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。     

极端耐盐放线菌白色普氏菌YIM 90005T四氢嘧啶及5-羟基四氢嘧啶合成相关基因的克隆

摘要：【目的】 为了研究耐盐放线菌对高盐环境的适应机理。【方法】 用HPLC定量检测了极端耐盐、丝状产孢放线菌——白色普氏菌（Prauserella alba） YIM 90005T在不同盐浓度下胞内相容性溶质的种类和含量。【结果】 结果发现,四氢嘧啶和5-羟基四氢嘧啶是其主要的相容性溶质。在培养基NaCl浓度为10%时,四氢嘧啶在胞内累积浓度最大,为18.77 μg/mg干菌体重。之后随NaCl浓度的升高,胞内的四氢嘧啶含量逐渐减少,而5-羟基四氢嘧啶的含量逐渐增加,在该菌耐受的最高NaCl浓度下（24% w/v）,胞内5-羟基四氢嘧啶含量达到最大值,为22.98 μg/mg干菌体重。设计兼并引物,利用染色体步移,克隆得到四氢嘧啶及5-羟基四氢嘧啶合成相关基因ectABCD。序列分析表明,ectABCD位于一个操纵子中。进一步对不同NaCl浓度培养条件下ectB,D的表达量进行定量分析,结果表明该基因簇表达量随着培养基中NaCl浓度的增加而增大。【结论】 研究结果证实5-羟基四氢嘧啶是P. alba YIM 90005T在极高盐浓度条件下起渗透调节及保护的相容性溶质。     

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。     

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。     

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。     

嗜盐细菌中四氢嘧啶和羟基四氢嘧啶的生物合成及其生物学功能

在嗜盐细菌盐适应中,四氢嘧啶(1,4,5,6-四氢-2-甲基-4-嘧啶羧酸)和羟基四氢嘧啶(1,4,5,6-四氢-2-甲基-5-羟基-4-嘧啶羧酸)发挥着十分重要的作用。四氢嘧啶的生物合成以L-天冬氨酸-β-半醛(ASA)为底物,依次由2,4-二氨基丁酸转氨酶(EctB),2,4-二氨基丁酸乙酰基转移酶(EctA)和四氢嘧啶合成酶(EctC)催化反应,分别生成L-2,4-二氨基丁酸(DABA),N-乙酰-L-2,4-二氨基丁酸(ADABA)和四氢嘧啶。羟基四氢嘧啶则由四氢嘧啶羟化酶(EctD)将四氢嘧啶羟基化产生。通常,四氢嘧啶合成基因以ectABC基因簇的形式存在,而羟基四氢嘧啶合成基因ectD单独存在。四氢嘧啶生物合成基因在微生物菌株和转基因经济作物中的表达可以提高其耐盐碱旱等抗逆能力,羟基四氢嘧啶合成基因的表达可以增强宿主耐热和耐干燥的能力。四氢嘧啶类相容性溶质的生物学功能及其潜在应用已成为前沿性研究热点。     

