pH值对运动发酵单胞菌葡萄糖代谢酶活性影响的研究

以运动发酵单胞菌(Zymomonas mobilis)ATCC31821为模式菌株,研究pH值条件对其葡萄糖代谢关键酶活力的影响.结果表明:发酵液pH5.5时,胞内乙醇脱氢酶、丙酮酸脱羧酶、葡萄糖激酶、葡萄糖-6-磷酸脱氢酶的活力较高,而异柠檬酸脱氢酶活性较低,能促进乙醇的生成;pH 5.0时,苹果酸脱氧酶的酶活力较低,使糖酵解反应向另一个方向发生偏移,促进乙醇的形成.pH 4.0～6.5时,丙酮酸激酶和甘油醛-3-磷酸脱氢酶的酶活力水平相差不大,说明pH值对这2种酶的活性影响甚微.因此,pH5.0～5.5,代谢途径(如糖酵解途径、ED途径等)中的胞内代谢酶活性增强,有利于乙醇的产生.     

不同代数啤酒酵母对胞内代谢关键酶活性的影响

对不同代数啤酒酵母在丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、苹果酸脱氢酶、异柠檬酸脱氢酶、乙醇脱氢酶的酶活性特征方面的差异进行了比较分析,发现酵母在不断传代过程中,胞内代谢关键酶的活性均呈现一定变化,而丙酮酸激酶、葡萄糖-6-磷酸脱氢酶和乙醇脱氢酶变化最为明显,并且与酵母活力有着紧密的联系,当3种酶活性高时,酵母活力旺盛,代谢物质的量较为协调.     

溶氧量对酿酒酵母及其工程菌的β-苯乙醇合成代谢的影响及调控效应

目的:研究溶氧量对酿酒酵母S288C及工程菌ARO8-10的β-苯乙醇合成代谢的影响及调控效应。方法:在5 L发酵罐中装液量60%,控制p H 5.0,温度30℃,通气比0.5~2.0 vvm,定时取样检测发酵液生物量、β-苯乙醇、L-苯丙氨酸转化率以及菌体相关代谢酶活性等。结果:通气条件在0.5~2.0 vvm范围时,野生型菌株S288C的生物量、异柠檬酸脱氢酶(ICDH)酶活力明显高于转氨酶基因ARO8与脱羧酶基因ARO10耦合过表达工程菌ARO8-10;ARO8-10的β-苯乙醇发酵产量、L-苯丙氨酸转氨酶、苯丙酮酸脱羧酶、G6PD、GK及PDC酶活力均明显高于S288C(P≤0.01)。在1.0 vvm通气条件发酵60 h时,ARO8-10的发酵液中葡萄糖已基本用尽,β-苯乙醇产量达到2.68 g/L,L-苯丙氨酸转化率达到47.3%,较S288C分别提高了44.4%和12.4%。1.0 vvm为β-苯乙醇发酵较适宜的通气条件。结论 :酿酒酵母的β-苯乙醇代谢与艾利希途径、莽草酸途径、EMP、PPP途径TCA循环等密切相关,构成其合成代谢网络,代谢途经多种酶相互影响;ARO8和ARO10基因耦合过表达,增强了L-苯丙氨酸氨基转移酶和苯丙酮脱羧酶活力,有利于提高G6PD、GK及PDC酶活力,β-苯乙醇代谢流量及产量。     

乙醇胁迫对乳酸杆菌关键酶活力的影响

分别以面包乳杆菌(Lactobacillus crustorum D2-5)和植物乳杆菌(Lactobacillus plantarum D5-5)为研究对象,进行不同乙醇体积分数胁迫后菌体活力的对比,测定了乙醇胁迫后菌体中己糖激酶(HK)、丙酮酸激酶(PK)、乳酸脱氢酶(LDH)及ATP酶的活力变化,分析了乙醇胁迫后造成菌体失活的主要因素。结果表明:5%乙醇胁迫处理有助于提高菌体存活率,但是效果不显著;8%乙醇胁迫后的菌体存活率明显降低,面包乳杆菌D2-5和植物乳杆菌D5-5存活率依次仅为26.61%和23.50%,菌体的生长受到严重抑制,致使相关酶活力降低。乙醇胁迫对乳酸杆菌己糖激酶影响不显著,对其丙酮酸激酶、乳酸脱氢酶及ATP酶具有极显著的影响。这一结果说明,丙酮酸激酶、乳酸脱氢酶及ATP酶是影响乙醇胁迫损失的关键酶,酶活力变化与菌体活力相关。     

