启动子替代构建克雷伯氏菌普鲁兰酶高产菌株

应用启动子替代技术将普鲁兰酶产生菌克雷伯氏菌(Klebsiella sp.)HN9的普鲁兰酶基因的启动子替换为枯草芽孢杆菌(Bacillus subtilis)的P43启动子,构建高产酶菌株。通过融合PCR,将由3个DNA片段融合得到启动子替代同源重组片段,该3个DNA片段分别是带有克雷伯氏菌普鲁兰酶基因启动子上游部分同源序列的四环素抗性基因(Tet)的上游片段Tet1、带有枯草芽孢杆菌P43启动子部分上游同源序列的Tet下游片段Tet2和带有克雷伯氏菌普鲁兰酶基因上游同源序列的P43启动子部分序列的Pro。经电转化将同源重组片段导入克雷伯氏菌中,转化子的普鲁兰酶基因表达水平比出发菌株提高10.23~11.45倍,摇瓶发酵酶活力提高8.97~10.52倍。结果表明,应用高效组成型启动子P43替代原始菌株的普鲁兰酶基因的启动子能够显著提高普鲁兰酶基因的表达量和发酵酶活力。     

Pyrococcus yayanosii L-天冬酰胺酶在枯草芽孢杆菌中分泌途径的鉴定及其分泌能力的提高

食品安全（GRAS）菌株枯草芽孢杆菌由于其具有良好的分泌表达能力及易于基因工程操作等特性被广泛用作外源蛋白质的表达菌株。枯草芽孢杆菌中蛋白质的分泌大多依赖于信号肽介导的Sec分泌途径（General secretion pathway）和Tat分泌途径（Twin-arginine translocation pathway）。在本研究中,通过信号肽预测、蛋白质N端测序等手段发现来自于Pyrococcus yayanosii L-天冬酰胺酶在枯草芽孢杆菌中的分泌并不依赖信号肽,该酶通过非经典蛋白质分泌途径（non-classical protein secretion pathway）进行分泌。通过信号肽筛选,发现最适合该酶在枯草芽孢杆菌中表达的信号肽为Tat分泌途径信号肽SP_phoD,并通过共表达分子伴侣PrsA的方式将该L-天冬酰胺酶的分泌量提高了72.11%。     

海藻糖合酶在枯草芽孢杆菌中的高效表达

以枯草芽孢杆菌(Bacillus subtilis)为表达平台,海藻糖合酶为表达蛋白,研究了单启动子和多个启动子串联对外源蛋白表达的影响。分别选择6个启动子构建P_(srfA)、P_(43)单启动子和P_(abrB-spoVG-LytR-mmgA)四个时期特异性启动子串联表达系统进行验证。根据实验结果分析,组成型启动子P_(43)转录强度最高,摇瓶中酶活达到3 381 U/g,串联启动子P_(abrB-spoVG-LytR-mmgA)强度次之。针对验证酶活最高的重组菌pHT01-P_(43)-treS进行发酵优化,并在5 L发酵罐中进行放大实验,酶活达到7 215 U/g。实现了海藻糖合酶在枯草芽孢杆菌中自诱导高效表达,为获得高效率制备海藻糖合成酶的表达系统奠定了基础。     

α-淀粉酶的耐酸性改造及在枯草芽孢杆菌中的分泌表达

α-淀粉酶基因经改造后,克隆到穿梭质粒pBE2中。在α-淀粉酶基因的上游连接枯草芽孢杆菌sacB基因的启动子-信号肽序列(sacR),构建了含突变α-淀粉酶基因的分泌型诱导表达载体pBSAT,转化蛋白酶三缺陷枯草芽孢杆菌菌株DB403。含有pBSAT的菌株可将突变α-淀粉酶分泌到胞外,表明sacB基因的启动子-信号肽序列(sacR)能很好的将枯草芽孢杆菌中的重组α-淀粉酶引导到胞外,完成分泌表达。分泌的α-淀粉酶具有较高的耐酸性及生物学活性。     

