Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene

被引:38
作者
Sommer, S
Köhle, A
Yazaki, K
Shimomura, K
Bechthold, A
Heide, L
机构
[1] Univ Tubingen, Inst Pharmazeut, D-72076 Tubingen, Germany
[2] Kyoto Univ, Grad Sch Agr, Div Appl Life Sci, Mol & Cellular Biol Lab, Kyoto 60601, Japan
[3] Natl Inst Hlth Sci, Tsukuba Med Plant Res Stn, Ibaraki, Osaka 3050843, Japan
关键词
genetic engineering; 4-hydroxybenzoic acid glucoside; Lithospermum erythrorhizon; menisdaurin; shikonin; ubiC;
D O I
10.1023/A:1006185806390
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation. The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-C-13(2)]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.
引用
收藏
页码:683 / 693
页数:11
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