高产谷胱甘肽的酵母菌选育及其培养条件研究

筛选到一株具有较高GSH产量的酵母菌株S.cerevisiae2165,然后以该菌株为出发菌,采用紫外照射,紫外照射 LiCl联合处理,亚硝基胍（MNNG）等诱变处理,获得一株高产GSH的酿酒酵母优良菌株S.cerevisiaeJN-5-8。该菌株具有稳定的遗传性能,在经过优化的培养条件下培养24h,其GSH产量达到339.1mg/L。比出发菌株提高2.2倍。     

等离子体-紫外线复合诱变选育高产谷胱甘肽酵母菌

以产还原型谷胱甘肽（glutathione,GSH）的啤酒酵母（Sacchromyces cerevisiae）SC-20为出发菌株,采用氩气等离子体射线、紫外照射及两者的复合诱变处理,获得一株高产GSH的酿酒酵母优良菌株（S．cerevisiae）DU-20．结果表明该菌株具有稳定的遗传性能,GSH含量与产量分别比出发菌株提高了78.33％和118．4％．     

高产虾青素红法夫酵母的选育及代谢通量分析

以野生型红法夫酵母As2.1557为出发菌株,依次进行两轮紫外线诱变和亚硝基胍诱变,并以10-4与10-3mol/Lβ-紫罗酮作为筛选剂,得到突变株UV-N2-7,其虾青素产量和含量分别较出发菌株提高81.7%和2.25倍。采用Plackett-Burman设计法及响应面分析法对发酵培养基的组分及发酵条件进行了优化,突变株的虾青素产量由从初始的2.674 mg/L提高到了6.338 mg/L。建立了红法夫酵母的代谢网络,并对比了突变株和野生株间歇培养条件下对数生长期的代谢通量分布。结果表明:突变株与野生株相比,PP途径通量有所减小,而EMP途径和TCA循环有所增强,突变株用于菌体生长的通量有所减小;二者的丙酮酸脱氢酶的活性均较低,分泌至胞外的丙酮酸的代谢通量约为32%。因此预计通过遗传改造和发酵控制提高丙酮酸脱氢酶的活性可能会进一步提高虾青素的产率。     

高产谷胱甘肽酿酒酵母的选育及发酵工艺的研究

以酿酒酵母SC-001为出发菌株,利用紫外诱变结合乙硫氨酸和氯化锌的底物选择筛选到1株有较高GSH产量的酿酒酵母菌株SC-001-24,其GSH产量为743 mg/L,比出发菌株提高了22%。进一步在10 L发酵罐中考察了不同发酵模式和前体氨基酸补加方式对酿酒酵母产GSH的影响,确定最佳发酵模式为补料分批发酵,最佳前体氨基酸补加策略为:初始培养基加入41 mmol/L半胱氨酸,发酵42 h后一次性补加半胱氨酸246 mmol,甘氨酸160 mmol,谷氨酸82 mmol。在最佳发酵条件下GSH产量达到1 280.4 mg/L。     

酵母菌中谷胱甘肽的主要生理功能及其代谢调控

谷胱甘肽是生物体内一种重要的三肽小分子 ,具有广泛的生理功能。对谷胱甘肽在酵母细胞中的作用及其代谢调控机制 ,做了较为详细的介绍。这一带有基础性研究的内容 ,对于以酵母为生产菌的谷胱甘肽的生产 ,或是酵母的其他工业化生产 ,具有重要的启示。     

谷胱甘肽高产菌株的选育

以编号为 346的酿酒酵母为出发菌株 ,通过紫外线和_{60Coγ射线诱变处理 ,运用推理育种技术 ,选育到一株抗氯化锌和乙硫氨酸的突变株 0 5Eth40 0 5。该菌株经摇瓶发酵谷胱甘肽产量为 1 65 96mg L ,较出发株提高 350 % ,每克干细胞含谷胱甘肽 1 9 76mg ,较出发株提高 31 8 6 %。菌株经 1 0次传代培养 ,谷胱甘肽产量下降 1 0 7% ,是一株性状较稳定可深入开发研究的优良菌株。}     

