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Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris

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Synthetic and Systems Biotechnology 2024年第1期9卷 26-32页

作者： Xueqin Lv Yuting Zhang Lingrui Wang Shixiu Cui Yanfeng Liu Jianghua Li guocheng du Long Liu Key Laboratory of Carbohydrate Chemistry and Biotechnology Ministry of EducationJiangnan UniversityWuxi 214122China Science Center for Future Foods Jiangnan UniversityWuxi 214122China Yixing Institute of Food Biotechnology Co. LtdYixing 214200China Jiaxing Institute of Future Food Jiaxing314015China Food Laboratory of Zhongyuan Jiangnan UniversityWuxi 214122China

Lactoferricin,a multifunctional peptide located in the N-terminal region of lactoferrin,has a broad-spectrum bacteriostatic *** is a promising candidate as a food additive and immune fortification agent and does not have the risks associated with drug residues and drug ***,we performed promoter and host cell screening to achieve the recombinant expression of lactoferricin in Pichia pastoris,showing an initial titer of 19.5 mg/L in *** X-33 using PAOX1 ***,we constructed a 0030-α hybrid signal peptide by fusing the 0030 signal peptide with the pro-sequence of α-factor secretory signal *** further increased the production of lactoferricin,with a titer of 28.8 mg/L in the fermentation supernatant in the shaking ***,we increased the expression of lactoferricin by fusing it with anionic antioxidant *** neutralization of positive charges yielded a titer of 55.3 mg/L in the shaking flask,and a highest titer of 193.9 mg/L in a 3-L *** antimicrobial activity analysis showed that recombinant-expressed lactoferricin exhibited potent antibacterial activity against Escherichia coli,Bacillus subtilis,and Staphylococcus *** study provides a reference for the construction of microbial cell factories capable of efficiently synthesizing antimicrobial peptides.

关键词： Antimicrobial peptide Lactoferricin Recombinant expression Pichia pastoris

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

A CRISPR/Cas9-based visual toolkit enabling multiplex integration at specific genomic loci in Aspergillus niger

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Synthetic and Systems Biotechnology 2024年第2期9卷 209-216页

作者： Yangyang Li Cen Li Yishan Fu Quan Zhang Jianing Ma Jingwen Zhou Jianghua Li guocheng du Song Liu National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing Jiangnan UniversityWuxi214122China Science Center for Future Foods Jiangnan UniversityWuxi214122China School of Biotechnology Jiangnan University1800 Lihu RoadWuxi214122China Jiangsu Provisional Research Center for Bioactive Product Processing Technology Jiangnan University1800 Lihu RoadWuxiJiangsu214122China Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education) College of Life Sciences/Institute of Agro-bioengineeringGuizhou UniversityGuiyang550025China Dalian Research Institute of Petroleum and Petrochemicals SINOPECDalian116000China School of Chemical Engineering Dalian University of TechnologyDalian116000China

Aspergillus niger is a highly versatile fungal strain utilized in industrial *** expression levels of recombinant genes in *** can be enhanced by increasing the copy ***,given the prolonged gene editing cycle of ***,a“one-step”strategy facilitating the simultaneous integration of recombinant genes into multiple genomic loci would provide a definitive *** our previous study,a visual multigene editing system(VMS)was designed to knock out five genes,employing a tRNA-sgRNA array that includes the pigment gene albA and the target *** upon this system,hybrid donor DNAs(dDNAs)were introduced to establish a clustered regularly interspaced short palindromic repeats(CRISPR)-based multiplex integration ***,a CRISPR-Cas9 homology-directed repair(CRISPR-HDR)system was constructed in *** by co-transforming the CRISPR-Cas9 plasmid(with a highly efficient sgRNA)and the dDNA,resulting in precise integration of recombinant xylanase gene xynA into the target loci(theβ-glucosidase gene bgl,the amylase gene amyA,and the acid amylase gene ammA).Subsequently,the length of homology arms in the dDNA was optimized to achieve 100%editing efficiency at each of the three gene *** achieve efficient multiplex integration in ***,the CRISPR plasmid pLM2 carrying a sgRNA-tRNA array was employed for concurrent double-strand breaks at multiple loci(bgl,amyA,ammA,and albA).Hybrid dDNAs were then employed for repair,including dDNA1-3(containing xynA expression cassettes without selection markers)and dDNAalbA(for albA knockout).Among the obtained white colonies(RLM2′),23.5%exhibited concurrent replacement of the bgl,amyA,and ammA genes with xynA(three copies).Notably,the xynA activity obtained by simultaneous insertion into three loci was 48.6%higher compared to that obtained by insertion into only the bgl ***,this multiple integration toolkit successfully enhanced the expression of endogenous pectinase pelA and

关键词： Multiplex integration Aspergillus niger CRISPR-Cas9 homologous direct repair White colony

