[Cloning and functional characterization of two oxidosqualene cyclase genes from Siraitia grosvenorii].

Oxidosqualene cyclases(OSCs), belonging to a multigene family, can convert a common precursor 2,3-oxidosqualene into various types of triterpene skeletons. In this study, primers were designed according to the analysis of Siraitia grosvenorii transcriptome data, and two OSC genes SgAS1(GenBank No. QDO67189.1) and SgAS2(GenBank No. QDO67190.1) were cloned. The open reading frame(ORF) of SgAS1 was 2 262 bp, encoding 754 amino acids, and the ORF of SgAS2 was 2 289 bp, encoding 762 amino acids. Real-time quantitative PCR results demonstrated that the two SgOSCs genes showed different expression patterns in stems, leaves, and different stages of fruits. Phylogenetic analysis showed that both SgAS1 and SgAS2 were clustered with ß-amyrin synthases into a branch, but further functional characterization using yeast heterologous expression found that SgAS1 was inactive and SgAS2 could produce ß-amyrin as the sole product. Multiple sequence alignments revealed that SgAS2 had a conserved MWCYCR sequence related to ß-amyrin biosynthesis, while SgAS1 had an unusual LFCYTR sequence, for which the authors performed site-directed mutagenesis analysis of this sequence and found that tryptophan residue(W) was the key amino acid residue that affected the function of SgOSCs. In addition, the authors transformed the monofunctional ß-amyrin synthase SgAS2 into the chassis strain GH1, which was previously modified by the research group, and increased the yield of ß-amyrin to 44.05 mg·L~(-1). This study first reported the monofunctional ß-amyrin synthase SgAS2 from S. grosvenorii and conducted site-directed mutagenesis and synthetic biology investigation on it, providing a valuable resource for the directed biosynthesis of triterpenoids.

Identification of a cytochrome P450 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzes polpunonic acid formation in celastrol biosynthesis.

Tripterygium hypoglaucum (Levl.) Hutch, a traditional Chinese medicinal herb with a long history of use, is widely distributed in China. One of its main active components, celastrol, has great potential to be developed into anti-cancer and anti-obesity drugs. Although it exhibits strong pharmacological activities, there is a lack of sustainable sources of celastrol and its derivatives, making it crucial to develop novel sources of these drugs through synthetic biology. The key step in the biosynthesis of celastrol is considered to be the cyclization of 2,3-oxidosqualene into friedelin under the catalysis of 2,3-oxidosqualene cyclases. Friedelin was speculated to be oxidized into celastrol by cytochrome P450 oxidases (CYP450s). Here, we reported a cytochrome P450 ThCYP712K1 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzed the oxidation of friedelin into polpuonic acid when heterologously expressed in yeast. Through substrate supplementation and in vitro enzyme analysis, ThCYP712K1 was further proven to catalyze the oxidation of friedelin at the C-29 position to produce polpunonic acid, which is considered a vital step in the biosynthesis of celastrol, and will lay a foundation for further analysis of its biosynthetic pathway.

Key Glycosyltransferase Genes of Panax notoginseng: Identification and Engineering Yeast Construction of Rare Ginsenosides.

Panax notoginseng is one of the most famous valuable medical plants in China, and its broad application in clinical treatment has an inseparable relationship with the active molecules, ginsenosides. Ginsenosides are glycoside compounds that have varied structures for the diverse sugar chain. Although extensive work has been done, there are still unknown steps in the biosynthetic pathway of ginsenosides. Here, we screened candidate glycosyltransferase genes based on the previous genome and transcriptome data of P. notoginseng and cloned the full length of 27 UGT genes successfully. Among them, we found that PnUGT33 could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides by extending the sugar chain. We further analyzed the enzymatic kinetics and predicted the catalytic mechanism of PnUGT33 by simulating molecular docking. After that, we reconstructed the biosynthetic pathway of rare ginsenoside Rg3 and gypenoside LXXV in yeast. By combining the Golden Gate method and overexpressing the UDPG biosynthetic genes, we further improved the yield of engineering yeast strain. Finally, the shake-flask culture yield of Rg3 reached 51 mg/L and the fed-batch fermentation yield of gypenoside LXXV reached 94.5 mg/L, which was the first and highest record.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Friedelin via Genetic Manipulation.

