过表达purA基因对腺苷积累的影响

利用穿梭质粒pBE43,在腺苷产生菌枯草芽孢杆菌Bacillus subtilis XGL (Xan^-+ Deam^-+8-AG^r+SG^r)中过表达腺苷生物合成途径中关键酶腺苷酸琥珀酸合成酶(ADSS)的基因purA,研究其对腺苷积累的影响。以腺苷产生菌XGL基因组为模板,扩增出purA基因,将其连接到穿梭载体pBE43上,获得重组质粒pBE43∷purA。将重组质粒转化入XGL中,构建新的产生菌XGL-SY。通过逆转录PCR和定量PCR测定purA基因在腺苷产生菌XGL和XGL-SY中的转录量,并利用5 L罐发酵实验测定发酵参数的变化。分析发酵对数期的基因转录发现,在添加质粒后,purA基因的转录量提高了约9倍。上罐实验表明改造后的产生菌XGL-SY,菌体生长虽然受到一定的影响,但在相同发酵周期内腺苷产量提高了10.8%。研究结果表明在腺苷产生菌中过表达purA基因可以促进腺苷的积累,这为腺苷工程菌的进一步改造奠定了基础。     

枯草芽孢杆菌嘌呤合成途径的修饰对腺苷积累的影响

【目的】研究嘌呤操纵子中purF、purM、purN、purH和purD基因的共同过表达对枯草芽孢杆菌发酵生产腺苷的影响。【方法】利用温敏质粒pKS1,以单交换的形式增加了purF基因在基因组上的拷贝数,同时将强启动子P43插入嘌呤操纵子中,使嘌呤合成途径中purF基因及其下游purM、purN、purH和purD基因的表达水平得到加强,通过实时定量PCR(Realtime Quantitative PCR,RT-qPCR)测定相关基因(purF、purM、purN、purH和purD)的转录水平;通过酶活性检测分析关键酶基因扩增对PRPP转酰胺酶活性的影响;通过发酵实验考察出发菌株与工程菌株的生长、耗糖和腺苷积累情况。【结果】实时定量PCR结果表明,purF、purM、purN、purH和purD基因的表达水平均有不同程度的提高,嘌呤合成途径中关键酶PRPP转酰胺酶的活性是出发菌株的2.4倍。摇瓶发酵实验发现工程菌腺苷产量较出发菌提高17.5%,糖苷转化率增加26.1%。5 L罐发酵实验表明,虽然工程菌的菌体生长受到一定的影响,但在相同发酵周期内腺苷产量比出发菌提高了9.7%。【结论】嘌呤操纵子中purF、purM、purN、purH和purD基因转录水平的增强能够提高腺苷的产量,为通过代谢工程技术改造腺苷生产菌提供了理论依据和研究思路。     

假丝酵母YQ5产S-腺苷甲硫氨酸的发酵培养基优化

利用单因子实验和正交实验对假丝酵母(Candida sp.)突变菌株YQ5摇瓶发酵产生S-腺苷甲硫氨酸的培养基成分进行了优化。单因子实验结果表明, 发酵最适pH值为6.0, 最佳碳源为 8%蔗糖, 最佳氮源为1.5%胰蛋白胨, 酵母粉最适浓度为2%, MgSO4·7H2O、CaCl2、FeSO4·7H2O、CoCl2、CuSO4·5H2O、H3BO3等作为无机离子对胞内S-腺苷甲硫氨酸的积累均有促进作用。利 用正交实验获得了最终的发酵培养基配方。在最适条件下发酵48 h, S-腺苷甲硫氨酸含量可达1740.0 mg/L。     

淡色生赤壳菌(Bionectria ochroleuca)Bo-1菌株产生抗菌物质的发酵条件优化

以淡色生赤壳菌(Bionectria ochroleuca)Bo-1菌株发酵液乙酸乙酯粗提物对水稻白叶枯病菌(Xanthomonas oryzaepv.oryzae,Xoo)抑菌活性为检测指标,采用单因素试验优化Bo-1菌株产生抗菌物质培养所需的碳源、氮源、无机盐;通过正交试验优化培养基配方和摇瓶发酵条件。研究结果表明,Bo-1菌株产生抗菌物质适宜的碳源、氮源和无机盐分别为淀粉、蛋白胨、MgSO4·7H2O;优化的培养基配方为:淀粉30 g/L,蛋白胨2 g/L,MgSO4·7H2O 0.5 g/L;适宜的发酵条件为:温度30℃,转速150 r/min,装液量80 mL/250 mL,pH 6.5。     