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

Characterization of the effects of hyaluronan oligosaccharides on human gut microbiota using a simulated batch fermentation system

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Food Science and Human Wellness 2024年

作者： Xinwei Gao Hongkang Zhu Litao Hu Jieyu Sun Wenjie Zuo Ping Liu Yang Wang guocheng du Zhen Kang The Science Center for Future Foods， Jiangnan University The Key Laboratory of Carbohydrate Chemistry and Biotechnology， Ministry of Education， Jiangnan University The Key Laboratory of Industrial Biotechnology， Ministry of Education， School of Biotechnology， Jiangnan University School of Food Science and Technology， Jiangnan University

The prebiotic effects of hyaluronan (HA) are widely recognized， contributing to improved gut health and immune modulation. Despite its extensive use as dietary supplement， the specific interactions between HA oligosaccharides (o-HAs) and the gut microbiome remain largely unexplored. To investigate its role and metabolic fate in gut homeostasis， 200 mg/day of o-HAs (average molecule weight 1k Da) were added to an automated computer-controlled SIMulator of the Gastrointestinal tract (SIMGI). The results revealed a significant reshaping of the intestinal flora composition by o-HAs， notably reducing the Firmicutes/Bacteroides ratio. Fermentation of o-HAs by gut microbiota significantly increased the abundance of Bifidobacteria， Prevotellaceae＿Prevotella， Dialister， Eubacterium， and Sutterella， but decreased that of Catenibacterium， Oscillospira， Klebsiella， and Citrobacter (P < 0.05). This corresponded with significant enhancements in the content of short-chain fatty acids (SCFAs) such as acetic acid， propionic acid and n-butyric acid， highlighting the significant impact of o-HAs at the genus level. Furthermore， analysis of microbial function predicted the downregulation of pathological events in nine human diseases， particularly infectious ones (parasitic and bacterial). Potential inhibitions were observed in metabolic pathways associated with pentose and glucuronate interconversions as well as cationic antimicrobial peptide resistance. These findings underscore the in vitro prebiotic effects of o-HAs and their potential relevance in managing diverticular diseases or preventing metabolic disorders through the regulation of gut microbiota.

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症状性孤立性肠系膜上动脉夹层保守治疗与腔内治疗效果的Meta分析

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血管与腔内血管外科杂志 2024年第6期10卷 696-700页

作者：陈寅逵杜果城川北医学院第二临床医学院南充市中心医院血管外科四川南充637000

目的探讨症状性孤立性肠系膜上动脉夹层(ISMAD)患者保守治疗与腔内治疗的效果。方法检索Web of Science、PubMed、中国知网、万方数据库中ISMAD患者保守治疗与腔内治疗效果,检索时间为建库至2022年10月1日,采用RevMan 5.3统计软件对文... 详细信息

目的探讨症状性孤立性肠系膜上动脉夹层(ISMAD)患者保守治疗与腔内治疗的效果。方法检索Web of Science、PubMed、中国知网、万方数据库中ISMAD患者保守治疗与腔内治疗效果,检索时间为建库至2022年10月1日,采用RevMan 5.3统计软件对文献进行Meta分析。结果共纳入文献11篇,664例ISMAD。Meta分析结果显示,腔内治疗组患者治疗有效率、远期夹层重塑率均明显高于保守治疗组患者,差异均有统计学意义(P﹤0.01)。两组患者腹痛复发率比较,差异无统计学意义(P﹥0.05)。结论腔内治疗ISMAD患者有效率、夹层重塑率均高于保守治疗,两者复发性腹痛发生率无差异,因此腔内治疗ISMAD值得在临床上进一步推广应用。

关键词：孤立性肠系膜上动脉夹层保守治疗腔内治疗

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细菌酪氨酸酶高效异源表达及其在生物染发和丝素多肽多巴修饰中的应用

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生物工程学报 2024年第9期40卷 3083-3102页

作者：郑依琳胥睿睿王阳方承格堵国成康振江南大学未来食品科学中心江苏无锡214122 江南大学生物工程学院糖化学与生物技术教育部重点实验室江苏无锡214122 江南大学生物工程学院工业生物技术教育部重点实验室江苏无锡214122 江南大学化学与材料工程学院江苏无锡214122

酪氨酸酶是一种含铜的多酚氧化酶,广泛应用于食品、日化、医药等领域。目前商品化酪氨酸酶主要依赖于真菌提取,存在价格高、纯度低、比酶活低和稳定性差等问题。本研究旨在获得高效表达并具有工业化应用前景的细菌酪氨酸酶。通过在大肠... 详细信息

