title: Scale-up of Artemisia annua L. hairy root cultures produces complex patterns of terpenoid gene expression.
abstract: Hairy roots grow quickly, reach high densities, and can produce significant amounts of secondary metabolites, yet their scale-up to bioreactors remains challenging. Artemisia annua produces a rich array of terpenoids, 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 environments 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 farnesyl diphosphate synthase (FPS) in the three culture conditions. In shake flasks we found that although all four genes showed temporal regulation, only FPS expression 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, transcriptional regulation of HMGR, DXS, DXR, and FPS was greatly affected by the position of the roots in each reactor. Thus, relying on a single reactor sample to characterize the gene activity in a whole reactor can be misleading, especially if the goal is to examine the difference between reactor types or operating parameters, steps essential in scaling up cultures for production.
authors.affiliation: Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA.
authors.forename: Frédéric F
authors.initials: FF
authors.lastname: Souret
authors.forename: Yoojeong
authors.initials: Y
authors.lastname: Kim
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Wyslouzil
authors.forename: Kristin K
authors.initials: KK
authors.lastname: Wobbe
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Weathers
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D031172:Artemisia annua / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D037621:Artemisinins / Q000138:chemical synthesis; D019149:Bioreactors; D018929:Cell Culture Techniques; D017077:Culture Media, Conditioned; D005573:Formycins / Q000378:metabolism; D015870:Gene Expression; D051228:Geranyltranstransferase; D045782:Hemiterpenes / Q000096:biosynthesis / Q000378:metabolism; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D008027:Light; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010100:Oxygen; D018517:Plant Roots / Q000201:enzymology / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism; D012717:Sesquiterpenes / Q000138:chemical synthesis; D013729:Terpenes / Q000138:chemical synthesis* / Q000378:metabolism; D014166:Transferases / Q000378:metabolism
chemical_list: D037621:Artemisinins; D017077:Culture Media, Conditioned; D005573:Formycins; D045782:Hemiterpenes; D009097:Multienzyme Complexes; D009943:Organophosphorus Compounds; D012265:Ribonucleotides; D012717:Sesquiterpenes; D013729:Terpenes; C020332:formycin diphosphate; C004809:isopentenyl pyrophosphate; C031327:artemisinin; D010088:Oxidoreductases; D006903:Hydroxymethylglutaryl CoA Reductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase; D019747:Aldose-Ketose Isomerases; D010100:Oxygen