Octavio T. Ramírez

Production of Recombinant Proteins

Efficient strategies for the production of recombinant proteins are gaining increasing importance, as more applications that require high amounts of high-quality proteins reach the market. Higher production efficiencies and, consequently, lower costs of the final product are needed for obtaining a commercially viable process. In this chapter, common problems in recombinant protein production are reviewed and strategies for their solution are discussed. Such strategies include molecular biology techniques, as well as manipulation of the culture environment. Finally, specific problems relevant to different hosts are discussed.

Living with heterogeneities in bioreactors: understanding the effects of environmental gradients on cells.

The presence of spatial gradients in fundamental culture parameters, such as dissolved gases, pH, concentration of substrates, and shear rate, among others, is an important problem that frequently occurs in large-scale bioreactors. This problem is caused by a deficient mixing that results from limitations inherent to traditional scale-up methods and practical constraints during large-scale bioreactor design and operation. When cultured in a heterogeneous environment, cells are continuously exposed to fluctuating conditions as they travel through the various zones of a bioreactor. Such fluctuations can affect cell metabolism, yields, and quality of the products of interest. In this review, the theoretical analyses that predict the existence of environmental gradients in bioreactors and their experimental confirmation are reviewed. The origins of gradients in common culture parameters and their effects on various organisms of biotechnological importance are discussed. In particular, studies based on the scale-down methodology, a convenient tool for assessing the effect of environmental heterogene ities, are surveyed.

Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

BackgroundEscherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) are capable of coutilizing glucose and other carbon sources due to the absence of catabolite repression by glucose. In these strains, the lack of this important regulatory and transport system allows the coexistence of glycolytic and gluconeogenic pathways. Strains lacking PTS have been constructed with the goal of canalizing part of the phosphoenolpyruvate (PEP) not consumed in glucose transport to the aromatic pathway. The deletion of the ptsHIcrr operon inactivates PTS causing poor growth on this sugar; nonetheless, fast growing mutants on glucose have been isolated (PB12 strain). However, there are no reported studies concerning the growth potential of a PTS- strain in mixtures of different carbon sources to enhance the production of aromatics compounds.ResultsPB12 strain is capable of coutilizing mixtures of glucose-arabinose, glucose-gluconate and glucose-glycerol. This capacity increases its specific growth rate (μ) given that this strain metabolizes more moles of carbon source per unit time. The presence of plasmids pRW300aroGfbrand pCLtktA reduces the μ of strain PB12 in all mixtures of carbon sources, but enhances the productivity and yield of aromatic compounds, especially in the glucose-glycerol mixture, as compared to glucose or glycerol cultures. No acetate was detected in the glycerol and the glucose-glycerol batch fermentations.ConclusionDue to the lack of catabolite repression, PB12 strain carrying multicopy plasmids containing tktA and aroGfbrgenes is capable of coutilizing glucose and other carbon sources; this capacity, reduces its μ but increases the production of aromatic compounds.

Advances in Bioprocess Engineering

Preface. Monitoring and Characterization of Glycoprotein Quality in Animal Cell Cultures X. Gu, et al. Insect Cell Culture: Recent Advances, Bioengineering Challenges and Implications in Protein Production L.A. Palomares, O.T. Ramirez. On-Line Monitoring of Intracellular Properties and its Use in Bioreactor Operation R. Mutharasan. Methods for Plant Genetic Transformation C. Medina. Production of a New Antimycotic Agent by Plant Cell Culture M.L. Villarreal, et al. Microbial Physiology Applied to Process Optimisation: Lactic Acid Bacteria J.M. Bruno-Barcena, et al. Optimization of Interferon Production in Yeast by Strict Ethanol Control M. Griot, et al. Strategies for the Selection of Mold Strains Geared to Produce Enzymes on Solid Substrates G. Viniegra-Gonzalez. The Effect of Hydrodynamics on Biological Materials C.R. Thomas, Z. Zhang. Novel Processes for the Enzymatic Synthesis of Surfactants P.F. Monsan. Hemoproteins as Biocatalysts for the Oxidation of Polycyclic Aromatic Hydrocarbons R. Vazquez-Duhalt. Bioremediation in the Marine Environment R.A. Zilinskas. Biotechnology in the Mining Industry. Fundamental and Applied Aspects of Processes for Metal Extraction R.W. Lawrence. An Assessment of Droplet-Air Contact and Spray Drying Performance in Bioprocess Engineering G. Gutierrez, et al. Index.

Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters

The temperature inducible expression system, based on the pL and/or pR phage lambda promoters regulated by the thermolabile cI857 repressor has been widely use to produce recombinant proteins in prokariotic cells. In this expression system, induction of heterologous protein is achieved by increasing the culture temperature, generally above 37°C. Concomitant to the overexpression of heterologous protein, the increase in temperature also causes a variety of complex stress responses. Many studies have reported the use of such temperature inducible expression system, however only few discuss the simultaneous stress effects caused by recombinant protein production and the up-shift in temperature. Understanding the integral effect of such responses should be useful to develop improved strategies for high yield protein production and recovery. Here, we describe the current status of the heat inducible expression system based on the pL and/or pR λ phage promoters, focusing on recent developments on expression vehicles, the stress responses at the molecular and physiological level that occur after heat induction, and bioprocessing factors that affect protein overexpression, including culture operation variables and induction strategies.

Effect of pH in the synthesis of ampicillin by penicillin acylase

Recombinant Escherichia coli cells with high penicillin acylase (PA) activity were immobilized by gel entrapment with agar. This biocatalyst was used to study the effect of pH on the synthesis of ampicillin from phenylglycine methylester (PGME) and 6-aminopenicillanic acid (6-APA). The parallel hydrolysis reactions of PGME and ampicillin were also studied. A selective inhibition of the hydrolysis of the ester was possible by controlling the pH at 6.0. At such conditions, and using 6-APA solutions ranging from 50–200 mm, a 75% conversion to ampicillin was obtained. This yield was higher than obtained with other strategies. The reaction kinetics was described by a second-order model for ampicillin synthesis with experimentally determined Michaelis-Menten constants of 27 and 25 mm for 6-APA and PGME, respectively. In addition, ampicillin and PGME were hydrolyzed by the enzyme following Michaelis-Menten kinetics with Km values of 40.5 and 30 mm, respectively. A good correlation was found between experimental results of synthesis reactions and the kinetic model derived from initial rate experiments with only slight deviations at high substrate concentrations. This is the first report where the specific effect of pH on the synthesis of ampicillin was studied in detail. It is shown that, by controlling the pH, it is possible to inhibit the lateral undesirable reactions increasing the yield of the main reaction.

Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

BackgroundShikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis.ResultsBatch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (μ) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the μ decreased, whereas aromatic intermediate production reached its maximum. The double aroK-aroL- mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in μ, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol).ConclusionsThe effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS- background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and pykF as part of a strategy to improve SA production and its aromatic precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.

New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol

BackgroundGlycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes.ResultsTranscriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroGfbrtktA, when growing on glycerol, as compared to glucose.ConclusionsThe overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.

The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture.

AbstractDissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated μmax and