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Facilitating stable gene integration expression and copy number amplification in Bacillus subtilis through a reversible homologous recombination switch

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Synthetic and Systems biotechnology 2024年第3期9卷 577-585页

作者： Haoyu Guo Rongzhen Tian Yaokang Wu Xueqin Lv Jianghua Li Long Liu a b Guocheng Du Jian Chen Yanfeng Liu Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China Science Center for Future foods Jiangnan UniversityWuxi214122China engineering Research Center of Ministry of Education on food Synthetic biotechnology Jiangnan UniversityWuxi214122China Jiangsu Province engineering Research Center of food Synthetic biotechnology Jiangnan UniversityWuxi214122China

Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic *** comparison to plasmid-based multi-copy expression,the utilization of chromosomal multi-copy genes offers increased stability of expression level,diminishes the metabolic burden on host cells,and enhances overall genetic *** this study,we developed the“BacAmp”,a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis,which was achieved by employing a combination of repressor and non-natural amino acids(ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous *** the reversible switch is turned on,genome editing and gene amplification can be ***,the reversible switch was turned off therefore stabilizing the gene copy *** stabilized gene amplification system marked by green fluorescent protein,achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 *** we implemented the gene amplification system for the regulation of N-acetylneuraminic acid(NeuAc)synthesis,the copy number of the critical gene increased to an average of 7.7,which yielded a 1.3-fold NeuAc *** research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in ***.

关键词： Bacillus subtilis Stabilized gene amplification system Homologous recombination recA

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Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

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food Science and Human Wellness 2024年第4期13卷 2310-2316页

作者： Yuqi Zhu Pengdong Sun Chunjian Li Yu Zhang Yu Wang Jingyuan Li Yanfeng Liu Jian Chen Yang Deng College of food Science and engineering Qingdao Agricultural UniversityQingdao 266109China Key Laboratory of Special food Processing(Co-construction by Ministry and Province) Ministry of Agriculture Rural AffairsQingdao Agricultural UniversityQingdao 266109China Qingdao Special food Research Institute Qingdao 266109China Science Center for Future foods Engineering Research Center of Ministry of Education on Food Synthetic BiotechnologyJiangsu Province Engineering Research Center of Food Synthetic BiotechnologyJiangnan UniversityWuxi 214122China Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi 214122China

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of *** this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-*** was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation *** original strain most likely only producedα-LA intracellular,but not *** improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the *** 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in *** YjcN was determined to be the best signal *** in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal *** addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and *** results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/*** study demonstrates successful expression and secretion of humanα-LA in *** and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

关键词： Bacillus subtilis Alpha-lactalbumin Bioengineering milk Signal peptide Promoter screening

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Efficient biosynthesis of creatine by whole-cell catalysis from guanidinoacetic acid in Corynebacterium glutamicum

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Synthetic and Systems biotechnology 2024年第1期9卷 99-107页

作者： Chunjian Li Pengdong Sun Guoqing Wei Yuqi Zhu Jingyuan Li Yanfeng Liu Jian Chen Yang Deng College of food Science and engineering Qingdao Agricultural UniversityQingdao 266109China Key Laboratory of Special food Processing(Co-construction by Ministry and Province) Ministry of Agriculture Rural AffairsQingdao Agricultural UniversityQingdao 266109China Shandong Technology Innovation Center of Special food Qingdao 266109China Qingdao Special food Research Institute Qingdao 266109China Qingdao Nuoan Baite biotechnology Co. Ltd.Qingdao 266109China Science Center for Future foods Engineering Research Center of Ministry of Education on Food Synthetic Biotechnologyand Jiangsu Province Engineering Research Center of Food Synthetic BiotechnologyJiangnan UniversityWuxi 214122China Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi 214122China

Creatine is a naturally occurring derivative of an amino acid commonly utilized in functional foods and ***,the current industrial synthesis of creatine relies on chemical processes,which may hinder its utilization in certain ***,a biological approach was devised that employs whole-cell biocatalysis in the bacterium Corynebacterium glutamicum,which is considered safe for use in food production,to produce safe-for-consumption *** objective of this study was to identify a guanidinoacetate N-methyltransferase(GAMT)with superior catalytic activity for creatine *** employing whole-cell biocatalysis,a gamt gene from Mus caroli(Mcgamt)was cloned and expressed in *** ATCC 13032,resulting in a creatine titer of 3.37 g/***,the study employed a promoter screening strategy that utilized nine native strong promoters in *** to enhance the expression level of *** highest titer was achieved using the P1676 promoter,reaching 4.14 g/*** conditions of whole-cell biocatalysis were further optimized,resulting in a creatine titer of 5.42 g/*** is the first report of successful secretory creatine expression in ***,which provides a safer and eco-friendly approach for the industrial production of creatine.

