The effects of green and chemically-synthesized copper oxide nanoparticles on the production and gene expression of morphinan alkaloids in Oriental poppy.

Oriental poppy (Papaver orientale L.) belonging to the Papaveraceae family, has the capacity to synthesize a wide range of benzylisoquinoline alkaloids (BIAs). This experiment was conducted to investigate the effects of green and chemical copper oxide nanoparticles (CuO NPs) elicitors on oxidative stress and the BIAs biosynthesis pathway in the cell suspension culture of P. orientale. This research shows that both green and chemical CuO NPs at concentrations of 20 mg/L and 40 mg/L, induce oxidative stress in the cell suspension of P. orientale by increasing the production of H2O2 and the activity of antioxidant enzymes. The comparison of treatments revealed that utilizing a lower concentration of CuO NPs (20 mg/L) and extending the duration of cell suspension incubation (up to 48 h) play a more influential role in inducing the expression of the BIAs biosynthesis pathway genes (PsWRKY, TYDC, SalSyn, SalR, SalAT, T6ODM, COR and CODM) and increasing the production of morphinan alkaloids (thebaine, codeine, and morphine). The overarching results indicate that the concentration of CuO NPs and the duration of cell treatment have a more significant impact than the nature of CuO NPs in inducing oxidative stress and stimulating the expression of the BIAs pathway genes.

Xylose and shikimate transporters facilitates microbial consortium as a chassis for benzylisoquinoline alkaloid production.

Plant-sourced aromatic amino acid (AAA) derivatives are a vast group of compounds with broad applications. Here, we present the development of a yeast consortium for efficient production of (S)-norcoclaurine, the key precursor for benzylisoquinoline alkaloid biosynthesis. A xylose transporter enables the concurrent mixed-sugar utilization in Scheffersomyces stipitis, which plays a crucial role in enhancing the flux entering the highly regulated shikimate pathway located upstream of AAA biosynthesis. Two quinate permeases isolated from Aspergillus niger facilitates shikimate translocation to the co-cultured Saccharomyces cerevisiae that converts shikimate to (S)-norcoclaurine, resulting in the maximal titer (11.5 mg/L), nearly 110-fold higher than the titer reported for an S. cerevisiae monoculture. Our findings magnify the potential of microbial consortium platforms for the economical de novo synthesis of complex compounds, where pathway modularization and compartmentalization in distinct specialty strains enable effective fine-tuning of long biosynthetic pathways and diminish intermediate buildup, thereby leading to increases in production.

Tetrandrine attenuates SNI-induced mechanical allodynia by inhibiting spinal CKLF1.

Neuropathic pain (NP) is a prevalent clinical problem for which satisfactory treatment options are unavailable. Tetrandrine (TET), a bisbenzylisoquinoline alkaloid extracted from Stephania tetrandra S. Moore, possesses anti-inflammatory and immune-modulatory properties. Chemokine-like factor 1 (CKLF1) is known to play a crucial role in both peripheral and central inflammatory processes. This study aimed to investigate the potential anti-NP effects of TET and the involvement of CKLF1 in the action of TET. A male C57BL/6J mice model of NP caused by spared nerve injury (SNI) was established and mechanical withdrawal thresholds were measured using von Frey filaments. The results showed that TET improved mechanical allodynia in SNI mice and the propofol-induced sleep assay demonstrated that the TET group did not exhibit central inhibition, while the pregabalin (PGB) group showed significant central inhibition. Western blotting and immunofluorescence staining showed that TET significantly inhibited spinal protein expression levels of CKLF1, p-NF-κB/NF-κB, p-IKK/IKK, pro-inflammatory cytokines IL-1ß and TNF-α, and increased protein expression levels of the anti-inflammatory cytokine IL-10, while inhibiting the expression levels of microglia and astrocyte markers IBA-1 and GFAP of SNI mice. Moreover, immunofluorescence double-labeling results revealed that CKLF1 was predominantly colocalized with microglia of the spinal cord (SC) in SNI mice. C19 (an antagonism peptide of CKLF1) alleviated SNI-induced mechanical pain hypersensitivity, while C27 (an analog peptide of CKLF1) induced mechanical allodynia in normal mice. TET significantly attenuated mechanical allodynia induced by C27 in mice. TET may effectively alleviate NP by reducing neuroinflammation and decreasing CKLF1.

