Search Results (9)

Cannabinoids are high-value bioactive compounds whose sustainable production remains challenging, prompting interest in biocatalytic and microbial platforms as alternatives to plant extraction. In this study, we investigated the heterologous expression and functionality of two key cannabinoid-related enzymes in the photosynthetic microalga Chlamydomonas reinhardtii: the aromatic prenyltransferase, NphB_G286S/Y288A from Streptomyces sp., and the plant-derived cannabidiolic acid synthase (CBDAS) from Cannabis sativa. Codon-optimized genes were introduced into the nuclear genome of C. reinhardtii using several construct configurations and promoters, and stable transformants were generated and characterized for genomic integration, transcript accumulation, protein production, enzymatic activity, and cannabinoid-related metabolite formation. While NphB protein accumulation was achieved under the PSAD promoter control, CBDAS was not detected at the protein level under any condition tested. In vitro enzymatic assays using soluble algal protein extracts from NphB-expressing lines confirmed catalytic activity, yielding cannabigerolic acid (CBGA), reaching up to 633 ± 58 µg L⁻¹. However, no CBGA production was detected in vivo, despite substrate supplementation. These results indicate that, although bacterial prenyltransferase can be functionally expressed in C. reinhardtii, efficient metabolic conversion in vivo is limited by cellular and biochemical constraints, including substrate availability, intracellular compartmentalization, and potential competition with endogenous pathways. In contrast, the absence of detectable CBDAS highlights the challenges associated with expressing complex plant oxidocyclases in this photosynthetic host. Overall, this work provides mechanistic insights into enzyme compatibility and metabolic bottlenecks in microalgal systems and outlines key considerations for the future development of photosynthetic platforms for cannabinoid biocatalysis. Full article

Prenylated aromatic compounds (PACs) are widely distributed in nature and have important applications in medicine, cosmetics, and food due to their antioxidant, anticancer, and anti-inflammatory activities as well as role in the prevention of neurological diseases. Traditional methods of PAC production such as plant extraction and chemical synthesis remain constrained by the low content of these compounds in plants and the complexity of the chemical processes. PACs are synthesized from aromatic compound receptors and prenyl side chain donors, which are in turn synthesized via the shikimate pathway and 2-C-methyl-D-erythritol-4-phosphate/mevalonic acid pathways, respectively. Increasing exploration and research on prenyltransferases (PTs), the key enzymes involved in PAC biosynthesis, have facilitated the emergence of microbial synthesis of PACs as a promising alternative to industrial production. The microbial biosynthesis of PACs is summarized herein, mainly from the perspective of screening and modification of the key enzymes PTs, selection of suitable host systems, and engineering the modification of microbial cell factories to enhance the yields of PACs. The future prospects and challenges of PAC biosynthesis are also discussed. Full article

Prenylation increases the structural diversity and biological activity of flavonoids. In this study, an aromatic prenyltransferase, FgPT1, was identified from Fusarium globosum. This enzyme was demonstrated to specifically catalyze the prenylation of flavanones, including naringenin, hesperitin, eriodictyol, liquiritigenin, rac-pinocembrin, and dihydrogenistein, and exhibited no activity toward other types of flavonoids, including chalcones, flavonols, isoflavonoids, and flavonols. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and nuclear magnetic resonance (NMR) analysis indicated that the majority of prenylated products were 6-C prenyl flavanones, with the exception of liquiritigenin, which was additionally transformed to 4′-O prenyl liquiritigenin. Enzyme kinetic analysis suggested that FgPT1 exhibited the highest catalytic efficiency towards naringenin, with a k_cat/K_M value determined as 61.92 s⁻¹ M⁻¹, and the lowest catalytic efficiency towards liquiritigenin, with a k_cat/K_M of 1.18 s⁻¹ M⁻¹. Biochemical characterization suggested that FgPT1 functioned as a metal-dependent enzyme with optimal activity in the presence of Ba²⁺ at pH 7.5 and 30 °C. Site-directed mutagenesis resulted in a series of mutants, including A325V with impaired prenylation activity and V116I, V181I, and V194I with enhanced activity. V194I displayed the highest enzymatic activity with a nine-fold increase compared to wild-type FgPT1. Full article

Hericium erinaceus (Lion’s Mane mushroom) is widely consumed for its numerous reported benefits for brain health. A growing body of evidence suggests that these benefits are likely attributable to aromatic compounds contained in its fruiting bodies, including the meroterpenoids hericenones. Here, we report the identification and reconstitution of the first two steps of the biosynthetic pathway of hericenones via heterologous expression of the polyketide synthase HerA and the carboxylic acid reductase HerB in Aspergillus oryzae. Furthermore, we investigated a putative prenyltransferase that might be responsible for the following biosynthetic step. Ongoing efforts to reconstitute the full pathway will enable large-scale production of hericenones and other meroterpenoids in heterologous hosts. Full article

Traditional isolation methods often lead to the rediscovery of known natural products. In contrast, genome mining strategies are considered effective for the continual discovery of new natural products. In this study, we discovered a unique prenyltransferase (PT) through genome mining, capable of catalyzing the transfer of a prenyl group to an aromatic nucleus to form C-C or C-O bonds. A pair of new hydroxyphenylacetic acid derivative enantiomers with prenyl units, (±)-peniprenydiol A (1), along with 16 known compounds (2–17), were isolated from a marine fungus, Penicillium sp. W21C371. The separation of 1 using chiral HPLC led to the isolation of the enantiomers 1a and 1b. Their structures were established on the basis of extensive spectroscopic analysis, including 1D, 2D NMR and HRESIMS. The absolute configurations of the new compounds were determined by a modified Mosher method. A plausible biosynthetic pathway for 1 was deduced, facilitated by PT catalysis. In the in vitro assay, 2 and 3 showed promising inhibitory activity against Escherichia coli β-glucuronidase (EcGUS), with IC₅₀ values of 44.60 ± 0.84 μM and 21.60 ± 0.76 μM, respectively, compared to the positive control, D-saccharic acid 1,4-lactone hydrate (DSL). This study demonstrates the advantages of genome mining in the rational acquisition of new natural products. Full article