解淀粉芽孢杆菌的强启动子的克隆与鉴定

利用启动子探针质粒,从解淀粉芽孢杆菌XH7基因组中分离得到1个强度较高的启动子P28,该启动子在大肠杆菌中表达的β-半乳糖苷酶酶活为2 350 Miller U/m L,在枯草芽孢杆菌中的酶活为3 340 Miller U/m L。启动子的序列分析表明该启动子由σK因子识别,由2个启动子串联而成,分别为编码羧基末端加工蛋白酶的基因(ctp A)和编码转醛酶的基因(tal)启动子,为芽孢杆菌启动子库提供了一个新的选择。     

重组普鲁兰酶在枯草芽孢杆菌中的高效表达

普鲁兰酶可特异性地水解支链淀粉得到直链淀粉,因而在淀粉加工过程中具有重要的应用。本研究从Bacillus naganoensis ATCC53909基因组中克隆了普鲁兰酶基因pul,并克隆到大肠杆菌-枯草芽孢杆菌穿梭载体p BE中,构建表达载体p BE-pul。在此基础上,将来源于枯草芽孢杆菌、地衣芽孢杆菌以及解淀粉芽孢杆菌中的17个高表达基因的启动子分别克隆到表达载体p BE-pul中,并转化至Bacillus subtilis ATCC6051?10,成功构建了十七株含有不同启动子介导普鲁兰酶分泌表达的重组菌株。对重组菌株的分泌表达比较发现,启动子P43和Pspov G介导的普鲁兰酶活性明显优于其他启动子,其中Pspov G介导的普鲁兰酶活性更高。同时,还使用了启动子Pspov G介导N端的108个氨基酸缺失的pul324突变体进行分泌表达。通过对17种启动子的比较和两个普鲁兰酶基因的比较,本研究构建的一株重组菌株的普鲁兰酶的表达更为高效,其活性高达389.85 U/mL,后者显著高于现有的相关报道。     

中温α-淀粉酶基因在枯草芽孢杆菌中的高效表达及其发酵条件优化

以枯草芽孢杆菌为表达系统,以中温α-淀粉酶为目标蛋白,研究了启动子和信号肽对外源蛋白表达分泌的影响。分别选取了4种启动子以及8种信号肽来进行筛选,结果表明,启动子PApr E的强度最高,信号肽SPnpr E的分泌效率最好。针对重组菌株1A751Q31进行系统的发酵优化,在72 h发酵液中α-淀粉酶酶活达到380U/m L。在7.5 L发酵罐中进行放大实验,发酵液中α-淀粉酶酶活最高达到853 U/m L。以上研究表明,α-淀粉酶适合在枯草芽孢杆菌中进行表达,同时表明枯草芽孢杆菌是良好的异源蛋白表达系统。     

高压热杀菌结合葡萄糖对枯草芽孢杆菌芽孢的杀灭效果

作者研究了葡萄糖结合高压热杀菌（HPTS）处理对枯草芽孢杆菌芽孢的杀灭效果及其内膜的影响,通过平板计数法、电导率测定、流式细胞术分析及傅里叶变换红外光谱分析等对枯草芽孢杆菌芽孢的灭菌效果、离子释放量、内膜通透性及膜结构稳定性进行了研究。结果表明：在600 MPa、75 ℃条件下,芽孢死亡量达到4.64个对数值,而在添加了质量分数60%的葡萄糖溶液后,芽孢的死亡量减少了约2个对数值。随着葡萄糖质量分数的升高,芽孢的离子释放量显著下降（P<0.05）;流式细胞术结果显示在600 MPa、75 ℃结合质量分数60%葡萄糖溶液处理下样品的阳性区域（M2）占比为92.88%,这表明芽孢内膜通透性显著增加（P<0.05）;通过分析样品在3 000~ 2 800 cm-1波段的傅里叶变换红外光谱,发现与单独HPTS处理相比,添加葡萄糖可以使芽孢内膜的—CH₂/—CH₃官能团吸收峰明显减弱,葡萄糖可有助于维持芽孢内膜结构的稳定性。由此可知,葡萄糖含量对HPTS灭活枯草芽孢杆菌芽孢的效果有重要影响。     

