Exploring the production of high-value compounds in plant Catharanthus roseus hairy roots and yeast Yarrowia lipolytica

Zhao, Le
Major Professor
Zengyi Shao
Committee Member
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Chemical and Biological Engineering

This dissertation focuses on studying the production of two categories of high-value compounds in bio organisms. The first group is terpenoid indole alkaloids (TIAs) in plant Catharanthus roseus, and the second group is wax esters, one of fatty acid derivatives.

TIAs belong to secondary metabolites in C. roseus and some of them have wide pharmaceutical applications. In particular, vinblastine and vincristine are two TIAs with anticancer properties and have been marked and used in chemotherapeutic reagents. The biggest issue for TIA production is that the content of those secondary metabolites in plant is extremely low. To improve the TIA production, we studied the regulation mechanism of the TIA pathway and explored the feasibility of valuable TIA production in hairy root culture.

Compared with the whole plant, plant tissue culture, such as hairy root culture, has many advantages, like fast growth, large-scale cultivation, and ease of genetic engineering. But the biggest issue for hairy root is that the vinblastine and vincristine synthetic pathway is blocked, mainly one of their precursors, vindoline, can not be synthesized in hairy root. To explore whether C. roseus hairy root could produce the intermediates in the vindoline pathway by overexpressing the pathway enzymes, we co-expressed the first two genes, tabersonine 16-hydrolase (T16H) and 16-O-methyltransferase (16OMT) in the vindoline pathway into hairy root. Transcriptional analysis and metabolic profiling were done to compare the difference between the parent hairy root lines and the engineered hairy root lines.

For the metabolic profiling, since the standards for those intermediates were not available, we prepared in-house standards by expressing the plant genes in Saccharomyces cerevisiae, fed substrate, and purified TIA compounds from yeast cell culture. Liquid chromatography (LC) coupled with either photodiode array detector (PDA) or mass spectrometry (MS) were applied to isolate and analyze the TIA compounds by their UV-Visible absorption spectra and molecular weights.

In addition, fundamental research was done in C. roseus hairy root to study the effects of transcription regulators on the transcript levels and metabolite levels of the TIA pathway. Two of the seven reported transcription activators of the TIA pathway, octadecanoid-responsive Catharanthus AP2-domain 2(ORCA3) and MYB-like DNA-binding protein (BPF1), were overexpessed in hairy root separately, Two of the transcription repressors, G-box binding factors (GBF1 and GBF2), were knocked down by RNA interface in hairy root. And the transcription analysis and metabolic profiling of the transcription regulator-engineered hairy root lines were done to see what were the effects caused by those regulators.

Wax esters have a lot of applications in lubricant, skin care products, cosmetics, inking, and coating industries. Currently the main bio source for high-performed wax ester is from the seeds of jojoba. The tight supply makes wax esters high-value compounds. To reduce the production cost, we introduced the wax ester biosynthetic pathway into an oleaginous yeast, Yarrowia lipolytica. The free fatty alcohol, and wax ester were quantified in the engineered Y. lipolytica.

To provide more substrate for wax ester synthesis, we knocked out some genes in the substrate competitive pathways, and constructed four strains with different combination of knockout genes. To utilize the most abundant fatty acid types in Y. lipolytica, we compared three fatty acyl-CoA reductases (FAR), the first enzyme in the wax ester pathway, from different species. It was found that those three FARs had different substrate specificity and the wax ester production varied a lot in those strains.

To solve the plasmid instability issue, we randomly integrated FAR gene and WS gene into Y. lipolytica genome, and studied the effect of nitrogen limited fermentation on the wax ester production in one of our best strains.