肌苷和鸟苷生产菌中嘌呤核苷合成途径三段基因序列的分析

为了研究肌苷和鸟苷生产菌中与产苷有关的嘌呤核苷合成途径的遗传背景,选择了pur操纵子的启动子序列、编码SAMP合成酶的purA基因和编码GMP合成酶的guaA基因,设计合适的引物,分别从野生菌、一株肌苷低产菌和肌苷鸟苷高产菌中扩增出相应片段,经克隆和测序后,对它们进行比较和分析。分析结果表明两株生产菌的purA基因发生了1个碱基缺失,导致阅读框发生移码突变;而鸟苷高产菌在pur操纵子的启动子部分和操纵子抑制蛋白结合区域发生了近10％的突变,可能影响整个操纵子的表达调控。     

柠檬酸钠对枯草杆菌生长代谢及肌苷积累的影响

研究柠檬酸钠对枯草杆菌生长代谢及产苷的影响 ,在基础料中添加浓度为 0 2g L的柠檬酸钠 ,肌苷产量提高 18% ,肌苷对葡萄糖得率增加 38%。通过分析糖代谢途径中关键酶的酶活 ,结果表明添加柠檬酸钠改变了一些关键酶的活力 ,可降低糖酵解途径中 6_磷酸果糖激酶和丙酮酸激酶的活力 ,从而减弱了糖酵解途径的通量。     

添加次黄嘌呤或腺嘌呤提高肌苷产量

肌苷生产菌No.226具有将次黄嘌呤转变为肌苷的能力,添加次黄嘌呤有利于肌苷的积累。腺嘌呤缺陷型的肌苷生产菌株,在培养液中腺嘌呤的浓度对肌苷积累有显著影响。我们采用中国科学院上海生物化学研究所等单位选育的枯草芽孢杆菌7171-9-1菌株,进行舔加次黄嘌呤或腺嘌呤与酵母粉试验,现报道如下。     

短乳杆菌DM9218肌苷水解酶基因的异源表达及活性检测

目的探讨短乳杆菌DM9218肌苷水解酶基因A0008的异源表达及其对肌苷的分解活性检测。方法克隆来源于短乳杆菌DM9218基因组的肌苷水解酶基因A0008,构建原核表达载体,转入大肠埃希菌BL21诱导重组蛋白表达并纯化,进行体外酶活检测。结果成功构建了肌苷水解酶A0008-pET28a原核表达载体,表达并纯化出重组蛋白,酶活结果显示该重组蛋白具有水解肌苷的能力。结论短乳杆菌DM9218基因A0008可能编码肌苷水解酶并参与DM9218对肌苷的分解。     

应用遗传转化选育肌苷产生菌枯草杆菌“S14”

枯草杆菌链霉素基因位点和腺嘌呤基因位点靠近,因此利用它们共同转化频率较高的特性,从链霉素转化体中筛选腺嘌呤缺陷型转化体。用此方法使受体菌株168获得了给体菌株18R_3腺嘌呤缺陷型与产肌苷的遗传特性,并从中进一步筛选获得肌苷产生菌“S14”,它的肌苷产量为给体菌株18R_3的119.5％。     

肌苷生产菌枯草芽孢杆菌ATCC 13952的全基因组测序及序列分析

摘要:【目的】枯草芽孢杆菌ATCC 13952是一株肌苷工业生产菌株。为深入研究ATCC 13952菌株积累肌苷的分子机制以及为进一步分子育种研究提供序列背景信息,有必要解析ATCC 13952菌株的基因组序列信息。【方法】本研究采用高通量测序和Sanger测序相结合对ATCC 13952菌株进行全基因组测序,然后使用相关软件对测序数据进行基因组组装、基因预测与功能注释、GO/COG 聚类分析、共线性分析等。【结果】枯草芽孢杆菌ATCC 13952整个基因组大小为3876276 bp,GC含量为45.8%,序列已提交至GenBank 数据库,登录号为CP009748。比较基因组及嘌呤代谢相关基因分析结果显示:枯草芽孢杆菌ATCC 13952与其他几株芽孢杆菌具有较好的基因组共线性关系,嘌呤代谢相关基因编码的蛋白与标准菌株比较发生了一些缺失和突变。【结论】本研究首次报道了一株肌苷生产菌枯草芽孢杆菌ATCC 13952的全基因组序列,分析了基因组基本特征,初步探讨了该菌株积累肌苷的分子机制,为后续的进一步分子育种提供了理论基础。     