医药其它

麦角菌（Clauiceps purpurea）硬粒产生麦角菌生物碱。每种菌株要求不同的培养基配方。一个常见的性质是,发酵终结时的生物大浓度随培养     

L-色氨酸生产菌的选育及其发酵条件的研究

以代谢控制发酵理论为指导 ,对L -色氨酸产生菌的定向选育、摇瓶发酵条件、30L发酵罐发酵条件进行了研究。以谷氨酸棒杆菌Tx5 - 32 (Phe- +Tyr- )为出发菌株 ,经硫酸二乙酯 (DES)多次诱变处理 ,定向选育出一株L -色氨酸产生菌TQ2 2 2 3(Phe- +Tyr- +5 -MTr+5 -FTr+SGr+CINr)。以摇瓶分批发酵最优条件为基础 ,对菌株TQ2 2 2 3进行了 30L发酵罐分批发酵试验 ,该菌株发酵 6 4h ,产L -色氨酸 7.2 8g·L- 1 。     

苏芸金杆菌85-10-6(S)发酵培养基的筛选

采用正交设计及方差分析法,筛选到Ⅰ号、Ⅱ号、Ⅲ号高浓度培养基配方。通过以85-10-6(s)为供试菌株、Ⅰ号配方在7吨发酵罐进行发酵试验,证实生产性能良好,发酵液平均含菌量71亿/ml,检查菌形、同步率、产晶率均属正常。     

甲醇利用型吡咯喹啉醌产生菌的筛选及鉴定

从虎杖内生细菌和黏细菌中筛选吡咯喹啉醌(PQQ)产生菌。采用3种以甲醇为唯一碳源的培养基对160株供试菌株进行摇瓶培养发酵,发酵产物采用光谱学分析法及HPLC法筛选。结果显示,通过初筛和复筛共得到甲醇利用型菌134株,PQQ产生菌4株,其中菌株083114的PQQ产量为64.34 mg/L。菌株083114的16S rRNA基因序列分析结果显示,其序列与酸快生芽孢杆菌(Bacillus acidiceler)的系统发育关系最近。虎杖内生细菌及黏细菌中存在PQQ产生菌。     

不同发酵条件对乳杆菌O-2菌株代谢产物的影响

目的:研究乳杆菌(Lactobacillus acidophilus sp.)O-2菌株生长和积累类细菌素所需的营养和温度条件.方法:采用分段控温发酵法,即在发酵后期,改变温度继续发酵;以发酵液抑菌圈直径为指标对发酵的营养和温度条件进行筛选.结果:后期32 ℃控温发酵有利于类细菌素积累,优化后的4号配方,发酵液菌数高达8.2×1010个/ml(细菌计数器法),抑菌圈直径最大,达到8.8 mm.结论:利用4号配方进行分段控温发酵能有效地促进O-2菌株的生长繁殖和积累类细菌素.     

发酵白酒糟生产饲料蛋白的优良菌种的筛选

采用常规方法从1000多株菌(包括丝状真菌、酵母菌、链霉菌、细菌)中筛选到一批优良菌种,并进行了单菌发酵、多菌株组合发酵,不同原料配方发酵试验。在实验室条件下,发酵产物的粗蛋白含量高达35.9％,比原料本身的粗蛋白含量高50％以上,比所用培养基的粗蛋白含量高30％,发酵产物的粗纤维含量降低率为15％;粗脂肪含量为5.5％左右;产率达80％以上。结果证明,筛选到的菌株确是发酵白酒糟生产饲料蛋白的优良菌种。     

腺苷高产菌株Bacillus subtilis DI4-24退化的遗传机制与控制研究

经人工诱变获得的腺苷高产菌株Bacillus subtilis DI4-24很容易发生退化,通过分析回变菌株的遗传性状和考察理化条件对回复突变的影响,研究退化的机制,找到控制的方法。在选择培养基上筛选回变菌株,生长谱法分析其遗传型;于743 nm条件下分光光度法测定回复突变细胞内积累的特有红色物质,研究各理化条件对回复突变的影响。退化原因是由于组氨酸缺陷型发生回复突变所致,突变菌株腺苷产量大幅度下降;降低保藏温度、减少传代次数可以有效地减缓菌种的衰退;种子制备过程中适当降低培养温度2℃~3℃、缩短培养时间(8 h)、降低培养基pH至中性偏酸性(6.5)以及加入胡萝卜素、叶酸等物质都可以有效降低回变率,在此条件下制备的种子生产性能得到提高,其腺苷产量可比对照提高9%。     

