Establishing a Pan-European Network on Computational Redesign of Enzymes (COZYME)
 
Contact: Dirk Tischler

 
The COZYME (COmputationally assisted design of enZYMEs) Action comprises a Pan-European collaborative network aimed at developing and implementing state-of-the-art computational tools for rapid enzyme improvement. This will solve a key bottleneck in biotechnology: the exploitation of industrially relevant enzymes. Specifically, the Action focuses on three issues:
1.  Improvement of generic enzyme properties such as stability and solubility;
2.  Optimization of catalytic properties e.g. activity and stereoselectivity;
3. Advancement of experimental approaches to generate and evaluate computational predictions;
These goals are intended to be of particular benefit in the context of training young researchers in the development and use of computational methods in biotechnology.
Duration: October 2022 – October 2026
Funding: European Cooperation in Science and Technology (COST)
Link: https://www.cost.eu/actions/CA21162/

BiodeCCodiNNg

DC 8 - Towards N-N bond formation via N-hydroxylation in a biocatalytic cascade
Contact: Dirk Tischler
 

From enzyme discovery to industrial implementation – creating novel biocatalysts for industrial and pharmaceutical biotechnology; these are the focal points in our EU funded doctoral network. Herein, we combine basic research and applied engineering to deliver new synthetic routes to chemically relevant products in more efficient and cleaner ways than the present ones. The overall project connects three work packages (WP1: Enzyme discovery and characterization of novel NN- and CCzymes; WP2: Structure elucidation and enzyme engineering towards novel reactivities; and WP3: Reaction engineering and biocatalytic applications) which are investigated both at research institutes as well as at industrial sites.
Our part is important for the NN-coupling enzymes as we focus on the activation of amine groups by means of N-hydroxylating enzymes. Along this we implement methods to describe enzymes from phylogenetic point of view, to uncover mechanistic details, to evolve them by rational design, and to stabilize enzymes by immobilizations techniques.
 
Funding: The doctoral network is funded in course of the MARIE SKŁODOWSKA-CURIE ACTIONS (ID: 101073065)
Link: https://biodeccodinng.eu/

Glutathione in degradative metabolism of Actinobacteria

Contact: Anna Christina Lienkamp, Dirk Tischler

The Actinobacterium Gordonia rubripertincta CWB2 has a novel styrene degradative pathway comprising a glutathione dependent step. In course of this project we study the novel enzymes from strain CWB2: glutathione S-transferase and glutathione reductase. Here, general cloning and protein production methods are used in combination with sophisticated UHPLC and LC-MS methods to characterize enzyme activities. Furthermore, we employ mutagenesis strategies to knockout selected genes in strain CWB2 or to bring others under selected regulators to allow gene silencing as well as expression in those Actinobacteria.

Funding: The project is supported by the DFG-funded Research Training Group MiCon (RTG 2341). In addition, we get support for the project by Mercator Research Center Ruhr (An-2018-0044).

Degradation industrially relevant azo dyes

Contact: Anna Christina Ngo, Selvapravin Kumaran, Dirk Tischler
Cooperation: Dr. Isabel Bento, EMBL Hamburg

In this project we use either isolated enzymes or novel bacterial isolates to convert azo dyes. With respect to the enzymes we employ various flavin-dependent and -independent azoreductases to break the azo bridge leading to various aromatic amines. Here the prototype enzyme is AzoRo from Rhodococcus opacus 1CP which is subject to protein engineering and structural investigations. In addition, we screen for bacteria with the capability to degrade various azo dyes or even use them as sole source of carbon. Here we employ methods of functional genome annotation and biodegradation techniques in combination with chromatographical analytics.

Funding: The project is supported by a KAAD predoctoral scholarship awarded to Anna Christina Ngo.

Biotransformation of lignin-monomers by flavin-dependent oxidases

Contact: Daniel Eggerichs, Dirk Tischler
Cooperation: Prof. Dr. Marco Fraaije, University of Groningen

Except the details we know about eugenol oxidase the application of flavin-dependent oxidases is limited. Herein, we screen in genomes and metagenomes for novel bacterial representatives. These are subject of general cloning, homology modelling and protein engineering to obtain a simple oxidase-based route towards ferulic acid derivatives as platform chemicals. Useful enzymes will be employed in multi-enzyme cascades either as cell-free immobilized or whole-cell biocatalysts.

