(553c) Metabolic and Epigenetic Engineering Enables Temporal Control of Taxane Metabolism in Taxus Chinensis Plant Cell Culture

Many medicinal plants have developed unique biosynthetic capabilities that make production of specialized metabolites in plant cell culture (PCC) an attractive alternative to extensive engineering of model hosts. However, rational engineering of PCC systems is limited by both a lack of robust metabolic engineering tools and temporal downregulation of secondary metabolism in continuously subcultured cell lines. To this end, we have developed two engineering strategies that can be utilized in parallel to achieve stable, high titers of the chemotherapeutic paclitaxel in Taxus chinensis PCC. First, using a pathway overexpression approach, four genes identified as potential rate-limiting steps in paclitaxel biosynthesis (TASY, DBAT, BAPT, and DBTNBT) were constitutively overexpressed in Taxus chinensis cell lines, resulting in up to a 9.9-fold increase in paclitaxel titer and differential accumulation of the pathway intermediates 10-deacetylbaccatin III (10-DAB) and baccatin III. These genes remained overexpressed and stably integrated in the genome even after 3 months of continuous subculturing. Second, in previous work we have demonstrated that increased DNA methylation of promoters, transcription factors, and other regulatory elements in continuously subcultured cell lines results in global downregulation of secondary metabolism, which can be rescued with DNA methyltransferase inhibitors. Thus, to enable finely tuned epigenetic control of secondary metabolism, we have developed CRISPR/dCas9-based tools for targeted methylation and demethylation of secondary metabolite biosynthesis genes using the catalytic domains of the Nicotiana tabacum DNA methyltransferase NtDRM1 and the human DNA demethylase TET1, respectively. These tools were applied to epigenetically downregulate PAL, which controls phenylpropanoid metabolism and competes with taxane biosynthesis, and upregulate BAPT, a gene in the taxane biosynthetic pathway hypothesized to be controlled by DNA methylation. This work is easily generalizable to other PCC systems and builds the foundation for use of rational epigenetic manipulation of metabolism as a tool for metabolic engineering.