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Project description:

Project no:
Project proposal “LIGAROM” (LIGnin-based AROMatic amino acids)
1st project leader:
Rudat, Jens - Institute of Engineering in Life Science Section of Technical Biology; Engler-Bunte-Ring 3, 76131 Karlsruhe, Germany
2nd project leader
The research group “Biocatalysis” at KIT, BLT_II: Technical Biology (Chair: Professor C. Syldatk) is investigating the enzymatic synthesis and conversion of aromatic amino acids which constitute the main building blocks of the lignin part of plant lignocellulose.

The proposed CSC project aims to continue the CSC funded project of the same title conducted in the Rudat lab since Sep/2017. The project aims the use of phenylpropane compounds (“monolignols”) and biogenic waste for chemoenzymatic syntheses of high-priced fine chemicals, e.g. non-canonical aromatic amino acids. Key elements will be the development of processes to directly use the aromatic compounds from liquefied lignocellulose (using the expertise from bioliq pilot plant at KIT) and the stabilization of enzymes for the expectedly harsh reaction conditions. Suitable enzymes identified in the project up to now (hydantoinases, ω-transaminases, ammonia lyases, amino mutases) are to be adapted to a wider substrate range by molecular engineering and stabilized for process development by immobilization techniques. Chemical synthesis routes that have been established so far are to be scaled up and optimized concerning product yield and purity.

Provisional working plan:
1. Isolation and/or synthesis of phenylpropane compounds from lignocellulose
2. Further synthesis to differently substituted aromatic acids and alcohols
3. Characterization of enzymes for the stereoselective biotransformation of these compounds
4. Engineering, stabilization and immobilization of the most suitable enzymes
5. Set-up and modeling of a bioprocess to realize a (chemo-)enzymatic cascade reaction
6. Development and application of analysis methods to qualitatively and quantitatively analyze all substrates, intermediates and products

Buß O, Buchholz PCF, Gräff M, Klausmann P, Rudat J, Pleiss J (2018) The ω-transaminase engineering database (oTAED): A navigation tool in protein sequence and structure space. Proteins 86 (5): 566

Buß O, Muller D, Jager S, Rudat J, Rabe KS (2017) Improvement of a β-amino acid converting ω-transaminase by using FoldX. ChemBioChem 19 (4): 379

Slomka C, Späth GP, Lemke P, Skoupi M, Niemeyer CM, Syldatk C, Rudat J (2017) Toward a cell-free hydantoinase process: screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase. AMB Express 7: 122

Dreßen A, Hilberath T, Mackfeld U, Rudat J, Pohl M (2017) Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids Part II: Application in different reactor concepts for the production of (S)-2-chloro-phenylalanine. J Biotechnol 258: 158

Slomka C, Zhong S, Fellinger A, Engel U, Syldatk C, Bräse S, Rudat J (2015) Chemical synthesis and enzymatic, stereoselective hydrolysis of a functionalized dihydropyrimidine for the synthesis of β-amino acids. AMB Express 5: 85.
Methods that will be used:
- organochemical synthesis,
- qualitative and quantitative chemical and biochemical analysis methods,
- molecular cloning and genetic engineering,
- protein purification and immobilization,
- enzyme modelling and optimization
Collaboration partners:
Prof. Stefan Bräse (KIT-CS, IOC) and Prof. Uwe Beifuß, University of Hohenheim, Bioorganic Chemistry: Isolation and/or synthesis of defined aromatic compounds from liquefied lignocellulose; instrumental analysis

Dr. Ulrike Engel, KIT-CS, BLT_II: Isolation, characterization and molecular cloning of suitable enzymes

Dr. Kersten Rabe, KIT-CN, IBG_I (Chair: Prof. C. Niemeyer): Identification of stable enzymes from genomic databases; enzyme engineering using directed evolution

Prof. Matthias Franzreb, KIT-CN, IFG – Bioengineering and Biosystems: Immobilization and stabilization of enzymes; process development and simulation
Expected candidate‘s qualification:
- MSc or comparable degree in Chemistry, Biochemistry, Bioprocess engineering or similar subjects.
- Excellent knowledge and lab experience in at least three of the above mentioned method fields.
- Communication and teamwork skills; fluent English.
monolignols, non-canonical amino acids, biocatalysis, enzyme engineering, enzyme immobilization