Dr. Felix Tobola

Carbohydrates are more than an energy-storage. They are ubiquitously found on cells and most proteins, where they encode biological information. Lectins bind these carbohydrates and are essential for translating the encoded information into biological functions and processes. Hundreds of lectins are known, and they are found in all domains of life. For half a century, researchers have been preparing variants of lectins in which the binding sites are varied. In this way, the traits of the… Mehr anzeigen

Carbohydrates are more than an energy-storage. They are ubiquitously found on cells and most proteins, where they encode biological information. Lectins bind these carbohydrates and are essential for translating the encoded information into biological functions and processes. Hundreds of lectins are known, and they are found in all domains of life. For half a century, researchers have been preparing variants of lectins in which the binding sites are varied. In this way, the traits of the lectins such as the affinity, avidity and specificity towards their ligands as well as their biological efficacy were changed. These efforts helped to unravel the biological importance of lectins and resulted in improved variants for biotechnological exploitation and potential medical applications. This review gives an overview on the methods for the preparation of artificial lectins and complexes thereof and how reducing or increasing the number of binding sites affects their function. Weniger anzeigen

Galectin-1 is a β-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced… Mehr anzeigen

Galectin-1 is a β-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced affinity for 3’-sulfated oligosaccharides. Their interaction with different ligands was further analyzed by fluorescence polarization competition assay. Using molecular modeling we provide structural clues that the change in affinities comes from modulated interactions and solvation patterns. Thus, we show that the introduction of subtle atomic mutations in the ligand binding site of galectin-1 is an attractive approach for fine-tuning its interactions with different ligands. Weniger anzeigen

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional… Mehr anzeigen

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an l-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate l-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors. Weniger anzeigen

Lectins are carbohydrate-binding proteins with specificity for their target ligands. They play diverse roles in cellular recognition and signalling processes, as well as in infections and cancer metastasis. Owing to their specificity, lectins find application in biotechnology and medicine, e.g. for blood group typing, purification of glycoproteins or lipids and as markers that target cancer cells. For some applications, lectins are immobilized on a solid support, or they are conjugated with… Mehr anzeigen

Lectins are carbohydrate-binding proteins with specificity for their target ligands. They play diverse roles in cellular recognition and signalling processes, as well as in infections and cancer metastasis. Owing to their specificity, lectins find application in biotechnology and medicine, e.g. for blood group typing, purification of glycoproteins or lipids and as markers that target cancer cells. For some applications, lectins are immobilized on a solid support, or they are conjugated with other molecules. Classical protein conjugation reactions at nucleophilic amino acids such as cysteine or lysine are often non-selective, and the site of conjugation is difficult to pre-define. Random conjugation, however, can interfere with protein function. Therefore, we sought to equip lectins with a unique reactive handle, which can be conjugated with other molecules in a pre-defined manner. We site-specifically introduced non-canonical amino acids carrying bioorthogonal reactive groups into several lectins. As a proof of principle, we conjugated these ‘clickable lectins’ with small molecules. Furthermore, we conjugated lectins with different ligand specificities with one another to produce superlectins. The ‘clickable lectins’ might be useful for any process where lectins shall be conjugated with another module in a selective, pre-defined and site-specific manner. Weniger anzeigen

Protein-carbohydrate interactions play crucial roles in biology. Understanding and modifying these interactions is of major interest for fighting many diseases. We took a synthetic biology approach and incorporated non-canonical amino acids into a bacterial lectin to modulate its interactions with carbohydrates. We focused on tryptophan that is prevalent in carbohydrate binding sites. The exchange of the tryptophan residues with analogs fluorinated at different positions resulted in three… Mehr anzeigen

Protein-carbohydrate interactions play crucial roles in biology. Understanding and modifying these interactions is of major interest for fighting many diseases. We took a synthetic biology approach and incorporated non-canonical amino acids into a bacterial lectin to modulate its interactions with carbohydrates. We focused on tryptophan that is prevalent in carbohydrate binding sites. The exchange of the tryptophan residues with analogs fluorinated at different positions resulted in three distinctly fluorinated variants of the lectin from Ralstonia solanacearum. We observed differences in stability and affinity towards fucosylated glycans and rationalized them by X-ray and modeling studies. While fluorination decreased the aromaticity of the indole ring and, therefore, the strength of carbohydrate-aromatic interactions, additional weak hydrogen bonds were formed between fluorine and the ligand hydroxyl groups. Our approach opens new possibilities to engineer carbohydrate receptors. Weniger anzeigen

Commercially available phenol derivatives were transformed with pyruvate to form a new C–C bond leading to the corresponding para-coumaric acids and only one molecule of water as an innocent side product in buffer. The reaction was catalysed by a biocatalytic system consisting of two enzymatic steps, which were run simultaneously: (i) in the first step catalysed by a tyrosine phenol lyase the C–C coupling of phenol derivatives with pyruvate and ammonia yielded L-tyrosine derivatives, (ii) which… Mehr anzeigen

Commercially available phenol derivatives were transformed with pyruvate to form a new C–C bond leading to the corresponding para-coumaric acids and only one molecule of water as an innocent side product in buffer. The reaction was catalysed by a biocatalytic system consisting of two enzymatic steps, which were run simultaneously: (i) in the first step catalysed by a tyrosine phenol lyase the C–C coupling of phenol derivatives with pyruvate and ammonia yielded L-tyrosine derivatives, (ii) which were transformed in the second step to the final product via ammonia elimination catalysed by a tyrosine ammonia lyase. The reactions proceeded with exquisite regio- and stereoselectivity yielding just the para-products with the (E)-configuration. The method represents an efficient approach for the direct alkenylation of phenols on a preparative scale (up to 0.6 mmol) affording (E)-para-coumaric acids in excellent isolated yields without requiring chromatographic purification. Co-expression of the involved enzymes in a single host gave access to single catalyst preparation. Weniger anzeigen

To efficiently use lignocellulosic biomass hydrolysates as fermentation media for bioethanol production, besides being capable of producing significant amount of ethanol, the fermenting host should also meet the following two requirements: (1) resistant to the inhibitory compounds formed during biomass pretreatment process, (2) capable of utilizing C5 sugars, such as xylose, as carbon source. In our laboratory, a screening was conducted on microorganisms collected from environmental sources for… Mehr anzeigen

To efficiently use lignocellulosic biomass hydrolysates as fermentation media for bioethanol production, besides being capable of producing significant amount of ethanol, the fermenting host should also meet the following two requirements: (1) resistant to the inhibitory compounds formed during biomass pretreatment process, (2) capable of utilizing C5 sugars, such as xylose, as carbon source. In our laboratory, a screening was conducted on microorganisms collected from environmental sources for their tolerance to hydrolysate inhibitors. A unique resistant strain was selected and identified as Pichia anomala (Wickerhamomyces anomalus), deposited as CBS 132101. The strain is able to produce ethanol in various biomass hydrolysates, both with and without oxygen. Besides, the strain could assimilate xylose and use nitrate as N source. These physiological characteristics make P. anomala an interesting strain for bioethanol production from lignocellulosic biomass hydrolysates. Weniger anzeigen