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Cupriavidus necator H16 gains increasing attention in microbial research and biotechnological application due to its diverse metabolic features. Here we present a tightly controlled gene expression system for C. necator including the pBBR1-vector that contains hybrid promoters originating from C. necator native tolC-promoter in combination with a synthetic tetO-operator. The expression of the reporter gene from these plasmids relies on the addition of the exogenous inducer doxycycline (dc). The novel expression system offers a combination of advantageous features as; (i) high and dose-dependent recombinant protein production, (ii) tight control with a high dynamic range (On/Off ratio), which makes it applicable for harmful pathways or for toxic protein production, (iii) comparable cheap inducer (doxycycline, dc), (iv) effective at low inducer concentration, that makes it useful for large scale application, (v) rapid, diffusion controlled induction, and (vi) the inducer does not interfere within the cell metabolism. As applications of the expression system in C. necator H16, the growth ability on glycerol was enhanced by constitutively expressing the E. coli glpk gene-encoding for glycerol kinase. Likewise, we used the system to overcome the expression toxicity of mevalonate pathway in C. necator H16. With this system, the mevalonate-genes were successfully introduced in the host and the recombinant strains could produce about 200 mg/l mevalonate.
The immobilization of NAD+-dependent dehydrogenases, in combination with a diaphorase, enables the facile development of multiparametric sensing devices. In this work, an amperometric biosensor array for simultaneous determination of ethanol, formate, d- and l-lactate is presented. Enzyme immobilization on platinum thin-film electrodes was realized by chemical cross-linking with glutaraldehyde. The optimization of the sensor performance was investigated with regard to enzyme loading, glutaraldehyde concentration, pH, cofactor concentration and temperature. Under optimal working conditions (potassium phosphate buffer with pH 7.5, 2.5 mmol L-1 NAD+, 2.0 mmol L-1 ferricyanide, 25 °C and 0.4% glutaraldehyde) the linear working range and sensitivity of the four sensor elements was improved. Simultaneous and cross-talk free measurements of four different metabolic parameters were performed successfully. The reliable analytical performance of the biosensor array was demonstrated by application in a clarified sample of inoculum sludge. Thereby, a promising approach for on-site monitoring of fermentation processes is provided.
Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM−1 (L-malate biosensor) and 0.4 μA mM−1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM−1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.
The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33–43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12 U/mg and 23 U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115 U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74 U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development.
Regardless of size or destination, synthetic biology starts with com-parably small information units, which need to be combined and properly arranged in order to achieve a certain goal. This may be the de novo synthesis of individual genes from oligonucleotides, a shuffling of protein domains in order to create novel biocatalysts, the assembly of multiple enzyme encoding genes in metabolic pathway design, or strain development at the production stage. The CoLibry concept has been designed in order to close the gap between recombinant production of individual genes and genome editing.
An enzyme-based multi-parameter biosensor is developed for monitoring the concentration of formate, d-lactate, and l-lactate in biological samples. The sensor is based on the specific dehydrogenation by an oxidized β-nicotinamide adenine dinucleotide (NAD+)-dependent dehydrogenase (formate dehydrogenase, d-lactic dehydrogenase, and l-lactic dehydrogenase, respectively) in combination with a diaphorase from Clostridium kluyveri (EC 1.8.1.4). The enzymes are immobilized on a platinum working electrode by cross-linking with glutaraldehyde (GA). The principle of the determination scheme in case of l-lactate is as follows: l-lactic dehydrogenase (l-LDH) converts l-lactate into pyruvate by reaction with NAD+. In the presence of hexacyanoferrate(III), the resulting reduced β-nicotinamide adenine dinucleotide (NADH) is then regenerated enzymatically by diaphorase. The electrochemical detection is based on the current generated by oxidation of hexacyanoferrate(II) at an applied potential of +0.3 V vs. an Ag/AgCl reference electrode. The biosensor will be electrochemically characterized in terms of linear working range and sensitivity. Additionally, the successful practical application of the sensor is demonstrated in an extract from maize silage.