基于代谢通量分析的琥珀酸放线杆菌高产选育研究

在对产琥珀酸放线杆菌代谢分析的基础上选育出高产突变株对琥珀酸的工业生物转化有重要意义.在矩阵分析代谢通量基础上,围绕柔性节点下的副产物乙酸及乙醇的降低分别实施软X诱变及定点突变选育,并对比分析了突变株与出发株相关酶活及基因序列变化.针对出发株的流量分析显示产物琥珀酸的代谢通量为1.78(mmol/g/h),主要副产物乙酸与乙醇的代谢通量分别为(0.60mmol/g/h)和(1.04 mmol/g/h),并发现乙醇代谢加剧了琥珀酸合成中的H电子供体的不足;筛选出的氟乙酸抗性突变株S.JST1的乙酸代谢通量降低了96%,为0.024(mmol/g/h),酶活检测表明磷酸乙酰转移酶(Pta)的酶比活力从602降低到74,进一步的序列对比分析发现pta突变基因中产生了一个突变位点:adh定点复合突变株S.JST2的乙醇代谢通量降低了98%,为0.020(mmol/g/h),酶活检测表明Adh的酶比活力从585降低到62.最终突变株S.JST2琥珀酸累积产量达65.7 g/L.围绕产琥珀酸放线杆菌Pta及Adh酶活的降低实施定向选育,在降低副产物流量的同时,有助于改善细胞H供体代谢平衡进而提高琥珀酸的流量.所获突变株具有工业应用潜力.     

高产谷胱甘肽新菌种的选育及其发酵条件的研究

通过对谷胱甘肽新产生菌——藤黄八叠球菌进行紫外诱变处理,筛选到一株抗乙硫氨酸和氯化锌的抗性菌株,该突变株发酵生产谷胱甘肽的产量比出发菌株提高268．9％。通过对碳源、氮源、温度、初始pH等发酵条件对该菌株生物合成谷胱甘肽的影响研究,表明突变株HY78在发酵温度37℃、初始pH7.0、摇床转速180r／min条件下,摇瓶发酵26h,该高产菌株在发酵液中合成谷胱甘肽的产量可达160．628mg／L。     

Actinobacillus succinogenes抗氟乙酸突变株的选育及其代谢流量分析

琥珀酸是一种用于合成树脂、可降解塑料及许多化学中间体的重要绿色化工原料。为了提高琥珀酸的发酵产率, 基于Actinobacillus succinogenes的代谢流量分布情况对其育种机制进行了研究。以Actinobacillus succinogenes CGMCC1593为原始菌株进行NTG诱变, 挑选在含有50~100 mmol/L氟乙酸平板生长较快的菌落, 经过初筛和复筛, 发现SF-9菌株产生更多琥珀酸且积累乙酸较少。以50 g/L的葡萄糖为碳源, 在5 L发酵罐上进行分批发酵, 该菌株发酵32 h时琥珀酸产量(34.8 g/L)提高了23.4%, 琥珀酸/乙酸比率为9:1, 副产物乙酸量比原始菌株降低了约50%。代谢流量分析(MFA)结果表明, PEP是影响琥珀酸合成的关键节点, PYR是影响乙酸等杂酸生成比例的关键节点, 并且这两个节点均非刚性节点。通过氟乙酸抗性诱变, 成功地筛选出了流向乙酸、甲酸和乳酸等杂酸的流量相对减少, 而流向琥珀酸的流量明显增强的突变菌株SF-9。     

