Abstract

The first objective is to overcome one of the main bottlenecks for the use of laccases and peroxidases from ligninolytic basidiomycetes (metalloenzymes with high technological potential because of their ability to oxidise aromatic compounds), which is related to the difficulties of having high amounts of active enzyme. Different strategies for their heterologous expression will be used including optimised refolding of protein from bacterial inclusion bodies, improved production and secretion using industrial ascomycetes, and the development of a new basidiomycete expression system. Simultaneously, several key aspects of their catalytic activity will be investigated using techniques for protein structural characterisation (X-ray and NMR) and confirmed by site-directed mutagenesis. With the above information in hand, tailor-made enzymes will be designed (to improved catalytic efficiency and/or stability) and produced using the best expression systems. Finally, fermentation logy for production of native and engineered enzymes will be optimised, and industrial applications evaluated with the collaboration of two enzyme-producing companies.

Objectives

n order to understand how ligninolytic basidiomycetes manage to degrade recalcitrant aromatic com-pounds and to take advantage of this to design new bio-processes, the following objectives are defined:

• To optimise an expression system for fungal peroxidases and laccases (to be used for protein engineering and industrial fermentation) by comparing refolding of protein from bacterial inclusion bodies, improved expression in industrial ascomycetes, and development of a basidiomycete expression system.
• To characterise some of the most relevant metalloenzymes produced by basidiomycetes in terms of structure-function relationships and, based on the above information, to improve the catalytic properties of some of them for industrial application by protein engineering tech-niques.
• To develop efficient fermentation processes for the production of the most promising metalloenzymes using the expression tools developed, and to evaluate their industrial applicability in food production, paper pulp manufacture (as alternative to contaminating processes) and/or other environmentally-sound applications.

Description of the work

The workplan includes the following workpackages:

• WP1: Cloning DNA encoding peroxidases and laccases (for optimisation of expression, site-directed mutagenesis, and recombinant enzyme fermentation).
• WP2: Expression in Escherichia coli and production of active enzyme (refolding of protein from inclusion bodies).
• WP3: Fungal metalloenzyme expression in Ascomycetes (using Emericella nidulans as a reference and Aspergillus oryzae as a fungal host for industrial expression).
• WP4: Metalloenzyme expression in Basidiomycetes (using Schizophyllum commune as a model to develop a new expression system in the industrial basidiomycete Pycnoporus cinnabarinus).
• WP5: Isolation and characterisation of relevant enzymes (including new peroxidase).
• WP6: NMR spectroscopy and crystallo-graphic analysis (to obtain molecular models and structural information on new and recombinant enzymes).
• WP7: Structure-function and protein modification studies (for elucidation of key aspects of enzyme catalysis and production of improved variants).
• WP8: Fermentation technology for metalloenzyme production (using the most adequate host systems).
• WP9: Evaluation of the industrial application of the enzymes studied (including food and pulp-paper sectors).

Because of high multi-disciplinarity required, the project consortium includes groups with expertise in: i) biotechnology (CIB-CSIC, ES; and UHEL, FI); ii) fungal genetics (RuG, NL; and INRA, FR); iii) peroxidase engineering (USussex, GB); and iv) protein NMR (UFIR, IT) and X-ray diffraction analyses (ETH, CH); together with v) two enzyme-producing companies with experience in recombinant protein production (Frimond, BE; and Novo Nordisk, DK). In this way the critical mass necessary to carry out the project, which cannot be performed at the national level because of the need of highly specialised scientific teams and instrumental facilities, is attained. Partner capabilities to carry out the work proposed are guaranteed by their significant contributions in the past (including the first description of both lignin-peroxidase and laccase crystal structures), as well as by the good position of the industrial partners in the food and industrial enzyme markets. The above consortium (with partners from eight EU members and Switzerland) gives the proposal a strong European dimension.

Deliverables

• Cloning of DNA encoding enzymes to be expressed, modified and evaluated.
• Active enzyme production in Escherichia coli.
• Improved expression in the industrial ascomycete Aspergillus oryzae.
• Basidiomycete transformation.
• Expression in Pycnoporus cinnabarinus, an industrial basidiomycete.
• Isolation of relevant enzymes.
• Preliminary structural characterisation of "wild" enzymes.
• Production of improved variants.
• Final structure-function of native and improved enzymes.
• Optimised fermentation.
• Evaluation of industrial applications.

Contacts

Coordinator

• Angel T MARTINEZ / CIB, CSIC Centro de Investigaciones Biológicas Consejo Superior de Investigaciones Científicas / SPAIN

Participant

• Thierry DAUVRIN / Beldem S.A. R & D Laboratory / BELGIUM
• Serge MOUKHA / Laboratoire de Biotechnologie des Champignons Filamenteux / FRANCE
• Han A B WOSTEN / Rijksuniversiteit Groningen (RuG Biomolecular Sciences and Biotechnology Institute / NETHERLANDS
• Klaus PIONTEK / Swiss Federal Institute of Technology (ETHZ) Institut für Biochemie / SWITZERLAND
• Lucia BANCI / University of Florence Dept of Chemistry and Magnetic Resonance Center / ITALY
• Taina LUNDELL / University of Helsinki (UHEL) Departent of Applied Chemistry and Microbiol. / FINLAND
• Andrew T SMITH / University of Sussex School of Biological Sciences / UNITED KINGDOM