渗透压对酿酒酵母胞内代谢关键酶活性的影响

对耐高渗酵母与普通酿酒酵母在丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、苹果酸脱氢酶、异柠檬酸脱氢酶、乙醇脱氢酶的酶活性特征的差异进行比较分析,建立酶活力测定方法.发现耐高渗酵母在高渗环境的诱导下,其EMP途径、磷酸戊糖途径的关键酶活力都高于普通酿酒酵母.耐高渗酵母具有高活力的乙醇脱氢酶,其能高效地将丙酮酸转化成乙醇.并且三羧酸循环中的关键酶异柠檬酸脱氢酶和苹果酸脱氢酶的活性也得到加强.这些酶活力的增强维持了耐高渗酵母在高渗、高酒精环境下的生长需要与能量代谢的平衡.     

乳酸乳球菌中双乙酰代谢支路的调控

描述了从柠檬酸代谢、糖酵解来形成双乙酰的途径及其调节思路，并提出乳酸酸乳球菌的混合酸发酵及其调控机理，对双乙酰代谢支路的的遗传修饰方法进行了综述。结果表明，在特定条件下，有些乳酸菌从同型乳酸发酵转道成为异型乳酸发酵而产生其他代谢物(如甲酸或CO2，乙酸和乙醇)，这种变化涉及到丙酮酸代谢的遗传修饰：降低乳酸脱氢酶(LDH)活力或α-乙酰乳酸脱羧酶(ALD)活力、增加丙酮酸甲酸盐裂解酶(PFL)(厌氧条件)或α-乙酰合成酶(ALS)或丙酮酸脱氢酶(PDH)活力(好氧条件)，这些方法会有效地将糖转化成为双乙酰。对双乙酰代谢支路的调控主要集中在：超产αls或ileBN，失活ldh，pft，aldB或与超产NADH-氧化酶方法相结合使用。     

重组大肠杆菌内PDC和ADH表达条件优化

丙酮酸脱羧酶和乙醇脱氢酶II可将丙酮酸定向转化成乙醇。将已构建的含有丙酮酸脱羧酶和乙醇脱氢酶II基因的表达载体p ET-28a(+)-pdc-RBS-adh B转入大肠杆菌BL21中,实现在大肠杆菌体内高产乙醇的目的。对该工程菌株进行定性检测,优化诱导表达条件,定量检测,SDS-PAGE分析和做发酵试验,结果表明丙酮酸脱羧酶和乙醇脱氢酶II的最佳表达条件为IPTG浓度1.0 mmol/L,诱导时间7 h,诱导温度37℃。其最大酶活分别为1.34 U/mg和3.88 U/mg。重组子利用葡萄糖、木糖和混合糖发酵。在葡萄糖中发酵72 h,获得最大乙醇产量6.86 g/L,最高乙醇得率0.40 g/g。     

重组毕赤酵母表达人血清白蛋白-人白介素2融合蛋白过程的代谢特性

分析了重组甲醇营养型毕赤酵母在5L多参数自控发酵罐上生产人血清白蛋白-人白介素2融合蛋白(HSA-IL-2)的发酵过程。在使用多种在线传感器和离线检测参数的基础上,应用相关性分析的方法将细胞生长的关键酶(乙醇氧化酶、葡萄糖-6-磷酸脱氢酶和异柠檬酸脱氢酶)酶活和代谢特性相关联。实验结果显示,甲醇诱导后葡萄糖-6-磷酸脱氢酶和异柠檬酸脱氢酶的酶活分别下降了46.1%和66.8%,说明胞内代谢流发生了迁移:三羧酸循环(TCA)和磷酸戊糖途径(HMP)中代谢流通量下降,甲醇完全氧化代谢成为主要代谢流。此外,在不同控制策略下,乙醇氧化酶的酶活和重组蛋白HSA-IL-2表达量也呈现出一定的差异。高溶氧低甲醇控制方式下HSA-IL2的表达量达到27mg/L,比低溶氧高甲醇条件下的表达量高33.7%。     

