. In this work, the transcriptional and metabolic responses of a recombinant Escherichia coli strain to temperature-induced synthesis of pre-proinsulin in high cell density cultures were examined at different heating rates. Heating rates of 6, 1.7, 0.8, and 0.4\ub0C/min were tested in a scale-down approach to mimic fermentors of 0.1, 5, 20, and 100 m3, respectively. The highest yield and concentration of recombinant protein was obtained for the slowest heating rate. As the heating rate increased, the yield and maximum recombinant protein concentration decreased, whereas a larger fraction of carbon skeletons was lost as acetate, lactate, and formate. Compared to 30\ub0C, the mRNA levels of selected heat-shock genes at 38 and 42\ub0C, as quantified by qRT-PCR, increased between 2- to over 42-fold when cultures were induced at 6, 1.7, and 0.8\ub0C/min, but no increase was observed at 0.4\ub0C/min. Only small increases (between 1.5- and 4-fold) in the expression of the stress genes spoT and relA were observed at 42\ub0C for cultures induced at 1.7 and 6\ub0C/min, suggesting that cells subjected to slow temperature increases can adapt to stress. mRNA levels of genes from the transcription–translation machinery (tufB, rpoA, and tig) decreased between 40% and 80% at 6, 1.7 and 0.8\ub0C/min, whereas a transient increase occurred for 0.4\ub0C/min at 42\ub0C. mRNA levels of the gene coding for pre-proinsulin showed a similar profile to transcripts of heat-shock genes, reflecting a probable analogous induction mechanism. Altogether, the results obtained indicate that slow heating rates, such as those likely to occur in conventional large-scale fermentors, favored heterologous protein synthesis by the thermo-inducible expression system used in this report. Knowledge of the effect of heating rate on bacterial physiology and product formation is useful for the rational design of scale-down and scale-up strategies and optimum recombinant protein induction schemes. Biotechnol. Bioeng. 2009;102: 468–482. \ua9 2008 Wiley Periodicals, Inc

High cell-density cultivation in batch mode for plasmid DNA production by a metabolically engineered E. coli strain with minimized overflow metabolism

Progress on plasmid-based (pDNA) vaccines requires simpler and efficient cultivation techniques for their production. A prevalent problem in the cultivation of Escherichia coli (the main host for pDNA vaccines production) is the aerobic production of acetate. In this work, a metabolically engineered Escherichia coli strain with strongly reduced acetate formation was tested for the production of a plasmid vaccine at high cell-densities. The wild type (W3110) and engineered (VH33) strains were cultivated in batch mode using 100g/L of initial glucose concentration. This elevated amount of glucose allowed attaining high cell-densities of strain VH33 without external substrate feeding, simplifying the cultivation process. While W3110 produced 17mg/L of pDNA and 5.3g/L of acetate, VH33 reached 40mg/L of pDNA and only 2g/L of acetate. While the plasmid supercoiling degree progressively decreased in W3110 cultivations, it remained nearly constant for VH33. These results show the successful application of cell engineering concepts for improving DNA vaccine production processes

High cell-density cultivation in batch mode for plasmid DNA production by a metabolically engineered E. coli strain with minimized overflow metabolism

Progress on plasmid-based (pDNA) vaccines requires simpler and efficient cultivation techniques for their production. A prevalent problem in the cultivation of Escherichia coli (the main host for pDNA vaccines production) is the aerobic production of acetate. In this work, a metabolically engineered Escherichia coli strain with strongly reduced acetate formation was tested for the production of a plasmid vaccine at high cell-densities. The wild type (W3110) and engineered (VH33) strains were cultivated in batch mode using 100g/L of initial glucose concentration. This elevated amount of glucose allowed attaining high cell-densities of strain VH33 without external substrate feeding, simplifying the cultivation process. While W3110 produced 17mg/L of pDNA and 5.3g/L of acetate, VH33 reached 40mg/L of pDNA and only 2g/L of acetate. While the plasmid supercoiling degree progressively decreased in W3110 cultivations, it remained nearly constant for VH33. These results show the successful application of cell engineering concepts for improving DNA vaccine production processes

Escalamiento descendente del proceso de producción de proteína heteróloga por termo-inducción de cultivos de alta densidad celular de Escherichia coli : estudio de la respuesta transcripcional al choque térmico /

 tesis que para obtener el grado de Doctor en Ciencias Bioquímicas, presenta Luis Caspeta Guadarrama ; asesor Octavio Tonatiuh Ramírez Reivich. 122 páginas : ilustraciones. Doctorado en Ciencias Bioquímicas UNAM, Instituto de Biotecnología, 200

The yeastGemMap: A process diagram to assist yeast systems-metabolic studies

Visualization is a key aspect of the analysis of omics data. Although many tools can generate pathway maps for visualization of yeast metabolism, they fail in reconstructing genome-scale metabolic diagrams of compartmentalized metabolism. Here we report on the yeastGemMap, a process diagram of whole yeast metabolism created to assist data analysis in systems-metabolic studies. The map was manually reconstructed with reactions from a compartmentalized genome-scale metabolic model, based on biochemical process diagrams typically found in educational and specialized literature. The yeastGemMap consists of 3815 reactions representing 1150 genes, 2742 metabolites, and 14 compartments. Computational functions for adapting the graphical representation of the map are also reported. These functions modify the visual representation of the map to assist in three systems-metabolic tasks: illustrating reaction networks, interpreting metabolic flux data, and visualizing omics data. The versatility of the yeastGemMap and algorithms to assist visualization of systems-metabolic data was demonstrated in various tasks, including for single lethal reaction evaluation, flux balance analysis, and transcriptomic data analysis. For instance, visual interpretation of metabolic transcriptomes of thermally evolved and parental yeast strains allowed to demonstrate that evolved strains activate a preadaptation response at 30 degrees C, which enabled thermotolerance. A quick interpretation of systems-metabolic data is promoted with yeastGemMap visualizations

Enzymatic hydrolysis at high-solids loadings for the conversion of agave bagasse to fuel ethanol

Agave bagasse is the lignocellulosic residue accumulated during the production of alcoholic beverages in Mexico and is a potential feedstock for the production of biofuels. A factorial design was used to investigate the effect of temperature, residence time and concentrations of acid and ethanol on ethanosolv pretreatment and enzymatic hydrolysis of agave bagasse. This method and the use of a stirred in-house-made mini-reactor increased the digestibility of agave bagasse from 30% observed with the dilute-acid method to 98%; also allowed reducing the quantity of enzymes used to hydrolyze samples with solid loadings of 30%. w/w and glucose concentrations up to 225. g/L were obtained in the enzymatic hydrolysates. Overall this process allows the recovery of 91% of the total fermentable sugars contained in the agave bagasse (0.51. g/g) and 69% of total lignin as co-product (0.11. g/g). The maximum ethanol yield under optimal conditions using an industrial yeast strain for the fermentation was 0.25. g/g of dry agave bagasse, which is 86% of the maximum theoretical (0.29. g/g). The effect of the glucose concentration and solid loading on the conversion of cellulose to glucose is discussed, in addition to prospective production of about 50. million liters of fuel ethanol using agave bagasse residues from the tequila industry as a potential solution to the disposal problems