Date of Award
Master of Science
Isatis tinctoria L. (woad) is a natural source of the anticancer compound indirubin, as well as the related precursors for this and other indigoid compounds like indigo. Indirubin and indigo are produced from the dimerization of indoxyl molecules, which are products of hydrolysis of glycosylated indigoid precursors present in woad and other indigo-producing plants. The plant biosynthesis of indigoid precursors is not yet completely understood due to the lack of information on enzymes responsible for formation and oxidation of indole, which is the starting molecule for indigoid biosynthesis. This study is based on the hypothesis that the indigoid precursors in plants share part of their biosynthetic pathway with tryptophan (Trp) biosynthesis. It is proposed that a Trp synthase subunit alpha (TSA)-like enzyme in woad can independently produce indole from indole-3-glycerol phosphate (I3GP). Unlike what occurs in Trp biosynthesis, this indole is not channeled to Trp synthase subunit beta (TSB), but rather serves as a substrate for an indole oxidase that forms the indigoid precursor, indoxyl. The second hypothesis is that the indole oxidizing enzyme in woad is a P450 monooxygenase. In this study, a woad TSA-like gene (ItTSA2) complemented the growth of trpA mutant E. coli cells on minimal media, which lacked TSA activity. ItTSA2 shared 73% amino acid sequence similarity with the alpha subunit of tryptophan synthase of I. tinctoria (ItTSA1), but was more closely related (76% amino acid sequence similarity) to indole synthase (INS) of Arabidopsis thaliana. Further, two woad P450 genes (ItB4 and ItB24) were coexpressed with an arabidopsis P450-reductase gene (AtR2), which produced indirubin and indigo in E. coli cultures. When ItB4 and ItB24 were each coexpressed with ItTSA1 or ItTSA2 in a tryptophanase mutant E. coli strain, the transformed E. coli produced indirubin and isatin, which is another precursor of indirubin. Based on the results of this study, a possible indirubin biosynthesis pathway involving genes ItTSA2, ItB4 and ItB24 is proposed. The identification of these plant genes has opened the avenues for engineering indigoid biosynthetic pathway in plants, as well as generating blue-colored plant organs (e.g. flowers and fibers) for the floriculture and textile industry.
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