镁、锰离子对黄原胶生物合成的影响

通过添加不同浓度镁、锰的一系列摇瓶实验,研究野油菜黄单胞菌代谢过程中的关键酶：葡萄糖-6- 磷酸脱氢酶(G-6-PDH)和丙酮酸激酶(PYK)的活性变化,确定最佳单因素金属离子添加方案：Mg2+ 添加量0.4g/100ml、Mn2+ 添加量0.04g/100ml,随后进行罐发酵实验,从而对摇瓶实验结果进行验证并研究金属离子对黄原胶罐发酵生物合成的影响。实验结果表明：0.4g/100ml Mg2+ 可以较大程度提高G-6-PDH 的活性,与摇瓶实验结果基本一致;0.04g/100ml Mn2+ 大大提高了PYK 的活性,与摇瓶实验结果一致;5L 发酵罐中发酵液残糖总体呈下降趋势,在60h以后趋向平稳,Mg2+、Mn2+ 会一定程度上增加发酵液中残糖含量,从而降低碳源的利用率。     

不同葡萄糖转运途径相关基因的过表达对菌株葡萄糖代谢及产物合成的影响

磷酸烯醇式丙酮酸-葡萄糖磷酸转移酶系统(phosphoenolpyruvate-dependent glucose phosphotransferase system,PTS~(Glc))和不依赖于PTS~(Glc)系统葡萄糖转运系统在葡萄糖转运和磷酸化过程中起重要作用。该文通过在葡萄糖代谢缓慢的L-赖氨酸产生菌Corynebacterium glutamicum(C.glutamicum)ZL-8中过表达肌醇透性酶基因(iol T1)、葡萄糖激酶基因(ppgk)和EIIGlc基因(pts G),研究葡萄糖转运途径相关基因对葡萄糖代谢速率、L-赖氨酸及副产物合成的影响。与出发菌株C.glutamicum ZL-8相比,重组菌C.glutamicum ZL-8/p ECXK-99E-ppgkiol T1(葡萄糖激酶酶活力提高了768%)和C.glutamicum ZL-8/p DXW-8-pts G(PTS~(Glc)系统酶活力提高了62.7%)葡萄糖消耗速率分别提高了16.7%和27.3%,L-赖氨酸产量分别提高了24.7%和16.4%。此外,有机酸和氨基酸分析结果表明,过表达不同葡萄糖转运途径基因对有机酸(丙酮酸、乳酸和乙酸)和副产物氨基酸(丙氨酸、缬氨酸、天冬氨酸)具有不同的影响。     

Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon

NADPH, a major reducing power in microorganisms, is mostly generated from the pentose phosphate (PP) pathway by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) expressed by the zwf and gnd genes, respectively. The characteristics of these two genes in Escherichia coli were compared after their re-introduction into the parent strain for over-expression. zwf encoding G6PDH increased the level of NADPH 3 folds compared to gnd encoding 6PGDH. An excess of NADPH depressed cell growth mainly due to the inhibition of citrate synthase in the TCA cycle. Recombinant plasmids containing zwf or gnd co-integrated with the phbCAB operon from Ralstonia eutropha were constructed, and introduced into E. coli for the oddball biosynthesis of PHB. The amount of PHB increased after enforcing the genes; especially the zwf gene an increase of around 41%, due to the rise in NADPH and the depressed TCA cycle, leading to the metabolic flux of intermediates to the pathway for the biosynthesis of PHB.     

‘印度青’苹果合成与分解代谢通量研究及贮藏温度的优化

本研究以‘印度青’苹果为原料，研究在不同贮藏温度下合成与分解代谢网络通量，并在此基础上优化其贮藏温度。将‘印度青’苹果放置在7 个不同温度（0、2、5、10、15、20、40 ℃）条件下，测定‘印度青’苹果不同部位糖酵解途径、三羧酸循环、蔗糖合成途径、磷酸戊糖途径的代谢通量。结果显示：2 ℃时蔗糖有较高的合成通量，由外到内4 个部位分别为32、42、47和33，乳酸盐也在2 ℃时有较高的代谢通量，由外到内4 个部位分别为1.87、1.86、1.86和1.86，在此温度下有较低的分解代谢和较高的蔗糖合成代谢，因此，2 ℃是‘印度青’苹果较好的贮藏温度。通过不同温度下‘印度青’苹果的各个部位之间代谢通量上的变化，可以建立以乳酸盐与蔗糖通量为评价标准的定量化模型，这可为今后更好地研究延长水果的贮藏寿命提供新的研究方法和理论参考。     

