Development of a Culture Sub-population Induction Model: Signaling Pathways Synergy and Taxanes Production by Taxus canadensis Ryan S. Senger, † Muenduen Phisalaphong, ‡ M. Nazmul Karim, † and James C. Linden* ,§ Department of Chemical Engineering, Texas Tech University, Lubbock, Texas 79409, Department of Chemical Engineering, Chulalongkorn University, Bangkok, Thailand, and Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, Colorado 80523 Cell cultures of Taxus canadensis were subjected to exogenously applied ethylene (ET) hormone and methyl jasmonate (MJ) elicitation in factorial design experiments. Levels of extracellular taxanes, including paclitaxel, were used with principal component analysis for fault detection and real-coded genetic algorithms for parameter optimization to construct a culture sub-population induction model. Culture sub-populations were identified by the model as (1) uninduced, (2) induced to unilateral function of the ET-signaling pathway, and (3) induced to cooperation between jasmonic acid (JA)- and ET-signaling pathways. Comprehensive model results suggested greater rates of cellular induction (resulting in exogenous taxane production) by ET gas as opposed to MJ elicitation. However, cellular induction of ET-signaling pathway genes increased the rate of induction of JA-signaling pathway genes by orders of magnitude. In addition, model results showed that induction of genes leading to extracellular production of the simple taxane 10- deacetylbaccatin III was regulated by the unilateral ET-signaling pathway. However, it was suggested that further processing of this simple taxane to complex taxane structures, such as paclitaxel, required further gene induction by the JA-signaling pathway. Thus, production rate constants of exogenous complex taxanes were predicted to be an order of magnitude lower than that for the simple taxane 10-deacetylbaccatin III. The fraction of the cell culture sub-population displaying unilateral ET-signaling pathway gene induction was found inversely proportional to levels of MJ elicitation. When coupled with simple non-growth product models, levels of all extracellular taxanes were effectively predicted using the culture sub-population induction model. Introduction and Background Clinical Properties and Commercial Production of Pacli- taxel. As a result of its therapeutic activity in treating many forms of cancer, Paclitaxel (Taxol, Bristol-Meyers Squibb, Princeton, NJ) is a valuable secondary metabolite product from plant cultures. In particular, paclitaxel is a naturally occurring diterpenoid alkaloid of many Taxus plant species and is therapeutically classified as a microtubule stabilizing agent, prompting its use in oncology to retard tumor growth (1-3). According to the U.S. Food and Drug Administration (FDA) (2005), paclitaxel is currently used in treatment of ovarian, node- positive breast and small lung cell cancers, and it is also used in treatment of AIDS-related Kaposi’s sarcoma. In addition, the U.S. National Institute of Health (NIH) (2005) reports paclitaxel is currently involved in multiple clinical trials, examining its incorporation into treatment procedures for over 30 different types of cancers. Due to the therapeutic properties and com- mercial value of paclitaxel and related taxanes, stimulated induction of genes leading to taxanes production in Taxus sp. plant cell cultures and chemical synthesis (4) of these taxanes have been active areas of research. Investigations of induction factors such as abiotic elicitors and medium components (5- 8), gas-phase composition (9), osmotic pressure (10), shear stress (11) and temperature (12) on the regulation of secondary metabolite pathways (leading to paclitaxel); plant cell culture selection (13); and culture viability (14) have been previously investigated. Reactor design and bioprocessing strategies (3), including total cell recycle (15) and endogenous paclitaxel liberation (16), have also led to significant increases in paclitaxel production from Taxus sp. cell cultures. Elicitors and Secondary Metabolite Induction. Taxanes, including paclitaxel, are produced in relatively low titers by Taxus sp. cell cultures in the absence of designed induction schemes. This has been determined unfeasible for commercial production (3), prompting further investigations into induction schemes of Taxus sp. cell cultures. Genes related to secondary metabolite (including taxanes) production and regulation have been found to be induced as part of highly regulated defense responses to biotic stresses such as pathogenic attack (17) or abiotic stresses such as wounding (18, 19), UV-B (20), ozone exposure (20) and temperature fluctuation (12). The common regulators of plant defense responses upon insect or pathogen attack are jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) (17, 21). In particular, these regulators vary in amount, timing and synergy, and specific responses have been found tied to transcription factors induced by signaling pathways regulated by these agents. Specific defense responses have also been observed to be dependent on specific pathogens or stresses encountered (17, 22, 23). The use of fungal elicitors was observed to increase secondary metabolite production, including paclitaxel, by Taxus chinensis; however, this form of biotic * To whom correspondence should be addressed. Ph: 970.491.6122. Fax: 970.491.1815. Email: jlinden@colostate.edu. † Texas Tech University. ‡ Chulalongkorn University. § Colorado State University. 1671 Biotechnol. Prog. 2006, 22, 1671-1682 10.1021/bp0602552 CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 10/19/2006