增强回补途径对谷氨酸棒状杆菌合成L-异亮氨酸的影响（英文）

该实验考察和比较增强回补途径对谷氨酸棒状杆菌合成Lacterium glutamicum-异亮氨酸的影响。通过以L-异亮氨酸生产菌Corynebppc YI为出发菌株,分别采用基因组整合和质粒的方式过表达磷酸烯醇式丙酮酸羧化酶编码基因及丙酮酸羧化酶编码基因pyc。结果表明,获得pyc基因组整合和质粒过表达菌株ILE01和ILE02,摇瓶条件下菌株L-异亮氨酸产量分别提高17. 3%(6. 1 g/L)和9. 6%(5. 7 g/L)、发酵罐条件下分别提高11. L7%(24. 8 g/L)和8. 1%(24. 0 g/L)。获得ppc基因组整合和质粒过表达菌株ILE03和ILE04,摇瓶条件下菌株-异亮氨酸产量分别提高(30. 8%(6. 8 g/L)和13. 5%(5. 9 g/L)、发酵罐条件下分别提高15. 8%(25. 7 g/L)和9. 5%24. 3 g/L)。此外,过表达pyc和ppc还可不同程度地提高L-异亮氨酸转化率。然而采用质粒过表达pyc和ppc均使得菌株生物量下降。因此,过表达pyc和ppc均能显著提高L-异亮氨酸产量和转化率,基因组整合的过表达方式效果优于质粒...     

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.     

基于基因转录和代谢物分析的异亮氨酸生产菌谷氨酸棒状杆菌YILW的理性改造

为获得异亮氨酸生产菌谷氨酸棒状杆菌（Corynebacterium glutamicum）YILW的理性改造策略,考察该菌株与出发菌株C. glutamicum ATCC 13032异亮氨酸合成途径中关键酶及代谢产物的差异。结果表明,C. glutamicumYILW丙酮酸羧化酶编码基因pyc的下调表达使得其胞内草酰乙酸含量降低,过表达该基因显著增加胞内草酰乙酸含量及异亮氨酸产量（分别从1.32 μmol/g（细胞干质量,下同）和5.18 g/L提高至3.32 μmol/g和5.81 g/L）,但副产物赖氨酸及胞内2-酮丁酸积累量提高。针对该问题采用强启动子替换手段过表达ilvBNC操纵子,使得其异亮氨酸产量提高至6.63 g/L。为进一步增加异亮氨酸合成,过表达输出载体编码基因brnE和brnF,其产量提高至7.31 g/L,较出发菌株C. glutamicum YILW提高41.1%,转化率提高40.0%。由此可见,在基因转录及代谢物分析结果指导下理性过表达pyc、ilvBNC操纵子及brnE和brnF能够显著提高异亮氨酸产量并降低副产物浓度。     

通过添加NaHCO_3和调节pH提高谷氨酸得率

研究了在谷氨酸发酵产酸期分别添加NaHCO3、调节pH及两者耦联操作条件下的发酵性能。结果表明:只有在升高pH值、添加NaHCO3同时进行或先升高pH值后添加NaHCO3的情况下,葡萄糖消耗量大幅下降,谷氨酸得率显著提高,分别比对照提高36%和34%,且谷氨酸产量可以达到正常水平(75 g/L以上)。酶学代谢分析表明,仅仅提高丙酮酸羧化酶活性不能提高谷氨酸得率,只有各个关键酶相互协调配合,即适度弱化丙酮酸脱氢酶和α-酮戊二酸脱氢酶活性的同时,适度提高丙酮酸羧化酶和异柠檬酸脱氢酶活性,才能有效提高谷氨酸发酵整体性能。     

