青海湖盐单胞菌QHL1 ectABC基因簇克隆鉴定及二氨基丁酸转氨酶基因ectB的异源表达

从青海湖20份水样筛选得到一株优势中度嗜盐菌QHL1,经初步鉴定为盐单胞菌属。设计保守基因ectB的引物,以基因组DNA为模板,PCR扩增获得ectB基因(1269 bp)。利用染色体步移技术,克隆获得四氢嘧啶合成基因簇ectABC及其上下游调控序列。DNAStar软件分析表明ectA、ectB和ectC位于同一个操纵子上,大小分别为579 bp、1269 bp和390 bp,预测分别编码192、422和129个氨基酸的肽链。同源性分析表明:Halomonas sp.QHL1ectABC基因簇所编码的二氨基丁酸转乙酰基酶(EctA)、二氨基丁酸转氨酶(EctB)和四氢嘧啶合成酶(EctC)与Halomonas sp.Nj223 ectABC基因簇所编码的酶蛋白相似性分别达57%、96%和85%。利用分子克隆技术构建二氨基丁酸转氨酶基因ectB的重组表达载体pET-28-ectB,通过限制性内切酶酶切和测序分析,结果表明其目的基因的插入位置、大小和读码框均正确。诱导表达重组菌,SDS-PAGE分析,目的蛋白条带约46 kDa。     

盐单胞菌属BYS1四氢嘧啶合成基因ectABC克隆及其盐激表达

利用SEFA-PCR技术从中度嗜盐菌Halomonassp.BYS-1总DNA中克隆了四氢嘧啶合成基因ectABC及其上游序列(GenBank accession number DQ017757);OMIGA软件分析结果显示ectA、ectB、ectC位于同一个操纵子上,大小分别为573bp1、251bp和387bp,预测编码的DAT(L-二氨基丁酸转氨酶)、DAA(L-二氨基丁酸乙酰转移酶)和ES(四氢嘧啶合酶)大小分别为21.1kDa(191 amino acid)、45.7kDa(417 amino acid)和14.5kDa(129 amino acid);将包含ectABC基因及其上游1000bp序列的片段克隆到pUC19中并转化E.coliDH5α,转化子E.coli(pUC19ECT)能够在盐激条件下合成四氢嘧啶,但其耐盐能力没有得到显著改善。     

中度嗜盐菌四氢嘧啶合成基因的克隆与功能分析

中度嗜盐菌Bacillus alcalophilus DTY1分离自晋西北黄土高原盐碱土壤, 能够产生耐盐相关的相容性溶质四氢嘧啶。为了研究四氢嘧啶的功能, 克隆了DTY1菌株四氢嘧啶合成基因簇ectABC。ectA、ectB和ectC分别编码169、428和132个氨基酸的肽链, 分别与B. halodurans C-125中的二氨基丁酸乙酰基转移酶(EctA)、二氨基丁酸氨基转移酶(EctB)、四氢嘧啶合成酶(EctC)同源性达59%、81%和81%。将携带该基因簇的4.0 kb片段转入蜡质芽孢杆菌B. cereus Z后, 芽孢杆菌的耐盐度显著提高。HPLC检测发现, 在1.0% NaCl浓度下, 转化菌B. cereus Z-E菌株生成70.1 mg/g四氢嘧啶, 而在5.0%的NaCl浓度下四氢嘧啶的产量高达118.6 mg/g, 显著高于B. alcalophilus DTY1的四氢嘧啶产量。而且随着盐浓度的提高, 四氢嘧啶的合成量也随之提高。由此证明四氢嘧啶参与中度嗜盐菌重要的渗透调节, ectABC的表达受盐诱导。     

