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Ultrahigh-throughput screening of antagonistic bacteria against Erwinia carotovora Ecc15 based on droplet microfluidics

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Food Science and Human Wellness 2024年

作者： Longfeng Wei Shuang Li Hang Pan Xingjin Jian Renli Ai Zhu Li Chong Zhang Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education)， College of Life Sciences/Institute of Agro-bioengineering， Guizhou University Guizhou Key Laboratory of Agricultural biotechnology Department of Chemical Engineering， Institute of Biochemical Engineering， Tsinghua University Key Laboratory of Industrial Biocatalysis， Ministry of Education， Tsinghua University

Soft rot is a destructive disease that inflicts significant losses on agricultural production and the economy post-harvest. Biocontrol strategies based on antagonistic microorganisms have a broad application prospect to fight against plant pathogens. This study utilized fluorescence-activated droplet sorting （FADS） technology as an alternative to traditional plate culture methods to isolate microorganisms with antagonistic activity against the soft rot pathogen Erwinia carotovora Ecc15. Initially， the culture performance of the FADS platform was evaluated by analyzing bacterial diversity in droplet culture samples and agar plate culture samples， our data showed that droplet culture exhibited higher species richness and diversity than plate culture， and more than 95% of the OTUs in the droplet samples belonged to the rare biosphere. Additionally， we developed a GFP-Ecc15-based FADS screening system， which achieved an enrichment ratio of up to 148. Using this system， we successfully screened 32 antagonistic bacteria from rhizosphere soil sample of healthy konjac plants， and some may be novel microbial resources， including the genera Lelliottia， Buttiauxella and Leclercia. Notably， strain D-62 exhibited the strongest antibacterial ability against Ecc15， with an inhibition zone diameter of （20.86 ± 1.56） mm. In vivo experiments conducted on the corms of Amorphophallus konjac demonstrated that strain D-62 could effectively reduce the infection ability of Ecc15 to the corms， indicating that strain D-62 has the potential to be developed as a biocontrol agent. Our findings suggested that the FADS screening system showed a screening efficiency approximately 3 × 103times higher than plate screening system， while significantly reducing costs of infrastructure， labor and consumables， it provides theoretical guidance for the screening of other plant pathogen biocontrol bacteria.

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Methane biotransformation in the ocean and its effects on climate change: A review

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Science China Earth Sciences 2018年第12期61卷 1697-1713页

作者： Mingyang NIU Wenyue LIANG Fengping WANG State Key Laboratory of microbial Metabolism School of Life Sciences and Biotechnology Shanghai Jiao Tong University State Key Laboratory of Ocean Engineering School of Naval Architecture Ocean and Civil Engineering Shanghai Jiao Tong University

Methane is a potent greenhouse gas. Continental margins contain large reservoirs of methane as solid gas hydrate and the dissolved and gaseous forms of methane. Submarine methane seeps along the global continental margins, including the coastal seas, have been estimated to contribute 0.01 to 0.05 Gt of carbon to the atmosphere annually, accounting for between 1%and 5% of the global methane emissions to the atmosphere. Much of this methane is exhausted via microbial anaerobic methane oxidation. Methane biotransformation in the ocean has effects on global climate change. This review mainly introduces the mechanisms of methanogenesis and methane oxidation and describes new findings that will provide information that will improve the understanding of the balance in terms of the generation, migration and consumption of methane in marine environments. Moreover, this review provides new insights into methane biogeochemical cycles and the effects of marine methane budgets on global climate.

关键词： Methane production Methane oxidization Marine Sediment Climate

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Production of N-acetylglucosamine with Vibrio alginolyticus FA2,an emerging platform for economical unsterile open fermentation

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Synthetic and Systems biotechnology 2023年第3期8卷 546-554页

作者： Yuan Peng Ping Xu Fei Tao State Key Laboratory of microbial Metabolism Joint International Research Laboratory of Metabolic and Developmental Sciencesand School of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China

Members of the Vibrionaceae family are predominantly fast-growing and halophilic microorganisms that have captured the attention of researchers owing to their potential applications in rapid *** them,Vibrio alginolyticus FA2 is a particularly noteworthy halophilic bacterium that exhibits superior growth *** has the potential to serve as a biotechnological platform for sustainable and eco-friendly open fermentation with *** evaluate this hypothesis,we integrated the N-acetylglucosamine(GlcNAc)pathway into *** *** nag genes were knocked out to obstruct the utilization of GlcNAc,and then 16 exogenous gna1s co-expressing with EcglmS were introduced to strengthen the flux of GlcNAc pathway,*** further enhance GlcNAc production,we fine-tuned promoter strength of the two genes and inactivated two genes alsS and alsD to prevent the production of ***,unsterile open fermentation was carried out using simulated seawater and a chemically defined medium,resulting in the production of 9.2 g/L GlcNAc in 14 *** is the first report for de-novo synthesizing GlcNAc with a Vibrio strain,facilitated by an unsterile open fermentation process employing seawater as a substitute for fresh *** development establishes a basis for production of diverse valuable chemicals using Vibrio strains and provides insights into biomanufacture.