BIOTIN AND THE METABOLISM OF BAKER'S YEAST

Based on experimental results and the results presented in the literature a model for the metabolism of baker's yeast under biotin deficiency is presented. In these conditions the most sensitive point in the metabolism is the carboxylation of pyruvate to oxaloacetate catalyzed by the biotin-containing pyruvate carboxylase. Because the rate of glycolysis is not affected by biotin-deficiency pyruvate accumulates in the cells and is partially excreted into the medium. The high pyruvate pool in the cells means that the metabolism is mainly fermentative even in vigorously aerated cultures. The oxidation of pyruvate to acetyl-CoA proceeds almost unaffected, as can be seen by the production of elevated amounts of ethyl acetate by yeast grown under sub-optimal biotin concentrations. Acetyl-CoA carboxylase, which also has biotin as the prosthetic group, is not as sensitive to a deficiency of this growth factor as is pyruvate carboxylase. In yeast grown without biotin the amounts of fatty acids and lipids are the same or even higher than in cells grown under optimal conditions. The metabolism from oxaloacetate towards the TCA cycle and glutamate is not affected as strongly as is the metabolism to aspartate, which is present in cells in strictly limited amounts. This causes an over-production of metabolic intermediates, e.g. diazotizable arylamine and hypoxanthine as well as citrulline. Their conversion to normal cell constituents, purine nucleotides and arginine, is dependent on the aspartate available and is thus depressed by lack of biotin.     

Changes in hepatic key enzymes of dairy calves in early weaning production systems

The objective of the present study was to describe plasma hormonal and metabolite profile and mRNA expression levels and activities of the enzymes pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and acetyl-coenzyme A (CoA) carboxylase in the liver of male Holstein calves before (1 and 3 wk of age) and after (8, 13, and 19 wk of age) weaning at 6 wk of age. The mean plasma concentration of acetate and β-hydroxybutyrate increased, and that of plasma lactate and nonesterified fatty acids decreased with week, particularly after weaning. Plasma glucose concentration was lowest at 8 wk of age. The mean plasma concentration of insulin and glucagon did not change with time, and that of cortisol was greatest at 1 wk of age. In the liver, enzyme activity of PC was greatest at 1 wk of age and decreased with time. There was a significant relationship between the activity and the mRNA level for PC. Activity of PEPCK also decreased with week. Acetyl-CoA carboxylase activity tended to decrease with week, and activity at 13 wk of age was lower than that at other times. Expression of PC mRNA, but not that of PEPCK and acetyl-CoA carboxylase α, decreased with week. We conclude that the hepatic gluconeogenic enzymes and acetyl-CoA carboxylase activities tend to decrease with age, reflecting changes in plasma metabolites in early weaning production systems.     

谷氨酰胺产生菌NS611的代谢流分析

建立并完善了谷氨酸棒杆菌NS611生产谷氨酰胺的中心碳代谢网络.研究了菌体生长和生产阶段的代谢流分布,结果说明该菌葡萄糖的代谢以糖酵解途径为主,在产酸阶段丙酮酸羧化反应的代谢流量较大。NADPH不是谷氨酸棒杆菌NS611代谢的限制性因素.研究了氮饥饿处理对代谢流分布的影响,提出要提高谷氨酰胺的产量必须适当保留α-酮戊二酸的向下氧化的功能,以保证能量供给.     

利用代谢网络模型和线性规划法在线预测谷氨酸发酵中产物浓度

在好氧型的谷氨酸发酵实验中发现，溶解氧（DO）对发酵性能有很大的影响，谷氨酸的生成方式也因此有很大不同：较低的DO水平能够延长产酸期、提高谷氨酸的最终浓度，但是代谢副产物——乳酸也有较大程度的积蓄；而DO水平过高，虽然代谢副产物不会生成积蓄，但菌体消亡过快导致产酸期缩短、谷氨酸的最终浓度降低．同时，谷氨酸的生成方式与发酵过程中摄氧率（OUR）和CO2的释放率（CER）有着非常紧密的关联．作者利用代谢网络模型并结合使用线性规划优化法，通过在线测定OUR和CER，比较准确地在线推定出发酵过程中谷氨酸的质量浓度变化。与传统的非构造式动力学模型相比，上述预测方法具有建模简单、模型物理意义明确、通用性能好等优点，为后续过程的在线控制和优化提供一种全新和有效的途径。     

