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Protoberberine alkaloids and benzophenanthridine alkaloids (BZDAs) are subgroups of benzylisoquinoline alkaloids (BIAs), which represent a diverse class of plant-specialized natural metabolites with many pharmacological properties. Microbial biosynthesis has been allowed for accessibility and scalable production of high-value BIAs. Here, we engineer Saccharomyces cerevisiae to de novo produce a series of protoberberines and BZDAs, including palmatine, berberine, chelerythrine, sanguinarine and chelirubine. An ER compartmentalization strategy is developed to improve vacuole protein berberine bridge enzyme (BBE) activity, resulting in >200% increase on the production of the key intermediate ( S )-scoulerine. Another promiscuous vacuole protein dihydrobenzophenanthridine oxidase (DBOX) has been identified to catalyze two-electron oxidation on various tetrahydroprotoberberines at N7-C8 position and dihydrobenzophenanthridine alkaloids. Furthermore, cytosolically expressed DBOX can alleviate the limitation on BBE. This study highlights the potential of microbial cell factories for the biosynthesis of a diverse group of BIAs through engineering of heterologous plant enzymes. Protoberberine alkaloids and benzophenanthridine alkaloids (BZDAs) are plant derived natural products with many pharmaceutical properties. Here, the authors report de novo synthesizing of a series of these compounds by engineered Saccharomyces cerevisiae .

The application and drug development of plant-derived natural products are often limited by their low abundance in medicinal plants and the lack of structural complexity and diversity. Herein, we design a concise enzyme cascade to efficiently produce natural and unnatural protoberberine alkaloids from cost-effective, readily available substrates. Through enzyme discovery and engineering, along with systematic optimization of the berberine bridge enzyme to address remaining manufacturing challenges in protoberberine alkaloid biosynthesis, the high production of drug Rotundine is achieved at an impressive gram-scale titer, demonstrating its industrial potential. More importantly, this cascade also enables the efficient biosynthesis of various unnatural halogenated protoberberine alkaloids. Thus, this work not only unlocks the potential of enzyme cascades in overcoming longstanding challenges in the efficient biosynthesis of plant-derived alkaloids, but also opens avenues to introduce structural complexity and diversity into alkaloids through synthetic biology, offering significant potential for drug development. Plant-derived alkaloids are an important class of natural products due to their diverse pharmacological properties. Here, the authors report a concise enzyme cascade for the biosynthesis of natural and halogenated protoberberine alkaloids at higher productivity levels.

Chinese goldthread ( Coptis chinensis Franch.), a member of the Ranunculales, represents an important early-diverging eudicot lineage with diverse medicinal applications. Here, we present a high-quality chromosome-scale genome assembly and annotation of C. chinensis . Phylogenetic and comparative genomic analyses reveal the phylogenetic placement of this species and identify a single round of ancient whole-genome duplication (WGD) shared by the Ranunculaceae. We characterize genes involved in the biosynthesis of protoberberine-type alkaloids in C. chinensis . In particular, local genomic tandem duplications contribute to member amplification of a Ranunculales clade-specific gene family of the cytochrome P450 (CYP) 719. The functional versatility of a key CYP719 gene that encodes the ( S )-canadine synthase enzyme involved in the berberine biosynthesis pathway may play critical roles in the diversification of other berberine-related alkaloids in C. chinensis . Our study provides insights into the genomic landscape of early-diverging eudicots and provides a valuable model genome for genetic and applied studies of Ranunculales. Coptis chinensis represents an early-diverging eudicot lineage with diverse medicinal applications. Here, the authors report its chromosome-scale genome assembly, infer a single ancient whole-genome duplication, and characterize the function of CYP719 in diversification of protoberberine-type alkaloids.