中度嗜盐菌四氢嘧啶合成基因的克隆与功能分析

中度嗜盐菌Bacillus alcalophilus DTY1分离自晋西北黄土高原盐碱土壤, 能够产生耐盐相关的相容性溶质四氢嘧啶。为了研究四氢嘧啶的功能, 克隆了DTY1菌株四氢嘧啶合成基因簇ectABC。ectA、ectB和ectC分别编码169、428和132个氨基酸的肽链, 分别与B. halodurans C-125中的二氨基丁酸乙酰基转移酶(EctA)、二氨基丁酸氨基转移酶(EctB)、四氢嘧啶合成酶(EctC)同源性达59%、81%和81%。将携带该基因簇的4.0 kb片段转入蜡质芽孢杆菌B. cereus Z后, 芽孢杆菌的耐盐度显著提高。HPLC检测发现, 在1.0% NaCl浓度下, 转化菌B. cereus Z-E菌株生成70.1 mg/g四氢嘧啶, 而在5.0%的NaCl浓度下四氢嘧啶的产量高达118.6 mg/g, 显著高于B. alcalophilus DTY1的四氢嘧啶产量。而且随着盐浓度的提高, 四氢嘧啶的合成量也随之提高。由此证明四氢嘧啶参与中度嗜盐菌重要的渗透调节, ectABC的表达受盐诱导。     

新喀里多尼亚弧菌CGJ02-2中四氢嘧啶合成基因簇ectABC的克隆及其功能鉴定

研究旨在克隆新的四氢嘧啶合成基因簇,并对其功能进行鉴定,为应用于四氢嘧啶的生产奠定基础。从新喀里多尼亚弧菌CGJ02-2中克隆获得四氢嘧啶合成基因簇ectABC,ectABC与表达载体pBAD连接后转化至大肠杆菌BW25113中,通过L-阿拉伯糖诱导表达。采用SDS-PAGE和液质联用鉴定重组表达蛋白,利用全细胞催化合成四氢嘧啶,通过高分辨质谱鉴定四氢嘧啶,并从天冬氨酸浓度、KCl浓度、温度和pH 4个方面优化催化条件。结果表明,来自新喀里多尼亚弧菌CGJ02-2 基因组的ectABC大小为2 235 bp。SDS-PAGE显示表达产物中有3个重组蛋白产生, 液质联用鉴定表明其分子量分别与ectA、ectB、ectC的理论分子量一致。高分辨质谱分析发现全细胞催化上清中有四氢嘧啶产生。优化后的最适全细胞催化条件为：天冬氨酸浓度100 mmol·L^-1,KCl浓度100 mmol·L^-1,温度30 ℃,pH 7.0,最优条件下产量为1.11 mg·mL^-1。研究从弧菌中克隆了四氢嘧啶合成基因簇ectABC,并在大肠杆菌BW25113中实现了异源表达和低盐环境下的四氢嘧啶合成,为大规模发酵生产四氢嘧啶奠定了基础。     

相容溶质四氢嘧啶与羟基四氢嘧啶的代谢调控研究进展

四氢嘧啶(Ectoine)及其衍生物羟基四氢嘧啶(5-hydroxyectoine,5-HE)是嗜盐微生物胞内合成的一类能够抵抗外界高盐胁迫的相容溶质,具有细胞、细胞膜、蛋白质和核酸的保护作用,可抵抗高盐、高温、冷冻和干燥等极端环境因素的刺激,从而倍受关注。本文对不同类型微生物Ectoine/5-HE(Ects)生物合成代谢、分解代谢以及吸收/转运系统涉及的调控机制进行综述,以期为Ects合成产量的提升与高效积聚策略的优化,提供一定的理论参考依据。     

