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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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KoNA:Korean Nucleotide Archive as A New Data Repository for Nucleotide Sequence Data

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Genomics, Proteomics & Bioinformatics 2024年第1期22卷 161-167页

作者： Gunhwan Ko Jae Ho Lee Young Mi Sim Wangho Song Byung-Ha Yoon Iksu Byeon Bang Hyuck Lee Sang-Ok Kim Jinhyuk Choi Insoo Jang Hyerin Kim Jin Ok Yang Kiwon Jang Sora Kim Jong-Hwan Kim Jongbum Jeon Jaeeun Jung Seungwoo Hwang Ji-Hwan Park Pan-Gyu Kim Seon-Young Kim Byungwook Lee Korea Bioinformation center Korea Research Institute of Bioscience&BiotechnologyDaejeon 34141Republic of Korea

During the last decade,the generation and accumulation of petabase-scale high-throughput sequencing data have resulted in great challenges,including access to human data,as well as transfer,storage,and sharing of enormous amounts of *** promote data-driven biological research,the Korean government announced that all biological data generated from government-funded research projects should be deposited at the Korea BioData Station(K-BDS),which consists of multiple databases for individual data ***,we introduce the Korean Nucleotide Archive(KoNA),a repository of nucleotide sequence *** of July 2022,the Korean Read Archive in KoNA has collected over 477 TB of raw next-generation sequencing data from national genome *** ensure data quality and prepare for international alignment,a standard operating procedure was adopted,which is similar to that of the International Nucleotide Sequence Database *** standard operating procedure includes quality control processes for submitted data and metadata using an automated pipeline,followed by manual *** ensure fast and stable data transfer,a high-speed transmission system called GBox is used in ***,the data uploaded to or downloaded from KoNA through GBox can be readily processed using a cloud computing service called *** seamless coupling of KoNA,GBox,and Bio-Express enhances the data experience,including submission,access,and analysis of raw nucleotide *** not only satisfies the unmet needs for a national sequence repository in Korea but also provides datasets to researchers globally and contributes to advances in *** KoNA is available at https://***/kona/.

关键词： Korea BioData Station Nucleotide sequence Next-generation sequencing repository Genomics Deposition and access of big data

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Autoimmune gastritis studies and gastric cancer: True renaissance or bibliometric illusion

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World Journal of Gastroenterology 2024年第32期30卷 3783-3790页

作者： Vasily Isakov Department of Gastroenterology and Hepatology Federal Research Center of NutritionBiotechnology and Food SafetyMoscow 115446Russia

A bibliometric analysis of studies dedicated to autoimmune gastritis(AIG)recently published demonstrated a noteworthy surge in publications over the last three *** can be explained by numerous publications from different regions of the world reporting the results of several studies that stimulated reassessment of our view of AIG as a precancerous ***-up studies and retrospective analyses showed that the risk of gastric cancer(GC)in AIG patients is much lower than expected if the patients ever being infected with Helicobacter pylori(***)were *** low prevalence of precancerous lesions,such as the incomplete type of intestinal metaplasia,may explain the low risk of GC in AIG patients because the spasmolytic polypeptide-expressing metaplasia commonly observed in AIG does not involve clonal reprogramming of the gastric gland and can be considered as an adaptive change rather than a true precancerous ***,changes in gastric secretion due to the progression of gastric atrophy during the course of AIG cause changes in the gastric microbiome,stimulating the growth of bacterial species such as streptococci,which may promote the development of precancerous lesions and ***,Streptococcus anginosus exhibited a robust proinflammatory response and induced the gastritis-atrophy-metaplasia-dysplasia sequence in mice,reproducing the wellestablished process for carcinogenesis associated with *** studies in ***-naïve patients evaluating gastric microbiome changes during the long-term course of AIG might provide an explanation for the enigmatic increase in GC incidence in the last decades in younger cohorts,which has been reported in economically developed countries.

关键词： Autoimmune gastritis Gastric cancer Type 1 neuroendocrine tumors Helicobacter pylori Streptococcus anginosus Intestinal metaplasia

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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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Fermented soybean meal improved laying performance and egg quality of laying hens by modulating cecal microbiota,nutrient digestibility,intestinal health,antioxidant and immunological functions

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Animal Nutrition 2024年第3期18卷 309-321页

作者： Uchechukwu Edna Obianwuna Lingling Huang Haijun Zhang Jing Wang Guanghai Qi Kai Qiu Shugeng Wu National Engineering research center of Biological Feed Institute of Feed ResearchChinese Academy of Agricultural SciencesBeijing 100081China Wilmar(Shanghai)biotechnology research&Development center Co. Ltd.Shanghai 200137China

