(228e) Exploration of Epigenetic Regulation and Development of Global and Targeted Epigenetic Engineering Tools for Taxus Plant Cell Culture

Plant-derived secondary metabolites have many applications as fragrances, food additives, dyes, and bioactive medicinal compounds. Many medicinal plants have developed unique biosynthetic capabilities that make production of these metabolites in plant cell culture (PCC) an attractive alternative to extensive engineering of model hosts. Yet, long-term continuous subculturing of PCCs can reduce productivity due to increased DNA methylation of promoters, transcription factors, and other regulatory elements, resulting in temporal downregulation of secondary metabolism. Innovative epigenetic engineering techniques that take advantage of new insights into epigenetic regulation have been developed for a variety of eukaryotes, but these tools have not yet been explored in the context of metabolic engineering for PCC production systems. Applying this epigenetic control to PCC could restore and maintain high productivity, even in cultures that have been in suspension for many years. Here, we present on our identification of epigenetic engineering targets and development of both global and targeted DNA methylation engineering tools for improving production of the chemotherapeutic drug paclitaxel (Taxol) in Taxus PCC.

We have demonstrated that treatment of Taxus cuspidata and Taxus chinensis PCCs with 5-azacytidine (5AC), a nonspecific DNA methyltransferase inhibitor, results in a 5 to 20-fold increase in accumulation of paclitaxel precursors 10-deacetylbaccatin (10-DAB) and baccatin III with no negative effects on cell viability or biomass accumulation. Yet, despite this increase in precursor production, treatment with 5AC had no significant effect on paclitaxel yield, indicating a need for a mechanistic understanding of epigenetic regulatory mechanisms and more targeted epigenetic engineering approaches to increase end-product yield. To this end, we seek to identify key epigenetic regulatory points in taxane biosynthesis by correlating gene expression of taxane biosythetic pathway genes and regulatory elements with DNA methylation of the corresponding promoters.

Differential transcriptome analysis of Taxus cuspidata PCCs in response to elicitation with methyl jasmonate (MeJA), a well-characterized activator of secondary metabolism in numerous plant systems, was used to identify potential gene targets for targeted epigenetic engineering. Studies revealed that phenylpropanoid metabolism (which competes for the paclitaxel precursor phenylalanine) was significantly upregulated under MeJA elicitation, which was confirmed by an observed increase in both lignin content and total flavonoids in MeJA-elicited cultures. Additionally, we have identified paclitaxel biosynthetic pathway genes that are either expressed at low levels or not significantly upregulated under elicitation (including BAPT and DBAT), indicating that these genes may be rate-limiting steps in paclitaxel biosynthesis and thus, good targets for targeted epigenetic engineering. Finally, we will discuss our development of CRISPR/dCas9-based tools for targeted demethylation of taxane biosynthetic pathway genes and targeted methylation of phenylpropanoid metabolism genes. This is the first known study to implement CRISPR-based epigenetic engineering approaches for metabolic engineering of plant cell cultures.

In summary, through both improving our fundamental understanding of epigenetic regulation in plant cell culture systems and developing tools to engineer DNA methylation, we have pioneered a novel approach to rational engineering of Taxus plant cell culture. These approaches are easily generalizable and can be applied in parallel with traditional metabolic engineering methods to achieve stable, high productivity in a variety of different plant cell culture systems.