Characterization of CYP82 genes involved in the biosynthesis of structurally diverse benzylisoquinoline alkaloids in Corydalis yanhusuo.

Benzylisoquinoline alkaloids (BIAs) represent a significant class of secondary metabolites with crucial roles in plant physiology and substantial potential for clinical applications. CYP82 genes are involved in the formation and modification of various BIA skeletons, contributing to the structural diversity of compounds. In this study, Corydalis yanhusuo, a traditional Chinese medicine rich in BIAs, was investigated to identify the catalytic function of CYP82s during BIA formation. Specifically, 20 CyCYP82-encoding genes were cloned, and their functions were identified in vitro. Ten of these CyCYP82s were observed to catalyze hydroxylation, leading to the formation of protopine and benzophenanthridine scaffolds. Furthermore, the correlation between BIA accumulation and the expression of CyCYP82s in different tissues of C. yanhusuo was assessed their. The identification and characterization of CyCYP82s provide novel genetic elements that can advance the synthetic biology of BIA compounds such as protopine and benzophenanthridine, and offer insights into the biosynthesis of BIAs with diverse structures in C. yanhusuo.

Identification of the cytochrome P450s responsible for the biosynthesis of two types of aporphine alkaloids and their de novo biosynthesis in yeast.

Aporphine alkaloids have diverse pharmacological activities; however, our understanding of their biosynthesis is relatively limited. Previous studies have classified aporphine alkaloids into two categories based on the configuration and number of substituents of the D-ring and have proposed preliminary biosynthetic pathways for each category. In this study, we identified two specific cytochrome P450 enzymes (CYP80G6 and CYP80Q5) with distinct activities toward (S)-configured and (R)-configured substrates from the herbaceous perennial vine Stephania tetrandra, shedding light on the biosynthetic mechanisms and stereochemical features of these two aporphine alkaloid categories. Additionally, we characterized two CYP719C enzymes (CYP719C3 and CYP719C4) that catalyzed the formation of the methylenedioxy bridge, an essential pharmacophoric group, on the A- and D-rings, respectively, of aporphine alkaloids. Leveraging the functional characterization of these crucial cytochrome P450 enzymes, we reconstructed the biosynthetic pathways for the two types of aporphine alkaloids in budding yeast (Saccharomyces cerevisiae) for the de novo production of compounds such as (R)-glaziovine, (S)-glaziovine, and magnoflorine. This study provides key insight into the biosynthesis of aporphine alkaloids and lays a foundation for producing these valuable compounds through synthetic biology.

Phylogenetic analysis and functional characterization of norcoclaurine synthase involved in benzylisoquinoline alkaloids biosynthesis in Stephania tetrandra.

Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites that possess diverse pharmaceutical properties and are exclusively accumulated in specific plant genera. The Pictet-Spengler condensation, catalyzed by norcoclaurine synthase (NCS), represents a key enzymatic reaction in the biosynthetic pathway of BIAs. While NCS genes have been identified in several plant families such as Papaveraceae, Berberidaceae, and Ranunculaceae, no NCS genes have been reported in Menispermaceae, which is another genus known to accumulate BIAs. Here, NCSs were isolated and functionally characterized from the Menispermaceae family plant Stephania tetrandra. In vitro enzyme assay identified two functional StNCSs which could catalyze the formation of (S)-norcoclaurine. These functionally characterized genes were then integrated into engineered yeast to enable the production of norcoclaurine. Phylogenetic analysis of the NCS enzymes revealed that the StNCSs predominantly clustered into two clades. The functional StNCSs clustered with known NCSs, highlighting the presence of a specific NCS catalytic domain. This study not only provides additional genetic components for the synthetic biology-based production of BIAs in yeast but also contributes to the understanding of the phylogenetic relationships and structure-function relationship of NCS genes involved in the origin and production of BIAs.

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.

The Differences of Nutrient Components in Edible and Feeding Coix Seed at Different Developmental Stages Based on a Combined Analysis of Metabolomics.