N-乙酰神经氨酸合成途径在枯草芽孢杆菌的构建

对食品安全认可的枯草芽孢杆菌进行菌株改造,利用生物发酵法制备N-乙酰神经氨酸。首先通过基因合成获取来自枯草芽孢杆菌溶源体的P_(holin)启动子并构建了p MK4-P_(holin)-GFP质粒,转入枯草芽孢杆菌,以GFP为报告基因,对P_(holin)及其它常见的强启动子进行了转录效率的比较,然后将优化后的P_(holin)用于构建N-乙酰神经氨酸表达质粒p MK4-P_(holin)-neu BC。研究结果显示:P_(holin)启动子是一种优异的枯草芽孢杆菌组成型强启动子,在利用LB进行发酵培养的实验中,P_(holin)的转录效率为同样方式构建下的P43启动子的2.62倍。通过N-乙酰神经氨酸表达质粒,可以成功地在枯草芽孢杆菌168菌株中实现N-乙酰神经氨酸的重组生产,摇瓶培养中N-乙酰神经氨酸的产量为0.226 g/L。本文为枯草芽孢杆菌进行N-乙酰神经氨酸的工业化发酵生产奠定了研究基础。     

重组β-淀粉酶在枯草芽孢杆菌中的高效表达及其在糖化反应中的应用

β-淀粉酶(β-amylase,Amy M)是一种重要的工业用酶,广泛应用在啤酒酿造、制糖等行业中。本研究利用Bacillus megatherium DSM 319来源的β-淀粉酶基因在枯草芽孢杆菌(Bacillus subtilis ATCC 6051?10)中分泌表达。首先,构建不同单启动子和双启动子介导β-淀粉酶表达的重组菌株,在摇瓶培养条件下,由P43-P43介导的双启动子重组菌株在发酵培养27 h时,分泌β-淀粉酶酶活力最高达2950.76 U/m L;随后研究碳代谢阻遏效应(Carbon Catabolite Repression,CCR)对重组β-淀粉酶表达的影响,发现对分解代谢控制蛋白(Catabolite control protein A,Ccp A)在DNA上顺式调控元件的碱基位点进行定点突变,可以有效缓解碳阻遏效应,β-淀粉酶酶活提高到4663.03 U/mL;最后,将重组β-淀粉酶应用在糖化反应过程中,结果表明麦芽糖转化率可以达到57.62%。综上,本研究成功构建出β-淀粉酶在枯草芽孢杆菌中的高效表达体系,为工业化生产β-淀粉酶提供了理论数据支撑。     

Enhancement of Recombinant Subtilisin YaB Production by Bacillus subtilis

Bacillus subtilis is an ideal host for the production of extracellular heterologous proteins. The use of a strong, constitutive promoter is a good means of optimizing the expression of heterologous proteins. In this study, high level extracellular production of subtilisin YaB, a potent meat tenderizer, was achieved by engineering the wild-type subtilisin YaB promoter into an artificial promoter and expressed in Bacillus subtilis host. This artificial promoter provides an efficient tool to produce mutant subtilisin YaB or other recombinant heterologous proteins by B. subtilis at high level.     

Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter P_AOX1 and constitutive promoter P_GAP. The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA‐seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol‐inducible promoter P₀₅₄₇ and the constitutive promoter P₀₄₇₂.     

Tandemly Repeated 147 bp Elements Cause Structural and Functional Variation in Divergent MAL Promoters of Saccharomyces cerevisiae

We have studied four novel MAL promoters isolated from a single strain of bakers' yeast. Within these promoters we have identified up to five tandem 147 bp repeats located between the MAL UAS region and the MALT TATA box. These repeats strongly reduce MALT (maltose permease) gene expression but only weakly reduce MALS (maltase) gene expression. Insertion of the 147 bp elements into the heterologous CYC1 promoter reduced expression when located between the CYC1 UAS and the TATA box, but not when located upstream of the UAS. We propose that these naturally occurring repeats have evolved as a mechanism to lower the level of MALT expression relative to MALS expression, thus avoiding possible toxic effects associated with over-expression from multiple copies of the permease gene. Accession numbers are: WIG1, U86359; WIG3, U86360; WIG4, U86361; WIG5, U86362. © 1997 by John Wiley & Sons, Ltd.     