转录组学分析盐单胞菌四氢嘧啶合成代谢相关的表达差异基因与qRT-PCR验证

【目的】探究盐适应条件下坎帕尼亚盐单胞菌(Halomonas campaniensis)的差异基因表达水平,挖掘四氢嘧啶(ectoine)合成代谢相关联的差异基因。【方法】设置无盐组NS (0 mol/L NaCl)、中盐组MS (1.5 mol/L NaCl)和高盐组HS (2.5 mol/L NaCl),培养H. campaniensis XH26菌株,利用IlluminaHiSeq测序进行转录组学分析,筛选四氢嘧啶合成代谢关联的差异基因,并进行qRT-PCR验证。【结果】菌株XH26胞内四氢嘧啶的积累量与盐度变化密切相关,1.5mol/LNaCl时积聚量最大为419.2mg/L。转录组测序(n=3/组)能定位到基因组测序序列的数量统计为87.24%–95.87%,共注释操纵子748个(涉及2 182个基因),转录起始-终止位点941个,预测新转录本456个,上调基因385个和下调基因326个(涉及245个KEGG通路)。组间NSvsMS分析表明,合成基因ectABC和lysC表达上调以促进四氢嘧啶生成,关联基因gltB、gltD、davT、hisD、alh-9、betA、acnB...     

大肠杆菌BL21(DE3)膜组分相关基因的敲除对重组蛋白胞外分泌的影响

摘要:【目的】探究Escherichia coli BL21(DE3)中膜组分相关的脂多糖合成基因waaF或msbB的敲除对重组蛋白胞外分泌的影响。【方法】运用Red重组技术将E.coli BL21 (DE3)染色体上的基因waaF或msbB敲除,构建敲除菌株E.coli BL21(ΔwaaF)、E.coli BL21(ΔmsbB)。将本实验室保存的带有β-呋喃果糖苷酶(β-fructofuranosidase,β-FFase)、青霉素G 酰化酶(penicillin G acylase,PGA)基因的重组质粒pET-ffase、pET-pga分别转入敲除菌株及出发菌株中,构建工程菌株E.coli BL21(ΔmsbB)/pET-ffase、E.coli BL21(ΔwaaF)/pET-ffase、E.coli BL21(DE3)/pET-ffase、E.coli BL21(ΔmsbB)/pET-pga、E.coli BL21(ΔwaaF)/pET-pga、E.coli BL21(DE3)/pET-pga。最后通过摇瓶发酵研究敲除菌株对β-FFase、PGA胞外分泌的影响。【结果】当诱导表达4 h,以出发菌株E.coli BL21(DE3)为宿主时,β-呋喃果糖苷酶β-FFase的胞外分泌量占总表达量的2.6%,以敲除菌株ΔmsbB为宿主时,胞外分泌量达到19.7%,而以敲除菌株ΔwaaF为宿主时,胞外分泌量达到50.9%。另外,当诱导表达24 h,以敲除菌株ΔwaaF为宿主时,青霉素G酰化酶PGA的胞外酶活是出发菌株中的4.1倍,达到1708 U/L。【结论】本研究成功构建了敲除菌株ΔmsbB和ΔwaaF,ΔmsbB能明显增强β-FFase的胞外分泌,而ΔwaaF对β-FFase和PGA的胞外分泌均有显著的强化作用。     

南极深海底泥色盐杆菌属NJS-2 ectABC基因克隆及二氨基丁酸转氨酶ectA基因的表达

从南极深海底泥中分离筛选得到一株中性嗜盐菌Chromhalobacter sp.NJS-2,以该菌株基因组为模板,利用PCR技术扩增出ectABC基因,基因全序列大小为2378bp。OMIGA软件分析该基因序列上含有三个阅读框,大小分别为576bp、1272bp和393bp,预测其分别编码二氨基丁酸乙酰转移酶（EctA）、二氨基丁乙酸转氨酶（EctB）和四氢嘧啶合酶(EctC)。将二氨基丁酸乙酰转移酶ectA基因的PCR扩增产物克隆至表达载体pET-his, 构建重组表达载体pET-his-ectA,并经酶切、PCR鉴定和测序验证,结果表明其目的基因的插入位置、大小和读码框均正确。SDS-PAGE分析,出现大小约21kDa的目的蛋白条带。     