Actinobacillus succinogenes抗氟乙酸突变株的选育及其代谢流量分析

琥珀酸是一种用于合成树脂、可降解塑料及许多化学中间体的重要绿色化工原料。为了提高琥珀酸的发酵产率, 基于Actinobacillus succinogenes的代谢流量分布情况对其育种机制进行了研究。以Actinobacillus succinogenes CGMCC1593为原始菌株进行NTG诱变, 挑选在含有50~100 mmol/L氟乙酸平板生长较快的菌落, 经过初筛和复筛, 发现SF-9菌株产生更多琥珀酸且积累乙酸较少。以50 g/L的葡萄糖为碳源, 在5 L发酵罐上进行分批发酵, 该菌株发酵32 h时琥珀酸产量(34.8 g/L)提高了23.4%, 琥珀酸/乙酸比率为9:1, 副产物乙酸量比原始菌株降低了约50%。代谢流量分析(MFA)结果表明, PEP是影响琥珀酸合成的关键节点, PYR是影响乙酸等杂酸生成比例的关键节点, 并且这两个节点均非刚性节点。通过氟乙酸抗性诱变, 成功地筛选出了流向乙酸、甲酸和乳酸等杂酸的流量相对减少, 而流向琥珀酸的流量明显增强的突变菌株SF-9。     

啤酒酵母代谢工程研究进展

啤酒工业上应用的啤酒酵母菌株在生产中都会存在着某些方面的缺陷。通过分析啤酒酵母某些代谢产物的代谢途径,寻找改变其代谢流量的方法,然后用分子生物学手段对其代谢流量加以改变,来调节啤酒酵母某些产物的代谢水平已经成为啤酒酵母育种的新方式。对酵母的底物利用、可操作性、控制有害副产物的产量及改善啤酒风味等方面的研究成果进行了综述。     

乙醇耐受性酿酒酵母菌株选育的研究进展

酿酒酵母是工业发酵生产乙醇的重要菌种,但是其发酵产物乙醇对酿酒酵母有明显的抑制作用.选育乙醇耐受性酿酒酵母是克服高浓度乙醇的抑制作用,提高乙醇产量的一条重要途径.本文对近年来国内外选育乙醇耐受性酵母的研究作一综述,旨在为乙醇耐受性酵母的选育提供参考.     

Metabolic flux analysis of a glycerol-overproducing Saccharomyces cerevisiae strain based on GC-MS, LC-MS and NMR-derived C-labelling data

This study focuses on unravelling the carbon and redox metabolism of a previously developed glycerol-overproducing Saccharomyces cerevisiae strain with deletions in the structural genes encoding triosephosphate isomerase (TPI1), the external mitochondrial NADH dehydrogenases (NDE1 and NDE2) and the respiratory chain-linked glycerol-3-phosphate dehydrogenase (GUT2). Two methods were used for analysis of metabolic fluxes: metabolite balancing and (13)C-labelling-based metabolic flux analysis. The isotopic enrichment of intracellular primary metabolites was measured both directly (liquid chromatography-MS) and indirectly through proteinogenic amino acids (nuclear magnetic resonance and gas chromatography-MS). Because flux sensitivity around several important metabolic nodes proved to be dependent on the applied technique, the combination of the three (13)C quantification techniques generated the most accurate overall flux pattern. When combined, the measured conversion rates and (13)C-labelling data provided evidence that a combination of assimilatory metabolism and pentose phosphate pathway activity diverted some of the carbon away from glycerol formation. Metabolite balancing indicated that this results in excess cytosolic NADH, suggesting the presence of a cytosolic NADH sink in addition to those that were deleted. The exchange flux of four-carbon dicarboxylic acids across the mitochondrial membrane, as measured by the (13)C-labelling data, supports a possible role of a malate/aspartate or malate/oxaloacetate redox shuttle in the transfer of these redox equivalents from the cytosol to the mitochondrial matrix.     

代谢工程改造酿酒酵母生产L-乳酸的研究进展

L-乳酸是一种重要的有机化合物,具有广泛的应用价值。微生物发酵法生产是当前L-乳酸的主要来源,但受限于精确的发酵条件、菌体产物耐受能力低及底物要求高等因素,导致L-乳酸供给不足且价格偏高。鉴于酿酒酵母利用廉价底物生产有价值物质方面的诸多优势,并随着分子生物学技术的发展,利用代谢工程改造酿酒酵母本身固有的代谢网络,使其高产L-乳酸已成为当前研究的热点。从L-乳酸的异源生产、关键途径改造及菌体生长能力恢复三个方面归纳了关于代谢工程改造酿酒酵母生产L-乳酸的研究进展。最后,指出了酿酒酵母异源生产L-乳酸存在的不足和今后研究的方向。     