Opium alkaloids, biosynthesis, pharmacology and association with cancer occurrence.

Papaver somniferum L. (Family: Papaveraceae) is a species well known for its diverse alkaloids (100 different benzylisoquinoline alkaloids (BIAs)). L-tyrosine serves as a precursor of several specific metabolites like BIAs. It has been used as an antitussive and potent analgesic to alleviate mild to extreme pain since ancient times. The extraction of pharmaceutically important alkaloids like morphine and codeine from poppy plant reflects the need for the most suitable and standard methods. Several analytical and extraction techniques have been reported in open literature for morphine, codeine and other important alkaloids which play a vital function in drug development and drug discovery. Many studies suggest that opioids are also responsible for adverse effects or secondary complications like dependence and withdrawal. In recent years, opium consumption and addiction are the most important risk factors. Many evidence-based reviews suggest that opium consumption is directly linked or acts as a risk factor for different cancers. In this review, we highlight significant efforts related to research which have been done over the past 5 decades and the complete information on Papaver somniferum including its phytochemistry, pharmacological actions, biosynthetic pathways and analytical techniques of opium alkaloid extraction and the link between opium consumption and cancer-related updates.

Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera).

Sacred lotus (Nelumbo nucifera) has been utilized as a food, medicine, and spiritual symbol for nearly 3000 years. The medicinal properties of lotus are largely attributed to its unique profile of benzylisoquinoline alkaloids (BIAs), which includes potential anti-cancer, anti-malarial and anti-arrhythmic compounds. BIA biosynthesis in sacred lotus differs markedly from that of opium poppy and other members of the Ranunculales, most notably in an abundance of BIAs possessing the (R)-stereochemical configuration and the absence of reticuline, a major branchpoint intermediate in most BIA producers. Owing to these unique metabolic features and the pharmacological potential of lotus, we set out to elucidate the BIA biosynthesis network in N. nucifera. Here we show that lotus CYP80G (NnCYP80G) and a superior ortholog from Peruvian nutmeg (Laurelia sempervirens; LsCYP80G) stereospecifically convert (R)-N-methylcoclaurine to the proaporphine alkaloid glaziovine, which is subsequently methylated to pronuciferine, the presumed precursor to nuciferine. While sacred lotus employs a dedicated (R)-route to aporphine alkaloids from (R)-norcoclaurine, we implemented an artificial stereochemical inversion approach to flip the stereochemistry of the core BIA pathway. Exploiting the unique substrate specificity of dehydroreticuline synthase from common poppy (Papaver rhoeas) and pairing it with dehydroreticuline reductase enabled de novo synthesis of (R)-N-methylcoclaurine from (S)-norcoclaurine and its subsequent conversion to pronuciferine. We leveraged our stereochemical inversion approach to also elucidate the role of NnCYP80A in sacred lotus metabolism, which we show catalyzes the stereospecific formation of the bis-BIA nelumboferine. Screening our collection of 66 plant O-methyltransferases enabled conversion of nelumboferine to liensinine, a potential anti-cancer bis-BIA from sacred lotus. Our work highlights the unique benzylisoquinoline metabolism of N. nucifera and enables the targeted overproduction of potential lotus pharmaceuticals using engineered microbial systems.

Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production.

Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32 mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.

Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL.

BACKGROUND: Tetrandrine, a bisbenzylisoquinoline (BBI) alkaloid extracted from Stephania tetrandra (S. Moore), and is widely used in several diseases such as tuberculosis, hyperglycemia, malaria, and tumors. Tetrandrine was recently shown to prevent bone loss in ovariectomized mice. However, the specific mechanism underlying osteoclastogenesis inhibition remains unclear. METHODS: Tetrandrine's cytotoxicity to cells was determined using the Cell Counting Kit-8 assay. Tartrate-resistant acid phosphatase staining, immunofluorescence and bone resorption assay were performed to evaluate osteoclasts' differentiation and absorption capacity. The bone-forming capacity was assessed using alkaline phosphatase and Alizarin red S staining. qPCR and Western blotting were applied to assess the related genes and protein expression. Tetrandrine's impact on TRAIL was demonstrated through a co-immunoprecipitation assay. Animal experiments were performed for the detection of the therapeutic effect of Tetrandrine on osteoporosis. RESULTS: Tetrandrine attenuated RANKL-induced osteoclastogenesis and decreased the related gene expression. The co-immunoprecipitation assay revealed that Tetrandrine administration accelerated the ubiquitination of TNF-related apoptosis-inducing ligand (TRAIL), which was subsequently degraded. Moreover, TRAIL overexpression was found to partially reverse the Tetrandrine-induced inhibition of osteoclastogenesis. Meanwhile, Tetrandrine significantly inhibited the phosphorylation of p38, p65, JNK, IKBα and IKKα/ß, while the TRAIL overexpression weakened this effect. In addition, Tetrandrine promoted osteogenesis and inhibited the TRAIL expression in osteoblasts. Tetrandrine consistently improved bone destruction by stimulating bone formation and inhibiting bone resorption in an OVX-induced mouse model. CONCLUSION: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting TRAIL degradation and promotes osteoblast differentiation, suggesting its potential in antiosteopenia pharmacotherapy.

