Biocatalysis

Broadening the substrate range of Triosphosphate Isomerase (TIM) by Structure based Directed Evolution towards novel chiral hydroxyaldehydes

Triosephosphate Isomerase (TIM)

© Neubauer

Directed evolution is a key technology in the field of molecular enzyme engineering. It is widely used to change properties of enzymes. If little is known about structure or catalytic mechanisms of the target enzyme the whole sequence is randomly mutated, or if the structure is known certain areas can be chosen for randomization through rational design. 

The target enzyme of this project, the TIM barrel protein Triosephosphate Isomerase, has been studied extensively, in structure and mechanism. Therefore, we cannot only randomly mutate our target sequence, but can use the knowledge to specifically target areas of interest in the enzyme and randomize them. TIM is small in size, easily crystallized, soluble highly expressed in E. coli, a stable molecule that does not require any cofactors and the TIM-barrel group represents the most common enzyme fold in the Protein Data Bank (PDB). It has been proven already in studies of the group around Saab-Rincon (2001) that an increase of activity in monoTIM through directed evolution gives promising results. Origninally TIM is a dimer, but a monomeric version has been created in earlier studies.
In this project knockout strains will be created, heterologous protein expression and tools of protein engineering will be used to develop new biocatalysts with new substrate activities, focusing on isomerise activities.


Involved researchers:

Mirja Krause, Peter Neubauer


Cooperation Partners:

Rik Wierenga, Structural Biochemistry, University of Oulu, Finland

Mari Ylianttila, Bioprocess Engineering, University of Oulu, Finland

Unicat Cluster of Excellence



Biocatalysis

Novel biocatalysts for the preparation of modified nucleosides

Purine nucleoside phosphorylase

© Neubauer

In this project heterologous protein expression and tools of protein engineering will be used to develop new biocatalysts with improved properties, focusing on thermostability and an extended substrate spectrum.

Biocatalysts play an essential role for the preparation of modified nucleosides that are widely used as pharmaceutical agents for the treatment of viral infections and cancer.
The facilitation of specific enzymes can be used in so-called chemo-enzymatic processes. Here, biocatalysts allow the performance of reactions with strict regio-and stereoselectivity, and the operation under environmentally friendly conditions. 


Involved researchers:

Kathleen Szeker, Xinrui Zhou, Peter Neubauer


Cooperation partners:

Unicat cluster of excellence

Recent Publications:

Szeker K, Niemitalo O, Casteleijn MG, Juffer AH, Neubauer P. 2011: High-temperature cultivation and 5' mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli. Journal of Biotechnology 20;156(4):268-74.

Nonribosomal Peptide Synthetases (NRPSs)

Model of the biosynthetic valinomycin assembly line

© Jaitzig

Nonribosomal Peptide Synthetases (NRPSs) - Heterologous expression of large multi-functional enzymes

Non-ribosomal peptide synthetases (NRPSs) are multimodular megaenzymes that produce a wide range of pharmaceutically relevant peptide secondary metabolites in bacteria and fungi. Even though non-ribosomal peptides display a remarkable structural diversity, the mode of action by which they are assembled from NRPSs follows a fundamental logic, with each enzyme module being responsible for the recognition, attachment and incorporation of a single building block into the growing peptide chain. The modular organization of NRPSs offers the potential to tailor the assembly line as a promising way to obtain novel bioactive compounds. This may be achieved by combinatorial approaches, in which the modular composition of the NRPS is changed, but also by random or directed mutagenesis to alter substrate specificities. The heterologous expression of NRPSs in organisms that are easy to cultivate, robust and amenable to genetic manipulation, represents the bottleneck for any NRPS engineering approach. Possible strategies for the soluble and functional expression will therefore be screened with valinomycin synthetase as a model NRPS.


Involved researchers:

Jennifer Jaitzig, Jian Li, Peter Neubauer


Cooperation partners:

AG Prof. R. Süssmuth

Unicat cluster of excellence

Optimization of Molybdoenzyme Expression Through Metabolic Engineering and Media Optimization

Molybdenum is biologically inactive unless it is complexed by a cofactor.
Two types of  cofactor that contain Mo exist:  1.) iron-molybdenum cofactor (FeMoco) and 2.) pterin-based molybdenum cofactor (Moco). Within the enzymes the Molybdenum shuttles between three oxidation states (+IV, +V, +VI), thereby catalyzing two-electron redox reactions.

Molybdoenzymes are devided into three families according to the structure of the active site and the reaction type they catalyze: Sulfite Oxidase, Xanthine Oxidase, Dimethyl sulphoxide reductase (DMSOR). All three are mononuclear.
The Sulfite Oxidase for example is currently used for the development of biosensors.

Within the expression of Molybdoenzymes the expression rate of the Moco (cofactor) is the limiting factor for high protein yield.

In this project the yield of recombinant protein expression is enhanced using tools of metabolic and protein engineering and also by media optimization.


Involved researchers:

Mirja Krause, Peter Neubauer


Cooperation partners:

AG Prof. Silke Leimkühler, Biochemistry-Protein Analytics, Universität Potsdam

Unicat cluster of excellence

Expression of Wnt proteins in E. coli

Wnt proteins constitute a family of cysteine-rich, acyl-modified glycoproteins, which control several key processes, such as cell proliferation, cell migration, and cell differentiation. Wnt signaling is furthermore involved in body axis specification and stem cell maintenance. Wnt are considered as morphogens exhibiting both short-range and long-range actions, whereby their actions depend on the availability of different receptors, on the stage of development and the type of tissue. Deregulation of Wnt signaling pathways implicate developmental disorders, degenerative diseases, and tumorgenesis.

Nineteen wnt genes have been identified in the human genome comprising two functional classes. Members of the Wnt-1 class are able of inducing the canonical signaling pathway leading to an intracellular stabilization and nuclear translocation of β-catenin. β-catenin associates with the transcriptions factors lymphoid enhancer factor/T-cell transcription factor (LEF/TCF) for activating target genes. Members of the Wnt-5a functional class act as antagonists for canonical signaling and regulate at least two different pathways involving G-proteins (with Ca2+/calmodulin, small GTPases, JNK).

Wnt morphogens are considered beneficial for the regulation of stem cell development (e.g. hematopoietic, intestinal epithelium, epidermal, mesenchymal, neuronal, and hair follicle stem cells) and for their maintenance. Possible applications for this are found in regenerative tissue processes for human therapy. However, the availability of Wnt proteins in pure and active form is very limited. Wnt expression in eukaryotic cell culture often results in accumulation of misfolded protein in the ER. Moreover, Wnts are associated with the extracellular matrix or the cell surface leading to a very small amount of secreted and soluble protein.

In this project Wnt proteins are expressed in E. coli applying libraries with different vectors for cytoplasmic and periplasmic expression comprising e.g. different fusion proteins enhancing the solubility or different leader peptides, respectively as well as different ribosome binding sites and promoters of different strength. Wnt expression and purification is optimized regarding a high amount of soluble and biologically active protein.

Involved researchers:

Kathrin Ralla, Peter Neubauer


Cooperation partners:

Unicat cluster of excellence