Scale-Up of Artemisia annua L. Hairy Root Cultures Produces Complex Patterns of Terpenoid Gene Expression Fre ´ de ´ ric F. Souret, 1* Yoojeong Kim, 2 Barbara E. Wyslouzil, 2 Kristin K. Wobbe, 3 Pamela J. Weathers 1 1 Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts 01609; telephone: 508-831-5196; fax: 508-831-5936; e-mail: weathers@wpi.edu 2 Department of Chemical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts 3 Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts Received 13 November 2002; accepted 14 March 2003 Published online 13 June 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10711 Abstract: Hairy roots grow quickly, reach high densities, and can produce significant amounts of secondary me- tabolites, yet their scale-up to bioreactors remains chal- lenging. Artemisia annua produces a rich array of terpe- noids, including the sesquiterpene, artemisinin, and transformed roots of this species provide a good model for studying terpenoid production. These cultures were examined in shake flasks and compared with cultures grown in two types of bioreactors, a mist reactor and a bubble column reactor, which provide very different en- vironments for the growing roots. Mist reactors have been shown previously to result in cultures that produce significantly more artemisinin per gram fresh weight of culture, while bubble column reactors have produced greater biomass. We have compared expression levels of four key terpenoid biosynthetic genes: 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMGR), 1-deoxy- D-xylulose-5-phosphate synthase (DXS), 1-deoxy- D- xylulose-5-phosphate reductoisomerase (DXR), and far- nesyl diphosphate synthase (FPS) in the three culture conditions. In shake flasks we found that although all four genes showed temporal regulation, only FPS ex- pression correlated with artemisinin production. Light also affected the transcription of all four genes. Although expression in reactors was equivalent to or greater than that of roots grown in shake flasks, no correlation was found between expression level within six different zones of each reactor and their respective oxygen levels, light, and root-packing density. Surprisingly, transcrip- tional regulation of HMGR, DXS, DXR, and FPS was greatly affected by the position of the roots in each re- actor. Thus, relying on a single reactor sample to char- acterize the gene activity in a whole reactor can be mis- leading, especially if the goal is to examine the difference between reactor types or operating parameters, steps es- sential in scaling up cultures for production. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 653–667, 2003. Keywords: immobilization; transformed roots; bioreac- tors INTRODUCTION Transformed “hairy” root cultures are a biochemically and genetically stable model for assessing biosynthesis and scale-up of pharmacologically important natural products (Flores et al., 1999). The ability of rapidly growing hairy roots to steadily produce a wide range of organic molecules offers additional advantages compared to conventional field production and unorganized cell suspension cultures (Shanks and Morgan, 1999). The latter, in particular, are generally unstable and unproductive over time. The ability of hairy roots to grow to high density and to produce sig- nificant amounts of secondary metabolites also makes them a suitable system for large-scale culture in reactors (Wilson, 1997). Many factors are severely affected during scale-up of any kind of cell culture. These include oxygen transfer rate, heat transfer, mixing and the associated shear stress, superficial air velocity associated with impeller-mixed cultures, and culture age and stability (Humphrey, 1998; Kim et al., 2002a). For example, inadequate mixing is believed to cre- ate gradients of nutrients including oxygen and carbon sources throughout the bioreactor and can rapidly lead to nonoptimal large-scale performance (Bylund et al., 1998; Humphrey, 1998). Indeed, Schweder et al. (1999) reported that within 60 seconds E. coli cells in specific zones of a bioreactor showed differential transcription of genes in re- sponse to changes in glucose and oxygen levels. Scale-up of hairy root cultures remains very challenging, and, despite recent advances, the objectives of optimal growth and production in bioreactors are far from being *Current address: Delaware Biotechnology Institute, 15 Innovation Way, Newark, Delaware 19711. Correspondence to: Pamela J. Weathers Contract grant sponsors: The National Science Foundation; National Institutes of Health Contract grant numbers: BES-9414858; R21 AI39170-01 © 2003 Wiley Periodicals, Inc.