能量代谢工程促地衣芽胞杆菌DW2高效合成杆菌肽

杆菌肽是一种主要由芽胞杆菌产生的广谱性环肽类抗生素,目前广泛应用于兽药领域。能量代谢在微生物高效合成目的代谢产物中具有重要作用。文中以杆菌肽工业生产菌株地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株,首先构建了呼吸链分支途径细胞色素bd泛醇氧化酶基因cydB缺失菌株,发现cydB缺失后杆菌肽效价和胞内ATP浓度相比于对照菌株分别提高了10.97%和22.96%。接着,证实了强化表达另外一条呼吸链分支途径——细胞色素aa3氧化酶基因qoxA能够提高杆菌肽合成水平,其杆菌肽效价和胞内ATP浓度相比于对照菌株分别提高了18.27%和34.00%。强化ADP合成供给也是促进胞内ATP积累的有效策略,结果表明强化表达腺苷激酶DcK和腺苷酸激酶AdK均可以提高杆菌肽效价和胞内ATP浓度,其中强化表达DcK效果较好,其杆菌肽效价相比对照提高16.78%。最后,通过组合代谢工程育种,在基因cydB缺失菌DW2ΔcydB基础上整合表达了qoxA和dck,得到工程菌株DW2-CQD(DW2ΔcydB::qoxA::dck),发酵结果表明,DW2-CQD杆菌肽效价达到954.25 U/mL,相比于对照菌株提高了21.66%,单位菌体杆菌肽效价为2.11 U/CFU,相比对照提高了11.05%。此外,DW2-CQD胞内ATP浓度为39.54 nmol/L,相比于对照提高了49.32%。结果证实能量代谢工程是提高杆菌肽发酵水平的有效策略,提供了一株具有工业化应用前景的杆菌肽生产菌株。     

代谢工程改造L-半胱氨酸供给模块促地衣芽胞杆菌高效合成杆菌肽

杆菌肽是一种主要由芽胞杆菌产生的广谱性抗生素,目前作为兽药广泛应用于畜禽养殖领域.前体氨基酸供应不足可能是限制微生物发酵高产杆菌肽的重要因素.文中以杆菌肽工业生产菌株——地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株,研究L-半胱氨酸供给模块强化对杆菌肽合成的影响.首先,构建了L-半胱氨酸合...     

地衣芽胞杆菌活菌制剂的临床应用进展

整肠生是应用二十多年的地衣芽胞杆菌活菌制剂,已有大量的研究报道其临床有效性和安全性。地衣芽胞杆菌大量分泌种类丰富的消化酶,通过抑制肠道有害菌生长和促进有益菌增殖调节肠道菌群,并产生杆菌肽、地衣素和乙酸等生物活性物质发挥益生作用。本文总结了益生菌的益生特点,并重点分析了芽胞杆菌的益生特点,归纳了整肠生的作用机制与临床研究现状,揭示了其在肝脏疾病、溃疡性结肠炎、肠易激综合征、幽门螺旋杆菌感染以及病毒感染等疾病中的治疗作用,并对整肠生未来的临床应用方向进行了展望。     

地衣芽胞杆菌培养条件的研究

应用单因素和正交试验设计试验法对地衣芽胞杆菌在摇瓶水平上进行了培养基配方和培养条件的研究。结果表明：最适培养基配方为山芋淀粉1．0％,豆粕0．6％,玉米芯粉0．8％,K2HPO4 0．1％,MgSO40．1％。最适培养条件为37℃,摇床转速150r／min,250mL三角瓶装液50mL,接种量5．0％,振荡培养48h,pH7．0。在20L自动发酵罐中进行了扩大培养试验,考察溶氧对菌体生长的影响,并根据试验结果进一步扩大至1m^3发酵罐,通过控制搅拌速度和通气量,1m^3发酵罐中地衣芽胞杆菌培养液菌浓为7．2×10^9efu／mL,芽胞率达到90％。     

地衣芽胞杆菌在植物病害生物防治中的应用

随着对地衣芽胞杆菌研究的不断深入和其杀虫、抗菌、生物降解等多种生物学活性的发现,地衣芽胞杆菌被认为是芽胞杆菌属中最具有生物防治应用价值的菌种之一。本文论述了地衣芽胞杆菌的生物学特性及其防治植物病害的四种主要作用机制,包括竞争、分泌抗菌物质、诱导植物系统抗性和促进植物生长;介绍了地衣芽胞杆菌在水稻、棉花、番茄、芒果、辣椒等多种大田作物和经济果蔬上的应用现状和生物防治效果;并讨论了其在生防应用过程中存在的主要问题,为今后地衣芽胞杆菌在生物防治方面的研究提供理论依据和应用参考。     