关键词： Vibrio Open fermentation Metabolic engineering N-acetylglucosamine

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The PilZ domain of MrkH represents a nove ）NA binding motif

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Protein & Cell 2016年第10期7卷 766-772页

作者： Feng Wang Qing He Kaixuan Su Fei Gao Yan Huang Zong Lin Deyu Zhu Lichuan Gu State Key Laboratory of microbial Technology Shandong University Jinan 250100 China Department of biotechnology and Biomedicine Yangtze Delta Region Institute of Tsinghua University Jiaxing 314006 China

MrkH is the first characterized c-di-GMP related transcrip- tional regulator which affects type 3 fimbrial expression in response to cellular c-di-GMP level, and thus plays an important role in the biofilm formation of Klebsiella pneu- moniae （Murphy and Clegg, 2012; Wilksch et al., 2011）. However, how MrkH recognizes c-di-GMP and the target DNA sequence remains obscure.

关键词： Fluorescence Polarization Fluorescence Polarization Measurement Small Positive Charge Fimbrial Expression Fluorescence Polarization Analysis

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Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco

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Protein & Cell 2014年第7期5卷 552-562页

作者： Zhen Cai Guoxia Liu Junli Zhang Yin Li CAS Key Laboratory of microbial Physiological and Metabolic Engineering Institute of Microbiology Chinese Academy of Sciences Beijing 100101 China Tianjin Institute of Industrial biotechnology Chinese Academy of Sciences Tianjin 300308 China

Photosynthetic CO2 fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration, Ribulose-1,5-bis- phosphate carboxylase/oxygenase （Rubisco） catalyzes the first step of photosynthetic CO2 fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving pho- tosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an effi- cient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coil-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G sub- stitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxyla- tion activity and a 45% improvement in catalytic efficiency towards CO2. The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.

关键词： carboxylation efficiency CO2 fixation directed evolution Rubisco Synechococcus sp. PCC7002

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The interactive effect of temperature and fertilizer types determines the dominant microbes in nitrous oxide emissions and the dicyandiamide efﬁcacy in a vegetable soil

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Soil Ecology Letters 2024年第3期6卷 121-133页

作者： Xiaoya Xu Haiyang Liu Yaowei Liu Didier Lesueur Laetitia Herrmann Hongjie Di Caixian Tang Jianming Xu Yong Li Zhejiang Provincial Key Laboratory of Agricultural Resources and Environment College of Environmental and Resource SciencesZhejiang UniversityHangzhou 310058China College of Geography and Environment Shandong Normal UniversityJinan 250014China Centre de Coopération Internationale en Recherche Agronomique pour le Développement(CIRAD) UMR Eco&SolsHanoiVietnam Eco&Sols Universitéde Montpellier(UMR)CIRADInstitut National de Recherche pour l’Agriculturel’Alimentation et l’Environnement(INRAE)Institut de Recherche pour le Développement(IRD)Montpellier SupAgro34060 MontpellierFrance Alliance of Biodiversity International and International Center for Tropical Agriculture(CIAT) Asia hubCommon Microbial Biotechnology Platform(CMBP)HanoiVietnam School of Life and Environmental Sciences Faculty of ScienceEngineering and Built Environment–Deakin UniversityMelbourneVIC 3125Australia Rubber Research Institute Chinese Academy of Tropical Agricultural SciencesHaikou 571737China La Trobe Institute for Sustainable Agriculture and Food Department of AnimalPlant&Soil SciencesLa Trobe UniversityBundooraVic 3086Australia

