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Cobalamin-dependent radical S-adenosyl-L-methionine protein functions with a partner to successively methylate tricyclic indole alkaloid for chuangxinmycin maturation and derivatization

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Science China Chemistry 2025年第1期 308-316页

作者： Sili Wang Jiancheng Huang Yanan Du Wei Huang Yufeng Xue Xiaokun Xu Guannan Zhong Yuanyuan Shi Bin Hong Xiaoying Bian Wen Liu State Key Laboratory of Chemical Biology Shanghai Institute of Organic Chemistry University of Chinese Academy of SciencesChinese Academy of Sciences State Key Laboratory of Microbial Technology Shandong University National Health Commission (NHC) Key Laboratory of biotechnology for Microbial Drugs Institute of Medicinal BiotechnologyChinese Academy of Medical Science & Peking Union Medical College

Cobalamin(Cbl)-dependent radical S-adenosyl-L-methionine(SAM) proteins constitute the largest collection of the radical SAM superfamily that has hundreds of thousands of individual members. Many of these proteins are involved in the biosynthesis of pharmaceutically important natural products to catalyze chemically demanding reactions. In the biosynthetic pathway of chuangxinmycin(CXM), a unique indole alkaloid antibiotic with potent anti-infective activity, functionalization of the characteristic thiopyrano[4,3,2-cd]indole scaffold by regio-and stereoselective C3-methylation is believed to rely on a Cbl-dependent radical process, which, however, remained to be reconstituted biochemically. We here report the dissection of this enzymatic process, which requires the incorporation of Cxm8, a Cbl-dependent radical SAM protein, with Cxm9, a DUF5825family protein that shares no homology to any proteins of known functions. Cxm8 and Cxm9 function together by forming an unexpected heterodimeric complex that selectively catalyzes C3-methylation of the tricyclic indole-S-hetero ring system in a successive manner, achieving CXM and a recently identified, C3-dimethylated congener. Detailed biochemical characterization,isotope labeling, structural simulation and bioinformatics analysis rationalized the catalysis of the Cxm8/Cxm9 complex and particularly the necessity of the DUF5825 protein for C3-methylase activity. This is the first example that a Cbl-dependent protein acts with a partner to exhibit radical SAM activity.

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Effects of Soil Depth and Season Variation on Community Structure of Arbuscular mycorrhizal Fungi in Greenhouse Soils Planted with Watermelon

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Pedosphere 2013年第3期23卷 350-358页

作者： LIU Run-Jin LI Yan DIAO Zhi-Kai LI Min LIN Xian-Gui institute of mycorrhizal biotechnology Qingdao Agricultural University State Key Laboratory of Soil and Sustainable Agriculture Institute of Soil Science Chinese Academy of Sciences

The characteristics of arbuscular mycorrhizal fungal (AMF) community structure in various soil depths and growing seasons of watermelon (Citrullus vulgaris) grown in commercial greenhouses in Daxing of Beijing and Weifang and Laiyang of Shandong, China were investigated using both morphological identification and denaturing gradient gel electrophoresis. The sampled soils had been used for continuous greenhouse production of watermelon for 0, 5, 10, 15, or 20 years. Glomus claroideum was the dominant species in the greenhouse soils planted for 5, 10, and 15 years in Laiyang, while Glomus mosseae and Glomus etunicatum were dominant in the nearby open farmland soil. Sorenson's similarity index of AMF community composition ranged from 0.67 to 0.84 in the soils planted for 5 years, and from 0.29 to 0.33 for 20 years among the three locations. Spore abundance, species richness, and the Shannon index were highest near the soil surface (0-10 cm) and decreased with soil depth, and higher in June and October than in August and December. Canonical correspondence analysis showed that available P and the number of years that soil had been used for greenhouse production were the main factors contributing to the variance of AMF community composition. It was concluded that the community structure of AMF was mainly influenced by soil available P and planting time of watermelon as well as by soil depth and seasonal variation in the commercial greenhouse.

关键词： canonical correspondence analysis Shannon index Sorenson's similarity index species richness spore abundance

Genes and proteins expressed at different life cycle stages in the model protist Euplotes vannus revealed by both transcriptomic and proteomic approaches

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Science China Life Sciences 2025年第1期 232-248页

作者： Yaohan Jiang Xiao Chen Chundi Wang Liping Lyu Saleh A.Al-Farraj Naomi A.Stover Feng Gao Key Laboratory of Evolution & Marine Biodiversity (Ministry of Education) and institute of Evolution & Marine Biodiversity Ocean University of China Marine College Shandong University Zoology Department College of ScienceKing Saud University Department of Biology Bradley University Laboratory for Marine Biology and biotechnology Qingdao Marine Science and Technology Center