Alkaloid binding to opium poppy major latex proteins triggers structural modification and functional aggregation.

Opium poppy accumulates copious amounts of several benzylisoquinoline alkaloids including morphine, noscapine, and papaverine, in the specialized cytoplasm of laticifers, which compose an internal secretory system associated with phloem throughout the plant. The contiguous latex includes an abundance of related proteins belonging to the pathogenesis-related (PR)10 family known collectively as major latex proteins (MLPs) and representing at least 35% of the total cellular protein content. Two latex MLP/PR10 proteins, thebaine synthase and neopione isomerase, have recently been shown to catalyze late steps in morphine biosynthesis previously assigned as spontaneous reactions. Using a combination of sucrose density-gradient fractionation-coupled proteomics, differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography, we show that the major latex proteins are a family of alkaloid-binding proteins that display altered conformation in the presence of certain ligands. Addition of MLP/PR10 proteins to yeast strains engineered with morphine biosynthetic genes from the plant significantly enhanced the conversion of salutaridine to morphinan alkaloids.

Characterization of benzylisoquinoline alkaloid methyltransferases in Liriodendron chinense provides insights into the phylogenic basis of angiosperm alkaloid diversity.

Benzylisoquinoline alkaloids (BIAs) are a class of plant secondary metabolites with great pharmacological value. Their biosynthetic pathways have been extensively elucidated in the species from the Ranunculales order, such as poppy and Coptis japonica, in which methylation events play central roles and are directly responsible for BIA chemodiversity. Here, we combined BIA quantitative profiling and transcriptomic analyses to identify novel BIA methyltransferases (MTs) from Liriodendron chinense, a basal angiosperm plant. We identified an N-methyltransferase (LcNMT1) and two O-methyltransferases (LcOMT1 and LcOMT3), and characterized their biochemical functions in vitro. LcNMT1 methylates (S)-coclaurine to produce mono- and dimethylated products. Mutagenesis experiments revealed that a single-residue alteration is sufficient to change its substrate selectivity. LcOMT1 methylates (S)-norcoclaurine at the C6 site and LcOMT3 methylates (S)-coclaurine at the C7 site, respectively. Two key residues of LcOMT3, A115 and T301, are identified as important contributors to its catalytic activity. Compared with Ranunculales-derived NMTs, Magnoliales-derived NMTs were less abundant and had narrower substrate specificity, indicating that NMT expansion has contributed substantially to BIA chemodiversity in angiosperms, particularly in Ranunculales species. In summary, we not only characterized three novel enzymes that could be useful in the biosynthetic production of valuable BIAs but also shed light on the molecular origin of BIAs during angiosperm evolution.

Tetrandrine Inhibits Skeletal Muscle Differentiation by Blocking Autophagic Flux.

Tetrandrine is well known to act as a calcium channel blocker. It is a potential candidate for a tumor chemotherapy drug without toxicity. Tetrandrine inhibits cancer cell proliferation and induces cell death through apoptosis and autophagy. As cancer patients usually experience complications with sarcopenia or muscle injury, we thus assessed the effects of tetrandrine on skeletal muscle cells. We report in this study that a low dose of tetrandrine (less than 5 µM) does not affect the proliferation of C2C12 myoblasts, but significantly inhibits myogenic differentiation. Consistently, tetrandrine inhibited muscle regeneration after BaCl2-induced injury. Mechanistic experiments showed that tetrandrine decreased the p-mTOR level and increased the levels of LC3 and SQSTM1/p62 during differentiation. Ad-mRFP-GFP-LC3B transfection experiments revealed that the lysosomal quenching of GFP signals was suppressed by tetrandrine. Furthermore, the levels of DNM1L/Drp1, PPARGA1 and cytochrome C (Cyto C), as well as caspase 3 activation and ROS production, were decreased following tetrandrine administration, indicating that the mitochondrial network signaling was inhibited. Our results indicate that tetrandrine has dual effects on autophagic flux in myoblasts during differentiation, activation in the early stage and blockade in the late stage. The ultimate blocking of autophagic flux by tetrandrine led to the disruption of mitochondria remodeling and inhibition of myogenic differentiation. The inhibitory effects of tetrandrine on skeletal muscle differentiation may limit its application in advanced cancer patients. Thus, great attention should be paid to the clinical use of tetrandrine for cancer therapy.