关键词： Creatine Corynebacterium glutamicum Whole-cell biocatalysis Guanidinoacetate N-Methyltransferase food additive

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The interaction mechanism between zein and folic acid in alkaline aqueous solutions:an experimental and molecular simulation study

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food Quality and Safety 2024年第1期8卷 166-178页

作者： Jun Zhu Yiling Yao Yuying Liao Lala Cai Wenqi Zhang Nuo Hong Wenlin Sun Chunhua Xiong Lanying Yao School of food Science and biotechnology Zhejiang Gongshang UniversityHangzhouChina

Objectives:Folic acid is a vitamin that is not highly soluble in water and is sensitive to the ***,it is important to find suitable *** study aimed to exemplify the interaction of folic acid with zein in alkaline aqueous solutions and shed light on how zein can be used as a carrier for folic *** and Methods:Zein and folic acid were separately dissolved in NaOH solutions with a PH of *** solution and folic acid solution were combined in specific *** methods including multi-spectroscopy,dynamic light scattering,and electron microscopy combining with molecular dynamics simulations were used to study the interaction mechanism between zein and folic acid in alkaline aqueous ***:Fluorescence spectroscopy showed that the quenching of zein by folic acid was mainly static,and the main driving force behind this interaction was van der Waals forces and hydrogen *** formation of zein–folic acid complexes was confirmed by ultraviolet–visible spectroscopy,Fourier transform infrared spectroscopy,and circular dichroism *** results also showed that the structure of zein changed when it interacted with folic *** light scattering analysis revealed that the addition of folic acid caused proteins to *** aggregates of the complexes had an irregular shape and were large,as observed by scanning electron microscopy and transmission electron *** simulation was used to further investigate the interaction *** to these findings,the folic acid molecule interacted with zein in a shallow recess near the protein *** dominant forces at play in the zein–folic acid interaction were van der Waals forces and electrostatic forces,including hydrogen ***:The zein alkali-soluble system is very suitable for folic acid delivery.

关键词： Zein folic acid alkaline aqueous solutions molecular simulation interaction mechanism.

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Characteristics and mechanism of Ni^(2+)and Cd^(2+)adsorption by recovered perlite from agar extraction residue

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Chinese Journal of Chemical engineering 2024年第8期72卷 141-152页

作者： Juan Wang Yanbing Zhu Zedong Jiang Xiping Du Mingjing Zheng Lijun Li Hui Ni Yuanpeng Wang Zhipeng Li Qingbiao Li College of Ocean food and Biological engineering Jimei UniversityXiamen 361021China Fujian Provincial Key Laboratory of food Microbiology and Enzyme engineering Xiamen 361021China Research Center of food biotechnology of Xiamen City Xiamen 361021China Xiamen Ocean Vocational College Xiamen 361021China College of Chemistry and Chemical engineering Xiamen UniversityXiamen 361021China

Ni^(2+)and Cd^(2+)in wastewater accumulated through the ecological chain and could jeopardize human *** of Ni^(2+)and Cd^(2+)from wastewater using recovered perlite was an important way to solve the problem of resource utilization of solid waste from agar *** previous study confirmed that recovered perlite from agar extraction residue had better pore size and specific surface area than commercial ***,the adsorption efficiency and adsorption mechanism of recovered perlite were the main factors limiting its adsorption *** adsorption process of Ni^(2+)and Cd^(2+)by recovered perlite in aqueous solution was described by the pseudo-second-order kinetic equation,and the relevant adsorption mechanism was mainly *** with commercial perlite,the adsorption removal rate of Ni^(2+)and Cd^(2+)by enzymatic recovered perlite could reach 92.9%and 89.2%,respectively,and were improved by 12.63%and 13.03%.Langmuir isothermal adsorption model could better describe the isothermal adsorption process of recovered perlite on heavy metal Ni^(2+)and Cd^(2+),and the relevant adsorption mechanism was mainly monolayer *** X-ray photoelectron spectroscopy(XPS)results indicated that the decrease of Si—O Si^(2+)hydroxyl coordination bond and the increase of C—Si bond might make the binding effect of recovered perlite with heavy metals *** competitive adsorption of Ni^(2+)and Cd^(2+)by recovered perlite was still dominated by chemisorption and monolayer *** study was expected to provide a theoretical basis and technical support for the removal of Ni^(2+)and Cd^(2+)from wastewater using recovered perlite from seaweed residue.