Berberis darwinii, known for its bioactive alkaloids like berberine and palmatine, has gained attention for its medicinal properties. However, comprehensive studies on the specific bioactive molecules of Michay are lacking, as previous research has primarily focused on wild plants. Therefore, this study proposes to evaluate the alkaloid content in various tissues of B. darwinii collected from different locations, aiming to identify high-yielding accessions suitable for consistent bioactive alkaloid production. This research focuses on plants from a cultivated Michay orchard established five years ago. Leaves, stems, roots, and fruits from 96 accessions of Michay were collected to obtain an alkaloidal extract used for the characterization and comprehensive analysis of bioactive alkaloids through high-performance liquid chromatography. Based on these results, a search for the main outliers was conducted to identify the accessions with the highest alkaloid production. The results showed that roots had the highest concentrations of both berberine and palmatine, followed by stems, while leaves and seeds had lower levels, and the pulp from fruits had no detectable alkaloids. Notably, alkaloid concentrations reached up to 30,806 µg/g in roots, with accession C2P18 standing out for its combined total of 20,827.74 µg/g of berberine and 9978.27 µg/g of palmatine. Accession C3P26 showed the highest berberine concentration at 26,482.20 µg/g. These values underscore the wide variation in alkaloid accumulation and highlight the potential for selecting elite accessions with exceptionally high yields. These findings highlight the importance of plant selection for optimal alkaloid extraction. Choosing high-yielding accessions and standardizing cultivation practices will ensure a stable supply of berberine and palmatine for pharmaceutical, nutraceutical, and food industry applications.

Protoberberine alkaloids are a characteristic group of natural products in Coptis plants known for their notable pharmacological activities. However, the structural similarity and the substrate promiscuity of their biosynthetic enzymes have left the precise synthetic pathways remain unclarified, posing challenges to regulate product formation. In this study, we identified CcOMT8, a key enzyme responsible for C2‐methoxylation in the biosynthesis of epiberberine in C. chinensis, through methyl jasmonate elicitation analysis and comparative genomics‐based microsynteny analysis. Functional characterisation demonstrated that CcOMT8 specifically catalyses 2‐O‐methylation of (S)‐scoulerine, as verified by heterologous expression in both microbial and plant systems. Its lack of activity toward (S)‐cheilanthifoline further confirmed the specific route for epiberberine biosynthesis. Structural investigations of CcOMT8 and its complexes revealed key aspects of substrate recognition and a catalytic mechanism mediated by the His253‐Asp254‐Glu312 triad. Comparative structural analysis with 9‐O‐methyltransferases indicated that hydrophilic residues and reduced steric hindrance in the substrate binding pocket govern the regioselectivity of CcOMT8. Using focused rational iterative site‐specific mutagenesis (FRISM), we developed an optimised mutant, S109L/C250A/L300A, with 4.88‐fold enhanced catalytic efficiency. This study elucidates the biosynthetic pathway of epiberberine in Coptis, clarifies the molecular basis of enzyme‐directed metabolic flux, and provides efficient biocatalysts for the synthetic biosynthesis of protoberberine alkaloids.

Corydalis yanhusuo W.T. Wang is a traditional herb. Benzylisoquinoline alkaloids (BIAs) are the main pharmacological active ingredients that play an important role in sedation, relieving pain, promoting blood circulation, and inhibiting cancer cells. However, there are few studies on the biosynthetic pathway of benzylisoquinoline alkaloids in Corydalis yanhusuo , especially on some specific components, such as tetrahydropalmatine. We carried out widely targeted metabolome and transcriptomic analyses to construct the biosynthetic pathway of benzylisoquinoline alkaloids and identified candidate genes. In this study, 702 metabolites were detected, including 216 alkaloids. Protoberberine-type and aporphine-type alkaloids are the main chemical components in C . yanhusuo bulbs. Key genes for benzylisoquinoline alkaloids biosynthesis, including 6-OMT , CNMT , NMCH , BBE , SOMT1 , CFS , SPS , STOX , MSH , TNMT and P6H , were successfully identified. There was no significant difference in the content of benzylisoquinoline alkaloids and the expression level of genes between the two suborgans (mother-bulb and son-bulb). The expression levels of BIA genes in the expansion stage (MB-A and SB-A) were significantly higher than those in the maturity stage (MB-C and SB-C), and the content of benzylisoquinoline alkaloids was consistent with the pattern of gene regulation. Five complete single genes were likely to encode the functional enzyme of CoOMT, which participated in tetrahydropalmatine biosynthesis in C . yanhusuo bulbs. These studies provide a strong theoretical basis for the subsequent development of metabolic engineering of benzylisoquinoline alkaloids (especially tetrahydropalmatine) of C . yanhusuo .