A functionally conserved STORR gene fusion in Papaver species that diverged 16.8 million years ago.

The STORR gene fusion event is considered essential for the evolution of the promorphinan/morphinan subclass of benzylisoquinoline alkaloids (BIAs) in opium poppy as the resulting bi-modular protein performs the isomerization of (S)- to (R)-reticuline essential for their biosynthesis. Here, we show that of the 12 Papaver species analysed those containing the STORR gene fusion also contain promorphinans/morphinans with one important exception. P. californicum encodes a functionally conserved STORR but does not produce promorphinans/morphinans. We also show that the gene fusion event occurred only once, between 16.8-24.1 million years ago before the separation of P. californicum from other Clade 2 Papaver species. The most abundant BIA in P. californicum is (R)-glaucine, a member of the aporphine subclass of BIAs, raising the possibility that STORR, once evolved, contributes to the biosynthesis of more than just the promorphinan/morphinan subclass of BIAs in the Papaveraceae.

The genome of Corydalis reveals the evolution of benzylisoquinoline alkaloid biosynthesis in Ranunculales.

Species belonging to the order Ranunculales have attracted much attention because of their phylogenetic position as a sister group to all other eudicot lineages and their ability to produce unique yet diverse benzylisoquinoline alkaloids (BIAs). The Papaveraceae family in Ranunculales is often used as a model system for studying BIA biosynthesis. Here, we report the chromosome-level genome assembly of Corydalis tomentella, a species of Fumarioideae, one of the two subfamilies of Papaveraceae. Based on comparisons of sequenced Ranunculalean species, we present clear evidence of a shared whole-genome duplication (WGD) event that has occurred before the divergence of Ranunculales but after its divergence from other eudicot lineages. The C. tomentella genome enabled us to integrate isotopic labeling and comparative genomics to reconstruct the BIA biosynthetic pathway for both sanguinarine biosynthesis shared by papaveraceous species and the cavidine biosynthesis that is specific to Corydalis. Also, our comparative analysis revealed that gene duplications, especially tandem gene duplications, underlie the diversification of BIA biosynthetic pathways in Ranunculales. In particular, tandemly duplicated berberine bridge enzyme-like genes appear to be involved in cavidine biosynthesis. In conclusion, our study of the C. tomentella genome provides important insights into the occurrence of WGDs during the early evolution of eudicots, as well as into the evolution of BIA biosynthesis in Ranunculales.

Effects of codon optimization, N-terminal truncation and gene dose on the heterologous expression of berberine bridge enzyme.

Morphine, sanguinarine and chelerythrine are benzylisoquinoline alkaloids (BIAs), and these compounds possess strong biological activities. (S)-scoulerine is a commonly shared precursor of these compounds, and berberine bridge enzyme (BBE) is a key rate-limiting enzyme in the synthesis of (S)-scoulerine. We isolated the BBE gene from Macleaya cordata (McBBE) and used CEN.PK2-1C as a chassis strain. We compared the catalytic efficiency of five codon-optimized McBBE genes in Saccharomyces cerevisiae and finally obtained a yeast strain (YH03) that exhibited a 58-fold increase in yield (1.12 mg/L). Then, we truncated the N-terminus of McBBE by 8 and 22 amino acids and found that with the increase in the number of N-terminal truncated amino acids, the production of (S)-scoulerine gradually decreased. Additionally, we used CRISPR-Cas9 to integrate the McBBE gene at the delta site of the S. cerevisiae genome to achieve stable genetic inheritance and found that the yield of (S)-scoulerine was not significantly increased in the integrated strain. In conclusion, our work achieved high-efficiency expression of McBBE in S. cerevisiae, explored the influence of N-terminal truncation on the yield of (S)-scoulerine, and obtained a genetically stable S. cerevisiae strain with high McBBE expression. This study provides a reference for further complex metabolic engineering optimization and lays a foundation for the efficient biosynthesis of BIAs.