Pyruvate Metabolism in Saccharomyces cerevisiae

In yeasts, pyruvate is located at a major junction of assimilatory and dissimilatory reactions as well as at the branch-point between respiratory dissimilation of sugars and alcoholic fermentation. This review deals with the enzymology, physiological function and regulation of three key reactions occurring at the pyruvate branch-point in the yeast Saccharomyces cerevisiae: (i) the direct oxidative decarboxylation of pyruvate to acetyl-CoA, catalysed by the pyruvate dehydrogenase complex, (ii) decarboxylation of pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase, and (iii) the anaplerotic carboxylation of pyruvate to oxaloacetate, catalysed by pyruvate carboxylase. Special attention is devoted to physiological studies on S. cerevisiae strains in which structural genes encoding these key enzymes have been inactivated by gene disruption.     

Partial biochemical characterization of tetrazolium red‐reactive Lactobacillus plantarum mutants

One thousand colonies derived from Lactobacillus plantarum ATCC 8014 cells that survived 34–43 × 10³ ergs cm^‐2 ultraviolet irradiation were screened on media containing tetrazolium red to detect fermentative mutants. Fermentation end‐products formed from pyruvate, glucose, or lactose catabolism were determined. All 37 stable tetrazolium red‐reactive mutants had increased pyruvate utilization compared to the wild‐type strain. Only two did not produce lactate from exogenous pyruvate. When glucose or lactose were substrates, these two mutants and six other representative mutants produced lactate at levels similar to the parent strain. Although the average lactate and acetoin production from pyruvate by the mutants and wild‐type strains were similar, 25% of the mutants had increased acetoin production.     

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。     

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.     

新型表面活性剂在谷氨酸发酵中的应用

通过对预先选择的8种表面活性剂和3种溶剂作为谷氨酸发酵促进剂效果的初步筛选,得知聚氧乙烯基失水山梨醇单脂肪酸酯三季铵盐(CTTE)和二甲基亚砜(DMSO)对产酸率提高有较为明显的效果;进而从加入时间,加入剂量两个方面研究了筛选出的两种表面活性剂对谷氨酸棒状杆菌(Corynebacterium glutamicum)T-613菌体生长和谷氨酸发酵产酸的影响,同时研究了CITE和DMSO同时加入的协同效果,从而得到最佳的发酵工艺参数. 研究结果表明:菌种生长对数期(10h)加入DMSO为0. 1%(V/V),发酵中期(18h)加入(CTTE)为0. 08%(V/V),在不影响菌种生长的前提下,促使谷氨酸发酵产酸率由4. 86%提高到6. 55%,较原产量提高了36. 46%,同时发酵的周期由44h下降为36h,缩短了8h,有效的提高了生产效率;最终结合染色显微照片,分析了表面活性剂促进谷氨酸发酵的机理.     

Cellular gluconeogenesis by lactating bovine mammary tissue.

The gluconeogenic capacity of mammary tissue of lactating cow was investigated by incubating mammary tissue slices with alanine, glutamate, lactate, pyruvate, or glycerol in conjunction with acetate and glucose (10mM or 1 mM). In no case was any substrate incorporated into glucose per se. In lactose synthesis, glucose was the major source of carbon although glycerol also was incorporated into lactose. Alanine, glutamate, lactate, or pyruvate were not incorporated into lactose at optimum (10 mM) or suboptimum (1 mM) concentrations of glucose. Activity of glucose-6-phosphatase was negligible in mammary tissue, less than 1% of the activity in liver or kidney tissue from the same cows. Pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and fructose-1,6-diphosphatase were in cow mammary tissue, but the activities were lower than in liver. Gluconeogenic substrates were not converted to glucose regardless of whether the incubation contained an optimum (10 mM) or a suboptimum (1 mM) glucose concentration. Consistent with the inability of cow mammary tissue to convert gluconeogenic metabolites to glucose is the virtual absence of glucose-6-phosphatase and the lack of excess gluconeogenic substrates available to the intact mammary gland of lactating cow.     