Antinutritional factors in feedstuffs may limit their utilization in livestock production,but fermentation process can be used to improve feed quality;however,studies on fermented soybeans for laying hens remain *** investigated the effect of fermented soybean meal(FSBM)at various inclusion levels as a partial replacement for soybean meal(SBM)on egg production,egg quality,amino acid digestibility,gut morphology and microbiota,antioxidant capacity and immune response of young laying hens.A total of 360 Hy-line Brown laying hens aged 18 weeks were selected and divided into 5 groups of 6 replicates each and 12 birds per *** control group received a basal diet while the trial group received the basal diet with FSBM included at 2.5%,5.0%,7.5%and 10.0%,respectively,for 12 *** findings revealed that the nutritional value of FSBM was higher compared to that of SBM in terms of reduced content of trypsin inhibitors and increased contents of crude protein,amino acids and *** enhanced egg production(P<0.05),feed-to-egg ratio(P<0.05),and albumen quality(albumen height and Haugh unit)(P<0.05).Furthermore,FSBM improved apparent fecal amino acid digestibility(P<0.05),gut morphology(increased villus height,villus width,villus height-to-crypt depth ratio and decreased crypt depth)(P<0.05),antioxidant capacity(reduced malondialdehyde and increased catalase,total superoxide dismutase,glutathione peroxidase and total antioxidant capacity)(P<0.05)and immune function(increased concentrations of IgG,IgA,and IgM;increased levels of transforming growth factor beta and Toll-like receptor 2;and reduced levels of interleukin 1βand tumor necrosis factor alpha)(P<0.05).Further analysis showed that FSBM altered the composition of the gut microbiota favoring beneficial *** findings suggest that probiotic fermentation improved the nutritional value of *** inclusion of FSBM in the diets of laying hens at 2.5%or 5.0%improved amino acid digestibility,gut

关键词： Fermented soybean meal Laying hen Egg production Egg quality Immune response

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A sustainable process to 100%bio-based nylons integrated chemical and biological conversion of lignocellulose

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Green Energy & Environment 2024年第2期9卷 390-402页

作者： Ruijia Hu Ming Li Tao Shen Xin Wang Zhuohua Sun Xinning Bao Kequan Chen Kai Guo Lei Ji Hanjie Ying Pingkai Ouyang Chenjie Zhu College of biotechnology and Pharmaceutical Engineering Nanjing Tech UniversityNanjingChina National Engineering research center for biotechnology Nanjing Tech UniversityNanjingChina Beijing Key Laboratory of Lignocellulosic Chemistry Beijing Forestry UniversityBeijingChina

Considerable progress has been made in recent years to the development of sustainable polymers from bio-based *** this study,100%bio-based nylons were prepared via an integrated chemical and biological process from *** novel nylons were obtained by the melt polymerization of 3-propyladipic acid derived from lignin and 1,5-pentenediamine/1,4-butanediamine derived from carbohydrate *** to the concept is a three-step noble metal free catalytic chemical funnelling sequence(Raney Ni mediated reductive catalytic fractionation-reductive funnelling-oxidative funnelling),which allowed for obtaining a single component 3-propyladipic acid from lignin with high *** structural and thermodynamic properties of the obtained nylons have been systematically investigated,and thus obtained transparent bio-based nylons exhibited higher Mw(>32,000)and excellent thermal stability(Td5%>265℃).Considering their moderate Tg and good melt strength,these transparent bio-based nylons could serve as promising functional additives or temperature-responsive materials.

关键词： Lignocellulose Lignin Reductive catalytic fractionation Bio-based nylon

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Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

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Journal of Integrative Agriculture 2024年第6期23卷 1897-1909页

作者： Yuting Liu Hanjia Li Yuan Chen Tambel Leila.I.M Zhenyu Liu Shujuan Wu Siqi Sun Xiang Zhang Dehua Chen Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation center for Modern Production Technology of Grain Crops Yangzhou UniversityYangzhou 225009China Agricultural research Cooperation Biotechnology and Biosafety Research CenterKhartoum 13314Sudan

Bacillus thuringiensis(Bt)cotton production is challenged by two main problems,i.e.,the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s ***,increasing the Bt protein concentration at the boll stage,especially in bolls,has become the main goal for increasing insect resistance in *** this study,two protein degradation inhibitors(ethylene diamine tetra acetic acid(EDTA)and leupeptin)were sprayed on the bolls,subtending leaves,and whole cotton plants at the peak flowering stage of two Bt cultivars(medium maturation Sikang 1(SK1))and early maturation Zhongmian 425(ZM425)in 2019 and *** Bt protein content and protein degradation metabolism were *** results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8%and 25.0 to 38.6%in the treated bolls of SK1 and ZM425 respectively,while they were decreased in the subtending leaves of these treated *** the treated leaves,the Bt protein concentrations increased by 7.6 to 23.5%and 11.2 to 14.9%in SK1 and ZM425,*** combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending *** Bt protein concentrations in bolls were higher,increasing by 22.5 to 31.0%and 19.6 to 32.5%for SK1 and ZM425,*** organs treated with EDTA or/and leupeptin showed reduced free amino acid contents,protease and peptidase activities and significant enhancements in soluble protein *** results indicated that inhibiting protein degradation could improve the protein content,thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton ***,the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