关键词： Agar extraction residue Recovered perlite Heavy metal ion Adsorption property

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Lactobacillus plantarum CCFM1180 attenuates obesity induced by estrogen deficiency by activating estrogen receptor alpha in abdominal adipose tissue and regulating gut microbiota-derived metabolites

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food Science and Human Wellness 2024年第2期13卷 765-777页

作者： Qian Chen Chunxia Mei Min Guo Botao Wang Haiqin Chen Jianxin Zhao Gang Wang Wei Chen State Key Laboratory of food Science and Resources Jiangnan UniversityWuxi 214122China School of food Science and Technology Jiangnan UniversityWuxi 214122China National engineering Research Center for Functional food Jiangnan UniversityWuxi 214122China (Yangzhou)Institute of food biotechnology Jiangnan UniversityYangzhou 225004China Bloomage biotechnology Co. Ltd.Jinan 250000China

Lipid metabolism disorders commonly occur during *** deficiency has been shown to lead to excessive energy intake and abnormal lipid metabolism in ovariectomized rats,resulting in *** exhibit anti-obesity properties,and their underlying mechanism has been widely *** this study,we demonstrated the metabolic benefits of Lactobacillus plantarum CCFM1180 in suppressing appetite,controlling body weight,correcting obesity-induced abnormalities,enhancing liver lipid metabolism,and protecting liver function in estrogen-deficient *** mechanisms associated with the anti-obesity and anti-dyslipidemia effects of CCFM1180 on estrogen-deficient rats were *** results showed that CCFM1180 dramatically reduced food intake by activating the expression of estrogen receptor alpha(ERα)and increasing the level of leptin in abdominal adipose *** changes,combined with the increased butyrate concentration and recovered bile acid structure,helped enhance lipid ***,CCFM1180 treatment was found to be safer than exogenous estrogen ***,*** CCFM1180 could be considered a new therapeutic strategy for preventing and alleviating menopausal lipid abnormalities.

关键词： Bile acid Gut microbiota Menopause Ovariectomy Short chain fatty acid

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CRISPR/Cpf1–FOKI-induced gene editing in Gluconobacter oxydans

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Synthetic and Systems biotechnology 2024年第2期9卷 369-379页

作者： Xuyang Wang Dong Li Zhijie Qin Jian Chen Jingwen Zhou engineering Research Center of Ministry of Education on food Synthetic biotechnology Jiangnan University1800 Lihu RoadWuxiJiangsu214122China Science Center for Future foods Jiangnan University1800 Lihu RoadWuxiJiangsu214122China Key Laboratory of Industrial biotechnology Ministry of Education and School of BiotechnologyJiangnan University1800 Lihu RoadWuxiJiangsu214122China Jiangsu Province engineering Research Center of food Synthetic biotechnology Jiangnan UniversityWuxi214122China

Gluconobacter oxydans is an important Gram-negative industrial microorganism that produces vitamin C and other products due to its efficient membrane-bound dehydrogenase *** incomplete oxidation system has many crucial industrial ***,it also leads to slow growth and low biomass,requiring further metabolic modification for balancing the cell growth and incomplete oxidation *** a non-model strain,*** lacks efficient genome editing tools and cannot perform rapid multi-gene editing and complex metabolic network *** the last 15 years,our laboratory attempted to deploy multiple CRISPR/Cas systems in different *** strains and found none of them as *** this study,Cpf1-based or dCpf1-based CRISPRi was constructed to explore the targeted binding ability of Cpf1,while Cpf1–FokI was deployed to study its nuclease activity.A study on Cpf1 found that the CRISPR/Cpf1 system could locate the target genes in *** but lacked the nuclease cleavage ***,the CRISPR/Cpf1–FokI system based on FokI nuclease was ***-gene knockout with efficiency up to 100%and double-gene iterative editing were achieved in *** this system,AcrVA6,the anti-CRISPR protein of *** was discovered for the first time,and efficient genome editing was realized.

关键词： Cell growth CRISPRi Anti-CRISPR protein Multi-gene editing Vitamin C

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Design-build-test of recombinant Bacillus subtilis chassis cell by lifespan engineering for robust bioprocesses

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Synthetic and Systems biotechnology 2024年第3期9卷 470-480页

作者： Kexin Ren Qiang Wang Jianghua Chen Hengwei Zhang Zhoule Guo Meijuan Xu Zhiming Rao Xian Zhang Key Laboratory of Industrial biotechnology of the Ministry of Education School of BiotechnologyJiangnan UniversityWuxiJiangsu214122China Yixing Institute of food and biotechnology Co. LtdYixing214200China