Sexual reproduction first appeared in unicellular protists and has continued to be an essential biological process in almost all *** protists, which contain both germline and somatic genomes within a single cell, have evolved a special form of sexual reproduction called conjugation that involves mitosis, meiosis, fertilization, nuclear differentiation, genome rearrangement, and the development of unique cellular structures. The molecular basis and mechanisms of conjugation vary dramatically among ciliates, and many details of the process and its regulation are still largely unknown. In order to better comprehend these processes and mechanisms from an evolutionary perspective, this study provides the first comprehensive overview of the transcriptome and proteome profiles during the entire life cycle of the newly-established marine model ciliate Euplotes vannus. Transcriptome analyses from 14 life cycle stages(three vegetative stages and 11 sexual stages) revealed over 26,000 genes that are specifically expressed at different stages, many of which are related to DNA replication,transcription, translation, mitosis, meiosis, nuclear differentiation, and/or genome rearrangement. Quantitative proteomic analyses identified 338 proteins with homologs associated with conjugation and/or somatic nuclear development in other ciliates, including dicer-like proteins, Hsp90 proteins, RNA polymerase Ⅱ and transcription elongation factors, ribosomal-associated proteins, and ubiquitin-related proteins. Four of these homologs belong to the PIWI family, each with different expression patterns identified and confirmed by RT-qPCR,which may function in small RNA-mediated genome rearrangement. Proteins involved in the nonhomologous end-joining pathway are induced early during meiosis and accumulate in the developing new somatic nucleus, where more than 80% of the germline sequences are eliminated from the somatic genome. A number of new candidate genes and proteins likely t

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Effects of Arbuscular mycorrhizal Fungi Communities on Soil Quality and the Growth of Cucumber Seedlings in a Greenhouse Soil of Continuously Planting Cucumber

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Pedosphere 2012年第1期22卷 79-87页

作者： LI Yan CHEN Ying-Long LI Min LIN Xian-Gui LIU Run-Jin institute of mycorrhizal biotechnology Qingdao Agricultural UniversityQingdao 266109(China) State Key Laboratory of Soil and Sustainable Agriculture Institute of Soil ScienceChinese Academy of SciencesNanjing210008(China) School of Earth and Environment The University of Western AustraliaPerth 6009(Australia)

A pot experiment was performed to determine the effects of arbuscular mycorrhizM fungi （AMF） communities on soil properties and the growth of cucumber seedlings in a degraded soil that had been used for continuous cucumber monoculture in a greenhouse for 15 years. In the experiment, AMF communities （created by combining various AMF species that were found to be dominant in natural farm soil） were inoculated into the degraded soil, and then the soil was planted with cucumber. Inoculation with AMF communities did not affect soil pH but increased soil aggregate stability and decreased the concentrations of salt ions and electrical conductivity （EC） in the soil. Inoculation with AMF communities increased the numbers of culturable bacteria and actinomycetes but reduced the number of fungi. AMF communities increased plant growth, soluble sugar content, chlorophyll content, and root activity compared to non-mycorrhizal or a single AMF species treatments. Improvements of soil quality and plant growth were greatest with the following two communities： Glomus etunicatum ＋ G. mosseae ＋ Gigaspora margarita ＋ Acaulospora lacunosa and G. aggregatum ＋ G. etunicatum ＋ G. mosseae ＋ G. versiforme ＋ G. margarita ＋ A. lacunosa. The results suggested that certain AMF communities could substantially improve the quality of degraded soil.

关键词： enzyme activities microbial community protected cultivation rhizosphere soluble sugar

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

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Journal of Integrative Agriculture 2014年第9期13卷 1943-1951页

作者： LIU Huan SONG Xi-jiao NI Yue-qun LU Li-na ZHOU Xue-ping WU Jian-xiang institute of biotechnology College of Agriculture & Biotechnology Zhejiang University

Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（DE3） using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody（MAb） against RRSV was obtained by fusing mouse myeloma cells（Sp 2/0） with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay（ACP-ELISA）, a dot enzyme-linked immunosorbent assay（dot-ELISA）, and immunocapture-RT-PCR（IC-RT-PCR） assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1：40 960, 1：1 280 and 1：655 360（w/v, g mL-1）, respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1：12 800 and 1：1 600（an individual planthopper μL-1）, respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.

关键词： Rice ragged stunt virus rice brown planthopper monoclonal antibody antigen-coated-plate enzyme-linked immunosorbent assay(ACP-ELISA) dot-blot ELISA immunocapture RT-PCR

Recombinant human B7-H4 expressed in Escherichia coli inhibits T lym-phocyte proliferation and IL-2 secretion in vitro

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Acta Pharmacologica Sinica 2006年第6期27卷 741-746页

作者： Yi-xiang MAO~(2,3)Yong-Jing CHEN~2 Yan GE~2 Hong-bing MA~2 Jian-feng YU~2 Hong-ya WU~2 Yu-min HU~2 Qin WANG~2 Qin SHI~2 Xue-guang ZHANG~(2,4)2 institute of biotechnology,Soochow University,Suzhou 215007,China 3 Department of Oncology,First Affiliated Hospital to Soochow University,Suzhou 215007,China institute of biotechnology Soochow University

