Journal Article

Genetic Engineering on Shikonin Biosynthesis: Expression of the Bacterial <i>ubiA</i> Gene in <i>Lithospermum erythrorhizon</i>

Robert Boehm, Susanne Sommer, Shu-Ming Li and Lutz Heide

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 41, issue 8, pages 911-919
Published in print August 2000 | ISSN: 0032-0781
Published online August 2000 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcd013
Genetic Engineering on Shikonin Biosynthesis: Expression of the Bacterial ubiA Gene in Lithospermum erythrorhizon

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The naphthoquinone pigment shikonin from Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) was the first plant secondary metabolite produced in industrial scale from plant cell cultures. We have now manipulated the biosynthetic pathway leading to shikonin in L. erythrorhizon by introduction of the bacterial gene ubiA. This gene of Escherichia coli encodes 4-hydroxybenzoate-3-polyprenyltransferase, a membrane-bound enzyme that catalyzes a key step in ubiquinone biosynthesis. Using geranyl diphosphate (GPP) as substrate, it is able to catalyze the formation of 3-geranyl-4-hydroxybenzoate (GBA), a principal step of shikonin biosynthesis. The prokaryotic ubiA gene was fused to two signal sequences for targeting of the resulting peptide to the endoplasmic reticulum (ER). Constructs with different constitutive promoters were introduced into L. erythrorhizon using Agrobacterium rhizogenes-mediated transformation. In the resulting hairy root lines, high UbiA enzyme activities could be observed, reaching 133 pkat mg–1. Expression of ubiA resulted in an accumulation of GBA in an amount exceeding that of the control culture by a factor of 50. However, the ubiA-transformed lines showed only a marginal (average 22%) increase of shikonin production in comparison to the control lines, and there was no significant correlation of UbiA enzyme activity and shikonin accumulation. This suggests that overexpression of ubiA alone is not sufficient to increase shikonin formation, and that further enzymes are involved in the regulation of this pathway.

Keywords: Key words: Endoplasmic reticulum — 4-Hydroxybenzoate-prenyltransferase — Lithospermum erythrorhizon — Shikonin — Targeting — ubiA.; Abbreviations: 4EB, 4-ethyl benzoate; 4HB, 4-hydroxybenzoate; DHEF, dihydroechinofuran; ER, endoplasmic reticulum; FBA, 3-farnesyl-4-hydroxybenzoate; FPP, farnesyl diphosphate; GBA, 3-geranyl-4-hydroxybenzoate; GPP, geranyl diphosphate; MSV, Murashige and Skoog with vitamins; WP medium, woody plant medium.

Journal Article.  6005 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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