利用IABS模型筛选L－异亮氨酸高产菌株

IABS模型是本研究中确立的理性化菌种选育方法,可用于高效率地筛选多种调控变株。大大减少摇瓶筛选工作量。利用IABS模型以L－异亮氨酸产生菌Brevibacterium flavumAslll为出发菌株,有目的地活化PC、HD和AHAS等酶,筛得变株23—10(α—AB rrt+Suc g+Eth rr),23—10在优化的培养条件下可产生20mg／ml以上的L-异亮氨酸,且无L-亮氨酸积累。23—10变株25代后,产酸能力毫不下降。     

大肠杆菌乙酰羟基酸合酶I调控亚基IlvN的结晶及其与配体缬氨酸的共结晶

乙酰羟基酸合酶(acetohydroxyacid synthase,AHAS)是生物体内支链氨基酸合成通路中的第一个通用酶,它是目前市售多种除草剂的靶标．AHAS通常由分子质量较大的催化亚基和分子质量较小的调控亚基组成．催化亚基结合催化必需的辅基(FAD、ThDP和Mg2+);调控亚基可以结合终产物(缬氨酸、亮氨酸或异亮氨酸)作为负反馈信号调节全酶的活性．大肠杆菌中AHAS有3个同工酶,每种同工酶都由催化亚基和调控亚基组成．大肠杆菌ilvN基因编码了AHAS同工酶Ⅰ的调控亚基．ilvN基因克隆到pET28a表达载体中,在大肠杆菌BL21(DE3)菌株中得到可溶性的大量表达．表达的蛋白质通过镍离子亲和层析和分子筛层析得到纯化．为了对调控亚基的调节机理有深入了解,对IlvN蛋白进行结晶并对蛋白质与其配体缬氨酸进行共结晶．IlvN蛋白晶体衍射能力为2.6 Å,IlvN与缬氨酸共结晶的晶体衍射能力为3.0 Å.     

基于途径分析的L-异亮氨酸发酵溶氧控制研究

利用途径分析方法对黄色短杆菌（Brevibacterium flavum）TC-21 生产L-异亮氨酸的途径进行了分析,确定了黄色短杆菌TC-21生产L-异亮氨酸的最佳途径的通量分布,根据途径分析的结果,TCA循环的代谢流量对L-异亮氨酸产量有明显影响,而TCA循环与发酵过程中的溶氧密切相关,因此可以通过控制溶氧来提高L-异亮氨酸产量。在发酵过程的不同阶段,根据菌体生长和产酸的需求,改变TCA代谢流量,可以有效提高产酸率。实验证明,通过溶氧分阶段控制发酵生产L-异亮氨酸,比溶氧恒定控制方式发酵产率提高了15.77％。实验结果说明,用途径分析的结果指导发酵过程中的溶氧可以大幅度提高L-异亮氨酸的产量。     

灵芝β-葡萄糖苷酶基因的克隆与功能分析

灵芝是我国著名的药用真菌,具有抗癌、抗肿瘤等功效。灵芝酸属于三萜类化合物,是灵芝的主要活性成分,并已成为评价灵芝品质的重要指标之一。β-葡萄糖苷酶(β-glucosidase,BG)是次生代谢产物合成途径中的关键限速酶,能够调节次生代谢产物的生物合成。本研究通过同源序列比对,注释获得了灵芝β-葡萄糖苷酶基因(GlBG),并通过RNAi技术对灵芝β-葡萄糖苷酶进行功能分析。序列分析结果显示GlBG基因的DNA全长为2 759 bp,包含7个外显子和6个内含子,编码793个氨基酸,其编码的蛋白序列中含有β-糖苷水解酶的2个保守结构域。灵芝β-葡萄糖苷酶基因的沉默转化子中灵芝酸含量比野生型菌株的灵芝酸含量平均降低了38%,并且灵芝酸生物合成途径中的关键酶基因(hmgs、hmgr、fps、sqs和osc)的表达量也显著下降,实验结果表明灵芝β-葡萄糖苷酶在灵芝酸生物合成过程中具有重要作用,并为灵芝次生代谢途径及其调控机制提供了参考。     

