Production and in situ separation of secondary metabolites in hairy root culture

Abstract

Plant hairy root cultures of Catharanthus roseus and Lithospermum erythrorhizon were performed to produce plant-derived secondary metabolites such as shikonin and indole alkaloids. Various hairy root cell lines were derived from two types of C. roseus plants (Catharanthus roseus cvs. Little Linda (LL) and Little Delicata (LD)) by transforming using Agrobacterium rhizogenes ATCC 15834. Hairy root clones derived from LD plants and LL plants showed contrasting morphologies. The hairy root derived from LD plants showed the characteristics of thick, straight, and fragile branches. In contrast to that, the hairy root derived from LL plants showed the characteristics of thin, coil-like, and unfragile branches. The production of alkaloids and root growth between hairy root clones were affected by their morphological characteristics. Hairy root clone LL 15 which showed the excellent growth and alkaloids productivity was selected for this study. The optimization of environmental condition was carried out both in C. roseus and L. erythrorhizon hairy root cultures. When sucrose was used as a carbon source in L. erythrorhizon hairy root culture, the maximum shikonin production and hairy root growth were obtained. However, when glucose or fructose was used instead, hairy root growth was severely inhibited. In contrast to this, hairy root culture of C. roseus, either glucose or fructose was a suitable carbon source as well as sucrose. Plant hairy root cultures of Lithospermum erythrorhizon were carried out to produce shikonin derivatives by employing in situ extraction with nhexadecane in a shake flask and a bubble column bioreactor. Over 95\% shikonin produced was recovered in the n-hexadecane layer. In flask cultures the concentration of shikonin with n-hexadecane extraction was 3 times than that obtained without extraction. To test the effect of elicitation on shikonin production and secretion, Penicillium species homogenate which was isolated from contaminated plant cell ...