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

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the nap hthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon ha iry root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that co nverts 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 introdu ced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformati on. The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vi vo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic a cid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experimen t with [1,7-C-13(2)]shikimate showed that in the absence of AIP the artific ially introduced CPL reaction contributed ca. 20% of the overall 4HB biosyn thesis in the transgenic cultures. ubiC transformation did not lead to a st atistically significant increase of shikonin formation, but to a 5-fold inc rease of the accumulation of menisdaurin, a nitrile glucoside which is pres umably related to aromatic amino acid metabolism.