酯类风味物质微生物代谢机理研究

本文对产酯微生物Ｃ．Ｐｅｎｉｃｉｌｌａｔｕｍ代谢机理进行了分析,并进行了实验证明,结果发现Ｇ．ｐｅｎｉｃｉｌｌａｔｕｍ产酯代谢经历了好氧与厌氧二个步骤,氨基酸既是醇的供体又是酸的供体,适宜的供氧量和添加异亮氨酸对酯的生产均有较大影响。     

脑特异脂肪酸活化酶的克隆

东北大学基因实验室助教授山本德男小组克隆了特异存在于小鼠脑内、控制脂肪酸代谢和脂质生物合成的脂肪酸活化酶的cDNA.大脑干重的48%以上由脂质构成,是弄清脑脂质合成和代谢的最关键的物质. 脂肪酸活化酶(酰基CoA合成酶)是由脂肪酸和ATP、辅酶A(CoA)合成脂肪酸CoA的酶.脂肪酸被活化,转化成脂肪酸CoA之后,才能作为脂肪酸β氧化反应的能量以及乙酰基CoA的合成反应和中性脂肪、磷脂质、胆固醇酯等合成脂质的底物.由此看来,脂肪酸活化酶是一种重要的特异性酶.     

奎尼酸生物合成的代谢工程

奎尼酸及其衍生物氢醌和苯醌等是一类重要的化工原料,可作为一些化学合成制剂和药物中间原料,且在食品和化学工业中有着广泛的应用。目前奎尼酸的制备方法有植物提取法、化学合成法、酶工程法和微生物发酵法,其中微生物发酵法是近年发展起来的一种十分经济有效的方法。在介绍奎尼酸的制备方法的基础上重点综述了应用代谢工程在生物合成奎尼酸基因工程菌的改造中的研究进展,其中涉及奎尼酸生物合成途径中相关基因及其酶的调控、中心代谢途径的改造和修饰等,并探讨了将来的发展前景。     

拟南芥乙酰羟酸合成酶(AHAS)点突变原核表达与活性测定

拟南芥乙酰羟酸合成酶(AHAS)参与支链氨基酸合成。为考察AHAS不同结构域对支链氨基酸合成的影响,分别对其大小亚基上特定位点进行点突变后进行原核表达,体外重组后对其全酶活性进行测定,并对其终端产物之一——缬氨酸对AHAS全酶活性的影响进行探讨。结果显示:AHAS小亚基G88D突变将解除其终端产物的反馈抑制作用,而大亚基E305D与E482D的突变降低AHAS全酶活性,且2种不同突变大亚基对AHAS全酶活性影响存在差异。AHAS大亚基E482D突变较E305D突变影响更大。研究结果表明:AHAS大小亚基间存在着相互作用,且大小亚基不同结构域突变对AHAS全酶活性具有不同的影响。     

地中海拟无枝菌酸菌U32染色体特性的脉冲场电泳分析

地中海拟无枝菌酸菌(Amycolatopsis mediterranei)U32是产力复霉素SV的工业生产菌株。采用脉冲场电泳分析发现,地中海拟无枝菌酸菌U32仅有一条约10 Mb的线性染色体, 没有内源性质粒。利用Southern杂交法,对11个编码力复霉素生物合成、相关初级、次级代谢关键酶以及调控蛋白的基因,在U32染色体DNA的PshBI酶切片段上进行了定位。分析发现在一条长度约700kb的PshBI酶切片段上,分别存在着力复霉素合成基因簇(rif)、氮代谢的亚硝酸还原酶小亚基基因(nasD)、衔接初级与次级代谢的甲基丙二酰变位酶基因(mcm)、脂肪酸代谢的乙酰辅酶A羧化酶生物素载体蛋白基因(accA)以及一套核糖体RNA转录单元。同时还发现U32至少有5套核糖体RNA转录单元。其余定位的基因均只出现单一杂交信号。     

