FG Biokatalyse

29 Items

Recent Submissions
Towards the creation of synthetic Escherichia coli via Tryptophan and Methionine substitutions

Tolle, Isabella (2021)

Billions of years of evolution have produced extant living organisms with a vast biodiversity and the ability to adapt to changing environments. At the foundation of it all lies the central dogma of molecular biology, according to which the information flows from the information storage polymer DNA to RNA (“informational polymers”), which is finally translated into proteins (“catalytic polymers...

Anticipating alien cells with alternative genetic codes: away from the alanine world!

Kubyshkin, Vladimir ; Budisa, Nediljko (2019-07-03)

Can we make life with a different genetic amino acid repertoire? Can we expect organisms which would keep newly given genetic code associations permanently? To address these questions, we would like to analyze the existent genetic code amino acid repertoire as formed from derivatives of alanine. Derivation from alanine leads to the α-helix based biological world, the Alanine World, whereas vari...

An Engineered Escherichia coli Strain with Synthetic Metabolism for in‐Cell Production of Translationally Active Methionine Derivatives

Schipp, Christian Johannes ; Ma, Ying ; Al‐Shameri, Ammar ; D'Alessio, Federico ; Neubauer, Peter ; Contestabile, Roberto ; Budisa, Nediljko ; di Salvo, Martino Luigi (2020-10-13)

In the last decades, it has become clear that the canonical amino acid repertoire codified by the universal genetic code is not up to the needs of emerging biotechnologies. For this reason, extensive genetic code re‐engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploi...

Conjugation of Synthetic Polyproline Moietes to Lipid II Binding Fragments of Nisin Yields Active and Stable Antimicrobials

Deng, Jingjing ; Viel, Jakob H. ; Kubyshkin, Vladimir ; Budisa, Nediljko ; Kuipers, Oscar P. (2020-11-20)

Coupling functional moieties to lantibiotics offers exciting opportunities to produce novel derivatives with desirable properties enabling new functions and applications. Here, five different synthetic hydrophobic polyproline peptides were conjugated to either nisin AB (the first two rings of nisin) or nisin ABC (the first three rings of nisin) by using click chemistry. The antimicrobial activi...

Synthesis of New Aza‐ and Thia‐Crown Ether Based Amino Acids

Schneider, Tobias ; Brüssow, Nico ; Yuvanc, Alev ; Budisa, Nediljko (2020-03-05)

Crown ethers have been known since the middle of the 20th century as metal chelating agents with numerous applications. For example, amino acids functionalized with crown ethers have been used to create artificial ion channels. However, only a few of those crown amino acids are known, all of which contain exclusively oxygen as heteroatoms in the crown ether moiety. Alternative structures may be...

Combating Antimicrobial Resistance With New-To-Nature Lanthipeptides Created by Genetic Code Expansion

Karbalaei-Heidari, Hamid Reza ; Budisa, Nediljko (2020-11-05)

Due to the rapid emergence of multi-resistant bacterial strains in recent decades, the commercially available effective antibiotics are becoming increasingly limited. On the other hand, widespread antimicrobial peptides (AMPs) such as the lantibiotic nisin has been used worldwide for more than 40 years without the appearance of significant bacterial resistance. Lantibiotics are ribosomally synt...

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

Tseng, Hsueh-Wei ; Baumann, Tobias ; Sun, Huan ; Wang, Yane-Shih ; Ignatova, Zoya ; Budisa, Nediljko (2020-09-25)

In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly wi...

Genetic code expansion for ageing study

Sun, Huan (2020)

Halogen modified Tyr analogs (HMYs) are typical products of the Tyr residue oxidation in proteins. Their occurrence may lead to destabilized protein integrity and functions, ultimately contributing to ageing processes[1]. In this study, I investigated the effects of the HMYs on model proteins. The samples were produced by exploiting the genetic code expansion methodology. The eukaryotic gene re...

Discovery and Characterization of a New Cold-Active Protease From an Extremophilic Bacterium via Comparative Genome Analysis and in vitro Expression

Perfumo, Amedea ; Saß, Georg Johannes Freiherr von ; Nordmann, Eva-Lena ; Budisa, Nediljko ; Wagner, Dirk (2020-05-13)

Following a screening of Antarctic glacier forefield-bacteria for novel cold-active enzymes, a psychrophilic strain Psychrobacter sp. 94-6PB was selected for further characterization of enzymatic activities. The strain produced lipases and proteases in the temperature range of 4–18°C. The coding sequence of an extracellular serine-protease was then identified via comparative analysis across Psy...

Bilayer thickness determines the alignment of model polyproline helices in lipid membranes

Kubyshkin, Vladimir ; Grage, Stephan L. ; Ulrich, Anne S. ; Budisa, Nediljko (2019-09-18)

Our understanding of protein folds relies fundamentally on the set of secondary structures found in the proteomes. Yet, there also exist intriguing structures and motifs that are underrepresented in natural biopolymeric systems. One example is the polyproline II helix, which is usually considered to have a polar character and therefore does not form membrane spanning sections of membrane protei...