Aim:To explore the biofunctions of human B7-H4 generated from ***:The gene of human B7-H4 extracellular region（IgV-like andIgC-like domains）was obtained by PCR from human cDNA FLJ22418 and theninserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathiones-transferase（GST）fusion *** being identified by restriction enzymedigestion and sequencing,the recombinant vector was transferred into host strainE coli BL21-RIL（DE3）.A 47 kDa fusion protein（GST/hB7-H4）was induced byisopropyl-beta-D-thiogalactopyranoside（IPTG）and purified by standard meth-ods reported in the prokaryotic *** inhibitory effect of GST/hBT-H4 onproliferation of T cells was observed in vitro by CD3mAb activated T-cell cultur-ing system and [3H]-thymidine incorporation *** concentrations ofinterleukin-2 and iterferon-g in the supernatants of T cells were determined ***:We successfully constructed the method for high-level expres-sion and purification of the hB7-H4 extracellular domain as GST fusion proteinfrom E *** GST/hB7-H4 fusion protein produced in bacteria had obviousbiological activity to inhibit T-lymphocyte proliferation and IL-2 ***:The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions,whichprovided an efficient and economical way for the preparation of this target protein.

关键词： B7-H4 glutathione s-transferase fusion protein inhibition

Screening and identification of Shigella flexneri 2a virulence-related genes induced after invasion of epithelial cells

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Science China(Life Sciences) 2004年第6期47卷 494-502页

作者： SHI Zhaoxing WANG Hengliang HU Kun FENG Erling YAO Xiao HUANG Liuyu SU Guofu HUNAG Peitang HUANG Cuifen Beijing institute of biotechnology Beijing 100071China Beijing Institute of BiotechnologyBeijing 100071China Beijing Institute of BiotechnologyBeijing 100071China College of Life Science Shandong Normal UniversityJinan 250014China Beijing Institute of BiotechnologyBeijing 100071China Beijing Institute of BiotechnologyBeijing 100071China College of Environmental and Chemical Engineering Xi'an Jiaotong UniversityXi'an 710049China Beijing Institute of BiotechnologyBeijing 100071China Beijing Institute of BiotechnologyBeijing 100071China Beijing Institute of BiotechnologyBeijing 100071China Beijing Institute of BiotechnologyBeijing 100071China

An in vivo expression technology (IVET) was applied to screen *** 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three ap- peared to be antisense to genes involved in the transmethylation,biosyntheseis, and phos- photransferase system;and three were predicted to encode polypeptides with unknown functions. Intracellular survival assavs showed that the mutants of alkA,citC and wcaJ genes had lower capability of intracellular replication or survival than the the wild-type *** results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intra- cellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.

关键词： Shigella flexneri 2a intracellular induced expression virulence-related genes in vivo expression technology.

Human pescadillo induces large-scale chromatin unfolding

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Science China(Life Sciences) 2005年第3期48卷 270-276页

作者： ZHANG Hao FANG Yan HUANG Cuifen YANG Xiao YE Qinong Beijing institute of biotechnology Beijing100850China Beijing Institute of Biotechnology Beijing100850China Beijing Institute of Biotechnology Beijing100850China Beijing Institute of Biotechnology Beijing100850China Beijing Institute of Biotechnology Beijing100850China

The human pescadillo gene encodes a protein with a BRCT domain. Pescadillo plays an important role in DNA synthesis, cell proliferation and transformation. Since BRCT do- mains have been shown to induce chromatin large-scale unfolding, we tested the role of Pesca- dillo in regulation of large-scale chromatin unfolding. To this end, we isolated the coding region of Pescadillo from human mammary MCF10A cells. Compared with the reported sequence, the isolated Pescadillo contains in-frame deletion from amino acid 580 to 582. Targeting the Pesca- dillo to an amplified, lac operator-containing chromosome region in the mammalian genome re- sults in large-scale chromatin decondensation. This unfolding activity maps to the BRCT domain of Pescadillo. These data provide a new clue to understanding the vital role of Pescadillo.

关键词： Pescadillo, large-scale chromatin unfolding, replication.

Construction of a trivalent candidate Shigella vaccine strain with host-vector balanced-lethal system

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Science China(Life Sciences) 1997年第1期40卷 52-59页

作者：芮贤良徐永强吴旭东苏国富黄翠芬 institute of biotechnology Beijing 100071 China

A trivalent live Shigella vaccine candidate FSD01 against S. flexneri 2a, S. sonnei and S. dysen-teriae I was constructed. This candidate strain was based on the S. flexneri 2a vaccine T32. By homologous recombi-nation exchange, the chromosomal asd gene of T32 was site-specifically inactivated, resulting in the strain unable to grow normally in LB broth, while another asd gene of S. mutans was employed to construct an Asd complementary vector. This combination of asd 'host/ Asd+ vector formed a balanced-lethal expression system in T32 strain. By use of this system, two important protective antigen genes coding for S. sonnei Form I antigen and Shiga toxin B subunit were cloned and expressed in T32, which led to the construction of trivalent candidate vaccine FSD01. Experimental results showed that this strain was genetically stable, but its recombinant plasmid was non-resistant. Moreover, it was able to effectively express trivalent antigens in one host and induce protective responses in mice against the challenges of the above three Shigella strains.

关键词： Shigella spp balanced-lethal system recombinant vaccine strain immune protection.