Metabolic engineering and elucidation of the terpenoid indole alkaloid pathway in Catharanthus roseus hairy roots
Date
2016
Authors
Sun, Jiayi, author
Peebles, Christie A. M., advisor
Snow, Christopher D., committee member
Pilon-Smits, Elizabeth A. H., committee member
Fisk, Nick, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Catharanthus roseus (Madagascar periwinkle) produces many pharmaceutically important chemicals such as vinblastine, vincristine, serpentine, and ajmalicine. They are synthesized through the highly branched and complex terpenoid indole alkaloids (TIA) pathway in C. roseus. Among these TIAs, vinblastine and vinblastine, which are solely extracted from C. roseus, are the efficient anti-cancer drugs widely used in the clinic. However, due to the low accumulation of these TIAs within the plant and the industrial infeasibility of production using chemical synthesis, the market price of these drugs still remain high, and the production is inconsistent. With the advanced knowledge of molecular biology, metabolic engineering and bioinformatics, building a robust and efficient alternative production platform by manipulating the TIA pathway has become a major trend and promising strategy in recent research. However, many biosynthetic enzymes in TIA pathway and the regulation of the pathway are still poorly understood which impedes the rational engineering of this plant for enhanced TIA production. This thesis first uses advanced high-throughput sequencing technology to study the global transcriptional alterations after overexpressing a rate-limiting enzyme anthranilate synthase (AS) in the pathway. This study helps to increase understanding of TIA regulation in this transgenic hairy root line from a broader perspective. Furthermore, transcriptome sequencing of this unique transgenic line under three different conditions (uninduced control, induced AS overexpression, and methyl jasmonate elicitation) is analyzed using hierarchical clustering. A 200 candidate transcripts set was identified for the pathway genes located around the tabersonine branch point. Six cytochrome P450 monooxygenase candidates are selected for the unknown tabersonine 6,7 epoxidase that can convert tabersonine to lochnericine in C. roseus hairy roots. Meanwhile, effort on genetic modification of C. roseus hairy roots for TIA production using two different strategies are reported here. The first strategy helps establish a transgenic hairy root line with significantly increased TIA accumulation of all measure alkaloids by co-expressing the positive transcription factor ORCA3 (AP2-domain DNA-binding protein 3) and a pathway gene strictosidine glucosidase (SGD) that is not controlled by ORCA3. Since C. roseus hairy roots do not produce detectable vinblastine and vincristine due to the absence of the vindoline pathway, the second strategy initiated the effort to introduce the pathway by engineering the first two enzymes in C. roseus hairy root. Overexpression of these two genes, tabersonine 16-hydroxylase (T16H) and 16-O-methyl transferase (16OMT), leads to the accumulation of the expected vindoline pathway intermediates 16-hydroxytabersonine and 16-methoxytabersonine but not vindoline. Interestingly, the overexpression of these two genes influences the root native metabolite levels, triggers the altered transcription of TIA genes, and leads to the production of two new unknown metabolites. Overall, studies in this thesis not only contribute new transcriptome information to current publicly available databases, but also facilitate elucidating the TIA pathway and its complex regulation. This thesis also provides a metabolic engineering approach to enhance alkaloid production in C. roseus hairy roots by simultaneously overexpressing ORCA3 and SGD. Genetic modification of T16H and 16OMT in C. roseus hairy roots promisingly leads to the production of vindoline pathway intermediates. It also emphasizes some potential complexities for the future attempts to express the full vindoline pathway in hairy roots.
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Subject
terpenoid indole alkaloid
Madagascar periwinkle
high-throughput sequencing
transcription factor
plant secondary metabolism