Biotechnological approaches to secondary metabolites production in Cannabis sativa cell cultures

Cannabis sativa is a rich source of different molecules with growing commercial interest due to their bioactive properties. The most abundant compounds produced by its secondary metabolism are cannabinoids, terpenes, and phenolic compounds. This complex mixture of biomolecules is primarily synthesized in the glandular trichomes of female inflorescences and exhibits significant variability depending on cultivar and growth conditions. Molecular techniques and genome editing offer the potential to manipulate the biosynthetic pathways of cannabis, thereby increasing the production of specific metabolites. However, cannabis is recalcitrant to transformation, and reliable protocols for its in vitro regeneration are currently lacking. In this context, the use of cell cultures represents an attractive alternative for the industrial production of specialized metabolites, given their growth in a controlled environment and the possibility of direct transformation without the need for whole plant regeneration. Through biolistic transformation, a protocol has been developed that enables the stable transformation of cannabis suspension cultures. Transcription factors involved in trichome development from the R2R3 MYB MIXTA and HD-ZIP IV families, were selected to generated different transformed cell lines, with the aim of inducing cellular differentiation and the subsequent activation of the cannabinoid biosynthetic pathway. These factors were expressed using a dexamethasone-inducible expression system. The effect of varying dexamethasone concentrations on transcriptional activation was evaluated utilizing reporter genes that allowed to identify the best induction treatment. Gene expression analyses on transformed cell lines showed that the expression of SlCD2, an HD-ZIP IV from tomato, and of PpMYB25, a MIXTA from peach, can transactivate endogenous cannabis transcription factors related to trichome development and structural genes of the cannabinoid biosynthetic pathway, especially when transgene induction was paired with methyl jasmonate elicitation. PpMYB25 was expressed also in tobacco plants to study its effects at the organism level. Taken together, results from both cannabis cells and tobacco support the idea that the function of some master regulators of glandular trichome development is conserved among different species, making them interesting biotechnological tools for secondary metabolism manipulation.