Adenosine 5′-Triphosphate Release and Membrane Collapse in Glycerol-Requiring Mutants of Bacillus subtilis

Glycerol-requiring mutants of Bacillus subtilis could not sporulate in nutrient sporulation medium even when additional glycerol was added from the beginning of growth. Sporulation could be partially restored either by the frequent addition of small amounts of glycerol during the developmental period or by the single addition of both 10 mM glycerol and 10 mM malate. But sporulation could be completely restored by the addition of 50 mM glycerol-phosphate from the beginning. At the end of growth of the glycerol mutants in nutrient sporulation medium, the cell membrane collapsed and separated from the cell wall, and much of the cellular adenosine 5'-triphosphate was released into the medium. These observations were made in two glycerol mutants, one derived from strain 168 containing glycerol-teichoic acid in the cell wall and the other derived from strain W23 containing ribitol-teichoic acid.     

A decrease in S-adenosylmethionine synthetase activity increases the probability of spontaneous sporulation.

Starting with a relaxed (relA) strain, mutants with reduced activity of adenosine triphosphate:L-methionine S-adenosyl transferase (EC 2.5.1.6; SAM synthetase) were isolated in Bacillus subtilis. One such mutant (gene symbol metE1) had only 3% of the normal SAM synthetase activity but grew almost as well as the parent strain. Another mutant was isolated (gene symbol spdC1) as being able to sporulate continually at a high frequency; it had one-half the normal SAM synthetase activity at 33 degrees C. Both mutants continually and spontaneously entered spore development at a higher frequency than the parent strain in a medium containing excess glucose, ammonium ions, and phosphate. Sporulation was prevented by a high concentration of SAM (1 mM or more) or by the combination of adenosine and methionine (0.5 mM or more each), both of which are precursors of SAM. In contrast to this continual increase in the spore titer, addition of decoyinine, an inhibitor of GMP synthetase, rapidly initiated massive sporulation. Various amino acid analogs also induced sporulation in the relA strain, the methionine analogs ethionine and selenomethionine being most effective.     

Response of Guanosine 5′-Triphosphate Concentration to Nutritional Changes and Its Significance for Bacillus subtilis Sporulation

We have investigated the changes in the guanosine 5'-triphosphate (GTP) and P-ribosyl-PP pools in stringent and relaxed strains of Bacillus subtilis under conditions frequently used to initiate sporulation. After a shift-down from a Casamino Acids-glutamate to a glutamate medium (Sterlini-Mandelstam shift-down), the pools of adenosine 5'-triphosphate and P-ribosyl-PP increased in both strains; in the stringent strain, ppGpp and pppGpp increased and GTP decreased rapidly, whereas in the relaxed strain, ppGpp and pppGpp increased only slightly and GTP decreased only slowly and less extensively. The stringent strain sporulated well, whereas the relaxed strain sporulated late and poorly. Addition of decoyinine, an inhibitor of guanosine 5'-monophosphate synthetase, caused a further decrease of GTP and initiated good sporulation of the relaxed strain. After a shift-down from a glucose-lactate to a lactate medium (Ramaley-Burden shift-down) the pool of P-ribosyl-PP (and GTP) decreased in both strains, indicating a shortage of purine precursors. This shift-down also caused a stringent response which prevented the consumption of nucleotides, as shown by the maintenance of adenosine 5'-triphosphate at a high concentration in the stringent strain but not in the relaxed strain. After a delay, the relaxed strain, in which GTP decreased as fast as in the stringent strain, sporulated also as efficiently. In nutrient sporulation medium the stringent strain and, less effectively, the relaxed strain accumulated ppGpp and pppGpp transiently towards the end of exponential growth. Eventually, the P-ribosyl-PP pool decreased drastically in both strains. In all cases the initiation of sporulation was correlated with a significant decrease of GTP. Granaticin, an antibiotic which prevents the charging of leucyl-transfer ribonucleic acid, was used to show that the stringent response inhibited the formation of xanthosine monophosphate from inosine monophosphate. It prevented the accumulation of xanthosine monophosphate in decoyinine-treated cultures of the stringent strain but not in those of the relaxed strain.     