Funding: The project is supported by a DBU predoctoral scholarship awarded to Daniel Eggerichs.

EnzymPrint

Contact: Fabian Schultes, Myra Schmidtke, Dirk Tischler
Cooperation: Hirsch Engineering Solutions GmbH & Co. KG, Eichstätt

The project aims at immobilizing enzymes on biocompatible materials to provide access towards cascades. Exemplarily, will be the synthesis of trehalsoe from glucose-moieties and polyphosphate investigated. 

external links: https://www.hirsch-es.de/

Funding: The project is funded as a cooperative project by the German Federal Ministry for Economic Affairs and Energy (KK5161101CS0).

  

Contact: Fabian Schultes, Carolin Mügge

The project BioCOnversion unites multidisciplinary expertise from academia and industry in a cross-border consortium to make CO-containing process gases available for the production of added-value chemicals. The German Federal Ministry of Education and Research (BMBF) funds this innovative bioprocess to convert syngas into a defined plastic precursor by evaluating different technology approaches.

Carbon monoxide (CO)-containing process gases, abundant in the BIG-Cluster region through numerous industrial sites, can be valuable feedstock streams for the biotechnical production of building blocks that are currently produced via petrochemical process routes. Mid-chain carbon compounds with multifunctional groups are of special industrial interest. Since they are conventionally generated from fossil resources, routes using renewable non-food feedstocks to provide such precursors would be a major step to establish a sustainable economy. Therefore, BioCOnversion aims at developing and implementing a sustainable process from CO to a defined polymer precursor by evaluating different technologies. An international consortium of industrial and academic partners join their high-level, multidisciplinary expertise to develop a process comprising the primary conversion of CO/syngas into an intermediate through gas fermentation and its enzymatic upgrading conversion to a defined plastic precursor.

RUB's part of the project focuses on enzymatic uprading of the intermediate produced by syngas-converting microorganisms. Within this, the full spectrum of strain and enzyme optimization as well as reaction tuning is tackled. The project is corroborated by mechanistic studies on the target enzymes.

external links: https://www.bigc-initiative.eu/bioconversion.php
Press release: pdf

Funding: The project is funded by the BMBF (03INT513BF) in frame of the BIG-cluster initiative.

ChemBioCat

Contact: Antje Kumpf, Artur Maier, Carolin Mügge, Dirk Tischler
Cooperation: Prof. Dr. Pablo Sobrado, Virginia Tech, Blacksburg

The project aims to establish novel chemo-enzymatic cascades towards valuable building blocks and can be divided into two subprojects:

• Production of N-hydroxytriazene derivatives from simple amino acids is the first target. Here we employ amino acid decarboxylases (ADCs) to produce amines, which can be converted by N-hydroxylating enzymes (NMOs). These NMOs use NADPH and a flavin-cofactor to selectively hydroxylate a single terminal nitrogen leading to N-hydroxy amines. However, NADPH needs to be recycled and this can be achieved by formate dehydrogenase (FDH). The product of this 3-enzyme cascade is a N-hydroxy amine which can subsequently be converted by chemical synthesis employing diazonium salts towards the desired N-hydroxytriazenes. Here we apply methods as protein production, enzyme immobilization and process optimization, among others.
  
  
• Lignin-monomers include styrene like molecules and those styrene derivatives can selectively be epoxidized by styrene and indole monooxygenases. In this part, we start with styrene and derivatives and study the chiral epoxidation. The used monooxygenases need reduced FAD to activate molecular oxygen and this can be achieved by FAD-reductases or by means of chemical reductants as NAD(P)H-mimics. We employ the latter and obtain a chemo-enzymatic cascade towards chiral products. In addition, we investigate whole-cell biotransformation to obtain chiral epoxides. And those can be further converted by metal- or metal-independent chemical catalysis using azide-like compounds towards hydroxytriazoles. 
  
  

Hydroxytriazenes and hydroxytriazoles are important fine chemicals with biological activity and those can be tested to affect bacteria, fungi or single enzymes.

Funding: The project is supported by the State North Rhine-Westphalia funding the Research Group Grant (PtJ-TRI/1411ng006).