利用淀粉直接发酵生产肌苷菌株的构建

以肌苷生产菌枯草芽孢杆菌(Bacillus subtilis)TF_2为受体菌,利用质粒DNA的原生质体转化法,将携带糖化型α-淀粉酶基因的重组质粒pBX96导入肌苷产生菌TF_2中,转化频率为5.7×10^(-6),获得一株能以淀粉为碳源生产肌苷的转化子T140(pBX96),该工程菌株能在以淀粉为碳源的培养基上平均积累肌苷4.64g/L,经过92代,质粒自发丢失率为0.78％。培养48h后,工程菌株的糖化型α—淀粉酶活力为受体菌的7.79倍。并对工程菌株TI40(pBX96)的发酵条件做了初步摸索。     

肌苷产生菌guaA基因的修饰

由于肌苷生产菌枯草杆菌JSIM-1019,腺嘌呤缺陷,但黄嘌呤并不缺陷。以鸟苷产生菌枯草杆菌JSIM-G-518为供体菌,得到编码IMP脱氢酶的guaA基因。并用氯氨苄抗性基因插入guaA基因,使之不产生活性IMP脱氢酶,从而可制备黄嘌呤缺陷的新菌株,从而可以制备肌苷产量高于JSIM-1019的新菌株。     

枯草杆菌JSIM-1019突变株肌苷发酵研究

以肌苷产生菌枯草杆菌7171-9-1为出发菌株,经物理、化学诱变剂连续处理,获得一株腺嘌呤、组氨酸、硫胺素三重营养缺陷型并对8-氮杂鸟嘌呤、6-巯基嘌呤有双重抗性的突变株JSIM-1019。在摇瓶和发酵罐试验中,该变异株的肌苷产量显著高于亲株。摇瓶试验产肌苷达20g/L,最高可达24.83g/L。工业生产试验最高达14.5g/L,稳定在12g/L。发酵周期平均为43.8小时。菌株遗传性状稳定。     

白腐菌紫外诱变选育高产漆酶突变株及其产酶条件研究

以白腐菌为出发菌株,利用紫外线(UV)进行诱变,筛选高产漆酶突变菌株。通过测定致死率绘制出发菌株的致死曲线,采用PDA-RBBR平板变色法进行初筛,ABTS检测酶活对突变株进行摇瓶复筛。结果表明:利用15 w紫外灯在照射距离为30 cm,照射时间为120 s,致死率为72.1%的条件下进行诱变处理,获得一株高产菌株,其酶活提高79.54%,经过5代传代培养,未见酶活下降,具有较好的遗传稳定性,进一步研究了初始pH值,接种量和培养基装液量等对诱变菌株产酶的影响,结果表明在最佳的培养条件pH值6.0,15%的装液量于28℃下,酶活达214.9 U/L。     

Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5′ untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.     

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.     

Regulation of hypoxanthine transport in Neurospora crassa.

Hypoxanthine uptake and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) were determined in germinated conidia from the adenine auxotrophic strains ad-1 and ad-8 and the double mutant strain ad-1 ad-8. The mutant strain ad-1 appears to lack aminoimidazolecarboximide ribonucleotide formyltransferase (EC 2.1.2.3) or inosine 5'monophosphate cyclohydrolase (EC 3.5.1.10) activities, or both, whereas the ad-8 strain lacks adenylosuccinate synthase activity (EC 6.3.4.4). Normal (or wild-type) hypoxanthine transport capacity was found to the ad-1 conidia, whereas the ad-8 strains failed to take up any hypoxanthine. The double mutant strains showed intermediate transport capacities. Similar results were obtained for hypoxanthine phosphoribosyl-transferase activity assayed in germinated conidia. The ad-1 strain showed greatest activity, the ad-8 strain showed the least activity, and the double mutant strain showed intermediate activity levels. Ion-exchange chromatography of the growth media revealed that in the presence of NH+/4, the ad-8 strain excreted hypoxanthine or inosine, the ad-1 strain did not excrete any purines, and the ad-1 ad-8 double mutant strain excreted uric acid. In the absence of NH+/4, none of the strains excreted any detectable purine compounds.     