The Alanine World Model for the Development of the Amino Acid Repertoire in Protein Biosynthesis

Kubyshkin, Vladimir ; Budisa, Nediljko (2019-11-05)

A central question in the evolution of the modern translation machinery is the origin and chemical ethology of the amino acids prescribed by the genetic code. The RNA World hypothesis postulates that templated protein synthesis has emerged in the transition from RNA to the Protein World. The sequence of these events and principles behind the acquisition of amino acids to this process remain elu...

Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine

Baumann, Tobias ; Hauf, Matthias ; Richter, Florian ; Albers, Suki ; Möglich, Andreas ; Ignatova, Zoya ; Budisa, Nediljko (2019-05-11)

Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5–7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome ...

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation

Nojoumi, Saba ; Ma, Ying ; Schwagerus, Sergej ; Hackenberger, Christian P. R. ; Budisa, Nediljko (2019-05-09)

In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine co...

Promotion of the collagen triple helix in a hydrophobic environment

Kubyshkin, Vladimir ; Budisa, Nediljko (2019-02-06)

In contrast to many other water-soluble peptide arrangements, the formation of a triple helix in collagen proceeds inside out: polar glycyl residues form the interior, whereas nonpolar prolyl side chains constitute the exterior. In our work, we decided to exploit this aspect of the peptide architecture in order to create hyperstable collagen mimicking peptides (CMPs). The key element of this st...

Dendronylation: Residue-specific chemoselective attachment of oligoglycerol dendrimers on proteins with noncanonical amino acids

Ma, Ying ; Thota, Bala N. S. ; Haag, Rainer ; Budisa, Nediljko (2015)

Polyglycerol dendrimers as an important class of polymeric materials especially attractive for covalent attachment to therapeutic proteins as a useful alternative to traditional PEGylation procedures. Herein, we combine in vivo noncanonical amino acid (ncAA) incorporation and chemoselective conjugation in vitro to produce novel hybrid protein–dendrimer conjugates with the defined architectures....

Construction of a polyproline structure with hydrophobic exterior using octahydroindole-2-carboxylic acid

Kubyshkin, Vladimir ; Budisa, Nediljko (2016)

The proline analogue (2S,3aS,7aS)-octahydroindole-2-carboxylic acid (Oic) has been previously applied as a proline substitute in pharmocologically active peptides and as a structural component of the antihypertensive drug Perindopril. Herein, we describe the formation of an oligoproline structure by an Oic oligomer. A series of Oic oligomers were investigated to show the structural and energeti...

Proteome characterization of Escherichia coli cells evolved to tolerate elongation factor P deletion

Wang, Mingjian (2017)

Elongation factor P (EF-P) is an essential translation factor in bacteria, it has been reported that EF-P plays an important role in rescuing the translation stalling caused by the oligo-proline containing peptides stretch. The growth rate of E. coli was significantly restricted when EF-P was knocked out. However, through a continuous directed evolution in the turbidostatic model, both the wild...

gamma-(S)-Trifluoromethyl proline: evaluation as a structural substitute of proline for solid state F-19-NMR peptide studies

Kubyshkin, Vladimir ; Afonin, Sergii ; Kara, Sezgin ; Budisa, Nediljko ; Mykhailiuk, Pavel K. ; Ulrich, Anne S. (2015)

gamma-(4S)-Trifluoromethyl proline was synthesised according to a modified literature protocol with improved yield on a multigram scale. Conformational properties of the amide bond formed by the amino acid were characterised using N-acetyl methyl ester model. The amide populations (s-trans vs. s-cis) and thermodynamic parameters of the isomerization were found to be similar to the corresponding...

Site-specific conjugation of 8-ethynyl-BODIPY to a protein by [2+3] cycloaddition

Albrecht, Marcel ; Lippach, Andreas ; Exner, Matthias P. ; Jerbi, Jihene ; Springborg, Michael ; Budisa, Nediljko ; Wenz, Gerhard (2015)

We report a straightforward synthesis of 8-ethynyl-BODIPY derivatives and their potential as fluorescent labeling compounds using an alkyne-azide click chemistry approach. The ethynyl substituted BODIPY dyes at the meso-position were reacted under Cu+ catalysis and mild physiological conditions in organic and biological model systems using benzyl azide and a Barstar protein which was selectivel...

Photochemical formation of quinone methides from peptides containing modified tyrosine

Husak, Antonija ; Noichl, Benjamin P. ; Ramljak, Tatjana Šumanovac ; Sohora, Margareta ; Škalamera, Đani ; Budiša, Nediljko ; Basarić, Nikola (2016)

We have demonstrated that quinone methide (QM) precursors can be introduced in the peptide structure and used as photoswitchable units for peptide modifications. QM precursor 1 was prepared from protected tyrosine in the Mannich reaction, and further used as a building block in peptide synthesis. Moreover, peptides containing tyrosine can be transformed into a photoactivable QM precursor by the...