地衣芽胞杆菌在植物病害生物防治中的应用

随着对地衣芽胞杆菌研究的不断深入和其杀虫、抗菌、生物降解等多种生物学活性的发现,地衣芽胞杆菌被认为是芽胞杆菌属中最具有生物防治应用价值的菌种之一。本文论述了地衣芽胞杆菌的生物学特性及其防治植物病害的四种主要作用机制,包括竞争、分泌抗菌物质、诱导植物系统抗性和促进植物生长;介绍了地衣芽胞杆菌在水稻、棉花、番茄、芒果、辣椒等多种大田作物和经济果蔬上的应用现状和生物防治效果;并讨论了其在生防应用过程中存在的主要问题,为今后地衣芽胞杆菌在生物防治方面的研究提供理论依据和应用参考。     

通过优化表达元件及培养基组分提高地衣芽胞杆菌中碱性丝氨酸蛋白酶产量

【背景】碱性丝氨酸蛋白酶(Subtilisin)是一种具有广泛用途的工业酶制剂。【目的】旨在通过优化启动子、信号肽及培养基组分来提高地衣芽胞杆菌中碱性丝氨酸蛋白酶产量。【方法】以地衣芽胞杆菌BL10为出发菌株,构建了含有4种不同类型启动子(PbacA、P43、PaprE和PsrfA)及4种不同类型信号肽(SPVpr、SPSacB、SPSacC和SPAprE)的碱性丝氨酸蛋白酶表达菌株,并在获得高产菌株的基础上进行培养基优化。【结果】4种启动子的表达水平为PbacAPaprEP43PsrfA,4种信号肽的分泌效率为SPAprESPSacCSPSacBSPVpr。其中,菌株BL10/pPbacA-aprE产生最高的碱性丝氨酸蛋白酶酶活(275.21 U/mL),相比于出发菌株BL10/pHY-aprE (167.98 U/mL)提高了64%。随后,通过对发酵培养基成分进行优化并结合正交优化,获得了一种高产碱性丝氨酸蛋白酶的培养基(g/L):玉米淀粉40.0,豆粕50.0,(NH4)2SO4 4.0,K2HPO4 3.0,CaCO3 1.0。最后,碱性丝氨酸蛋白酶酶活提高到747.37 U/mL,是初始酶活的4.45倍。【结论】为工业化高产碱性丝氨酸蛋白酶提供了一种有效策略。     

地衣芽孢杆菌9945a转运蛋白YdgF1的功能鉴定

【目的】本研究对地衣芽孢杆菌(Bacillus licheniformis) 9945a菌株中ydgF1基因编码的转运蛋白进行功能鉴定。【方法】分别构建ydgF1基因过表达株9945a/pHY300-Shu-ydgF1和敲除株9945aΔydgF1,设计了以D-丙氨酸为唯一氮源的磷酸盐D-丙氨酸(phosphate D-alanine, PDA)培养基来考察菌株的生长能力,并进行细胞吸收实验。利用实时荧光定量PCR(real-time quantitative polymerase chain reaction, RT-qPCR)评价菌株9945a与9945aΔydgF1在LB培养基中不同生长时期ydgF1的相对表达量,然后对9945a与9945aΔydgF1后期的培养基平板活菌计数,计算菌落形成单位(colony-forming units, CFU)。【结果】在PDA培养基中,9945aΔydgF1的比生长速率始终低于9945a,而9945a/pHY300-Shu-ydgF1最大比生长速率为0.336h–1,是9945a/pHY300-Shu的1.98倍,并且培养15 h后9945...     

地衣芽胞杆菌（CMCC63519）及代谢产物对部分细菌的抑菌作用

本文通过两个试验,证实了地衣芽杆菌的代谢产物对某些细菌有类似抗生素样作用。抑菌作用与代谢产物有关。     

来自猪肠道芽胞杆菌培养液中氨基酸成分分析

从动物肠道分离得到112株芽胞杆菌,采用氨基酸全自动分析仪对分离得到的芽胞杆菌的上清进行氨基酸分析。结果表明,不同芽胞杆菌菌株上清中各氨基酸水平相差较大。其中,动物的几种限制性必需氨基酸中,赖氨酸的含量分布在0.18~0.32g·L~(-1)之间,蛋氨酸的含量分布在0.05~0.09g·L~(-1)之间。精氨酸的含量分布在0.004~0.02g·L~(-1)之间。所有氨基酸种类中,赖氨酸、亮氨酸、苯丙氨酸、异亮氨酸含量较为丰富,超过了0.1g·L~(-1),而天冬氨酸、苏氨酸、丝氨酸含量较少,不到0.01g·L~(-1)。这些结果为开发提高饲料氮利用效率的饲用益生菌提供参考。     

Amplification of bacitracin transporter genes in the bacitracin producing Bacillus licheniformis

We have amplified the previously cloned and sequenced genes of the bacitracin exporter (bcr), a member of the ATP-binding transport protein family, within the chromosome of the bacitracin producing Bacillus licheniformis. Amplification of the transporter genes was followed by greatly increased bacitracin resistance. Antibiotic production was enhanced at a low level of bcr genes amplification. An enlarged increase in the copy number of the bcr genes negatively affects the overall growth of bacteria.     

