High level expression of chorismate pyruvate-lyase (UbiC) and HMG-CoA reductase in hairy root cultures of Lithospermum erythrorhizon.
Shikonin, a red naphthoquinone pigment, is produced by cell cultures of Lithospermum erythrorhizon (Boraginaceae). It is biosynthetically derived from two key precursors, 4-hydroxybenzoate (4HB) and geranyldiphosphate (GPP). The bacterial ubiC gene, encoding chorismate pyruvate-lyase (CPL) which converts chorismate to 4-hydroxybenzoate, was expressed in L. erythrorhizon under the control of the strong (ocs)(3)mas-promoter. This introduced an efficient biosynthetic pathway to 4HB, i.e. a one-step reaction from chorismate, in addition to the endogeneous multi-step phenylpropanoid pathway. Feeding experiments with [1,7-(13)C(2)]shikimic acid showed that in the most active transgenic line, 73% of 4HB was synthesized via the genetically introduced pathway. However, there was no correlation between CPL activity and 4HB glucoside or shikonin accumulation in the transgenic lines. HMG-CoA reductase (HMGR) is involved in the biosynthesis of GPP in L. erythrorhizon. Two forms of HMGR1 of Arabidopsis thaliana were expressed in Lithospermum under control of the (ocs)(3)mas promoter. Only moderate increases in enzyme activity were obtained with the complete enzyme, but high activity was achieved using the soluble cytosolic domain of HMGR1. Shikonin accumulation remained unchanged even upon high expression of soluble HMGR.
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany.
Annegret
A
Köhle
Susanne
S
Sommer
Kazufumi
K
Yazaki
Albert
A
Ferrer
Albert
A
Boronat
Shu-Ming
SM
Li
Lutz
L
Heide
D017360:Arabidopsis / Q000201:enzymology; D015152:Blotting, Northern; D015139:Blotting, Southern; D002247:Carbon Isotopes; D022081:Cell Surface Extensions / Q000201:enzymology; D003001:Cloning, Molecular; D046508:Culture Techniques; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics; D005960:Glucosides / Q000096:biosynthesis / Q000737:chemistry; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics* / Q000378:metabolism; D029684:Lithospermum / Q000201:enzymology* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D009285:Naphthoquinones / Q000737:chemistry / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000378:metabolism; D010226:Parabens / Q000737:chemistry / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology* / Q000235:genetics; D030821:Plants, Genetically Modified; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D012765:Shikimic Acid / Q000378:metabolism
D002247:Carbon Isotopes; D005960:Glucosides; D009285:Naphthoquinones; D010226:Parabens; D011106:Polyisoprenyl Phosphates; D012765:Shikimic Acid; C016101:shikonin; D006903:Hydroxymethylglutaryl CoA Reductases; D007652:Oxo-Acid-Lyases; C075960:chorismate pyruvate lyase; C038193:4-hydroxybenzoic acid