Advances in research methods for biosynthetic pathway analysis of active ingredients in traditional Chinese medicine / 中国中药杂志

The active ingredients in traditional Chinese medicine(TCM)are the foundation for the efficiency of TCM and the key to the formation of Dao-di herbs. It is of great significance to study the biosynthesis and regulation mechanisms of these active ingredients for analyzing the formation mechanism of Daodi herbs and providing components for the production of active ingredients in TCM by synthetic biology. With the advancements in omics technology, molecular biology, synthetic biology, artificial intelligence, etc., the analysis of biosynthetic pathways for active ingredients in TCM is rapidly progressing. New methods and technologies have promoted the analysis of the synthetic pathways of active ingredients in TCM and have also made this area a hot topic in molecular pharmacognosy. Many researchers have made significant progress in analyzing the biosynthetic pathways of active ingredients in TCM such as Panax ginseng, Salvia miltiorrhiza, Glycyrrhiza uralensis, and Tripterygium wilfordii. This paper systematically reviewed current research me-thods for analyzing the biosynthetic functional genes of active ingredients in TCM, elaborated the mining of gene elements based on multiomics technology and the verification of gene functions in plants in vitro and in vivo with candidate genes as objects. Additionally, the paper summarized new technologies and methods that have emerged in recent years, such as high-throughput screening, molecular probes, genome-wide association studies, cell-free systems, and computer simulation screening to provide a comprehensive reference for the analysis of the biosynthetic pathways of active ingredients in TCM.

Gene clone and functional identification of sterol glycosyltransferases from Paris polyphylla var. yunnanensis / 中国中药杂志

In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl β-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.

Advances of synthetic biology of flavonoids / 药学学报

Flavonoids is one of the biggest families of the plant-derived secondary metabolites with structural diversity. Until now, over 10 000 kinds of flavonoids with distinct structures have been purified and identified from plants, and some of them possess a range of important pharmacological effects, such as anticancer, anti-inflammatory and so on. So far, a number of genes and enzymes responsible for the biosynthesis of flavonoids have been reported, especially, a great of progress has been achieved in the synthetic biology of flavonoids in the recent years. Herein, based upon a brief introduction on the biosynthesis of flavonoids, this review summarizes the research advances in synthetic biology of flavonoids in the past two decades (2001-2021), highlighting the cell factories construction of the representative flavonoids. And, a brief discussion and prospects of the relevant metabolic bottlenecks and optimizing strategies are proposed.

Biopathway construction of plant natural products / 药学学报

Plant natural products (PNPs) are important sources of innovative drugs. They are mainly obtained by isolation or extraction from plants. Low content and with structural analogues in plants result in high production cost, which restricts the research and application of PNPs. While biopathway construction by synthetic biology provides an alternative for production of PNPs. By biosynthetic pathway analysis of PNPs and reconstructing the biopathway in microorganisms, we can produce PNPs in cell factories efficiently. Recently, several predominantly international reports about biosynthesis of PNPs and its synthetic biology production, triggered the researches of PNPs. Abundant traditional Chinese medicine resources and profound cultural heritage of Chinese medicine make biosynthesis pathway analysis of PNPs to be a research hotspot. And some of the studies have achieved significant progress. Here, recent progress in the biosynthesis of plant natural products and its synthetic biology was reviewed. In particular, the application of new methods and technologies in recent years were summarized and discussed. This will provide reference for the biopathway construction of plant natural products.

Ginsenosides in

Ginsenosides are a series of glycosylated triterpenoids which belong to protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, ocotillol (OCT)- and oleanane (OA)-type saponins known as active compounds of

Research progress of polycyclic polyprenylated acylphloroglucinols natural products / 中国中药杂志

Due to their fascinating chemical structures and extensive pharmacological activities, polycyclic polyprenylated acylphloroglucinols(PPAPs) have become one of the current research hotspots of natural products. In particular, some of the PPAPs not only have novel non-traditional skeleton types, but also contain more unknown possible activities, which are of great significance for the development of lead compounds. The structure, source, biosynthetic pathway and pharmacological activities of PPAPs with non-traditio-nal skeleton types isolated and identified in recent years are reviewed, in order to provide references for further research on such compounds.