Machine learning discovery of missing links that mediate alternative branches to plant alkaloids.

Engineering the microbial production of secondary metabolites is limited by the known reactions of correctly annotated enzymes. Therefore, the machine learning discovery of specialized enzymes offers great potential to expand the range of biosynthesis pathways. Benzylisoquinoline alkaloid production is a model example of metabolic engineering with potential to revolutionize the paradigm of sustainable biomanufacturing. Existing bacterial studies utilize a norlaudanosoline pathway, whereas plants contain a more stable norcoclaurine pathway, which is exploited in yeast. However, committed aromatic precursors are still produced using microbial enzymes that remain elusive in plants, and additional downstream missing links remain hidden within highly duplicated plant gene families. In the current study, machine learning is applied to predict and select plant missing link enzymes from homologous candidate sequences. Metabolomics-based characterization of the selected sequences reveals potential aromatic acetaldehyde synthases and phenylpyruvate decarboxylases in reconstructed plant gene-only benzylisoquinoline alkaloid pathways from tyrosine. Synergistic application of the aryl acetaldehyde producing enzymes results in enhanced benzylisoquinoline alkaloid production through hybrid norcoclaurine and norlaudanosoline pathways.

Phloem-specific localization of benzylisoquinoline alkaloid metabolism in opium poppy.

Opium poppy is the only commercial source of the narcotic analgesics morphine and codeine, and semi-synthetic derivatives of the natural opiate precursor thebaine, including oxycodone and the opioid antagonist naloxone. The plant also accumulates the vasodilator and antitussive agents papaverine and noscapine, respectively, which together with morphine, codeine and thebaine comprise the major benzylisoquinoline alkaloids (BIAs) in opium poppy. A majority of enzymes involved in the highly branched BIA metabolism in opium poppy have now been discovered, with many specifically localized to sieve elements of the phloem based on immunofluorescence labeling techniques. Transcripts corresponding to sieve element-localized biosynthetic enzymes were detected in companion cells, as expected. The more recent application of shotgun proteomics has shown that several enzymes operating late in the morphine and noscapine biosynthetic pathways occur primarily in laticifers that are adjacent or proximal to sieve elements. BIA biosynthesis and accumulation in opium poppy involves three phloem cell types and implicates the translocation of key pathway intermediates between sieve elements and laticifers. The recent isolation of uptake transporters associated with laticifers supports an apoplastic rather than a symplastic route for translocation. In spite of the extensive elucidation of BIA biosynthetic enzymes in opium poppy, additional transporters and other auxiliary proteins are clearly necessary to support the complex spatial organization and dynamics involved in product formation and sequestration. In this review, we provide an update of BIA metabolism in opium poppy with a focus on the role of phloem in the biosynthesis of the major alkaloids.

Evaluating cepharanthine analogues as natural drugs against SARS-CoV-2.

Cepharanthine (CEP) is a natural biscoclaurine alkaloid of plant origin and was recently demonstrated to have anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) activity. In this study, we evaluated whether natural analogues of CEP may act as potential anti-coronavirus disease 2019 drugs. A total of 24 compounds resembling CEP were extracted from the KNApSAcK database, and their binding affinities to target proteins, including the spike protein and main protease of SARS-CoV-2, NPC1 and TPC2 in humans, were predicted via molecular docking simulations. Selected analogues were further evaluated by a cell-based SARS-CoV-2 infection assay. In addition, the efficacies of CEP and its analogue tetrandrine were assessed. A comparison of the docking conformations of these compounds suggested that the diphenyl ester moiety of the molecules was a putative pharmacophore of the CEP analogues.

Complete biosynthesis of the bisbenzylisoquinoline alkaloids guattegaumerine and berbamunine in yeast.

