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GREEN BIOTECHNOLOGY Fig. 1: The moss Physcomitrella patens The complete sequencing of the human genome has significantly increased the number of pharmaceutically important proteins (“biopharmaceuticals”) to be produced at large scale. As this tendency is expected to continue, production capacity of biopharmaceuticals has become a strategic issue for the pharmaceutical industry. Mammalian cells such as those of baby hamster kidney and Chinese hamster ovary have been used for more than two decades. However, these systems require major investments in production facilities and running costs of production are very high. Among other technologies, e. g. yeasts and transgenic animals, plants are an alternative for the production of recombinant proteins [1]. So far seed plants like tobacco, corn, and Alfalfa, have been tested for molecular farming. We here will focus on a moss, Physcomitrella patens, as an innovative tool. Physcomitrella has a moderately small genome of 511 Mbp [2]. Mosses are higher multicellular eukaryotes and therefore perform extensive posttranslational processing of proteins including the formation of disulfide bridges and complex glycosylation. Their major developmental stage is the haploid gametophyte (Fig. 1). In contrast, the dominating developmental stage of seed plants is the diploid or polyploid sporophyte [3]. Moss is unique among all multicellular plants analyzed to date in exhibiting a very effective homologous recombination in its nuclear DNA. This allows targeted knockouts of genes [4, 5], which is a highly attractive tool for production strain design. Thus, the manipulation of the glycosylation pathway is 2 • BIOforum Europe 2/2003 Moss – An Innovative Tool for Protein Production Eva L. Decker, Gilbert Gorr, Ralf Reski GREEN BIOTECHNOLOGY Keywords biotechnology, homologous recombination, moss, Physcomitrella patens, recombinant proteins feasible in moss, as opposed to seed plants. Xylose and fucose residues linked to the core structure are characteristic for plant glycans. These plant-specific residues are associated with allergenic risk and thus because of drug safety reasons their removal is essential for production of biopharmaceuticals [1]. Physcomitrella is grown under photoautotrophic conditions in a simple medium essentially consisting of water and minerals. Light and carbon dioxide serve as the only energy and carbon sources. Furthermore, cultivation in glass bioreactors is well established (Fig. 2) [6]. As compared to animal cell systems requirements for extensive process technology are reduced significantly. Physcomitrella grows under broad pH and temperature ranges. Our production system is based on the secretion of recombinant proteins into the medium. As newly synthesized extracellular proteins have to pass the endoplasmic reticulum and the Golgi apparatus where posttranslational modifications including glycosylation and disulfide bonding occur, secreted proteins are completely posttranslationally processed. Additionally, secretion into such simple medium has major advantages for protein purification in greatly reducing the costs for downstream processing. Proof of concept has been achieved by expression and secretion of biologically active human vascular endothelial growth factor (VEGF) using the human leader peptide for targeting the recombinant protein to the secretion pathway of the moss cells [7]. Above that Physcomitrella has an excellent safety profile as a production system for biopharmaceuticals. As in all plant-based production systems the risk of contamination with human or animal pathogens is minimized. As a contained system the moss bioreactor is not related to environmental release of transgenic plants and therefore allows production in an environmentally responsible manner. In conclusion, the moss Physcomitrella patens offers a paramount of important features that make it highly attractive not only as a model organism for the scientific community but also as an organism for the production of biopharmaceuticals. Literature Fig. 2: Photoautotrophic cultivation of moss in the bioreactor [1] W. L. Larrick, D. W. Thomas: Current Opinion in Biotechnology 12, 411–418 (2001) [2] G. Schween, G. Gorr, A. Hohe, R. Reski: Plant Biology, in press [3] R. Reski: Planta 208, 301–309 (1999) [4] T. Girke, H. Schmidt, U. Zähringer, R. Reski, E. Heinz: Plant J.15, 39–48 (1998) [5] A. Koprivova, A. J. Meyer, G. Schween, C. Herschbach, R. Reski, S. Kopriva: J. Biol. Chem. 277, 32195–32201 (2002) [6] A. Hohe, E. L. Decker, G. Gorr, G. Schween, R. Reski: Plant Cell Rep. 20, 1135–1140 (2002) [7] G. Gorr, E. Decker, M. Kietzmann, R. Reski: Naunyn-Schmiedeberg’s Arch. Pharmacol. 363(4) Suppl.,R 85 (2001) Dr Eva Decker was appointed Assistant Professor at the Chair Plant Biotechnology, Albert-Ludwigs-University of Freiburg (Germany) in 2000. She received her PhD in 1999 for her work on the transcriptional regulation of human cytokine gene expression at the Bernhard Nocht Institute for Tropical Medicine in Hamburg (Germany). Before that, she studied biology at the universities of Bielefeld and Hamburg. Prof Dr Ralf Reski was appointed Full Professor (C4) and head of the newly founded Chair Plant Biotechnology at Albert-Ludwigs-University Freiburg (Germany) in 1999. He was Assistant Professor at Hamburg University and received his Habilitation in “General Botany”. For his pioneering work on moss genetics he was awarded a Heisenberg-Fellowship by the Deutsche Forschungsgemeinschaft in 1996. He is co-inventor of the moss bioreactor and co-founder of greenovation Biotech GmbH. Since 2002 Prof Reski serves this company as chairman of the advisory board. Albert-Ludwigs-University Plant Biotechnology Schänzlestrasse 1 79104 Freiburg, Germany [email protected] www.plant-biotech.net Dr. Gilbert Gorr was nominated CSO on June 19th, 2002. Dr. Gorr is co-inventor of the moss bioreactor. He joined greenovation Biotech GmbH in October 2000 as Senior Scientist. He has studied biology and chemistry and received his PhD from Hamburg University for his pioneering work on the use of a moss for the expression of human VEGF. Before coming to greenovation Biotech GmbH he worked as scientist at the Institute for Pharmacology, Toxicology, and Pharmacy, School of Veterinary Medicine, Hannover. greenovation Biotech GmbH Boetzingerstrasse 29b 79112 Freiburg, Germany www.greenovation.com BIOforum Europe 2/2003 • 3