Advance in biosynthesis and metabolic regulation of ginkgolides / 中国中药杂志

Ginkgolides,the unique terpenoids in Ginkgo biloba,have a significant effect on the prevention and treatment of cardiovascular and cerebrovascular diseases. Metabolic regulation and synthetic biology strategies are efficient methods to obtain high-quality ginkgolides. The present study reviewed the cloning and functions of genes related to the biosynthetic pathway of ginkgolides,as well as relevant studies of omics,genetic transformation,and metabolic regulation in recent years,and predicted the research trends and prospects,aiming to provide a reference for discovering the key genes related to the biosynthetic pathway and the biosynthesis of ginkgolides.

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin III to baccatin III

10-Deacetylbaccatin III (10-DAB) C10 acetylation is an indispensable procedure for Taxol semi-synthesis, which often requires harsh conditions. 10-Deacetylbaccatin III-10-

Pathway design and key enzyme analysis of diosgenin biosynthesis / 生物工程学报

As a naturally occurring steroid sapogenin, diosgenin acts as the precursor of hundreds of steroid medicines, and thereby has important medicinal value. Currently, industrial production of diosgenin relies primarily on chemical extraction from plant materials. Clearly, this strategy shows drawbacks of excessive reliance on plant materials and farmland as well as environment pollution. Due to development of metabolic engineering and synthetic biology, bio-production of diosgenin has garnered plenty of attention. Although the biosynthetic pathways of diosgenin have not been completely identified, in this review, we outline the identified biosynthetic pathways and key enzymes. In particular, we suggest heterologous biosynthesis of diosgenin in Saccharomyces cerevisiae. Overall, this review aims to provide valuable insights for future complete biosynthesis of diosgenin.

Antibacterial activity of phytopathogenic Streptomyces strains against bacteria associated to clinical diseases / Atividade antimicrobiana de Streptomyces fitopatogênicas contra bactérias associadas a doenças de importância clínica

The genus Streptomyces is associated with the ability to produce and excrete a variety of bioactive compounds, such as antibiotic, antifungal and antiviral. Biological active polyketide and peptide compounds with applications in medicine, agriculture and biochemical research are synthesized by PKS-I and NRPS genes. The evaluation of the presence of these genes associated with the biosynthesis of secondary metabolites in different phytopathogenic Streptomyces strains were performed using degenerated primers. The positive signal was observed in 58/63 Streptomyces strains for NRPS gene, 43/63 for PKS-I, and for PKS-II all the 63 strains showed positive signal of amplification. These strains also were tested with double layer agar-well technique against bacterial with clinical importance, and it was possible to observe the Streptomyces spp. strains were able to inhibit the growth of 14, 20, 13 and 3 isolates Gram-positive and Gram-negative bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 11775) respectively. The Streptomyces sp. strains IBSBF 2019 and IBSBF 2397 showed antibacterial activity against all four bacteria-target tested.(AU)

O gênero Streptomyces apresenta alta capacidade de produzir e excretar uma grande variedade de compostos biologicamente ativos, como antibióticos, antifúngicos e antivirais. Compostos biologicamente ativos de policetídeos e peptídeos com aplicações na medicina, agricultura e pesquisas bioquímicas são sintetizados pelos genes PKS-I e NRPS. A avaliação da presença desses genes associados à biossíntese de metabólitos secundários em diferentes linhagens de Streptomyces fitopatogênicas foi realizada através do uso de primers degenerados. O sinal positivo foi observado em 58/63 linhagens de Streptomyces para o gene NRPS, 43/63 para o gene PKS-I e, para o gene PKS-II, todas as 63 linhagens apesentaram o sinal positivo de amplificação. Essas linhagens também foram testadas através da técnica de dupla camada contra bactérias de importância clínica e foi possível observar que as linhagens de Streptomyces spp. foram capazes de inibir o crescimento de 14, 20, 13 e 3 isolados de bactérias Gram-positivas e Gram-negativas, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) e Escherichia coli (ATCC 11775), respectivamente. As linhagens de Streptomyces sp. ISBSF 2019 e 2397 apresentaram atividade antibacteriana contra todas as bactérias-alvo testadas.(AU)

