Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures

Background: The E. coli lac operon and its components have been studied for decades, and lac-derived systems are widely used for recombinant protein production. However, lac operon dynamics and induction behavior remain the paradigm of gene regulation. Recently, an HPLC-MS-based method to quantify IPTG in the medium and inside the biomass has been established, and this tool may be useful to uncover the lack of knowledge and allow optimization of biotechnological processes. Results: The results obtained from the study of IPTG distribution profiles in fed-batch, high cell density cultures allowed discrimination between two different depletion patterns of an inducer from the medium to the biomass in E. coli-expressing rhamnulose-1-phosphate aldolase (RhuA). Moreover, we could demonstrate that active transport mediates the uptake of this gratuitous inducer. Additionally, we could study the induction behaviors of this expression system by taking into account the biomass concentration at the induction time. Conclusions: In the bistable range, partial induction occurred, which led to intermediate levels of RhuA activity. There was a direct relationship between the initial inducer concentrations and the initial inducer transport rate together with the specific activity. A majority of the inducer remains in the medium to reach equilibrium with the intracellular level. The intracellular inducer accumulation was a further evidence of bistability of the lac operon

Sistema de control per a la producció microbiana de proteïnes

El Departament d'Enginyeria Química de la UAB treballa en el desenvolupament de noves tecnologies per millorar, tant qualitativament com quantitativa, la producció de proteïnes recombinants -obtingudes mitjançant modificació genètica d'un organisme-. Ara acaba de presentar un sistema de control per automatitzar tot el procés.El Departament d'Enginyeria Química de la UAB trabaja en el desarrollode nuevas tecnologías para mejorar, tanto cualitativa comocuantitativamente, la producción de proteínas recombinantes -obtenidasmediante modificación genética de un microorganismo-. Ahora acaba depresentar un nuevo sistema de control para automatizar todo el proceso

Chloroperoxidase-catalyzed aminoalcohol oxidation : substrate specificity and novel strategy for the synthesis of N-Cbz-3-aminopropanal

The ability of chloroperoxidase (CPO) to catalyze amino alcohol oxidations was investigated. The oxidations of compounds with different configurations with respect to the amine position towards hydroxyl - using H₂O₂ and tert-butyl hydroperoxide (t-BuOOH) - were analyzed in terms of the initial reaction rate, substrate conversion, and CPO operational stability. It was observed that the further the amino group from the hydroxyl, the lower the initial reaction rate. The effect of the amino-protecting group and other substituents (i.e., methyl and hydroxyl) was also examined, revealing an increase in steric hindrance due to the effect of bulky substituents. The observed reaction rates were higher with t-BuOOH, whereas CPO was more stable with H₂O₂. Moreover, CPO stability had to be determined case by case as the enzyme activity was modulated by the substrate. The oxidation of N-Cbz-3-aminopropanol (Cbz, carboxybenzyl) to N-Cbz-3-aminopropanal was investigated. Main operational conditions such as the reaction medium, initial amino alcohol concentration, and peroxide nature were studied. The reaction kinetics was determined, and no substrate inhibition was observed. By-products from a chemical reaction between the formed amino aldehyde and the peroxide were identified, and a novel reaction mechanism was proposed. Finally, the biotransformation was achieved by reducing side reactions and identifying the key factors to be addressed to further optimize the product yield

Multi-reaction kinetic modeling for the peroxidase-aldolase cascade synthesis of a D-fagomine precursor

Altres ajuts: Acord transformatiu CRUE-CSICThe feasibility of a peroxidase-aldolase cascade reaction for the synthesis of therapeutically-valuable iminocyclitols is discussed herein. A two-enzyme system consisting of chloroperoxidase (CPO) and D-fructose-6-phosphate aldolase (FSA) was evaluated for the synthesis of a D-fagomine precursor (preFagomine) from a N-Cbz-3-aminopropanol. An in-depth, systematic, step-by-step kinetic modeling of seven reactions and two inactivation decays was proposed to elucidate the reaction mechanism, prepare suitable stabilized biocatalysts, and find the optimal conditions for its application. The model described accurately the data and predicted the outcome at different experimental conditions. The inactivation of FSA caused by CPO was identified as the main bottleneck in the reaction. A two-step reaction approach and the use of immobilized enzymes on magnetic nanoparticle clusters and functionalized agarose carriers increased the stability of FSA, with an 1839-fold higher preFagomine formation per mol of enzyme in comparison to a one-pot reaction using soluble enzymes

HLADH-catalyzed synthesis of β-amino acids, assisted by continuous electrochemical regeneration of NAD+ in a filter press microreactor

This work focuses on the use of electrochemical microreactors, applied to the direct electroregeneration of nicotinamide dinucleotide (NAD+) used in-situ for the enzymatically-assisted oxidation of the β-alanine. Mechanistic and chemical/electrochemical reactor approaches were investigated, to achieve satisfactory conversions (>80%) of the substrate in reasonable (1–2 days) reaction times; no fouling observed of the gold anode. An original method was proposed to limit the complexation effect of the produced β-alanine to the enzyme. Simulations of the overall system were achieved and an estimation of the required kinetic and Michaelis constants was proposed

Zoonotic "Enterocytozoon bieneusi" genotypes in free-ranging and farmed wild ungulates in Spain

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animal

Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli

First paragraph (this article has no abstract) Antibiotics and antibiotics resistance genes have been traditionally used for the selection and maintenance of recombinant plasmids in hosts such as E. coli. Although a powerful selection tool, their use has been considered unacceptable in many areas of biotechnology by regulatory authorities. Indeed, there is much international scientific and regulatory focus on this issue [1]. For instance, the use of selection markers that confer resistance to antibiotics in vaccine plasmids may introduce the risk of transforming the patient's microflora and spread resistance genes. Moreover, in recombinant protein production for therapeutic use, the antibiotic must be eliminated from the final product. Another problem arises from the potential loss of selective pressure as a result of antibiotic degradation i.e. ampicillin can be degraded by β-lactamases in less than 30 minutes in high cell density cultures [2].Peer reviewe

Immobilized l-aspartate ammonia-lyase from Bacillus sp. YM55-1 as biocatalyst for highly concentrated l-aspartate synthesis

L-Aspartate ammonia-lyase from Bacillus sp. YM55-1 (AspB, EC 4.3.1.1) catalyzes the reversible conversion of L-aspartate (Asp) into fumarate and ammonia with a high specific activity toward the substrate. AspB was expressed in Escherichia coli and partially purified by heat precipitation and saturation with ammonium sulfate reaching purification factor of 7.7 and specific activity of 334 U/mg of protein. AspB was immobilized by covalent attachment on Eupergit® C (epoxy support) and MANA-agarose (amino support), and entrapment in LentiKats® (polyvinyl alcohol) with retained activities of 24, 85 and 63 %, respectively. Diffusional limitations were only observed for the enzyme immobilized in LentiKats® and were overcome by increasing substrate concentration. Free and immobilized AspB were used for the synthesis of aspartate achieving high product concentration (≥450 mM) after 24 h of reaction. Immobilized biocatalysts were efficiently reused in 5 cycles of Asp synthesis, keeping over 90 % of activity and reaching over 90 % of conversion in all the cases