University of Groningen Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast

Background: β-Lactams like penicillin and cephalosporin are among the oldest known antibiotics used against bacterial infections. Industrially, penicillin is produced by the filamentous fungus Penicillium chrysogenum. Our goal is to introduce the entire penicillin biosynthesis pathway into the methylotrophic yeast Hansenula polymorpha. Yeast species have the advantage of being versatile, easy to handle and cultivate, and possess superior fermentation properties relative to filamentous fungi. One of the fundamental challenges is to produce functionally active enzyme in H. polymorpha. Results: The P. chrysogenum pcbC gene encoding isopenicillin N synthase (IPNS) was successfully expressed in H. polymorpha, but the protein produced was unstable and inactive when the host was grown at its optimal growth temperature (37°C). Heterologously produced IPNS protein levels were enhanced when the cultivation temperature was lowered to either 25°C or 30°C. Furthermore, IPNS produced at these lower cultivation temperatures was functionally active. Localization experiments demonstrated that, like in P. chrysogenum, in H. polymorpha IPNS is located in the cytosol. Conclusion: In P. chrysogenum, the enzymes involved in penicillin production are compartmentalized in the cytosol and in microbodies. In this study, we focus on the cytosolic enzyme IPNS. Our data show that high amounts of functionally active IPNS enzyme can be produced in the heterologous host during cultivation at 25°C, the optimal growth temperature for P. chrysogenum. This is a new step forward in the metabolic reprogramming of H. polymorpha to produce penicillin. Background β-lactam antibiotics like penicillins and cephalosporins belong to one of the largest-selling classes of drugs worldwide with a production of forty-five thousand tons in the year 2000 [1]. Penicillins and cephalosporins are produced by the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum, respectively, as well as some filamentous bacteria. These antibiotics possess as common structural motif the β-lactam ring [2]. Not surprisingly, the penicillin and cephalosporin biosynthetic Published: 19 March 2008 BMC Biotechnology 2008, 8:29 doi:10.1186/1472-6750-8-29 Received: 25 October 2007 Accepted: 19 March 2008 This article is available from: http://www.biomedcentral.com/1472-6750/8/29 © 2008 Gidijala et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Biotechnology 2008, 8:29 http://www.biomedcentral.com/1472-6750/8/29