Metabolic Engineering Strategies of Industrial Hemp (Cannabis sativa L.): A Brief Review of the Advances and Challenges

Abstract

Industrial hemp (Cannabis sativa L.) is a diploid (2n = 20), dioecious plant that is grown for fiber, seed, and oil. Recently, there has been a renewed interest in this crop because of its panoply of cannabinoids, terpenes, and other phenolic compounds. Specifically, hemp contains terpenophenolic compounds such as cannabidiol (CBD) and cannabigerol (CBG), which act on cannabinoid receptors and positively regulate various human metabolic, immunological, and physiological functions. CBD and CBG have an effect on the cytokine metabolism, which has led to the examination of cannabinoids on the treatment of viral diseases, including COVID-19. Based on genomic, transcriptomic, and metabolomic studies, several synthetic pathways of hemp secondary metabolite production have been elucidated. Nevertheless, there are few reports on hemp metabolic engineering despite obvious impact on scientific and industrial sectors. In this article, recent status and current perspectives on hemp metabolic engineering are reviewed. Three distinct approaches to expedite phytochemical yield are discussed. Special emphasis has been placed on transgenic and transient gene delivery systems, which are critical for successful metabolic engineering of hemp. The advent of new tools in synthetic biology, particularly the CRISPR/Cas systems, enables environment-friendly metabolic engineering to increase the production of desirable hemp phytochemicals while eliminating the psychoactive compounds, such as tetrahydrocannabinol (THC).

Keywords: CRISPR/Cas; Cannabis sativa; RNA interference; cannabinoid; metabolic engineering.

Figure 1

Biosynthetic pathways for cannabinoids and terpenoids in hemp. CBCA, cannabichromenic acid; CBDA, cannabidiolic acid; CBGA, cannabigerolic acid; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl pyrophosphate; GPP, geranyl diphosphate; HMBPP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate; IPP, isopentenyl diphosphate; MEP, methylerythritol phosphate; MVA, mevalonate; THCA, tetrahydrocannabinolic acid. Created with BioRender.

Figure 2

A schematic strategy of transgenic and transient gene delivery system. These gene delivery systems aim to alter gene expression in hemp female flower, where cannabinoids and terpenes are preferentially synthesized and stored. (A) Transgenic gene delivery systems. Specialized metabolites and their precursor synthesis genes, transcription factor genes, and other hemp genes related to the supply of energy and reducing power might be engineered by introducing conventional binary vectors or CRISPR/Cas vectors. Hemp SE might be activated by the overexpression of positive regulator genes such as BBM and WUS2 or the downregulation of negative regulator genes such as CLAVATA3. (B) Three transient gene delivery systems. To overcome the instability of dsRNA, clay nanoparticles, liposomes, viruses, or bacteria might be used for the effective delivery of dsRNA. For successful agroinfiltration, vacuum infiltration is likely to be more efficient than syringe infiltration based on our preliminary experiments. Created with BioRender. CRISPR/Cas-mediated genome editing is likely to be most powerful method for hemp metabolic engineering.