关键词： Bt cotton Bt protein inhibition of protein degradation protein degradation metabolism

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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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Highly efficient expression of human extracellular superoxide dismutase(rhEcSOD)with ultraviolet-B-induced damage-resistance activity in transgenic silkworm cocoons

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Insect Science 2024年第4期31卷 1150-1164页

作者： Feng Wang Ri-Yuan Wang De-Bin Zhong Ping Zhao Qing-You Xia Integrative Science center of Germplasm Creation in Western China(CHONGQING)Science City Biological Science Research CenterSouthwest UniversityChongqingChina Century Legend biotechnology research Institute(Chongqing)Co. Ltd.ChongqingChina

Extracellular superoxide dismutase(EcSOD)protects tissues from oxidative stress,and thus is considered as a therapeutic agent for many diseases such as atherosclerosis,hypertension,and ***,cost-effective production of bioactive recombinant human EcSOD(rhEcSOD)remains a ***,we developed an efficient strategy for producing active rhEcSOD by transgenic *** was successfully synthesized as homodimers and homotetramers in the middle silk gland and spun into the cocoons with a concentration of 9.48±0.21 mg/*** of rhEcSOD from the cocoons could be conveniently achieved with a purity of 99.50%and a yield of 3.5±0.5 mg/***,N-glycosylation at the only site of N89 in rhEcSOD with 10 types were *** purified rhEcSOD gained the potent enzymatic activity of 4162±293 U/mg after Cu/Zn ions *** importantly,rhEcSOD was capable of penetrating and accumulating in the nuclei of cells to maintain cell morphology and attenuate ultraviolet B-induced cell apoptosis by eliminating reactive oxygen species and inhibiting the C-Jun N-terminal kinase signaling *** results demonstrated that the transgenic silkworm could successfully produce rhEcSOD with enzymatic and biological activities for biomedical applications.

关键词： antioxidant human extracellular superoxide dismutase recombinant expression silkworm silk gland

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Targeted anti-tumor synergistic effects of Myc decoy oligodeoxynucleotides-loaded selenium nanostructure combined with chemoradiotherapy on LNCaP prostate cancer cells

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Oncology research 2024年第1期32卷 101-125页

作者： ROGHAYEH GHORBANI MAHMOUD GHARBAVI ALI SHARAFI ELHAM RISMANI HAMED REZAEEJAM YOUSEF MORTAZAVI BEHROOZ JOHARI Department of Medical biotechnology School of MedicineZanjan University of Medical SciencesZanjanIran Nanotechnology research center Ahvaz Jundishapur University of Medical SciencesAhvazIran Zanjan Pharmaceutical biotechnology research center Zanjan University of Medical SciencesZanjanIran Department of Pharmaceutical biotechnology School of PharmacyZanjan University of Medical SciencesZanjanIran Molecular Medicine Department Biotechnology Research CenterPasteur Institute of IranPasteur AvenueTehranIran Department of Radiology Technology School of Allied Medical SciencesZanjan University of Medical SciencesZanjanIran

In the present study,we investigated the synergistic effects of targeted methotrexate-selenium nanostructure containing Myc decoy oligodeoxynucleotides along with X-irradiation exposure as a combination therapy on LNCaP prostate cancer *** decoy ODNs were designed based on the promoter of Bcl-2 gene and analyzed by molecular docking and molecular dynamics *** were loaded on the synthesized Se@BSA@Chi-MTX *** physicochemical characteristics of nanostructures were determined by FTIR,DLS,UV-vis,TEM,EDX,in vitro release,and hemolysis ***,the cytotoxicity properties of them with and without X-irradiation were investigated by uptake,MTT,cell cycle,apoptosis,and scratch assays on the LNCaP cell *** results of DLS and TEM showed negative charge(−9 mV)and nanometer size(40 nm)for Se@BSA@Chi-DEC-MTX NPs,*** results of FTIR,UV-vis,and EDX showed the proper interaction of different parts and the correct synthesis of *** results of hemolysis showed the hemocompatibility of this nanoparticle in concentrations less than 6 mg/*** ODNs release from the nanostructures showed a pH-dependent manner,and the release rate was 15%higher in acidic *** targeted Se@BSA@Chi-labeled ODN-MTX NPs were efficiently taken up by LNCaP cells by targeting the prostate-specific membrane antigen(PSMA).The significant synergistic effects of nanostructure(containing MTX drug)treatment along with X-irradiation showed cell growth inhibition,apoptosis induction(~57%),cell cycle arrest(G2/M phase),and migration inhibition(up to 90%)compared to the *** results suggested that the Se@BSA@Chi-DEC-MTX NPs can potentially suppress the cell growth of LNCaP *** nanostructure system can be a promising approach for targeted drug delivery and chemoradiotherapy in prostate cancer treatment.

关键词： Chemoradiotherapy Combination therapy Decoy oligodeoxynucleotides Myc transcription factor Selenium nanoparticle Prostate cancer

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