Search Results (4)

The bioproduction of high-value molecules offers a sustainable and cost-effective alternative to traditional extraction and chemical synthesis, particularly for complex metabolites like cannabinoids (CBs), which have therapeutic potential for neurodegenerative diseases. The marine diatom Phaeodactylum tricornutum presents a promising chassis for CB biosynthesis due to its high lipid content, essential building blocks to biosynthesize CBs. In this study, we explored the feasibility of producing olivetolic acid (OA), the key CB precursor, using a hybrid-type polyketide synthase, SteelyA, from Dictyostelium discoideum. Unlike the native Cannabis sativa enzymes—tetraketide synthase and olivetolic acid cyclase—which exhibit low productivity and stability in diatoms, SteelyA was expected to offer an alternative biosynthetic route. Heterologous production in P. tricornutum resulted in a C-terminal fragment of the SteelyA enzyme, suggesting partial expression or processing of the very high-molecular-weight (352 kDa) SteelyA protein over six months without affecting cellular growth. However, HPLC-MS analysis did not detect intracellular OA or its derivatives in vivo and in vitro, suggesting enzymatic inactivity or metabolic limitations. These negative findings highlight the need for further investigation into the metabolic and proteomic requirements for CB precursor biosynthesis in diatoms, guiding future optimization strategies for sustainable cannabinoid production. Full article

Substrate channeling could be very useful for plant metabolic engineering; hence, we propose that functionalized supramolecular self-assembly scaffolds can act as enzymatic hubs able to perform reactions in close contiguity. Virus nanoparticles (VNPs) offer an opportunity in this context, and we present a functionalization strategy to display different enzymes on the outer surface of three different VNPs produced in plants. Tomato bushy stunt virus (TBSV) and Potato virus X (PVX) plant viruses were functionalized by the genetic fusion of the E-coil peptide coding sequence to their respective coat proteins genes, while the enzyme lichenase was tagged with the K-coil peptide. Immobilized E-coil VNPs were able to interact in vitro with the plant-produced functionalized lichenase, and catalysis was demonstrated by employing a lichenase assay. To prove this concept in planta, the Hepatitis B core (HBc) virus-like particles (VLPs) were similarly functionalized by genetic fusion with the E-coil sequence, while acyl-activating enzyme 1, olivetolic acid synthase, and olivetolic acid cyclase enzymes were tagged with the K-coil. The transient co-expression of the K-coil-enzymes together with E-coil-VLPs allowed the establishment of the heterologous cannabinoid precursor biosynthetic pathway. Noteworthy, a significantly higher yield of olivetolic acid glucoside was achieved when the scaffold E-coil-VLPs were employed. Full article

The increasing demand for novel natural compounds has prompted the exploration of innovative approaches in bioengineering. This study investigates the bioengineering potential of the marine diatom Phaeodactylum tricornutum through the introduction of cannabis genes, specifically, tetraketide synthase (TKS), and olivetolic acid cyclase (OAC), for the production of the cannabinoid precursor, olivetolic acid (OA). P. tricornutum is a promising biotechnological platform due to its fast growth rate, amenability to genetic manipulation, and ability to produce valuable compounds. Through genetic engineering techniques, we successfully integrated the cannabis genes TKS and OAC into the diatom. P. tricornutum transconjugants expressing these genes showed the production of the recombinant TKS and OAC enzymes, detected via Western blot analysis, and the production of cannabinoids precursor (OA) detected using the HPLC/UV spectrum when compared to the wild-type strain. Quantitative analysis revealed significant olivetolic acid accumulation (0.6–2.6 mg/L), demonstrating the successful integration and functionality of the heterologous genes. Furthermore, the introduction of TKS and OAC genes led to the synthesis of novel molecules, potentially expanding the repertoire of bioactive compounds accessible through diatom-based biotechnology. This study demonstrates the successful bioengineering of P. tricornutum with cannabis genes, enabling the production of OA as a precursor for cannabinoid production and the synthesis of novel molecules with potential pharmaceutical applications. Full article

The revised legislation on medicinal cannabis has triggered a surge of research studies in this space. Yet, cannabis proteomics is lagging. In a previous study, we optimised the protein extraction of mature buds for bottom-up proteomics. In this follow-up study, we developed a top-down mass spectrometry (MS) proteomics strategy to identify intact denatured protein from cannabis apical buds. After testing different source-induced dissociation (SID), collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) parameters on infused known protein standards, we devised three LC-MS/MS methods for top-down sequencing of cannabis proteins. Different MS/MS modes produced distinct spectra, albeit greatly overlapping between SID, CID, and HCD. The number of fragments increased with the energy applied; however, this did not necessarily translate into greater sequence coverage. Some precursors were more amenable to fragmentation than others. Sequence coverage decreased as the mass of the protein increased. Combining all MS/MS data maximised amino acid (AA) sequence coverage, achieving 73% for myoglobin. In this experiment, most cannabis proteins were smaller than 30 kD. A total of 46 cannabis proteins were identified with 136 proteoforms bearing different post-translational modifications (PTMs), including the excision of N-terminal M, the N-terminal acetylation, methylation, and acetylation of K resides, and phosphorylation. Most identified proteins are involved in photosynthesis, translation, and ATP production. Only one protein belongs to the phytocannabinoid biosynthesis, olivetolic acid cyclase. Full article