Benzylisoquinoline alkaloids, such as the analgesic compounds morphine and codeine, and the antibacterial agents berberine, palmatine, and magnoflorine, are synthesized from tyrosine in the Papaveraceae, Berberidaceae, Ranunculaceae, Magnoliaceae, and many other plant families. It is difficult to produce alkaloids on a large scale under the strict control of secondary metabolism in plants, and they are too complex for cost-effective chemical synthesis. By using a system that combines microbial and plant enzymes to produce desired benzylisoquinoline alkaloids, we synthesized (S)-reticuline, the key intermediate in benzylisoquinoline alkaloid biosynthesis, from dopamine by crude enzymes from transgenic Escherichia coli. The final yield of (S)-reticuline was 55 mg/liter within 1 h. Furthermore, we synthesized an aporphine alkaloid, magnoflorine, or a protoberberine alkaloid, scoulerine, from dopamine via reticuline by using different combination cultures of transgenic E. coli and Saccharomyces cerevisiae cells. The final yields of magnoflorine and scoulerine were 7.2 and 8.3 mg/liter culture medium. These results indicate that microbial systems that incorporate plant genes cannot only enable the mass production of scarce benzylisoquinoline alkaloids but may also open up pathways for the production of novel benzylisoquinoline alkaloids.

A comprehensive study of the interactions of human serum albumin (HSA) and α-1-acid glycoprotein (AAG) with two isoquinoline alkaloids, i.e., allocryptopine (ACP) and protopine (PP), was performed. The UV-Vis spectroscopy, molecular docking, competitive binding assays, and circular dichroism (CD) spectroscopy were used for the investigations. The results showed that ACP and PP form spontaneous and stable complexes with HSA and AAG, with ACP displaying a stronger affinity towards both proteins. Molecular docking studies revealed the preferential binding of ACP and PP to specific sites within HSA, with site 2 (IIIA) being identified as the favored location for both alkaloids. This was supported by competitive binding assays using markers specific to HSA’s drug binding sites. Similarly, for AAG, a decrease in fluorescence intensity upon addition of the alkaloids to AAG/quinaldine red (QR) complexes indicated the replacement of the marker by the alkaloids, with ACP showing a greater extent of replacement than PP. CD spectroscopy showed that the proteins’ structures remained largely unchanged, suggesting that the formation of complexes did not significantly perturb the overall spatial configuration of these macromolecules. These findings are crucial for advancing the knowledge on the natural product–protein interactions and the future design of isoquinoline alkaloid-based therapeutics.

Background Protoberberine alkaloids are bioactive molecules abundant in plant preparations for traditional medicines. Yeast engineered to express biosynthetic pathways for fermentative production of these compounds will further enable investigation of the medicinal properties of these molecules and development of alkaloid-based drugs with improved efficacy and safety. Here, we describe the optimization of a biosynthetic pathway in Saccharomyces cerevisiae for conversion of rac -norlaudanosoline to the protoberberine alkaloid ( S )-canadine. Results This yeast strain is engineered to express seven heterologous enzymes, resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. The seven enzymes include three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase. A number of strategies were implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization, that led to an over 70-fold increase in canadine titer up to 1.8 mg/L. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines, for the first time in a microbial host. We also demonstrate that this strain is viable at pilot scale. Conclusions By applying metabolic engineering and synthetic biology strategies for increased conversion of simple benzylisoquinoline alkaloids to complex protoberberine alkaloids, this work will facilitate chemoenzymatic synthesis or de novo biosynthesis of these and other high-value compounds using a microbial cell factory.

Stylopine is a protoberberine-type alkaloid that has potential biological activities. Based on the successful microbial production of ( S )-reticuline, we attempted to produce stylopine from ( S )-reticuline by the reaction of berberine bridge enzyme, cheilanthifoline synthase (CYP719A5) and stylopine synthase (CYP719A2). Biosynthetic enzyme expression was examined in a methanol-utilizing yeast ( Pichia pastoris ) and both a “consolidated” system with all genes expressed in one cell and a “co-culture” system with three cell lines that each express a single gene were examined. Although both systems efficiently converted reticuline to stylopine, the consolidated system was more rapid and efficient than the co-culture system. However, substrate-feeding experiments revealed a decrease in the conversion efficiency in the consolidated system during successive cultures, whereas the conversion efficiency in the co-culture system remained constant. Thus, the final amount of stylopine produced from reticuline after successive feedings in the co-culture system was more than 150 nmoles from 750 nmoles of ( R, S )-reticuline (375 nmoles of ( S )-reticuline). The advantages and drawbacks of the “consolidated” system and the “co-culture” system are discussed.