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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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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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Single carbon metabolism–A new paradigm for microbial bioprocesses?

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Synthetic and Systems biotechnology 2024年第2期9卷 322-329页

作者： Michael Baumschabl Özge Ata Diethard Mattanovich Austrian Centre of Industrial biotechnology(ACIB) Vienna1190Austria university of Natural Resources and Life Sciences Department of BiotechnologyInstitute of Microbiology and Microbial BiotechnologyViennaAustria

Increasing atmospheric carbon dioxide levels,a reduction of arable land area and the dependence of first and second generation biotechnology feedstocks on agricultural products,call for alternative,sustainable feedstock sources for industrial *** direct use of CO_(2)or conversion of CO_(2)into other single carbon(C1)sources have great potential as they might help to reduce carbon emissions and do not compete with agricultural land *** we discuss the microbial use of C1 carbon sources,their potential applications in biotechnology,and challenges towards sustainable C1-based industrial biotechnology *** focus on methanol,formic acid,methane,syngas,and CO_(2)as feedstocks for bioprocesses,their assimilation pathways,current and emerging applications,and limitations of their *** mini-review is intended as a first introduction for researchers who are new to the field of C1 biotechnology.

关键词： Sustainability BioeconomyCarbon dioxide Methanol Formate Methane

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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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Regulatory RNAs in Bacillus subtilis:A review on regulatory mechanism and applications in synthetic biology

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Synthetic and Systems biotechnology 2024年第2期9卷 223-233页

作者： Anqi Peng Guobin Yin Wenjie Zuo Luyao Zhang Guocheng Du Jian Chen Yang Wang Zhen Kang The Science center for Future Foods Jiangnan UniversityWuxi214122China The Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China The Key Laboratory of Industrial biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China

Bacteria exhibit a rich repertoire of RNA molecules that intricately regulate gene expression at multiple hierarchical levels,including small RNAs(sRNAs),riboswitches,and antisense ***,the majority of these regulatory RNAs lack or have limited protein-coding capacity but play pivotal roles in orchestrating gene expression by modulating transcription,post-transcription or translation *** and redesigning these regulatory RNA elements have emerged as pivotal strategies in the domains of metabolic engineering and synthetic *** previous investigations predominantly focused on delineating the roles of regulatory RNA in Gram-negative bacterial models such as Escherichia coli and Salmonella enterica,this review aims to summarize the mechanisms and functionalities of endogenous regulatory RNAs inherent to typical Gram-positive bacteria,notably Bacillus ***,we explore the engineering and practical applications of these regulatory RNA elements in the arena of synthetic biology,employing *** as a foundational chassis.

关键词： Regulatory RNA devices Small RNAs Riboswitches Synthetic biology Bacillus subtilis

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Minimizing endogenous cryptic plasmids to construct antibiotic-free expression systems for Escherichia coli Nissle 1917

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Synthetic and Systems biotechnology 2024年第1期9卷 165-175页

作者： Siyan Zhou Linlin Zhao Wenjie Zuo Yilin Zheng Ping Zhang Yanan Sun Yang Wang Guocheng Du Zhen Kang The Science center for Future Foods Jiangnan UniversityWuxi214122China The Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China The Key Laboratory of Industrial biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China

The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological ***,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its *** this study,we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and *** non-essential elements were removed to create more stable derivatives pMUT1NR△and pMUT2HBC△.Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinaseⅢand the biosynthesis of *** to traditional antibiotic-dependent expression systems,our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.

关键词： Cryptic plasmids Antibiotic-free Gene expression Plasmid stability E.coli Nissle 1917

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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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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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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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