Studies on Production of Nucleic Acid and its Related Compounds by Microorganisms

Adenosine was produced from adenine by means of fermentation using Bacillus subtilis Marburg 160-88 (st^r, try^-, pur^-). For the study on adenosine production, experiments concerning with pre-culture age, inoculum size, fermentation period, concentration of adenine, carbon source, nitrogen source and supplement of vitamins were carried out by test tube shaker. Furthermore the time course of fermentation was observed using jar fermentor. And it was proved that adenosine was produced about 1 mg/ml during the first 40 hrs of fermentation in the glucose mineral medium containing 1~2 mg/ml of adenine. This fermentation procedure seems to be one of economical methods for adenosine production.     

Isolation and identification of Bacillus sphaericus strains pathogenic for mosquito larvae

Three selective media for the isolation of Bacillus sphaericus have been compared. BATS medium and a formulation employing adenosine as the principal carbon source were the most effective for the recovery of spores of strain 1593. Anthranilic acid as the principal carbon source was less efficient. Eighty-four strains were isolated from mud samples using these media and were identified by computer. Identifications were confirmed for representative strains using DNA sequence homology. Most were B. sphaericus sensu stricto or members of an unnamed group. However, one strain (BSE 18) was identified as the DNA homology group IIB and this organism was found to be highly toxic toward larvae of Culex pipiens. Southern hybridization of BSE 18 DNA to a probe prepared from the cloned toxin gene from strain 1593 revealed that BSE 18 contained a typical gene for the 41.9-kDa toxin.     

纳豆杆菌对白假丝酵母菌的拮抗作用

目的探讨纳豆杆菌对白假丝酵母菌的拮抗作用。方法将纳豆杆菌和白假丝酵母菌混合培养24 h后,应用沙保弱平板培养基分离白假丝酵母菌,计数菌落,计算纳豆杆菌对白假丝酵母菌的拮抗率。结果纳豆杆菌对白假丝酵母菌的拮抗作用明显,拮抗率高达91.91%;纳豆杆菌肉汤培养物的除菌滤液对白假丝酵母菌也有明显的拮抗作用,拮抗率为79.05%。结论纳豆杆菌对白假丝酵母菌具有明显拮抗作用,是白假丝酵母菌的理想拮抗菌株。     

Production of PHB and P (3HB-co-3HV) biopolymers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain OU303A isolated from municipal sewage sludge

Bacillus megaterium strain OU303A isolated from municipal sewage sludge was selected for the study of biosynthesis of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate P (HB-co-HV) copolymer. The strain yielded a maximum of 62.43% DCW polymer in the medium containing glycerol as carbon source, which was followed by 58.63% DCW polymer in glucose containing medium. We found that this strain was capable of producing 2.5% hydroxyvalerate copolymer from a single carbon substrate, glucose. The strain showed an increase in the amount of HV monomer content, when the precursor for the copolymer was included in the fermentation medium. The characterization of the biopolymers was carried out using FTIR, GC-MS, H¹ NMR and DSC. This is the first report of B. megaterium strain producing HV copolymer, without the addition of any precursor in the fermentation medium.     

Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain.

Symbiobacterium thermophilum is a strictly symbiotic thermophile, the growth of which is dependent on the coexistence of an associating thermophilic Bacillus sp., strain S. S. thermophilum grows only in mixed culture with the Bacillus strain in liquid media, and does not form visible colonies on solid media. To measure the growth of this symbiotic bacterium and to analyze its growth requirements, we developed a quantitative PCR method by using its specific sequences in a putative membrane translocator gene tnaT as primers. According to this method, independent growth of S. thermophilum was first confirmed in a dialyzing culture physically separated from Bacillus strain S with a cellulose membrane. Independent growth of S. thermophilum was also managed by adding conditioned medium prepared from the culture filtrate of the Bacillus strain, but the growth in the conditioned medium stopped at a very limited extent with appearance of filamentous cells, suggesting the uncoupling of cellular growth and cell division. Formation of micro-colonies of S. thermophilum was observed on the conditioned agar medium under both aerobic and anaerobic conditions, but the colony-forming efficiencies remained below 1%. Several other bacterial species, such as Bacillus stearothermophilus, Bacillus subtilis, Thermus thermophilus, and even Escherichia coli, were also found to support the growth of S. thermophilum. These results indicate that S. thermophilum essentially requires some ubiquitous metabolite(s) of low molecular weight produced by various bacterial species as growth factor(s) but coexistence of the living partner cells is still required, probably to maintain an effective level of the putative factor(s) in the medium.     

The Self-Assembly of a Cyclic Lipopeptides Mixture Secreted by a B. megaterium Strain and Its Implications on Activity against a Sensitive Bacillus Species

Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300–800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.