Benzylisoquinoline alkaloids (BIAs) are a diverse class of medicinal plant natural products. Nearly 500 dimeric bisbenzylisoquinoline alkaloids (bisBIAs), produced by the coupling of two BIA monomers, have been characterized and display a range of pharmacological properties, including anti-inflammatory, antitumor, and antiarrhythmic activities. In recent years, microbial platforms have been engineered to produce several classes of BIAs, which are rare or difficult to obtain from natural plant hosts, including protoberberines, morphinans, and phthalideisoquinolines. However, the heterologous biosyntheses of bisBIAs have thus far been largely unexplored. Here, we describe the engineering of yeast strains that produce the Type I bisBIAs guattegaumerine and berbamunine de novo. Through strain engineering, protein engineering, and optimization of growth conditions, a 10,000-fold improvement in the production of guattegaumerine, the major bisBIA pathway product, was observed. By replacing the cytochrome P450 used in the final coupling reaction with a chimeric variant, the product profile was inverted to instead produce solely berbamunine. Our highest titer engineered yeast strains produced 108 and 25 mg/L of guattegaumerine and berbamunine, respectively. Finally, the inclusion of two additional putative BIA biosynthesis enzymes, SiCNMT2 and NnOMT5, into our bisBIA biosynthetic strains enabled the production of two derivatives of bisBIA pathway intermediates de novo: magnocurarine and armepavine. The de novo heterologous biosyntheses of bisBIAs presented here provide the foundation for the production of additional medicinal bisBIAs in yeast.

Three chromosome-scale Papaver genomes reveal punctuated patchwork evolution of the morphinan and noscapine biosynthesis pathway.

For millions of years, plants evolve plenty of structurally diverse secondary metabolites (SM) to support their sessile lifestyles through continuous biochemical pathway innovation. While new genes commonly drive the evolution of plant SM pathway, how a full biosynthetic pathway evolves remains poorly understood. The evolution of pathway involves recruiting new genes along the reaction cascade forwardly, backwardly, or in a patchwork manner. With three chromosome-scale Papaver genome assemblies, we here reveal whole-genome duplications (WGDs) apparently accelerate chromosomal rearrangements with a nonrandom distribution towards SM optimization. A burst of structural variants involving fusions, translocations and duplications within 7.7 million years have assembled nine genes into the benzylisoquinoline alkaloids gene cluster, following a punctuated patchwork model. Biosynthetic gene copies and their total expression matter to morphinan production. Our results demonstrate how new genes have been recruited from a WGD-induced repertoire of unregulated enzymes with promiscuous reactivities to innovate efficient metabolic pathways with spatiotemporal constraint.

Structural studies of codeinone reductase reveal novel insights into aldo-keto reductase function in benzylisoquinoline alkaloid biosynthesis.

Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are two closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the final and penultimate steps in the biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). However, the structural determinants that mediate substrate recognition and catalysis are not well defined. Here, we describe the crystal structure of apo-COR determined to a resolution of 2.4 Å by molecular replacement using chalcone reductase as a search model. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveal a novel conformation in the ß1α1 loop adjacent to the BIA-binding pocket. The proximity of this loop to several highly conserved active-site residues and the expected location of the nicotinamide ring of the NADP(H) cofactor suggest a model for BIA recognition that implies roles for several key residues. Using site-directed mutagenesis, we show that substitutions at Met-28 and His-120 of COR lead to changes in AKR activity for the major and minor substrates codeinone and neopinone, respectively. Our findings provide a framework for understanding the molecular basis of substrate recognition in COR and the closely related 1,2-dehydroreticuline reductase responsible for the second half of a stereochemical inversion that initiates the morphine biosynthesis pathway.

Development of an artificial biosynthetic pathway for biosynthesis of (S)-reticuline based on HpaBC in engineered Escherichia coli.

Benzylisoquinoline alkaloids (BIAs) are an important class of plant secondary metabolites with a variety of pharmacological activities. Although they are widely used, traditionally these compounds are extracted from natural sources because their structure is too complicated to achieve economically feasible chemical synthesis. Thus, microbial biosynthesis of BIAs is expected to reduce dependence on natural extracts. (S)-Reticuline is an important precursor for BIAs biosynthesis. Therefore, it is an attractive engineering target. In this study, we reported the development of a novel (S)-reticuline biosynthetic pathway based on 4-hydroxyphenylacetate 3-hydroxylase (HpaBC) in Escherichia coli. Then, we further improved the (S)-reticuline production to 307 ± 26.8 mg/L by increasing the availability of the precursor 3, 4-dihydroxyphenylacetaldehyde. The E. coli cell factory developed in this study can be used as a potential platform for further efficient biosynthesis of BIAs derivatives.