Research progress on synthesis of anthraquinones based on shikimic acid/o-succinylbenzoic acid pathway and polyketone pathway / 中草药

Anthraquinone and its derivatives are very important secondary metabolites in plants. They have many functions such as photoprotection and improvement of plant disease resistance. They also have very important applications in the fields of medicine and chemical engineering. Efficiently and quickly obtaining anthraquinones and improving the synthesis efficiency of anthraquinones in plants have become one of the research focuses of modern synthetic biology. However, the synthetic pathway of anthraquinones is more complicated. At present, it is generally believed that anthraquinones are formed in plants by the shikimic acid/o- succinylbenzoic acid pathway and polyketone pathway. This article focuses on the recent research advances in the skeleton synthesis of anthraquinone via shikimic acid/o-succinylbenzoic acid pathway and polyketone pathway in plants, and provides a certain theoretical basis for studying the synthesis and regulation of anthraquinone metabolites in plants.

Discussion on research idea of quality marker of Salvia miltiorrhiza based on biosynthetic pathway of tanshinone compounds / 中国中药杂志

Based on the theory of Q-marker, the hairy root of Salvia miltiorrhiza and S. miltiorrhiza in many provinces were studied. The relative expressions of SmCPS, SmKSL and CYP76AH1 genes in hairy roots were detected by real-time fluorescence quantitative PCR and the contents of tanshinoneⅡ_A, cryptotanshinone, tanshinoneⅠ, 1,2-dihydrotanshinone, ferruginol and miltiradiene were detected by UPLC and GC-MS, respectively. Statistical analysis shows as fllows: in the hairy root of S. miltiorrhiza, the content of miltiradiene and ferruginol is positively correlated with the content of tanshinone compounds in the downstream, and the relative expression of important genes in the biosynthetic pathway of tanshinone can reflect the content of tanshinone compounds to a certain extent; in many provinces of S. miltiorrhiza, the content of ferruginol and tanshinone compounds can also be found that there is a positive correlation between the contents. Based on the biosynthetic pathway of tanshinone compounds, which is a special index component in S. miltiorrhiza, this study focused on the important relationship between the upstream gene, the middle intermediate compound and the downstream tanshinone compound content of the biosynthetic pathway, and explored the possible research ideas of improving the quality marker system of S. miltiorrhiza, and then provided the possible research ideas for understanding and studying the quality marker of traditional Chinese medicine from the biosynthetic pathway.

Research progress of P450 in the biosynthesis of bioactive compound of medicinal plants / 药学学报

Most of the active ingredients of herbs are secondary metabolites of plants. Cytochrome P450s (P450s) are hemoglobin-containing monooxygenases encoded by a super-gene family, which play important roles in the metabolic network of plants. This review focuses on the role of P450s on biosynthesis of secondary metabolites such as terpenoids, alkaloids, flavonoids and phenylpropanoids. This will provide references for biosynthesis and regulation of secondary metabolites in medicinal plants.

Research progress in Atractylodes macrocephala and predictive analysis on Q-marker / 中草药

To study the quality marker (Q-marker) of Atractylodes macrocephala based on the concept, standard, and research model of Q-marker. The chemical constituents of A. macrocephala were identified by UPLC-Q-TOF-MS/MS. The source and specificity of chemical constituents were confirmed by analyzing biosynthetic pathway and component specificity. The major effective components were clarified through efficacy, drug property, and correlation analysis of chemical constituents. The possible Q-markers of A. macrocephala are estimated based on the results of the study.