Heat waves associated with global warming and extreme climates would arouse serious consequences on nitrogen(N)***,the responses of the functional guilds to different temperatures,especially high temperature and the cascading effect on N_(2)O emissions remain *** incubation study was conducted to examine the effect of different temperatures(20°C,30°C,and 40°C)and fertilizer types(urea and manure)on N_(2)O-producers and N_(2)O-reducers,as well as the efficacy of dicyandiamide(DCD)on N_(2)O emissions in a vegetable *** showed that ammonia oxidizers and nirS-type denitrifiers were well adapted to high temperature(40°C)with manure application,while the fungal nirK-denitrifiers had better tolerance with urea *** nosZ clade I microbes had a strong adaptability to various temperatures regardless of fertilization type,while the growth of nosZ clade II group microbes in non-fertilized soil(control)were significantly inhibited at higher *** N_(2)O emissions were significantly decreased with increasing temperature and DCD application(up to 60%,even at 40°C).Under high temperature conditions,fungal denitrifiers play a significant role in N-limited soils(non-fertilized)while nirS-type denitrifiers was more important in fertilized soils in N_(2)O emissions,which should be specially targeted when mitigating N_(2)O emissions under global warming climate.

关键词： nitrogen fertilizer microorganisms nitrification inhibitor bacteria archaea fungi

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Screening of Highly Effective Sinorhizobium meliloti Strains for 'Vector' Alfalfa and Testing of Its Competitive Nodulation Ability in the Field

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Pedosphere 2007年第2期17卷 219-228页

作者： ZENG Zhao-Hai CHEN Wen-Xin HU Yue-Gao SUI Xin-Hua CHEN Dan-Ming College of Agronomy and biotechnology China Agricultural University Beijing 100094 （China） College of Biology/Key Laboratory of Agro-microbial Resource and Application Ministry of Agriculture China Agricultural University Beijing 100094 （China）

Seventeen Sinorhizobium meliloti strains from seven provinces in China were used to screen highly effective strains for alfalfa cultivar in a greenhouse study and their symbiotic relationship and competitive ability were studied in the field. CCBAU30138 was the most effective strain, as evidenced by increase in dry weights. A field experiment showed that the inoculation of alfalfa with CCBAU30138 resulted in increases of 11.9% and 19.6% of dry matter production and crude protein production, respectively, in forage of monocultured plants. The total dry matter yields of alfalfa and tall fescue in binary culture were increased by 16.3% by inoculation of alfalfa with this strain. These results showed that S. rneliloti strain CCBAU30138 was an effective inoculant both in the greenhouse and in the field. The analysis of randomly amplified polymorphic DNA （RAPD） by polymerase chain reaction （PCR） from nodule extracts showed that the strain CCBAU30138 had high competitiveness in the field. It occupied 47.5% of nodules in alfalfa monoculture and 44.4% of nodules in alfalfa-tall fescue binary culture after 20 weeks of growth. In conclusion, a simple system to select highly effective and competitive symbiotic strains specific to alfalfa was established. Using this system, a *** suitable for the alfalfa cultivar ‘Vector＇ grown in Wuqiao County of Hebei Province was obtained.

关键词： alfalfa inoculant selection Sinorhizobium meliloti tall fescue

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Raising the production of phloretin by alleviation of by-product of chalcone synthase in the engineered yeast

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Science China(Life Sciences) 2020年第11期63卷 1734-1743页

作者： Chunmei Jiang Xiaonan Liu Xianqing Chen Yi Cai Yibin Zhuang Tian Liu Xiaoxi Zhu Hui Wang Yuwan Liu Huifeng Jiang Wen Wang Key Laboratory for Space Bioscience and Space biotechnology School of Life SciencesNorthwestern Polytechnical UniversityXi’an 710072China Key Laboratory of Systems microbial biotechnology Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin 300308China University of Chinese Academy of Sciences Beijing 100049China Center for Ecology and Environmental Sciences Northwestern Polytechnical UniversityXi’an 710072China Jiaxing Synbiolab biotechnology Co. Ltd.Jiaxing 314006China

Phloretin is an important skin-lightening and depigmenting agent from the peel of apples. Although de novo production of phloretin has been realized in microbes using the natural pathway from plants, the efficiency of phloretin production is still not enough for industrial application. Here, we established an artificial pathway in the yeast to produce phloretin via assembling two genes of p-coumaroyl-CoA ligase(4CL) and chalcone synthase(CHS). CHS is a key enzyme which conventionally condenses a CoA-tethered starter with three molecules of malonyl-CoA to form the backbone of flavonoids. However, there was 33% of byproduct generated via CHS by condensing two molecules of malonyl-CoA during the fermentation process. Hence, we introduced a more efficient CHS and improved the supply of malonyl-CoA through two pathways;the by-product ratio was decreased from 33% to 17% and the production of phloretin was improved from 48 to 83.2 mg L^(-1). Finally, a fed-batch fermentation process was optimized and the production of phloretin reached 619.5 mg L^(-1), which was 14-fold higher than that of the previous studies. Our work established a platform for the biosynthesis of phloretin from the low-cost raw material 3-(4-hydroxyphenyl) propanoic acid and also illustrated the potential for industrial scale bio-manufacturing of phloretin.

