Production of Halogenated Indigo Precursors in Metabolically Engineered Tobacco Plants

Indigogenic glycosides are useful chromogenic substrates for histochemistry and molecular biology. Most prominent is X-Gal (5-Bromo-4-chloroindoxyl-ß-D-galactoside) used for blue white selection during molecular cloning procedures in bacteria. Although the precursor indican is a natural product widespread in a large number of indigo plants, the halogenated derivatives mainly utilized as enzyme substrates needs to be chemically synthesized. Synthesis of halogenated aromatic components require numerous toxic precursors and intermediates as well as organic solvents, rendering the whole process potentially harmful to the environment. We established a system for the in-planta biosynthesis of a large array of indigogenic glucosides. Engineering of a three different bacterial halogenases along with tryptophanase and a monooxygenase lead to the formation of specifically chlorinated indican derivatives. Moreover, combination of halogenases enabled chlorination at multiple sites and co-administration of bromine enabled the formation of brominated indican derivatives. Also different subcellular compartments for optimal precursor supply as well as product formation were evaluated. Taken together, plants provide a suitable platform for the production of halogenated fine chemicals.