Sub-cellular localization and overexpressing analysis of hydroxylase gene TcCYP725A22 of Taxus chinensis / 生物工程学报

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.

Bioactive phenazines from an earwig-associated Streptomyces sp / 中国天然药物

Three new phenazine-type compounds, named phenazines SA-SC (1-3), together with four new natural products (4-7), were isolated from the fermentation broth of an earwig-associated Streptomyces sp. NA04227. The structures of these compounds were determined by extensive analyses of NMR, high resolution mass spectroscopic data, as well as single-crystal X-ray diffraction measurement. Sequencing and analysis of the genome data allowed us to identify the gene cluster (spz) and propose a biosynthetic pathway for these phenazine-type compounds. Additionally, compounds 1-5 exhibited moderate inhibitory activity against acetylcholinesterase (AChE), and compound 3 showed antimicrobial activities against Micrococcus luteus.

Polyhydroxyalkanoate biosynthesis by oxalotrophic bacteria from high Andean soil / Biosíntesis de polihidrxialcanoato por bacterias oxalotróficas de suelos altoandinos / Biossínteses de polihidroxialcanoato por bactérias oxalotróficas de solos andinos

Abstract Oxalate is a highly oxidized organic acid anion used as a carbon and energy source by oxalotrophic bacteria. Oxalogenic plants convert atmospheric CO2 into oxalic acid and oxalic salts. Oxalate-salt formation acts as a carbon sink in terrestrial ecosystems via the oxalate-carbonate pathway (OCP). Oxalotrophic bacteria might be implicated in other carbon-storage processes, including the synthesis of polyhydroxyalkanoates (PHAs). More recently, a variety of bacteria from the Andean region of Colombia in Narino have been reported for their PHA-producing abilities. These species can degrade oxalate and participate in the oxalate-carbonate pathway. The aim of this study was to isolate and characterize oxalotrophic bacteria with the capacity to accumulate PHA biopolymers. Plants of the genus Oxalis were collected and bacteria were isolated from the soil adhering to the roots. The isolated bacterial strains were characterized using biochemical and molecular biological methods. The consumption of oxalate in culture was quantified, and PHA production was monitored in batch fermentation. The polymeric composition was characterized using gas chromatography. Finally, a biosynthetic pathway based on our findings and on those from published sources is proposed. Strains of Bacillus spp. and Serratia sp. were found to metabolize calcium oxalate and synthesize PHA.

Resumen El oxalato es un anión de ácido orgánico altamente oxidado usado como fuente carbono y energía por bacterias oxalotróficas. Las plantas oxalogénicas convierten CO2 atmosférico en ácido oxálico y sales oxálicas. La formación de sales de oxalato actúa como un sumidero de carbono en ecosistemas terrestres via oxalato-carbonato (OCP). Las bacterias oxalotróficas podrían estar implicadas en otros procesos de almacenamiento de carbono, incluyendo la síntesis de polihidroxialcanoatos (PHAs). Recientemente, una variedad de bacterias de la región andina colombiana en Nariño ha sido reportada por su habilidad para producir PHAs. Estas especies pueden degradar oxalato y participar en la vía del oxalato-carbonato. El objetivo de este estudio fue aislar y caracterizar bacterias oxalotróficas con capacidad de acumular biopolímeros PHA. Se colectaron plantas del genero Oxalis y se aislaron bacterias del suelo adheridas a las raíces. Las cepas bacterianas aisladas se caracterizaron usando métodos bioquímicos y de biología molecular. Se cuantificó el consumo de oxalato en cultivo, y se monitoreó la producción de PHA en fermentación por lotes. La composición polimérica se caracterizó usando cromatografía de gases. Finalmente, se propone una via biosintética basada en nuestros hallazgos y en los de otras fuentes publicadas. Se encontró que las cepas de Bacillus spp. y Serratia sp. metabolizan oxalato de calcio y sintetizan PHA.