Microbial cell factories utilize renewable raw materials for industrial chemical production,providing a promising path for sustainable *** subtilis is widely used in industry for its food safety properties,but challenges remain in the limitations of microbial *** study proposes a novel strategy based on lifespan engineering to design robust *** chassis cells to supplement traditional metabolic modification strategies that can alleviate cell autolysis,tolerate toxic substrates,and get a higher mass transfer *** modified chassis cells could produce high levels of L-glutaminase,and tolerate hydroquinone to produceα-arbutin *** a 5 L bioreactor,the L-glutaminase enzyme activity of the final strain CRE15TG was increased to 2817.4±21.7 U m L^(-1),about 1.98-fold compared with that of the wild ***α-arbutin yield of strain CRE15A was increased to 134.7 g L^(-1),about 1.34-fold compared with that of the *** our knowledge,both of the products in this study performed the highest yields reported so *** chassis modification strategy described in this study can Improve the utilization efficiency of chassis cells,mitigate the possible adverse effects caused by excessive metabolic modification of engineered strains,and provide a new idea for the future design of microbial cell factories.

关键词： Bacillus subtilis Chassis cell Lifespan engineering Robustness Industrial production

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Environmental enrichment in combination with Bifidobacterium breve HNXY26M4 intervention amplifies neuroprotective benefits in a mouse model of Alzheimer's disease by modulating glutamine metabolism of the gut microbiome

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food Science and Human Wellness 2024年第2期13卷 982-992页

作者： Guangsu Zhu Min Guo Jianxin Zhao Hao Zhang Gang Wang Wei Chen State Key Laboratory of food Science and Resources Jiangnan UniversityWuxi 214122China School of food Science and Technology Jiangnan UniversityWuxi 214122China (Yangzhou)Institute of food biotechnology Jiangnan UniversityYangzhou 225004China National engineering Center of Functional food Jiangnan UniversityWuxi 214122China

The gut microbiota-brain axis has emerged as a novel target for Alzheimer's disease(AD),a neurodegenerative disease characterised by behavioural and cognitive ***,most previous microbiome-based intervention studies have focused on single factors and yielded only modest cognitive ***,we proposed a multidomain intervention strategy that combined Bifidobacterium breve treatment with environmental enrichment(EE)*** this study,we found that compared with EE or *** treatment alone,*** intervention combined with EE amplified its neuroprotective effects on AD mice,as reflected by improved cognition,inhibited neuroinflammation and enhanced synaptic ***,using microbiome and metabolome profiling,we found that the combination of *** and EE treatment restored AD-related gut microbiota dysbiosis and reversed microbial metabolite ***,by integrating behavioural and neurological data with metabolomic profiles,we revealed that the underlying mechanism may involve the modulation of microbiota-derived glutamine metabolism via gut-brain ***,combined *** intervention with EE treatment can alleviate AD-related cognitive impairment and improve brain function by regulating glutamine metabolism of the gut *** findings provide a promising multidomain intervention strategy,with a combination of dietary microbiome-based and lifestyle-targeted interventions,to promote brain function and delay the progression of AD.

关键词： Alzheimer’s disease Bifidobacterium breve Environmental enrichment Glutamine metabolism Microbiota-gut-brain axis

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Probiotics administration alleviates cognitive impairment and circadian rhythm disturbance induced by sleep deprivation

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food Science and Human Wellness 2024年第4期13卷 1951-1961页

作者： Peijun Tian Yunfei Hou Zheng Wang Jiaona Jiang Xin Qian Zhihao Qu Jianxin Zhao Gang Wang Wei Chen State Key Laboratory of food Science and Resources Jiangnan UniversityWuxi 214122China School of food Science and Technology Jiangnan UniversityWuxi 214122China National engineering Research Center for Functional food Jiangnan UniversityWuxi 214122China Institute of food biotechnology(Yangzhou) Jiangnan UniversityYangzhou 225004China

Gut microbiome is indispensable for maintaining normal brain ***,gut microbiota plays a causal role in sleep deprivation(SD)-induced cognitive *** this study,neurobehavioral effects of the Bifidobacterium breve strain(CCFM1025)were assessed in sleep-deprived ***1025 improved the body weight and food and water intake of the *** also alleviated SD-induced cognitive behavioural abnormalities(in the novel object recognition test),but did not show beneficial effects on mood-and spatial memory-related ***1025 significantly altered the gut microbial composition and genome *** microbial metabolites that may regulate sleep function were also identified,such as isovaleric acid andγ-aminobutyric acid in the gut and purine metabolites in the *** metabolites may participate in gutbrain communication by acting on the striatal melatonin system,for example to increase melatonin levels,and by regulating the expression of circadian clock genes such as those encoding the adenosine A2A receptor and period circadian regulator ***,administration of probiotics alleviated cognitive impairment and circadian rhythm disturbance induced by SD via modulation of gut microbiome and its *** findings may help guide the treatment of insomnia or other sleep disorders via dietary strategies.

关键词： Bifidobacterium Sleep Circadian rhythm Purine Gut microbiome

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