关键词： phloretin Saccharomyces cerevisiae metabolic engineering microbial cell factories fed-batch fermentation

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Trithorax Group Proteins Act Together with a Polycomb Group Protein to Maintain Chromatin Integrity for Epigenetic Silencing during Seed Germination in Arabidopsis

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Molecular Plant 2018年第5期11卷 659-677页

作者： Fan Xu Tony Kuo Yenny Rosli Mao-Sen Liu Limin Wu Long-Fang Oliver Chen Jennifer C. Fletcher Zinmay Renee Sung Li Pu biotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing 100081 China Department of Plant and microbial Biology University of California Berkeley CA 94720 USA Institute of Plant and microbial Biology Academia Sinica Taipei 11529 Taiwan China College of Life and Environmental Sciences Hangzhou Normal University Hangzhou 310036 China

Polycomb group （PcG） and trithorax group （trxG） proteins have been shown to act antagonistically to epigenetically regulate gene expression in eukaryotes. The trxG proteins counteract PcG-mediated floral repression in Arabidopsis, but their roles in other developmental processes are poorly understood. We investigated the interactions between the trxG genes, ARABIDOPSIS HOMOLOG OF TRITHORAX1 （ATX1） and ULTRAPETALA1 （ULT1）, and the PcG gene EMBRYONIC FLOWER 1 （EMF1） during early development. Unexpectedly, we found that mutations in the trxG genes failed to rescue the early-flowering phenotype of emfl mutants. Instead, emfl atxl ultl seedlings showed a novel swollen root phenotype and massive deregulation of gene expression. Greater ectopic expression of seed master regulatory genes in emfl atxl ultl triple than in emfl single mutants indicates that PcG and trxG factors together repress seed gene expression after germination. Furthermore, we found that the widespread gene derepression is asso- ciated with reduced levels of H3K27me3, an epigenetic repressive mark of gene expression, and with globally altered chromatin organization. EMF1, ATXl, and ULT1 are able to bind the chromatin of seed genes and ULT1 can physically interact with ATX1 and EMF1, suggesting that the trxG and EMF1 proteins directly associate at target gene loci for EMFl-mediated gene silencing. Thus, while ATXl, ULT1, and EMF1 interact antagonistically to regulate flowering, they work together to maintain chromatin integrity and prevent precocious seed gene expression after germination.

关键词： trxG PcG histone modification chromatin integrity seed gene repression

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Spatial analysis of bacteria in brackish lake sediment

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International Journal of Sediment Research 2020年第3期35卷 227-236页

作者： Jean-Sebastien Beaulne Samir R.Mishra Mrutyunjay Suar Ananta Narayan Panda Gurdeep Rastogi Ajit Kumar Pattnaik Vishakha Raina Timothy M.Vogel Environmental microbial Genomics Ecole Centrale de LyonUniversite de Lyon ISARA Department of Agroecology and Environment Environmental biotechnology Laboratory School of BiotechnologyKIIT University Wetland Research and Training Center Chilika Development AuthorityDepartment of Forest and EnvironmentGovernment of Odisha

The spatial distribution of bacterial communities inhabiting sediment is heterogeneous at different spatial scales,but mostly ***,it is postulated that the heterogeneity of the bacterial community composition varies at the same scale of the heterogeneity of sediment chemical *** large spatial scale(km) diversity in sediment from a brackish water lagoon(Chilika Lake,India) is studied,considering the large scale physical and chemical characteristics of land cover,climate,pH,and ***-two samples(24 stations,3 seasons:winter,rainy,and summer) of sediment from Chilika Lake were analyzed by 16 S ribosomal ribonucleic acid(rRNA) gene sequencing,and the relations with land cover and other physico-chemical paramete rs are *** re samples were collected after a severe cyclonic storm(Phailin) passed near the lagoon in 2013,to see the impact of the tropical storm on the spatial and tempo ral distribution of bacteria in the *** results demonstrate clear spatial relations between physico-chemical parameters(e.g.,salinity),land surfaces(e.g.,drainage area),and the distribution of sediment microbial communities.

关键词： Salinity 16S rRNA Macro-scale Sediment GIS

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