Enzymatic synthesis of glycosylated puerarin using maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis

BACKGROUND: The maltogenic amylase from Bacillus stearothermophilus (BSMA) is a valuable biocatalyst that has been used to transglycosylate natural glycosides to improve solubility. To ensure safety, BSMA was produced in Bacillus subtilis, using new shuttle vector‐based expression vectors. The transglycosylation of puerarin was also conducted with crude BSMA and analyzed. RESULTS: Two expression systems, each containing one of the promoters from the genes encoding Bacillus licheniformis maltogenic amylase (BLMA) and an α‐amylase from B. subtilis NA64 (amyR2), were constructed. The amyR2 promoter system was chosen as the best system; it yielded 107 mg of pure BSMA from a 2 L culture. In the transglycosylation reactions of puerarin using crude BSMA, relative amounts for maltosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 3)‐puerarin, and puerarin were determined as 26:18:7:49. A two‐step purification process, including gel permeation chromatography, yielded 1.7 g of the transfer products from 3 g of puerarin. CONCLUSION: The crude BSMA produced from a host generally recognized as safe (B. subtilis) can be used to transglycosylate various functional compounds. The expression system developed in this study will be helpful for the production of other food‐grade enzymes by B. subtilis. Copyright © 2010 Society of Chemical Industry     

16S与gyrB基因联合建树快速鉴定一株蜡样芽孢杆菌

开发一种可用于保守序列相似度较高的菌群快速鉴定的方法,并用其鉴定一株从土壤采集的芽孢杆菌。从厦门市同安国家农业科技园区施用动物肥的种植区采集土壤,稀释涂布平板,分离纯化菌株后,扩增16S与gyrB基因序列并测序,在Genbank选择序列相似的ATCC菌株进行比对,将16S序列与gyrB序列线性拼接后,利用Paup* 4.0构建进化树,通过生理生化实验对鉴定结果进行验证。在16S序列与gyrB基因序列单独建树均无法鉴定到种的情况下,通过16S与gyrB碱基线性拼接序列联合建树,将土壤中分离得到的芽孢杆菌HX2016002鉴定为蜡样芽孢杆菌,该方法所构建的进化树自展值高,鉴定结果与生理生化实验一致。对Genbank中已知的蜡样芽孢杆菌序列建树分析表明,该方法在蜡样芽孢杆菌中具有普适性。利用16S与gyrB基因拼接序列联合建树,在保守序列相似度高的属内菌种鉴定中具有进一步研究的价值。     

Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae

The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose‐containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose‐induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC–URA plasmid except that gene expression is mediated by constitutive promoters. Copyright © 2010 John Wiley & Sons, Ltd.     

Aflatoxin B1-degrading activity from Bacillus subtilis BCC 42005 isolated from fermented cereal products

ABSTRACT

Aflatoxin B₁ is a naturally occurring mycotoxin that is produced as secondary metabolite by Aspergillus spp., especially A. flavus and A. parasiticus. This is the most severe toxin due to its carcinogenic, mutagenic, and teratogenic properties. Hence, methods for toxin degradation have been received increasing interest from both scientific communities and industries. In this study, 32 isolates of Bacillus spp. from various fermented cereal products were screened for their aflatoxin B₁ degradation ability. The results indicated the extracellular fraction of Bacillus subtilis BCC 42005 isolated from Iru (African locust bean) potentially possessed aflatoxin B₁-degrading ability. The maximum activity of the active fraction was at 50°C and pH 8.0. The activity was stable in a wide range of pH (5.0–8.0) and temperature (25–60°C). The aflatoxin B₁-degrading mechanisms of this strain may be possibly involved by enzyme(s). This extracellular fraction was not toxic at IC₅₀ 4 mg/ml and it can be combined with water as a soaking agent for maize, which results in 54% of aflatoxin B₁ reduction after contact time 120 min. Hence, the extracellular fraction of Bacillus subtilis BCC 42005 can be further applied as an effective soaking agent in a pretreatment process with a practical and easy-to-implement condition and also probably used to reduce the aflatoxin B₁ contamination in other foods and feeds commodities.