Appl Microbiol Biotechnol (2004) 64: 782–786 DOI 10.1007/s00253-003-1524-z BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING I. A. Parshikov . K. M. Muraleedharan . M. A. Avery . J. S. Williamson Transformation of artemisinin by Cunninghamella elegans Received: 28 September 2003 / Revised: 5 November 2003 / Accepted: 21 November 2003 / Published online: 21 January 2004 # Springer-Verlag 2004 Abstract Semi-synthetic derivatives of the anti-malarial drug artemisinin hold great promise in the search for an effective and economical treatment of chloroquine-resis- tant forms of malaria. Unfortunately, synthetic functiona- lization of the artemisinin skeleton is often tedious and/or impractical. We seek to utilize 7β-hydroxyartemisinin, obtained from microbial transformation, as a semi-syn- thetic precursor for the synthesis of novel 7β-substituted artemisinin anti-malarial agents. Here we employ liquid cultures of Cunninghamella elegans as a means for the rational and economical bioconversion of artemisinin to 7β-hydroxyartemisinin in 78.6% yield. In addition, there were three other bioconversion products: 7β-hydroxy-9α- artemisinin (6.0%), 4α-hydroxy-1-deoxoartemisinin (5.4%), and 6β-hydroxyartemisinin (6.5%). Introduction Each year, it is estimated that almost 2 million people die from malaria, most of these victims being African children (Hu et al. 1992; Ekthawatchai et al. 2001). Although generally considered a tropical disease, many cases of malaria have been detected throughout the world as people migrate from regions of Southeast Asia and Africa (Mattelli et al. 2001). There is little doubt that the social and economic consequences of malaria make it a disease of global proportions. Artemisinin (Fig. 1), a naturally occurring sesquiterpene lactone, shows promising attributes as the basis of an anti- malarial. Artemisinin is effective against chloroquine- resistant parasites; however, toxicities and water insolubi- lity limit its usefulness. Quantitative structural activity relationship (QSAR) studies of its skeleton (Avery et al. 1999; Posner et al.1999) suggest that structural manipu- lation of the sesquiterpene lactone may yield more desirable anti-malarial analogs. The complexity of the artemisinin molecule renders many synthetic manipula- tions impossible and/or impracticable, yet there are reports indicating that the sequiterpene is susceptible to biocon- version by a variety of microorganisms (Fiaux de Medeiros et al. 2002; Lee and Hufford 1990; Lee et al. 1989; Zhan et al. 2002a, 2002b; Ziffer et al. 1992). It has been our goal to identify and develop a semi-preparative methodology for the bioconversion of the methylene carbon at position 7 of artemisinin. QSAR and molecular modeling studies suggest that the addition of steric bulk near the 7β-position of artemisinin will increase anti- malarial activity. Therefore, we have examined a variety of different microorganisms for their ability to efficiently convert artemisinin to 7β-hydroxyartemisinin, which will be used to develop novel 7β-substituted artemisinin-based anti-malarials. Materials and methods Certain strains of mycelial fungi obtained from collections of microorganisms in the United States and Russia served as subjects of the investigation. American type culture collection (ATCC): Aspergillus adametzi ATCC 10407, Cunninghamella elegans ATCC 9245, Mucor rammanianus ATCC 9624, and M. rammanianus ATCC MYA-883. The University of Mississippi (UM) collection: M. rammanianus 1839UM. Trichoderma viride T-58, Pestalotiopsis guepini P-8 and Penicillium purpurescens P-10 were collected from forest in Jefferson County, Arkansas. Cunninghamella verticillata VKPM F-430 was from the All-Russian Collection of Industrial Microorganisms; and Penicillium simplicissimum KM-16 from the Biological Faculty of Moscow State University. Well-developed fungal mycelia were removed from the surface of agar slants, suspended in 5 ml sterilized water, and used to inoculate 50 ml medium (20.0 g Sabouraud-dextrose, 15.0 g sucrose, 10.0 g peptone, in 1,000 ml deionized water) in a 125 ml shake flask. The pH was adjusted to 6.5 using 0.1 N NaOH. Cultures were grown for 48 h on a rotary shaker at 28°C with shaking at 180 rpm. The resulting biomass was used as inocula for 500 ml medium contained in 2.5 l shake flasks that were again incubated for 48 h before the addition of biotransformation substrate (artemisinin). I. A. Parshikov . K. M. Muraleedharan . M. A. Avery . J. S. Williamson (*) Department of Medicinal Chemistry, University of Mississippi, University, MS 38677-1848, USA e-mail: mcjsw@olemiss.edu Tel.: +1-662-9157142 Fax: +1-662-9155638