基于GC-MS分析刺五加和短梗五加不同器官的代谢差异特性

通过研究刺五加（Acanthopanax senticosus）与短梗五加（A. sessiliflorus）不同器官的初级代谢差异特性,探讨两者药效差异、药用价值及资源利用结构。利用代谢组学的研究策略,使用GC-MS技术从初生代谢的角度比较刺五加与短梗五加不同器官代谢差异特性。结果表明：在2种药用植物不同器官中共鉴定出186种初生代谢物,总体研究结果显示2物种在初生代谢上存在差异。进一步分析得到根中52种差异代谢物、茎中34种差异代谢物、叶中39种差异代谢物、叶柄中48种差异代谢物。差异代谢物涉及到的主要代谢途径有氨酰基-tRNA生物合成途径,缬氨酸、亮氨酸和异亮氨酸的生物合成途径,甘氨酸、丝氨酸和苏氨酸代谢途径,精氨酸的生物合成途径,丙氨酸、天冬氨酸和谷氨酸代谢途径,丙酮酸代谢途径等。此外,发现4种连接初生代谢和次生代谢的关键性代谢物在两者不同器官中的水平存在差异。刺五加和短梗五加在不同器官初生代谢方面显著不同,这可能是造成2物种不同器官药理效应和次生代谢存在差异的原因之一。     

Evidence for isoleucine as a positive effector of the ilvBN operon in Salmonella typhimurium

Concerted efforts were directed towards understanding the control of acetohydroxy acid synthase (AHAS) in the gyrB mutant hisU1820 of Salmonella typhimurium. A media shift from valine to valine plus isoleucine causes a dramatic 4 to 5 fold burst of AHAS valine sensitive activity which appears to be dependent on translation. DJ19, an isolated valine sensitive derivative of the gyrB mutant, maintains a dramatic increase in AHAS valine sensitive activity upon the addition of isoleucine to valine supplemented cultures, suggesting that the isoleucine effect is specific for valine sensitive AHAS. Evidence supports isoleucine as a positive effector on valine sensitive AHAS expression and that the gyrB mutation accentuates the isoleucine effect.     

Acetohydroxy acid synthase I is required for isoleucine and valine biosynthesis by Salmonella typhimurium LT2 during growth on acetate or long-chain fatty acids.

Salmonella typhimurium LT2 normally expresses two acetohydroxy acid synthases (AHAS I and AHAS II). The function of AHAS I in this organism was unclear, since AHAS I-deficient (ilvBN) mutants of LT2 grew well on glucose or succinate minimal media, whereas AHAS II-deficient (ilvGM) mutants requried isoleucine for normal growth on glucose minimal media. We report that AHAS I-deficient mutants of S. typhimurium required isoleucine and valine for growth on acetate or oleate minimal media, whereas AHAS II-deficient mutants were able to grow on these media without isoleucine supplementation.     

Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium: a Quantitative Analysis

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.     

Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium.

The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II. One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI). The effects of the inhibitors on growth, levels of several enzymes of the pathway, and levels of intermediates of the pathway were measured. The intracellular concentration of the AHAS substrate 2-ketobutyrate increased on addition of SMM, but a lack of correlation between increased ketobutyrate and growth inhibition suggests that the former is not the immediate cause of the latter. The levels of the keto acid precursor of valine, but not of the precursor of isoleucine, were drastically decreased by SMM, and valine, but not isoleucine, partially overcame SMM inhibition. This apparent stronger effect of SMM on the flux into the valine arm, as opposed to the isoleucine arm, of the branched-chain amino acid pathway is explained by the kinetics of the AHAS reaction, as well as by the different roles of pyruvate, ketobutyrate, and the valine precursor in metabolism. The organization of the pathway thus potentiates the inhibitory effect of SMM. IpOHA has strong initial effects at lower concentrations than does SMM and leads to increases both in the acetohydroxy acid substrates of KARI and, surprisingly, in ketobutyrate. Valine completely protected strain TV105 from IpOHA at the MIC. A number of explanations for this effect can be ruled out, so that some unknown arrangement of the enzymes involved must be suggested. IpOHA led to initial cessation of growth, with partial recovery after a time whose duration increased with the inhibitor concentration. The recovery is apparently due to induction of new KARI synthesis, as well as disappearance of IpOHA from the medium.     

Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum

The enzyme activities of the valine biosynthetic pathway and their regulation have been studied in the valine-producing strain, Corynebacterium glutamicum 13032DeltailvApJC1ilvBNCD. In this micro-organism, this pathway might involve up to five enzyme activities: acetohydroxy acid synthase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxyacid dehydratase and transaminases B and C. For each enzyme, kinetic parameters (optimal temperature, optimal pH and affinity for substrates) were determined. The first enzyme of the pathway, AHAS, was shown to exhibit a weak affinity for pyruvate (K(m)=8.3 mM). It appeared that valine and leucine inhibited the three first steps of the pathway (AHAS, AHAIR and DHAD). Moreover, the AHAS activity was inhibited by isoleucine. Considering the kinetic data collected during this work, AHAS would be a key enzyme for further strain improvement intending to increase the valine production by C. glutamicum.     

Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.     

Mutation affecting regulation of synthesis of acetohydroxy acid synthetase in Escherichia coli K-12.

Altered regulation of synthesis of acetohydroxy acid synthetase (AHAS) was previously reported in a mutant of Escherichia coli strain K-12. The mutant strain, growing in minimal medium, exhibits a partial growth limiatation and derepression of AHAS, owing to deficient synthesis of isoleucine. The genetic lesion (ilvE503) causing the isoleucine limitation was shown to cause derepression of a valine-sensitive AHAS activity. The derepression effect of the ilvE503 mutation upon synthesis of AHAS was conclusively demonstrated by introducing both the ilvE503 allele and an altered AHAS (ilv-521) into the same cell. Evidence is presented that suggests the presence of multiple genetic regions for synthesis and control of the valine-sensitive AHAS activity.     

Effects of amino acids on Thiobacillus acidophilus. I. Growth studies with special reference to valine.

The heterotrophic growth of Thiobacillus acidophilus was inhibited by branched-chain amino acids; valine, isoleucine, and leucine. The inhibition by valine and leucine were partially reversed by isoleucine, and the inhibition by isoleucine was partially reversed by valine. Inhibitions by methionine or threonine were partially reversed when both amino acids were present in the growth medium. Inhibition by tyrosine was increased by phenylalanine or tryptophan. Cystine completely inhibited growth. Other amino acids tested produced little or no inhibition. Acetohydroxy acid synthetase (AHAS) activity was demonstrated in crude extracts of T. acidophilus. In crude extracts the optimum pH was 8.5 with a shift to 9.0 in the presence of valine. Valine was the only branched-chain amino acid which inhibited the AHAS activity. The presence of only one peak of AHAS activity upon centrifugation in linear glycerol density gradients demonstrated that the AHAS activity sediments as one component.     

Imidazolinone-induced loss of acetohydroxyacid synthase activity in maize is not due to the enzyme degradation

Acetohydroxyacid synthase (AHAS), the first enzyme leading to the biosynthesis of valine, leucine, and isoleucine, is inhibited by different chemical classes of herbicides. There is a loss in the extractable AHAS activity in imidazolinone-treated plants. Immunological studies using a monoclonal antibody against AHAS revealed no degradation of AHAS protein in imidazolinone-treated maize (Zea mays) plants. Therefore, the loss in AHAS activity is not due to the loss of AHAS protein.     

Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms.

Acetohydroxy acid synthase (AHAS; EC 4.1.3.18) catalyzes the following two parallel, physiologically important reactions: condensation of two molecules of pyruvate to form acetolactate (AL), in the pathway to valine and leucine, and condensation of pyruvate plus 2-ketobutyrate to form acetohydroxybutyrate (AHB), in the pathway to isoleucine. We have determined the specificity ratio R with regard to these two reactions (where VAHB and VAL are rates of formation of the respective products) as follows: VAHB/VAL = R [2-ketobutyrate]/[pyruvate] for 14 enzymes from 10 procaryotic and eucaryotic organisms. Each organism considered has at least one AHAS of R greater than 20, and some appear to contain but a single biosynthetic AHAS. The implications of this for the design of the pathway are discussed. The selective pressure for high specificity for 2-ketobutyrate versus pyruvate implies that the 2-ketobutyrate concentration is much lower than the pyruvate concentration in all these organisms. It seems important for 2-ketobutyrate levels to be relatively low to avoid a variety of metabolic interferences. These results also reinforce the conclusion that biosynthetic AHAS isozymes of low R (1 to 2) are a special adaptation for heterotrophic growth on certain poor carbon sources. Two catabolic "pH 6 AL-synthesizing enzymes" are shown to be highly specific for AL formation only (R less than 0.1).