Introduction of the cannabinoid metabolic pathway into Nicotiana tabacum via combinatorial transformation

Multigene engineering presents significant challenges but is crucial for successfully undertaking engineering, especially when aiming at reconstructing complex biochemical pathways. While previous studies using tobacco have managed to integrate up to five genes using biolistic methods, further progress is needed to enable the simultaneous introduction of entire metabolic pathways. This research addresses this gap by using combinatorial transformation to introduce the genes necessary for the cannabinoid biosynthetic pathway from Cannabis sativa into the nuclear genome of Nicotiana tabacum (tobacco) in a single-step biolistic genetic transformation. Nine genes were chosen to reconstitute the cannabinoid biosynthetic pathway. All genes were successfully integrated into the nuclear genome of tobacco in a single transformation. Combinatorial transformation resulted in a variety of lines, with differences in transgene uptake, expression, and metabolite production. By regenerating hundreds of transgenic tobacco lines, plants capable of synthesising cannabinoids were successfully produced, achieving yields of up to 40.73 µg g-1 DW CBDA and 31.58 µg g-1 DW CBD, levels that have not been previously reported in tobacco. Out of 213 regenerated lines, 33 lines successfully integrated the full set of nine genes as confirmed by PCR. In addition to CBDA and CBD, other minor cannabinoids such as CBGA were detected, although in lower concentrations. Key pathways upregulated in transformants, in response to the insertion of the cannabinoid
metabolic pathway, included isoprenoid biosynthesis and plant defence mechanisms, while pathways related to light response and sulphur metabolism were downregulated. This study highlights the efficacy and effectiveness of combinatorial transformation for multigene integration, providing a streamlined approach to the introduction of novel ii secondary metabolite production pathways between species. The ability to introduce complex metabolic pathways into Nicotiana tabacum opens new avenues for biotechnological applications, including the potential large-scale production of valuable pharmaceuticals and other high-value compounds in a standardised plant
system.