柽柳翻译起始因子(eIF-5A)基因的克隆及原核表达

根据柽柳cDNA文库中获得的eIF-5A基因片段,用RACE技术克隆出其全长cDNA序列.cDNA长度为799 bp,编码159个氨基酸.将该cDNA序列克隆到原核表达载体pET28a中,获得重组质粒pET28a-eIF5A.不同浓度NaCl胁迫下大肠杆菌(Escherichia coli)BL21(pET28a-eIF5A)比E.coli BL21(pET28a)有明显的抗盐性,前者菌株存活率在1.0 mo1·L-1NaCl盐胁迫下是后者的9.3倍,据此认为E.coliBL21(pET28a-eIF5A)的耐盐性可能与eIF-5A基因的表达相关.该基因的GenBank登录号为AY587771(基因)、AAT01416(蛋白).     

Cloning and characterization of the gene cluster for biosynthesis of ectoine from <Emphasis Type="Italic">Nesterenkonia halobia</Emphasis> DSM 20541

The ectABC genes encoding the biosynthesis of ectoine were identified from Nesterenkonia halobia DSM 20541. The intergenic regions of the ectABC genes from N. halobia DSM 20541 were more loosely spaced than those that had been reported before. The amino acid sequence deduced from ectABC of the strain was highly homologous to the EctABC of Brevibacterium linens BL2 (EctA 50%, EctB 70%, and EctC 68% identities). The osmoprotection of ectABC was studied in the Escherichia coli KNabc and E. coli XL1-Blue. The results revealed that ectABC could shorten the lag phase and enhance the final OD600 of E. coli XL1-Blue in MM63 medium containing 0.68 M NaCl, and could initiate KNabc growth in 0.2 M NaCl. Ectoine was proven to be accumulated in E. coli KNabc/pGEM-Nect using HPLC-UV, and validated by LC-MSD-Trap-VL.     

Growth and nitrogen removal characteristics of Halomonas sp. B01 under high salinity

At present, the nitrogen (N) removal efficiency of the microbial treatment in the high-salinity nitrogenous wastewaters is relatively low. Study on the N removal behavior and properties of moderately halophilic bacteria Halomonas under high salinity is of great significance for the microbial treatment of high-salinity nitrogenous wastewater. The response mechanism of Halomonas sp. B01 to high osmotic pressure stress was investigated by measuring the compatible solute ectoine concentration and superoxide dismutase (SOD) activity. The salt tolerance during growth and N removal of the strain was evaluated by measuring the activities of growth-related and N removal–related enzymes and the mRNA expression abundance of ammonia monooxygenase-encoding gene (amoA). The process of simultaneous heterotrophic nitrification and aerobic denitrification (SND) under high salinity was described by measuring the concentration of inorganic N. Halomonas sp. B01 synthesized ectoine under NaCl stress, and the intracellular ectoine concentration increased with increased NaCl concentration in the growth medium. When the NaCl concentration of the medium reached 120 g L−1, the malondialdehyde concentration and SOD activity were significantly increased to 576.1 μg mg−1 and 1.7 U mg−1, respectively. The growth-related and N removal–related enzymes of the strain were active or most active in medium with 30–60 g L−1 NaCl. The amoA of the strain cultured in medium with 60 g L−1 NaCl had the highest mRNA expression abundance. In the N removal medium containing 60 g L−1 NaCl and 2121 mg L−1 NH4+-N, SND by Halomonas sp. B01 was performed over 96 h and the N removal rate reached 98.8%. In addition to the protective mechanism of synthetic compatible solutes, Halomonas sp. B01 had the repair mechanism of SOD for lipid peroxidation. The growth-related and N removal–related enzymes of the strain were most active at a certain salt concentration; amoA also had the highest mRNA expression abundance under high salinity. Halomonas sp. B01 could efficiently perform N removal by SND under high salinity.