Coix lachryma-jobi L. is an excellent plant resource that has a concomitant function for medicine, foodstuff and forage in China. At present, the commonly used cultivar for both medicine and foodstuff is Xiaobaike, and the cultivar for foraging is Daheishan. However, differences in the internal composition of plants lead to the expression of different phenotypic traits. In order to comprehensively elucidate the differences in nutrient composition changes in Coix seeds, a non-targeted metabolomics method based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) was used to analyze the metabolic changes in Coix seeds at different developmental stages. An edible Coix relative (Xiaobaike) and a feeding Coix relative (Daheishan) were selected as the research subjects. In the metabolome analysis of Coix seed, 314 metabolites were identified and detected, among which organic acids, carbohydrates, lipids, nucleotides and flavonoids were the main components. As an important standard for evaluating the quality of Coix seed, seven lipids were detected, among which fatty acids included not only even-chain fatty acids, but also odd-chain fatty acids, which was the first time detecting a variety of odd-chain fatty acids in Coix seed. The analysis of the compound contents in edible and feeding-type Coix lachryma-jobi L. and the lipid content at the mature stage showed that, among them, arachidic acid, behenic acid, heptadecanoic acid, heneicosanoic acid and pristanic acid may be the key compounds affecting the lipid content. In addition, in the whole process of semen coicis maturation, edible and feeding Coix show similar trends, and changes in the third period show clear compounds in the opposite situation, suggesting that edible and feeding Coix not only guarantee the relative stability of species but also provide raw materials for genetic breeding. This study provides valuable information on the formation of the edible and medicinal qualities of Coix.

Functional Study and Efficient Catalytic Element Mining of CYP76AHs in Salvia Plants.

Salvia is a large genus with hundreds of species used in traditional Chinese medicine. Tanshinones are a highly representative class of exclusive compounds found in the Salvia genus that exhibit significant biological activity. Tanshinone components have been identified in 16 Salvia species. The CYP76AH subfamily (P450) is crucial for the synthesis of tanshinone due to its catalytic generation of polyhydroxy structures. In this study, a total of 420 CYP76AH genes were obtained, and phylogenetic analysis showed their clear clustering relationships. Fifteen CYP76AH genes from 10 Salvia species were cloned and studied from the perspectives of evolution and catalytic efficiency. Three CYP76AHs with significantly improved catalytic efficiency compared to SmCYP76AH3 were identified, providing efficient catalytic elements for the synthetic biological production of tanshinones. A structure-function relationship study revealed several conserved residues that might be related to the function of CYP76AHs and provided a new mutation direction for the study of the directed evolution of plant P450.

[Modification of C20 oxidase in tanshinone biosynthesis pathway].

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Proteomic analysis and identification reveal the anti-inflammatory mechanism of clofazimine on lipopolysaccharide-induced acute lung injury in mice.

BACKGROUND AND OBJECTIVE: Acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS) was increasingly recognized as one of the most severe acute hyperimmune response of coronavirus disease 2019 (COVID-19). Clofazimine (CFZ) has attracted attention due to its anti-inflammatory property in immune diseases as well as infectious diseases. However, the role and potential molecular mechanism of CFZ in anti-inflammatory responses remain unclear. METHODS: We analyze the protein expression profiles of CFZ and LPS from Raw264.7 macrophages using quantitative proteomics. Next, the protective effect of CFZ on LPS-induced inflammatory model is assessed, and its underlying mechanism is validated by molecular biology analysis. RESULTS: LC-MS/MS-based shotgun proteomics analysis identified 4746 (LPS) and 4766 (CFZ) proteins with quantitative information. The key proteins and their critical signal transduction pathways including TLR4/NF-κB/HIF-1α signaling was highlighted, which was involved in multiple inflammatory processes. A further analysis of molecular biology revealed that CFZ could significantly inhibit the proliferation of Raw264.7 macrophages, decrease the levels of TNF-α and IL-1ß, alleviate lung histological changes and pulmonary edema, improve the survival rate, and down-regulate TLR4/NF-κB/HIF-1α signaling in LPS model. CONCLUSION: This study can provide significant insight into the proteomics-guided pharmacological mechanism study of CFZ and suggest potential therapeutic strategies for infectious disease.

Functional Characterization of a 2OGD Involved in Abietane-Type Diterpenoids Biosynthetic Pathway in Salvia miltiorrhiza.

Salvia miltiorrhiza is one of the most commonly used Chinese medicinal herbs. Tanshinones, the most abundant lipid-soluble bioactive constituents of S. miltiorrhiza, are a class of structural highly oxidized abietane-type diterpenoids with multiple pharmacological activities. Although several enzymes, including diterpene synthase, cytochrome P450, and Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD), have been functionally characterized in biosynthesis of abietane-type diterpenoids, the highly oxidized structure and complex secondary metabolic network of tanshinones imply that more oxidases should be characterized. Here, we identified a new 2OGD (Sm2OGD25) from S. miltiorrhiza. Molecular cloning and functional studies in vitro showed that Sm2OGD25 could catalyze the hydroxylation of sugiol at C-15 and C-16 positions to produce hypargenin B and crossogumerin C, respectively. The phylogenetic analysis of the DOXC family demonstrated that Sm2OGD25 belongs to the DOXC54 clade. Furthermore, structural modeling and site-directed mutagenesis characterization revealed the importance of the hydrogen-bonding residue Y339 and the hydrophobic residues (V122, F129, A144, A208, F303, and L344) in substrate binding and enzyme activity. This study will promote further studies on the catalytic characterization of plant 2OGDs and the secondary metabolic biosynthesis network of diterpenoids.

Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids.

O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, Corydalis yanhusuo, a plant used in traditional Chinese medicine due to the analgesic effects of its BIA-active compounds, was employed to analyze the catalytic characteristics of O-methyltransferases in the formation of BIA diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an O-methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4'- and 6-O-methylations of 1-BIAs was found. CyOMT6 was found to perform two sequential methylations at both 9- and 2-positions of the essential intermediate of tetrahydroprotoberberines, (S)-scoulerine. Two O-methyltransferases (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with the 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2-O-methyltransferase) and the 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, results of integrated phylogenetic molecular docking analysis and site-directed mutation suggested that residues at sites 172, 306, 313, and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at the 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of the 6-position and to expand substrate scopes. This work provides insight into O-methyltransferases in producing BIA diversity in C. yanhusuo and genetic elements for producing BIAs by metabolic engineering and synthetic biology.

Functional characterization of (S)-N-methylcoclaurine 3'-hydroxylase (NMCH) involved in the biosynthesis of benzylisoquinoline alkaloids in Corydalis yanhusuo.

Benzylisoquinoline alkaloids (BIAs) are compounds naturally found in plants and can have significant value in clinical settings. Metabolic engineering and synthetic biology are both promising approaches for the heterologous acquisition of benzylisoquinoline alkaloids. (S)-N-methylcoclaurine 3'-hydroxylase (NMCH), a member of the CYP80 family of CYP450, is the penultimate catalytic enzyme that forms the central branch-point intermediate (S)-reticuline and plays a key role in the biosynthesis of BIAs. In this study, an NMCH gene was cloned from Corydalis yanhusuo, while in vitro reactions demonstrated that CyNMCH can catalyze (S)-N-methylcoclaurine to produce (S)-3'-hydroxy-N-methylcoclaurine. The Km and Kcat of CyNMCH were estimated and compared with those identified in Eschscholzia californica and Coptis japonica. This newly discovered CyNMCH will provide alternative genetic resources for the synthetic biological production of benzylisoquinoline alkaloids and provides a foundation to help analyze the biosynthetic pathway of BIAs biosynthesis in C. yanhusuo.

Characterization of O-methyltransferases involved in the biosynthesis of tetrandrine in Stephania tetrandra.

Tetrandrine is the most effective small molecule that has been found to inhibit the Ebola virus. It is a typical bisbenzylisoquinoline alkaloid and is the main active ingredient in Stephania tetrandra. Metabolic engineering and synthetic biology are potential methods for efficient and rapid acquisition of tetrandrine. S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase (6OMT) is a rate-limiting step involved in the biosynthesis of tetrandrine. In this study, we identify S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase from S. tetrandra, which catalyzes the conversion of (S)-norcoclaurine to (S)-coclaurine. Four 6OMT-like genes were cloned from S. tetrandra. An in vitro enzyme assay showed that St6OMT1 could catalyze the conversion of (S)-norcoclaurine to produce (S)-coclaurine. St6OMT2 can catalyze the production of very few (S)-coclaurine molecules, accompanied by more by-products with m/z 300, compared to St6OMT1. The newly discovered 6OMTs will provide an optional genetic component for benzylisoquinoline alkaloid (BIA) synthetic biology research. This work will lay the foundation for the analysis of the biosynthetic pathway of tetrandrine in S. tetrandra.

Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology.

Cytochrome P450s (CYPs) are important enzymes in the secondary metabolism of plants and have been recognized as key players in bioengineering and synthetic biology. Previously reported CYP76AH1 and CYP76AH3, having greater than 80% sequence homology, played a continuous catalytic role in the biosynthesis of tanshinones in Salvia miltiorrhiza. Homology modeling indicates that four sites might be responsible for differences in catalytic activity between the two enzymes. A series of modeling-based mutational variants of CYP76AH1 were designed to integrate the functions of the two CYPs. The mutant CYP76AH1D301E,V479F, which integrated the functions of CYP76AH1 and CYP76AH3, was found to efficiently catalyze C11 and C12 hydroxylation and C7 oxidation of miltiradiene substrates. Integration and utilization of CYP76AH1D301E,V479F by synthetic biology methods allowed the robust production of 11-hydroxy ferruginol, sugiol, and 11-hydroxy sugiol in yeast. The functionally integrated CYP gene after active site modifications improves catalytic efficiency by reducing the transfer of intermediate metabolites between component proteins. This provides a synthetic biology reference for improving the catalytic efficiencies of systems that produce plant natural products in microorganisms.