渗透压冲击下中度嗜盐菌Halomonas sp. Y四氢嘧啶类相容性溶质的合成与释放

【背景】四氢嘧啶类物质在高温、冷冻和干燥等逆境条件下,对酶、蛋白质、核酸及整个细胞具有良好的保护作用,已经应用于酶制剂、生物医药及护肤品等相关领域。目前此类物质只能依赖中度嗜盐菌采用细菌泌乳工艺进行商业化生产,因此四氢嘧啶类高产菌株及其发酵技术的研究日益受到国内外研究者关注。【目的】分离获得高产合成四氢嘧啶类相容性溶质的中度嗜盐细菌,研究渗透压冲击对其胞内四氢嘧啶合成与释放的影响,探索细菌泌乳法制备四氢嘧啶的可行性。【方法】采用涂布平板法分离中度嗜盐菌,对分离菌株进行形态、生理生化和16S rRNA基因序列分析,鉴定其种属;采用高效液相色谱法(HPLC)和质谱法(MS)分析四氢嘧啶类物质,细菌泌乳法制备四氢嘧啶类物质。【结果】从盐池土样中分离到一株以四氢嘧啶类物质为主要相容性溶质的中度嗜盐菌Y,鉴定为盐单胞菌(Halomonas sp.)Y。盐单胞菌Y能在NaCl质量浓度为10-250 g/L的培养基中生长,最适生长的NaCl浓度为100 g/L;HPLC-MS测试结果证明盐单胞菌Y可同时合成四氢嘧啶和羟基四氢嘧啶2种相容性溶质,在最适生长的盐浓度下其合成量分别达175.5 mg/g和47.9 mg/g;在NaCl质量浓度为0-30 g/L的低渗溶液中胞内四氢嘧啶类物质经5 min即可达到最大释放率,而细菌泌乳工艺中最适合诱导四氢嘧啶释放的低渗溶液为质量浓度为10 g/L的NaCl溶液;采用细菌泌乳工艺制备四氢嘧啶,经连续11轮的高渗/低渗冲击,四氢嘧啶总合成量为6.0 g/L,总释放量为5.7 g/L,平均释放率为64.5%,底物转化率为128.9 mg/g。【结论】盐单胞菌Y是一株较高产合成四氢嘧啶类的中度嗜盐菌,能够耐受反复的渗透压冲击,采用细菌泌乳工艺显著提高了四氢嘧啶的制备效率。     

古盐井中嗜盐菌的分离及四氢嘧啶的制备

[目的]分离鉴定古盐井中的嗜盐菌,确定相容性溶质,增加相容性溶质的合成。[方法]从盐井卤水中分离嗜盐菌,对菌株进行形态、生理生化和16S rRNA分析,用高效液相色谱法(HPLC)和质谱法(MS)确定相容性溶质,以谷氨酸钠为唯一碳氮源来制备四氢嘧啶。[结果]菌株S2为色盐杆菌属;相容性溶质为四氢嘧啶; 1. 5 mol/L Na Cl时四氢嘧啶达到最适合成浓度,为0. 168 3 mg/mL,单位质量合成量为238. 201 mg/g;以谷氨酸钠为唯一碳氮源时,相同体积发酵液中四氢嘧啶的总合成量与单位质量合成量均增加,四氢嘧啶合成量为0. 184 8 mg/mL,单位质量合成量为390. 389 mg/g。[结论]成功分离出了产四氢嘧啶的嗜盐菌,并且以谷氨酸钠为唯一碳氮源时四氢嘧啶合成量大大增加。     

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。     

青海湖盐单胞菌Ectoine合成基因簇ectABC的重组共表达

盐单胞菌属(Halomonas)通过胞内积聚有机相容溶质(Compatible solutes)来抵抗胞外的高盐渗透压。为了探究相容溶质Ectoine合成代谢相关基因的结构特征和异源共表达的可能性, 以青海湖盐单胞菌Halomonas sp. QHL1为材料, 通过高效液相色谱(HPLC)分析不同盐梯度下QHL1胞内Ectoine的积聚量, 并借助于染色体步移技术(Genome walking)捕获QHL1菌株的Ectoine生物合成基因簇ectABC, 利用分子克隆技术分析ectABC基因簇的异源重组表达(E.coli BL21)。研究结果表明: 胞内Ectoine的积聚量随着培养基中Na+浓度的增加而增加, 最大积聚量为167.1 mg/g细胞干重(1.0 mol/L Na+), 但菌体生长却受到高浓度Na+的强烈抑制作用。QHL1的ectABC操纵子全长序列为3580 bp, 结构基因ectA(579 bp)、ectB(1269 bp)与ectC (390 bp)串联排列。基于生物信息学预测分析, 两个启动子(70与38因子控制)和若干未知功能的保守模序(Motifs)存在于QHL1的ect操纵子上游。构建重组表达载体pET-28-ectABC, 并在E.coli BL21中异源表达ectABC基因簇(2438 bp)。SDS-PAGE结果显示EctA、EctB和EctC分别为27.2、52.5 和 20.8 kD, 与预测结果一致, 表明ectA、ectB和ectC基因能在E. coli BL21中实现异源共表达, 为构建Ectoine合成代谢基因整合的系统代谢工程, 并实现低盐发酵控制和过量化生产提供了重要的理论基础。       

Accumulation and role of compatible solutes in fast-growing Salinivibrio costicola subsp. yaniae