Resumo O oxalato é um ânion de ácido orgânico altamente oxidado utilizado como fonte de carbono e nergía por bactérias oxalotróficas. As plantas oxalogênicas convertem CO2 atmosférico em ácido oxálico e sais oxálicos. A formação de sais de oxalato atua como um sumidouro de carbono em ecossistemas terrestres via oxalato-carbono (OCP). As bactérias oxalotróficas poderiam estar envolvidas em outros processos de armazenamento de carbono, incluindo a sínteses de polihidroxialcanoatos (PHAs). Recentemente, uma variedade de bactérias da região Andina colombiana no Departamento de Nariño foi reportada devido a sua habilidade para produzir PHAs. Estas espécies podem degradar oxalato e participar na via oxalato-carbono. O objetivo de esse estudo foi isolar e caracterizar bactérias oxalotróficas com capacidade de acumular biopolímeros PHA. Plantas do género Oxalis foram coletadas e se isolaram bactérias do solo aderido a suas raízes. As cepas bacterianas isoladas se caracterizaram utilizando métodos bioquímicos e de biologia molecular. O consumo de oxalato em cultivo foi quantificado, e a produção de PHA foi monitorada em fermentação por lotes. A composição polimérica se caracterizou utilizando Cromatografia de Gases. Finalmente, se propõe uma via biossintética baseada em nossos resultados juntamente com resultados da literatura. Se encontrou que as cepas de Bacillus spp. e Serratia sp. metabolizam oxalato de cálcio e sintetizam PHA.

Research progress on biosynthetic pathways and related enzymes of iridoid glycosides / 中草药

Iridoids glycosides are a class of compounds which have pharmacological functions of anti-inflammatory, antitumor, hepato-protection, cardio-protection, etc. This review summarizes the biosynthetic pathway, related enzymes (GPPS, GES, G10, G10H, 10-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, and SLS) and the application of functional genes in iridoids glycosides, in hopes of regulating the production of metabolites and providing the valuable reference for discovering new drugs.

Research progress on biosynthetic pathway of apo-carotenoids in Crocus sativus / 中草药

The apo-carotenoid compounds represented by crocins are the main medicinal components of Crocus sativus, which have extensive anti-oxidation, anti-inflammatory, anti-atherosclerosis, anticancer, antidepressant, and other pharmacological activities. Biosynthetic pathways of apo-carotenoids in C. sativus include the traditional upstream route of the synthesis of geranylgeranyl pyrophosphate to zeaxanthin starting from mevalonate, and downstream pathway for the specific synthesis of crocetin and crocin by cleavage of zeaxanthin. This article reviews the recent research of key enzymes involved in the metabolism of apo-carotenoids in C. sativus, which facilitates further analysis of downstream pathways for the synthesis of apo-carotenoid derivatives such as crocin, and further provides a theoretical basis for the use of metabolic engineering methods to increase the production of crocins and other pharmacodynamic substances.

Research Development in Biosynthesis and Metabolic Regulation of Tetracyclic Triterpenoid Saponins in Medicinal Plants / 世界科学技术-中医药现代化

Triterpenoid saponins is an important secondary metabolites in medicinal plants, and the tetracyclic triterpenoid saponins, as one of the main categories, have very high medicinal value and market demand. However, there is no systematic review on the research. Thus, the elucidation of the biosynthetic pathway and metabolism of the medicinal plant tetracyclic triterpenoid saponins has important theoretical significance and broad application prospects.In this review, the biosynthetic pathway and metabolic regulation of medicinal plant of tetracyclic triterpenoid saponins were discussed. My focus in this paper was to introduce the research development of several metabolic biosynthetic pathways of tetracyclic triterpenoid saponins centered on dammarane type, and the gene improvement by methods such as metabolic pathway and other technological methods. This study provides references on secondary metabolic framework of medicinal plants of tetracyclic triterpenoid saponins, accurately locating secondary metabolism and its key enzymes, and promoting the sustainable uses of medicinal plant resources.