Genetic manipulation of transcriptional regulators alters nicotine biosynthesis in tobacco.

The toxic alkaloid nicotine is produced in the roots of in Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explore the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. Transient overexpression of NtERF189 led to alkaloid production in the leaves of N. benthamiana and N. alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a bHLH-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.