酿酒酵母单倍体菌株分离筛选及其产腺苷甲硫氨酸的初步研究

获得产腺苷甲硫氨酸的二倍体酿酒酵母CGMCC 2842遗传育种单倍体亲本。采用不同产孢培养基考察了酿酒酵母的产孢率,并对酿酒酵母CGMCC 2842进行了生孢培养分离子囊孢子得到单倍体菌株,确定单倍体配型,测定不同单倍体菌株腺苷甲硫氨酸含量。从分离的七株单倍体菌株(6株a型和1株α型)中筛选出一株产腺苷甲硫氨酸较高的a配型的单倍体菌株,经250 m L摇瓶发酵48 h后产腺苷甲硫氨酸1.10 g/L。筛选得到了一株产腺苷甲硫氨酸较高a型的单倍体菌株,为菌株的进一步遗传育种改良和腺苷甲硫氨酸微生物发酵法规模化生产奠定了基础。     

酿酒酵母乙酸耐受性相关的微卫星分子标记筛选

【目的】筛选与酵母乙酸耐受性状紧密相关的微卫星分子标记。【方法】以两株表型差异菌株YHA和YLA作为亲本构建F2代菌株共计160株,选取15个微卫星位点通过PCR方法在40株子代中扩增产物,利用SPSS 11.5软件分析耐酸性状与微卫星序列间的相关性。【结果】找到3个与乙酸耐受性性状相关的微卫星位点,其中位点14P2与酵母乙酸耐受性状有极显著的正相关性(P<0.01),15P2和15P3与酵母乙酸耐受性具有显著的负相关性(P<0.01和P<0.05);此外对于微卫星位点14P2,耐酸亲本YHA在该位点的基因片段(344 bp)在子代耐酸菌株中出现频率达到70.6%,而不耐酸亲本YLA的基因片段(331 bp)在子代不耐酸菌株中出现的频率达91.3%。【结论】微卫星14P2的等位基因在子代菌株中的遗传具有明显的偏好性,该微卫星位点与某种耐酸基因存在一定的连锁遗传,为酵母分子标记辅助育种提供了有价值的遗传标记。     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

Metabolic flux analysis of the sterol pathway in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model system for examining the biosynthesis of sterols in eukaryotic cells. To investigate underlying regulation mechanisms, a flux analysis of the ergosterol pathway was performed. A stoichiometric model was derived based on well known biochemistry of the pathway. The model was integrated in the Software COMPFlux which uses a global optimization algorithm for the estimation of intracellular fluxes. Sterol concentration patterns were determined by gas chromatography in aerobic and anaerobic batch cultivations, when the sterol metabolism was suppressed due to the absence of oxygen. In addition, the sterol concentrations were observed in a cultivation which was shifted from anaerobic to aerobic growth conditions causing the sterol pools in the cell to be filled. From time-dependent flux patterns, possible limitations in the pathway could be localized and the esterification of sterols was identified as an integral part of regulation in ergosterol biosynthesis.     

杂交瘤细胞的代谢流量分析

应用代谢流量平衡模型定量分析了杂交瘤细胞的代谢流量分布。结果表明,在连续培养的杂交瘤细胞中,当葡萄糖和谷氨酰胺的流加浓度分别为13.8和2.6 mmol·L^-1时,86.2%的葡萄糖通过糖酵解生成乳酸,7.5%的生成脂类,进入TCA循环的仅占0.83%;谷氨酰胺中的氮有3%用于核酸的合成,54.5%生成氨,另有38.2%生成非必需氨基酸,碳骨架61.6%生成非必需氨基酸,34.1%进入TCA循环。     

L-色氨酸生物合成的代谢流量分析

建立了谷氨酸棒杆菌合成L-色氨酸(L-Try)的代谢流量平衡模型,应用该模型计算出发酵中后期的代谢流分布并通过MATLAB软件线性规划得到Try理想代谢流分布。结果表明75．15％的碳架进入糖酵解,24．85％的碳架进入HMP途径;但与理想代谢流相比,应从遗传改造和发酵控制方面降低TCA循环的代谢流,减少副产氨基酸的生成,摸索最适的溶氧控制对提高Try产率至关重要。