元阳豆豉中高产蛋白酶乳酸菌的筛选及其产酶条件的研究

从云南省元阳地区采集豆豉样品,并从中分离得到62株乳酸菌菌株。通过脱脂乳平板试验,从中初步筛得到21株具有高蛋白酶活力的菌株,采用茚三酮法测定其蛋白酶活力,复筛出高产蛋白酶菌株YY-1-6L,并对其产酶条件进行研究。结果表明,YY-1-6L在以葡萄糖为碳源、多聚蛋白胨为氮源、起始pH 5.0的明胶诱导培养基中,接种量为3%,35℃发酵培养36 h,其蛋白酶活力高达32.50 U/mL,且K2HPO4、MgSO4能促进蛋白酶产生。     

Mutation of an inosine-producing strain of Bacillus subtilis to DL-methionine sulfoxide resistance for guanosine production.

An inosine-producing strain of Bacillus subtilis was mutated to resistance against the antagonist of glutamine, DL-methionine sulfoxide. Among the mutants derived, guanosine producers were observed frequently. The best strain, 14119, produced 9.6 g of guanosine per liter at a weight yield of 12% from consumed sugar. Inosine production decreased concomitantly. When resistance was increased further by exposure to higher doses of DL-methionine sulfoxide, another strain, AG169, was obtained that did not excrete inosine but produced increased amounts of xanthosine. In these strains, the specific activity of 5'-nucleotidase was lower and that of inosine 5'-monophosphate (IMP) dehydrogenase was higher than the parent strain. It is speculated that the metabolic flow from IMP to xanthosine 5'-monophosphate proceeds more smoothly than that from IMP to inosine and yields more xanthosine and guanosine.     

Mutation of an inosine-producing strain of Bacillus subtilis to DL-methionine sulfoxide resistance for guanosine production.

An inosine-producing strain of Bacillus subtilis was mutated to resistance against the antagonist of glutamine, DL-methionine sulfoxide. Among the mutants derived, guanosine producers were observed frequently. The best strain, 14119, produced 9.6 g of guanosine per liter at a weight yield of 12% from consumed sugar. Inosine production decreased concomitantly. When resistance was increased further by exposure to higher doses of DL-methionine sulfoxide, another strain, AG169, was obtained that did not excrete inosine but produced increased amounts of xanthosine. In these strains, the specific activity of 5'-nucleotidase was lower and that of inosine 5'-monophosphate (IMP) dehydrogenase was higher than the parent strain. It is speculated that the metabolic flow from IMP to xanthosine 5'-monophosphate proceeds more smoothly than that from IMP to inosine and yields more xanthosine and guanosine.     

Relationship between macrotetrolide production and specific activity of some hydrolases in a high and a low producing strain of Streptomyces griseus

The activities of five hydrolytic enzymes in the culture filtrate and in cell-free extracts from strains of Streptomyces griseus, differing in macrotetrolide production, have been determined over a fermentation period of 200 h. The specific activities of phosphatase, phosphodiesterase, and adenosine triphosphatase in the medium, and phosphatase and phosphodiesterase in the cell-free extract were lower in the low than in the high producing strain. No significant difference was found between the strains, for adenosine triphosphatase and protease activity in the cell-free extract or protease activity in the medium. The specific activity of esterase was higher in the low than in the high producing strain.     

Investigation of various genotype characteristics for inosine accumulation in Escherichia coli W3110

For the derivation of an inosine-overproducing strain from the wild type microorganism, it is known that the addition of an adenine requirement, removal of purine nucleoside hydrolyzing activity, removal of the feedback inhibition, and repression of key enzymes in the purine nucleotides biosynthetic pathway are essential. Thus, the disruption of purA (adenine requirement), deoD (removal of purine nucleosides phosphorylase activity), purR (derepression of the regulation of purine nucleotides biosynthetic pathway), and the insensitivity of the feedback inhibition of phosphoribosylpyrophosphate (PRPP) amidotransferase by adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) were done in the Escherichia coli strain W3110, and then the inosine productivity was estimated. In the case of using a plasmid harboring the PRPP amidotransferase gene (purF) that encoded a desensitized PRPP amidotransferase, purF disrupted mutants were used as the host strains. It was found that the innovation of the four genotypes brought about a small amount of inosine accumulation. Furthermore, an adenine auxotrophic mutant of E. coli showed inappropriate adenine use because its growth could not respond efficiently to the concentration of adenine added. As the presence of adenosine deaminase is well known in E. coli and it is thought to be involved in adenine use, a mutant disrupted adenosine deaminase gene (add) was constructed and tested. The mutant, which is deficient in purF, purA, deoD, purR, and add genes, and harboring the desensitized purF as a plasmid, accumulated about 1 g of inosine per liter. Although we investigated the effects of purR disruption and purF gene improvement, unexpectedly an increase in the inosine productivity could not be found with this mutant.