4-Hydroxycoumarin (4HC) has been used as a lead compound for the chemical synthesis of various bioactive substances and drugs. Its prenylated derivatives exhibit potent antibacterial, antitubercular, anticoagulant, and anti-cancer activities. In doing this, E. coli BL21(DE3)pLysS strain was engineered as the in vivo prenylation system to produce the farnesyl derivatives of 4HC by coexpressing the genes encoding Aspergillus terreus aromatic prenyltransferase (AtaPT) and truncated 1-deoxy-D-xylose 5-phosphate synthase of Croton stellatopilosus (CstDXS), where 4HC was the fed precursor. Based on the high-resolution LC-ESI(±)-QTOF-MS/MS with the use of in silico tools (e.g., MetFrag, SIRIUS (version 4.8.2), CSI:FingerID, and CANOPUS), the first major prenylated product (named compound-1) was detected and ultimately elucidated as ferulenol, in which information concerning the correct molecular formula, chemical structure, substructures, and classifications were obtained. The prenylated product (named compound-2) was also detected as the minor product, where this structure proposed to be the isomeric structure of ferulenol formed via the tautomerization. Note that both products were secreted into the culture medium of the recombinant E. coli and could be produced without the external supply of prenyl precursors. The results suggested the potential use of this engineered pathway for synthesizing the farnesylated-4HC derivatives, especially ferulenol. Full article

The medicinal properties of cannabis and the its legal status in several countries and jurisdictions has spurred the massive growth of the cannabis economy around the globe. The value of cannabis stems from its euphoric activity offered by the unique phytocannabinoid tetrahydrocannabinol (THC). However, this is rapidly expanding beyond THC owing to other non-psychoactive phytocannabinoids with new bioactivities that will contribute to their development into clinically useful drugs. The discovery of the biosynthesis of major phytocannabinoids has allowed the exploration of their heterologous production by synthetic biology, which may lead to the industrial production of rare phytocannabinoids or novel synthetic cannabinoid pharmaceuticals that are not easily offered by cannabis plants. This review summarizes the biosynthesis of major phytocannabinoids in detail, the most recent development of their metabolic engineering in various systems, and the engineering approaches and strategies used to increase the yield. Full article

Prenylated natural products have interesting pharmacological properties and prenylation reactions play crucial roles in controlling the activities of biomolecules. They are difficult to synthesize chemically, but enzymatic synthesis production is a desirable pathway. Cyclic dipeptide prenyltransferase catalyzes the regioselective Friedel–Crafts alkylation of tryptophan-containing cyclic dipeptides. This class of enzymes, which belongs to the dimethylallyl tryptophan synthase superfamily, is known to be flexible to aromatic prenyl receptors, while mostly retaining its typical regioselectivity. In this study, seven tryptophan-containing cyclic dipeptides 1a–7a were converted to their C7-regularly prenylated derivatives 1b–7b in the presence of dimethylallyl diphosphate (DMAPP) by using the purified 7-dimethylallyl tryptophan synthase (7-DMATS) as catalyst. The HPLC analysis of the incubation mixture and the NMR analysis of the separated products showed that the stereochemical structure of the substrate had a great influence on their acceptance by 7-DMATS. Determination of the kinetic parameters proved that cyclo-l-Trp–Gly (1a) consisting of a tryptophanyl and glycine was accepted as the best substrate with a K_M value of 169.7 μM and a turnover number of 0.1307 s⁻¹. Furthermore, docking studies simulated the prenyl transfer reaction of 7-DMATS and it could be concluded that the highest affinity between 7-DMATS and 1a. Preliminary results have been clearly shown that prenylation at C7 led to a significant increase of the anticancer and antimicrobial activities of the prenylated derivatives 1b–7b in all the activity test experiment, especially the prenylated product 4b. Full article

In plants, prenylation of metabolites is widely distributed to generate compounds with efficient defense potential and distinct pharmacological activities profitable to human health. Prenylated compounds are formed by members of the prenyltransferase (PT) superfamily, which catalyze the addition of prenyl moieties to a variety of acceptor molecules. Cell cultures of Hypericum calycinum respond to elicitor treatment with the accumulation of the prenylated xanthone hyperxanthone E. A cDNA encoding a membrane-bound PT (HcPT) was isolated from a subtracted cDNA library and transcript preparations of H. calycinum. An increase in the HcPT transcript level preceded hyperxanthone E accumulation in cell cultures of H. calycinum treated with elicitor. The HcPT cDNA was functionally characterized by expression in baculovirus-infected insect cells. The recombinant enzyme catalyzed biosynthesis of 1,3,6,7-tetrahydroxy-8-prenylxanthone through regiospecific C–8 prenylation of 1,3,6,7-tetrahydroxyxanthone, indicating its involvement in hyperxanthone E formation. The enzymatic product shared significant structural features with the previously reported cholinesterase inhibitor γ-mangostin. Thus, our findings may offer a chance for semisynthesis of new active agents to be involved in the treatment of Alzheimer’s disease. Full article