热处理对哈密瓜蔗糖和乳酸通量的影响

对哈密瓜进行热处理会更好地提高其贮藏品质,从蔗糖积累和呼吸代谢的角度研究热处理更能揭示其作用的本质。以哈密瓜为材料,研究热处理对哈密瓜代谢网络通量的影响。将哈密瓜分别经过45、50、55 ℃热处理3 h之后立即置于最适贮藏温度5 ℃条件下贮藏21 h,以单一5 ℃处理24 h为对照,得到在不同温度条件下哈密瓜不同空间位置糖酵解途径、三羧酸循环、蔗糖合成途径、戊糖磷酸途径的代谢通量。结果显示：45 ℃和50 ℃处理组都有较高的蔗糖和乳酸通量,但以45 ℃处理组通量最高,作为热处理温度最为合适;而55 ℃处理组与空白对照无较大差别。本实验从代谢的角度深入研究果实的热处理贮藏,以期为提高果实的贮藏品质提供参考。     

通过体外代谢途径构建确定乳酸合成关键酶

基于代谢控制分析理论提出了一种通过体外代谢途径构建和分析确定关键酶的方法并应用其确定乳酸高产菌株中的关键酶。首先获得高产菌株的粗酶液并测定葡萄糖到乳酸合成途径中各种蛋白的绝对浓度,进而通过向粗酶液中分别添加同等比例的各纯酶对途径进行扰动,并由扰动前后的途径通量变化计算出各酶的通量控制系数以确定关键酶。结果表明,该菌株乳酸合成途径中丙酮酸激酶和甘油醛-3-磷酸脱氢酶对途径通量影响最大,由此预测在该菌株基础上进一步过表达这两个酶对提高乳酸生成速率可能最为有效。     

Changes in enzyme activities at the pyruvate node in glutamate-overproducing Corynebacterium glutamicum

Glutamate is industrially produced by fermentation using Corynebacterium glutamicum. The key factor for efficient glutamate production by this microorganism has been considered to be a metabolic change at the 2-oxoglutarate dehydrogenase (ODH) branch point caused by a decrease in ODH activity under glutamate-overproducing conditions. However, this change would be insufficient because the ODH branch is merely the final branch in the glutamate biosynthetic pathway, and efficient glutamate production requires a balanced supply of acetyl-CoA and oxaloacetate (OAA), which are condensed to form a precursor of glutamate, namely, citrate. Therefore, there must be another (other) change(s) in metabolic flux. In this study, we demonstrated that a decrease in pyruvate dehydrogenase (PDH) activity catalyzes the conversion of pyruvate to acetyl-CoA. It is speculated that carbon flux from pyruvate to acetyl-CoA decreases under glutamate-overproducing conditions. Furthermore, an increase in pyruvate carboxylase (PC) activity, which catalyzes the reaction of pyruvate to OAA, is evident under glutamate-overproducing conditions, except under biotin-limited condition, which may lead to an increase in carbon flux from pyruvate to OAA. These data suggest that a novel metabolic change occurs at the pyruvate node, leading to a high yield of glutamate through adequate partitioning of the carbon flux.     

呼吸代谢参与粉红单端孢侵染厚皮甜瓜果实不同阶段的防御反应

目的：探讨三羧酸（tricarboxylic acid cycle，TCA）循环和磷酸戊糖途径（pentose phosphate pathway，PPP）在病原真菌侵染果实中的作用。方法：用粉红单端孢（Trichothecium roseum）接种‘玉金香’厚皮甜瓜果实，观察（22±2）℃、相对湿度55%～60%条件下病斑直径的变化，测定接种果实病健交界处组织TCA循环和PPP关键酶活力以及中间产物含量的变化，分析不同侵染阶段TCA循环和PPP发挥的作用。结果：果实病斑直径在接种后24 h内无明显变化，48 h时明显增大，72 h时显著高于对照组（P＜0.05）。与对照组相比，接种粉红单端孢显著降低了早期果实的异柠檬酸脱氢酶活力（P＜0.05），而显著提高了苹果酸脱氢酶（malate dehydrogenase，MDH）活力（P＜0.05），24 h时MDH活力是对照组的2.14 倍；显著提高了早期果实烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide，NAD）和还原型烟酰胺腺嘌呤二核苷酸含量（P＜0.05），24 h时分别高出对照组50.37%和50.31%；激活了果实的NAD激酶，显著提高了后期葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶的活力（P＜0.05），48 h时分别是对照组的1.83 倍和2.51 倍；显著促进了后期果实烟酰胺腺嘌呤二核苷酸磷酸和葡萄糖-6-磷酸的合成（P＜0.05）；显著降低了早期和中期果实核糖-5-磷酸异构酶活力和核酮糖-5-磷酸含量（P＜0.05）。结论：粉红单端孢侵染厚皮甜瓜早期诱导了果实组织TCA循环，后期促进了PPP，说明TCA循环参与了更多果实早期的抗病防御，而PPP在后期寄主防御反应中发挥更大的作用。     

Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae

Succinate is the main taste component produced by yeasts during sake (Japanese rice wine) fermentation. The pathway leading to accumulation of succinate was examined in liquid culture in the presence of a high concentration (15%) of glucose under aerobic and anaerobic conditions using a series of Saccharomyces cerevisiae strains in which various genes that encode the expression of enzymes required in TCA cycle were disrupted. When cultured in YPD medium containing 15% glucose under aerobic conditions, the KGD1 (alpha-ketoglutarate dehydrogenase) gene disrupted mutant produced a lower level of succinate than the wild-type strain, while the SDH1 (succinate dehydrogenase) gene-disrupted mutant produced an increased level of succinate. On the other hand, the FUM1 (fumarase) gene disrupted mutant produced significantly higher levels of fumarate but did not form malate at all. These results indicate that succinate, fumarate and malate are mainly synthesized through the TCA cycle (oxidative direction) even in the presence of glucose at a concentration as high as 15%. When the growth condition was shifted from aerobic to anaerobic, the increased level of succinate in SDH1 disruptants was no longer observed, whereas the decreased level of succinate in the KGD1 diruptant was still observed. A double mutant of the two fumarate reductase isozyme genes (OSM1 and FRDS) showed a succinate productivity of 50% as compared to the parent when cells were incubated in glucose-buffered solution. These results indicate that succinate could be synthesized through two pathways, namely, alpha-ketoglutarate oxidation via the TCA cycle and fumarate reduction under anaerobic conditions.     

乳酸代谢通量调控规律的研究

选取了8株嗜酸乳杆菌进行分批发酵培养,分别测定了它们对数生长期乳酸发酵途径中10种相关酶的活性,分析了乳酸的代谢通量,并首次引入数量遗传学方法利用通径分析研究了酶活性对乳酸通量的直接和间接影响,建立了类似于代谢控制系数的决定系数R2(i)。该系数实际上反映了酶对乳酸通量的控制程度,研究结果显示,丙酮酸激酶(R2(PK)=0.3705)、己糖激酶(R2(HK)=0.3053)、磷酸丙糖异构酶(R2(TPI)=0.2733)和乳酸脱氢酶(R2(LDH)=0.2601)对乳酸通量具有相对较高的决定系数,对乳酸通量起主要控制作用;3-磷酸甘油醛脱氢酶(R2(GAPDH)= -0.1320)的决定系数为负值,对乳酸通量具有较为明显的负控制作用。本文首次应用数量遗传学方法研究相关酶对乳酸代谢通量的控制作用,这将为代谢控制分析提供新的思路。     

Metabolic flux analysis for efficient pyruvate fermentation using vitamin-auxotrophic yeast of Torulopsis glabrata

The metabolism of a vitamin-auxotrophic pyruvate-producing microorganism, Torulopsis glabrata IFO 0005, was investigated by metabolic flux analysis. Particular attention was focused on the effect of culture conditions, such as dissolved oxygen (DO) concentration and thiamine concentration, on specific pathway activities. The results of metabolic flux analysis indicate that the thiamine concentration significantly affected pyruvate dehydrogenase and pyruvate decarboxylase activities, and plays an important role in cell growth and pyruvate production. Metabolic flux analysis was also utilized to clarify the metabolism of this strain during pyruvate fermentation under different oxygen supply conditions, and the reason for the enhanced pyruvate production under conditions of 30-40% DO concentration was clarified from the viewpoint of intracellular flux distributions. Based on the analysis of the effect of thiamine concentration on the metabolic fluxes, we conducted a fed-batch experiment where the initial thiamine concentration was reduced to 30 microg/l and thiamine was added at 10 microg/l during fermentation when the cell growth rate decreased to 0.2 h(-1). With separate addition of thiamine, the overall pyruvate yield could be improved by 15% due to the decrease of ethanol production.