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).

Selection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp

“…To validate the reliability of the selected reference genes, we measured the relative expression of two cannabinoids pathway genes using EF-1α and TUB as the most stable reference genes and CHAL as the least stable reference gene ( Fig 4 ). Since CBDA content is decreased by the influence of osmotic stress [ 41 ], we measured the expression of AAE1 and CBDAS genes that are involved in the rate-determining enzymatic reactions leading to CBDA synthesis under drought and salinity stresses [ 51 , 52 ]. The expression of these two genes was significantly reduced under drought and salinity stresses when qRT-PCR data were normalized by EF-1α , TUB , and the combination of EF-1α and TUB .…”

Cannabis sativa: From Therapeutic Uses to Micropropagation and Beyond

“…Successful genetic transformation using Agrobacterium-mediated technology is often challenged by different responses associated with using a variety of different strains and the study by Sorokin et al [97] showed positive transient expression when seedling explants were transformed with a strain of A. tumefaciens (EHA105) harbouring the pCAMBIA130-uidA GUS reporter gene. Although many of these studies aim to ultimately increase metabolite yields in plant systems, other researchers and biotechnology companies intend to focus on using microbial systems for the heterologous manufacture of desired compounds for en masse production of cannabinoids that occur in nature in trace quantities [98]. With this in mind, it becomes critical to better understand the regulatory mechanisms that control specialised metabolism in Cannabis.…”

“…The application of CRISPR Cas9 gene editing techniques also holds promise for use in both C. sativa and C. indica and with future applications being imminent as CRISPR Cas 9-mediated editing has several advantages for Cannabis metabolite engineering. As detailed by Deguchi et al [98], gene knockouts that target multiple branch pathways may alter metabolic flux, leading to increased production of cannabinoids and terpenoids of pharmaceutical interest. Base editing of single-nucleotide polymorphisms associated with the Cannabis genome also offers superior methods that will likely have a higher public acceptance than conventional genetic modification strategies for the production of elite chemotypes of C. sativa [98].…”

“…As detailed by Deguchi et al [98], gene knockouts that target multiple branch pathways may alter metabolic flux, leading to increased production of cannabinoids and terpenoids of pharmaceutical interest. Base editing of single-nucleotide polymorphisms associated with the Cannabis genome also offers superior methods that will likely have a higher public acceptance than conventional genetic modification strategies for the production of elite chemotypes of C. sativa [98]. Such technologies are also highly suitable for molecular breeding for traits not only associated with phytochemical composition of Cannabis strains but also to impart better disease and pest resistance, polyploidy manipulation plus the alterations to general patterns of plant and growth development of Cannabis plants [83,98].…”

Biosynthesis of cannabinoid precursor olivetolic acid by overcoming rate-limiting steps in genetically engineered Yarrowia lipolytica

“…Almost all CBDs are sourced from farm-harvested hemp or Cannabis sativa -related species. Recent molecular breeding has resulted in special hemp cultivars which synthesize less tetrahydrocannabinol (THC) -- the principle psychoactive constituent of cannabis ( Marijuana ) 5 . CBD oils free of THCs will not cause human addiction.…”