Friedelin, the most rearranged pentacyclic triterpene, also exhibits remarkable pharmacological and anti-insect activities. In particular, celastrol with friedelin as the skeleton, which is derived from the medicinal plant Tripterygium wilfordii, is a promising drug due to its anticancer and antiobesity activities. Although a previous study achieved friedelin production using engineered Saccharomyces cerevisiae, strains capable of producing high-level friedelin have not been stably engineered. In this study, a combined strategy was employed with integration of endogenous pathway genes into the genome and knockout of inhibiting genes by CRISPR/Cas9 technology, which successfully engineered multiple strains. After introducing an efficient TwOSC1T502E, all strains with genetic integration (tHMG1, ERG1, ERG20, ERG9, POS5, or UPC2.1) showed a 3.0∼6.8-fold increase in friedelin production compared with strain BY4741. Through further double knockout of inhibiting genes, only strains GD1 and GD3 produced higher yields. Moreover, strains GQ1 and GQ3 with quadruple mutants (bts1; rox1; ypl062w; yjl064w) displayed similar increases. Finally, the dominant strain GQ1 with TwOSC1T502E was cultured in an optimized medium in shake flasks, and the final yield of friedelin reached 63.91 ± 2.45 mg/L, which was approximately 65-fold higher than that of the wild-type strain BY4741 and 229% higher than that in ordinary SD-His-Ura medium. It was the highest titer for friedelin production to date. Our work provides a good example for triterpenoid production in microbial cell factories and lays a solid foundation for the mining, pathway analysis, and efficient production of valuable triterpenoids with friedelin as the skeleton.

[Meta analysis on the treatment of coronavirus disease 2019 by traditional Chinese and Western medicine].

OBJECTIVE: To evaluate the clinical efficacy and safety of combination of traditional Chinese and Western medicine in the treatment of coronavirus disease 2019 (COVID-19) by Meta analysis. METHODS: The clinical randomized controlled trials (RCT) and cohort studies on the treatment of COVID-19 with combination of Chinese traditional and Western medicine published on CNKI, Wanfang database, VIP database and PubMed were searched by computer from January 2020 to June 2020. Patients in the simple Western medicine treatment group were treated with routine treatment of Western medicine, and the patients in integrated traditional Chinese and Western medicine treatment group were treated with traditional Chinese medicine on the basis of routine treatment of Western medicine. The main outcome was the total effective rate of treatment. The secondary outcome were the antipyretic rate, chest CT recovery rate, lymphocyte count (LYM), C-reactive protein (CRP) level and safety. The Cochrane manual and the Newcastle Ottawa Scale (NOS) were used to evaluate the quality of the literature; the RevMan5.3 software was used to analyze the articles that meets the quality standards, and a funnel chart was drawn to evaluate the total effective publication bias. RESULTS: Thirteen articles were analyzed, including 1 039 COVID-19 patients, 559 in integrated traditional Chinese and Western medicine treatment group and 480 in simple Western medicine treatment group. The results of Meta- analysis showed that compared with the simple Western medicine treatment group, the combination of routine treatment of Western medicine and traditional Chinese medicine Qingfei Paidu decoction, Lianhua Qingwen granule, Shufeng jiedu capsule, Xuebijing injection or Reyanning mixture could significantly improve the total effective rate, antipyretic rate and chest CT recovery rate [total effective rate: odds ratio (OR) = 2.95, 95% confidence interval (95%CI) was 2.10-4.14, P < 0.000 01; antipyretic rate: OR =3.01, 95%CI was 1.64-5.53, P = 0.000 4; chest CT recovery rate: OR = 2.53, 95%CI was 1.83-3.51, P = 0.000 1], increase LYM levels [mean difference (MD) = 0.26, 95%CI was 0.02-0.50, P = 0.03], and reduce of CRP content (MD = -17.68, 95%CI was -33.14 to -2.22, P = 0.02). Based on the funnel chart analysis of 12 articles with total efficiency, the result showed that the funnel chart distribution was not completely symmetrical, indicating that there might be publication bias. CONCLUSIONS: On the basis of routine treatment with Western medicine, combined with traditional Chinese medicine can significantly improve the total effective rate of COVID-19 and improve the laboratory results and clinical symptoms of patients. Compared with the routine treatment of Western medicine alone, the combination of traditional Chinese and Western medicine has better clinical efficacy and safety.