The moderately halophilic bacterium Salinivibrio costicola subsp. yaniae showed an extremely fast growth rate. Optimal growth was observed in artificial seawater containing 1.4 mol/L NaCl and in MM63 media containing 0.6 mol/L NaCl. We analyzed a variety of compatible solutes that had accumulated in this strain grown in the media. The supplementation effect of the compatible solutes glycine betaine, glutamate, and ectoine to the growth of S. costicola subsp. yaniae was examined. Glycine betaine and glutamate had no supplementation effect on the fast growth rate. Growth of salt-sensitive mutants MU1 and MU2, both of which were defective in the ability to synthesize ectoine, was not observed in MM63 medium in the presence of more than 1.0 mol/L NaCl. From these data, we conclude that ectoine was the predominant compatible solute synthesized in this bacterium that effected an extremely fast growth rate.     

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.     

Ectoines as compatible solutes and carbon and energy sources for the halophilic bacterium Chromohalobacter salexigens

AIMS: To investigate the catabolism of ectoine and hydroxyectoine, which are the major compatible solutes synthesized by Chromohalobacter salexigens. METHODS AND RESULTS: Growth curves performed in M63 minimal medium with low (0.75 mol l(-1) NaCl), optimal (1.5 mol l(-1) NaCl) or high (2.5 mol l(-1) NaCl) salinity revealed that betaine and ectoines were used as substrate for growth at optimal and high salt. Ectoine transport was maximal at optimal salinity, and showed 3- and 1.5-fold lower values at low and high salinity respectively. The salt-sensitive ectA mutant CHR62 showed an ectoine transport rate 6.8-fold higher than that of the wild type. Incubation of C. salexigens in a mixture of glucose and ectoine resulted in a biphasic growth pattern. However, CO(2) production due to ectoine catabolism was lower, but not completely abolished, in the presence of glucose. When used as the sole carbon source, glycine betaine effectively inhibited ectoine and hydroxyectoine synthesis at any salinity. CONCLUSIONS: The catabolic pathways for ectoine and hydroxyectoine in C. salexigens operate at optimal and high (although less efficiently) salinity. Endogenous ectoine(s) may repress its own transport. Ectoine utilization was only partially repressed by glucose. Betaine, when used as carbon source, suppresses synthesis of ectoines even under high osmolarity conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is a previous step to the subsequent isolation and manipulation of the catabolic genes, so as to generate strains with enhanced production of ectoine and hydroxyectoine.     

Efficient cyclic system to yield ectoine using Brevibacterium sp. JCM 6894 subjected to osmotic downshock

Brevibacterium sp. JCM 6894 cells grown in the presence of 1.5-2.5 M NaCl for 24 h at 30 degrees C were subjected to the osmotic downshock. Downshocked cells after ectoine release were grown for further 24 h in the fresh medium with same salinity as before shock. When this cyclic system was applied to the strain JCM 6894, the amount of ectoine in the cells increased with an increase of incubation time, which indicates that the cells manipulated by the present conditions were enough active to survive and synthesize ectoine after several times of osmotic downshock. In the presence of 2 M NaCl, the highest yield of ectoine released was achieved in this cyclic system, more than 2.4 g/L during 7 days of incubation. (1)H and (13)C-NMR analyses of solutes released from the cells by the osmotic downshock showed the presence of only ectoine with high purity. Release of ectoine from the cells was carried out within 5 min and its rates were increased by the dilution in the downshock treatment. For the convenience of operations, non-sterilized medium containing 2 M NaCl was examined for the cell growth in the present system, in which almost same level of ectoine yield, release rates, and cell viability were observed as those of sterilized medium.     

Ectoine accumulation in Brevibacterium epidermis

As a halotolerant bacterial species, Brevibacterium epidermis DSM 20659 can grow at relatively high salinity, tolerating up to 2 M NaCl. It synthesizes ectoine and the intracellular content increases with the medium salinity, with a maximum of 0.14 g ectoine/g CDW at 1 M NaCl. Sugar-stressed cells do not synthesize ectoine. Ectoine synthesis is also affected by the presence of external osmolytes. Added betaine is taken up and completely replaced ectoine, while L-proline is only temporarily accumulated after which ectoine is synthesized. The strain can metabolize ectoine; L-glutamate is a better carbon source for ectoine synthesis than L-aspartate.