Development of an asporogenic Bacillus licheniformis for the production of keratinase

AIMS: Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A sporulation-deficient mutant of B. licheniformis PWD-1, named B. licheniformis WBG, was developed and characterized. METHODS AND RESULTS: The mutation was generated using the splicing by overlap extension PCR method (Gene SOEing) to create 256 bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise the nucleotide fragment 256 bp from the B. licheniformis chromosome. PCR analysis and DNA sequencing confirmed the spoIIAC gene deletion. Heat-treatment assays and electron microscopy verified the absence of spores. CONCLUSIONS: This asporogenic strain is able to express normal levels of keratinase when compared with its wild-type host. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a method of constructing a stable sporulation-defective strain was developed. It can be potentially useful as a tool to generate asporogenic strains of Bacillus that retain their industrial capabilities for production of exoproteases and other exozymes.     

Cloning and characterization of the glutamate dehydrogenase gene in Bacillus licheniformis

The gdhA genes of IRC-3 GDH-strain and IRC-8 GDH+ strain were cloned,and they both successfully complemented the nutritional lesion of an E.coli glutamate auxotroph,Q100 GDH-.However,the gdhA gene from the mutant IRC-8 GDH+ strain failed to complement the glutamate deficiency of the wild type strain IRC-3.The gdhA genes of the wild type and mutant origin were sequenced separately.No nucleotide difference was detected between them.Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant.Additionally,no GDH inhibitor was found in the wild type strain IRC-3.It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression.Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the familyⅠ-type hexameric protein,while the GDH of Bacillus subtilis belongs to family II.     

地衣芽孢杆菌碱性果胶酶基因PelA的克隆与原核表达

通过PCR扩增的方法,从本实验室筛选保存的Bacillus licheniformis DG-3 菌株中扩增出碱性果胶酶的结构基因pelA , 序列分析表明,所获PelA 基因与已报道的B.licheniformis 14A菌株的pelA基因的同源性为100%. 将pelA 基因在大肠杆菌中表达,发酵液菌体的碱性果胶酶酶活为12U/mL. 4～8 mmol/L的Ca2 对碱性果胶酶具有显著的激活作用.     

Enhanced secretion of heterologous cyclodextrin glycosyltransferase by a mutant of Bacillus licheniformis defective in the D-alanylation of teichoic acids

AIMS: To examine whether inactivation of the dlt operon and increased charge density of the wall enhances secretion of heterologous proteins in industrial strains of Bacillus licheniformis. METHODS AND RESULTS: The dltA gene of B. licheniformis was cloned, sequenced and mutated by inserting a chloramphenicol acetyl transferase (cat) gene cassette. The mutation facilitated growth in the late exponential growth phase, increased endogenous autolysis and decreased resistance to a cationic peptide, polylysine. It was observed that dltA mutation increased the production of cyclodextrin glycosyltransferase (CGTase) by 1.5- to sevenfold depending on the growth phase, but decreased the production of penicillinase by twofold. CONCLUSIONS AND SIGNIFICANCE: The results suggest that the d-alanylation of teichoic acids is an element that can be used to improve the production of some secretory proteins in industrial applications based on this important industrial microorganism.     

微生态系统中地衣芽孢杆菌消长的研究

采用不同接种培养方法,研究了地衣芽孢杆菌在微生态系统的消长状况．结果表明,在健康人体内该菌能迅速增殖,菌数为１．４９×１０８～１．８３×１０８个·ｇ－１,并能较长时间定植下来,３０ｄ后菌数为１．２７×１０７～１．５１×１０７个·ｇ－１．在病人体内增殖相对较慢些,菌数为１．４０×１０８～１．６７×１０８个·ｇ－１,３０ｄ后菌数为１．１５×１０７～１．３１×１０７个·ｇ－１．在人工模拟微生态系统中,当ｐＨ为５．０～９．０,营养物为食物匀浆培养基、牛肉膏蛋白胨培养基时,其菌数为３．０５×１０８、３．４２×１０８个·ｍｌ－１．