酪氨酸酶是一种含铜的多酚氧化酶,广泛应用于食品、日化、医药等领域。目前商品化酪氨酸酶主要依赖于真菌提取,存在价格高、纯度低、比酶活低和稳定性差等问题。本研究旨在获得高效表达并具有工业化应用前景的细菌酪氨酸酶。通过在大肠杆菌(Escherichia coli)BL21(DE3)中异源表达5种不同来源的细菌酪氨酸酶,筛选获得了巨大芽孢杆菌(Bacillus megaterium)和多刺疣微菌(Verrucomicrobium spinosum)来源的酪氨酸酶TyrBm和TyrVs,酶活分别为(16.1±0.2)U/mL和(48.6±0.9)U/mL。纯化后通过比较TyrBm与TyrVs的酶学性质,证明了TyrVs具有更高的热稳定性和底物专一性。在表征TyrVs高催化性能的基础上,建立了基于TyrVs催化的酶法生物染发体系,实现了原位催化染发,水洗色牢度实验测得模拟14 d清洁后的色差值∆E低于7.38±0.64。为便于催化产物与酶快速分离,成功构建了依赖于自组装标签CipA的固定化酶TyrVs-CipA催化体系并应用于水解丝素多肽(hydrolysed silk fibroin,HSF)的多巴修饰(DOPA modification),酶连续催化达7次以上,单次多巴修饰度超过70.00%。进一步研究表明,多巴修饰使HSF对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-trinitrophenylhydrazine,DPPH)自由基与O2-自由基清除活性分别提高了507.80%与78.23%。本研究为基于酪氨酸酶的绿色生物染发剂及组织工程生物材料开发提供了技术基础。

关键词：细菌酪氨酸酶固定化酶生物染发剂水解丝素多巴修饰

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覆膜支架与裸支架在主髂动脉闭塞症中的疗效分析

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血管与腔内血管外科杂志 2024年第1期10卷 76-79,102页

作者：魏美娇杜果城川北医学院第二临床医学院四川南充637000 川北医学院第二临床医学院•南充市中心医院甲状腺乳腺血管外科四川南充637000

主髂动脉闭塞症(AIOD)是以盆腔缺血和/或下肢缺血为主要表现的疾病,其发病率、就诊率呈逐年上升趋势,已成为血管外科的常见疾病。随着介入材料不断发展和手术医师介入技术的不断提高,腔内治疗凭借其创伤小、操作方便、效果明确、周期短... 详细信息

主髂动脉闭塞症(AIOD)是以盆腔缺血和/或下肢缺血为主要表现的疾病,其发病率、就诊率呈逐年上升趋势,已成为血管外科的常见疾病。随着介入材料不断发展和手术医师介入技术的不断提高,腔内治疗凭借其创伤小、操作方便、效果明确、周期短、病死率低、术后并发症少等优点,逐步成为临床医师治疗AIOD的首选治疗方式。目前关于裸支架和覆膜支架在AIOD患者中的疗效研究较少,本综述旨在探讨覆膜支架与裸支架在AIOD中的疗效,以期为AIOD患者的腔内治疗提供参考。

关键词：主髂动脉闭塞症腔内治疗覆膜支架裸支架

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Influence of aeration intensity on the performance of A/0-type sequencing batch MBR system treating azo dye wastewater

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Frontiers of Environmental Science & Engineering 2011年第4期5卷 615-622页

作者： Xinhua WANG Jingmei LI Xiufen LI guocheng du Laboratory of Environmental Biotechnology School of Environmental and Civil EngineeringJiangnan UniversityWuxi 214122China Key Laboratory of Industrial Biotechnology Ministry of EducationJiangnan UniversityWuxi 214122China

Among the numerous parameters affecting the membrane bioreactor(MBR)performance,the aeration intensity is one of the most important *** the present investigation,an anoxic/aerobic-type(A/O-type)sequencing batch MBR system,added anoxic process as a pretreatment to improve the biodegradability of azo dye wastewater,was investigated under different aeration intensities and the impact of the aeration intensity on effluent quantity,sludge properties,extracellular polymeric substances(EPS)amount generated as well as the change of permeation flux were *** lower nor higher aeration intensities could improve A/O-type sequencing batch MBR *** results showed 0.15 m^(3)·h^(-1)aeration intensity was promising for treatment of azo dye wastewater under the conditions *** this aeration intensity,chemical oxygen demand(COD),ammonium nitrogen and color removal as well as membrane flux amounted to 97.8%,96.5%,98.7%and 6.21 L·m^(-2)·h^(-1),*** effluent quality,with 25.0 mg·L^(-1)COD,0.84 mg·L^(-1)ammonium nitrogen and 8 chroma,could directly meet the reuse standard in *** the meantime,the sludge relative hydrophobicity,the bound EPS,soluble EPS and EPS amounts contained in the membrane fouling layer were 70.3%,52.0 mg·g^(-1)VSS,38.8 mg·g^(-1)VSS and 90.8 mg·g^(-1)VSS,respectively,which showed close relationships to both pollutant removals and membrane flux.

关键词： batch membrane bioreactor azo dye aeration intensity extracellular polymeric substances sludge properties