[Visual analysis of influenza treated by traditional Chinese medicine based on CiteSpace].

OBJECTIVE: To analyze the research status, research hotspots and frontier trends of traditional Chinese medicine (TCM) in the treatment of influenza in the past 20 years through the knowledge graph, so as to provide reference basis for further research. METHODS: The related literatures of TCM in the treatment of influenza were collected in China National Knowledge Infrastructure (CNKI) from 2000 to 2019. The relevant graphs of authors, research institutions and key words were drawn by CiteSpace 5.6, the distribution and cooperation of main research forces in this field were analyzed, and the research frontiers and hot spot information in this field were discussed. RESULTS: A total of 3 048 related literatures were obtained, involving 949 authors and 242 research institutions. The analysis of the number of articles showed that the volume of articles related to the treatment of influenza with TCM fluctuated greatly in the past 20 years, which was obviously affected by the sudden hot spots around 2010, but showed an overall upward trend, with an average annual volume of about 152 articles. The analysis of the author's cooperation map showed that a total of 77 core authors had published more than 5 articles, accounting for only 8.1% of all authors, and 5 authors had published more than 30 articles. Five major teams had been formed with Gu Ligang, Liu Qingquan, Lu Fangguo, Cui Xiaolan and Zhang Fengxue as the core. The analysis of the cooperation map of research institutions showed that the cooperation among institutions was not good, and only the scientific research institutes in Beijing and Guangzhou had formed a closely related cooperation network. The keyword co-occurrence map showed that 8 keywords appeared more than 100 times, especially ultra-high-frequency keywords, influenza virus ranked first (n = 518). There were 14 key nodes, such as influenza virus, TCM treatment, viral pneumonia and so on, which supported the current research field of TCM in the treatment of influenza. Fourteen clusters were formed to classify the current research hotspots, including the nomenclature of influenza, virus type, TCM treatment, western medicine knowledge, etc., and the map showed that the clustering was reasonable and the structure was significant. Timeline graph showed that parainfluenza virus, virus disease, pharmacodynamics, heat-clearing and detoxifying drugs, bacteriostasis and experimental research had all been studied for more than 8 years, revealing the research hotspots and trends of TCM in the treatment of influenza. CONCLUSIONS: The overall research related to the treatment of influenza with TCM is relatively perfect. In the future, the close cooperation among authors and institutions should be strengthened. The molecular mechanism research, clinical and animal trials of TCM should be further studied, so as to improve the research system of TCM treatment of influenza.

Chinese Herbal Formula (CHF03) Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) Through Inhibiting Lipogenesis and Anti-Oxidation Mechanisms.

Nonalcoholic fatty liver disease (NAFLD) is a hepatic ailment with a rapidly increasing incidence in the human population due largely to dietary hyper nutrition and subsequent obesity. Discovering effective natural compounds and herbs against NAFLD can provide alternative and complementary medical treatments to current chemical pharmaceuticals. In this study, ICR male mice were fed a high-fat diet (HFD) in vivo and the AML12 cells were treated with palmitic acid (PA) in vitro. We explore the protective effect and potential mechanism of Chinese Herbal Formula (CHF03) against NAFLD by HE staining, transmission Electron Microscopy assay, Western blotting, and gene expression. In vivo, oxidative stress markers (GSH, GSH-px, MDA, SOD, and CAT) confirmed that CHF03 alleviated oxidative stress and abundance of NF-κB proteins indicating a reduction in inflammation and oxidative stress. The lower protein abundance of ACACA and FASN indicated a preventive effect on lipogenesis. Histological and ultrastructural observations revealed that CHF03 inhibited NAFLD. Expression of Srebf1, Fasn, and Acaca, which are associated with lipogenesis, were downregulated. In vitro, genes and proteins are expressed in a dose-dependent manner, consistent with those in the liver. CHF03 inhibited lipid accumulation and expression of NF-κB, nuclear transfer, and transcriptional activity in AML12 cells. The CHF03 might have a beneficial role in the prevention of hepatic steatosis by altering the expression of lipogenic genes and attenuating oxidative stress.