Changes in mRNA expression for gluconeogenic enzymes in liver of dairy cattle during the transition to lactation

The objective of this study was to profile phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) mRNA expression in the liver of dairy cattle during the peripartum transition and determine changes in abundance of these mRNA in response to protein fed during the prepartum period. Thirty-eight multiparous Holstein cows were fed diets containing either 12% crude protein (CP) and 26% rumen undegradable protein (RUP), 16% CP and 26% RUP, 16% CP and 33% RUP, or 16% CP and 40% RUP on a dry-matter basis beginning 28 d before expected calving. After calving, all cows were fed a common diet through 56 d in milk (DIM). Northern analysis of RNA from liver biopsy samples obtained on days -28, -14, +1, +28, and +56 relative to calving indicated that PC and PEPCK mRNA expression were responsive to onset of lactation but not to prepartum protein or RUP concentration. Abundance of PEPCK mRNA was similar at -28, -14, and +1 DIM but was elevated by +28 and +56 DIM relative to precalving levels. Liver PC mRNA abundance was elevated on +1 DIM, remained elevated through 28 DIM, and declined to precalving levels by 56 DIM. The activity of PC enzyme was correlated (r2 = 0.89) with PC mRNA abundance. The data demonstrate increased abundance of PC mRNA during the early transition period followed by increased abundance of PEPCK mRNA during the postpartum period and suggest increased potential metabolism of lactate, pyruvate, and amino acids that contribute to the liver pyruvate pool.     

Metabolite channeling and compartmentation in the human cell line AGE1.HN determined by 13C labeling experiments and 13C metabolic flux analysis

This study focused on analyzing active pathways and the metabolic flux distribution in human neuronal AGE1.HN cells that is a desirable basis for a rational design and optimization of producing cell lines and production processes for biopharmaceuticals. ¹³C-labeling experiments and ¹³C metabolic flux analysis were conducted using glucose, glutamine, alanine and lactate tracers in parallel experiments. Connections between cytosolic and mitochondrial metabolite pools were verified, e.g., flux from TCA cycle metabolite ¹³C to glycolytic metabolites. It was also found that lactate and alanine are produced from the same pyruvate pool and that consumed alanine is mainly directly metabolized and secreted as lactate. Activity of the pentose phosphate pathway was low being around 2.3% of the glucose uptake flux. This might be compensated in AGE1.HN by high mitochondrial malic enzyme flux producing NADPH. Mitochondrial pyruvate transport was almost zero. Instead pyruvate carbons were channeled via oxaloacetate into the TCA cycle which was mainly fed via α-ketoglutarate and oxaloacetate during the investigated phase. The data indicate that further optimization of this cell line should focus on the improved substrate usage which can be accomplished by an improved connectivity between glycolytic and mitochondrial pyruvate pools or by better control of the substrate uptake.     

Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identification of a suppressor of the pyc phenotype

We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source. Growth was possible with aspartate or glutamate as nitrogen source. The gene PpPYC1 expressd from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. In a P. pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions. The sequence of the PpPYC1 gene has been entered in the EMBL nucleotide sequence databank: Accession Number Y11106. © 1998 John Wiley & Sons, Ltd.     

基于响应面法优化谷氨酸棒杆菌发酵条件

该研究以谷氨酸棒杆菌（Corynebacterium glutamicum）P169为研究对象,以谷氨酸产量为主要评价指标,采用单因素试验和响应面法对其发酵条件进行优化,并进行摇瓶和20 L罐分批补料发酵验证。结果表明,谷氨酸棒杆菌P169产谷氨酸的最佳发酵条件为酵母粉41.0 g/L、葡萄糖27.0 g/L、尿素12.0 g/L和pH 7.0。在此优化条件下,谷氨酸产量达25.1 g/L,比优化前（16.5 g/L）提高了52.1%。以此为基料进行20 L罐分批补料发酵,谷氨酸产量达155 g/L,比优化前（142 g/L）提高了9.2%。该研究为提高谷氨酸棒杆菌谷氨酸产量提供了一种技术解决方案。     

高效合成L-高丝氨酸大肠杆菌基因工程菌株的构建

针对现有L-高丝氨酸生产菌株因营养缺陷需在发酵过程中添加L-苏氨酸等物质的问题,以大肠杆菌为底盘细胞,构建1株非营养缺陷型L-高丝氨酸高产菌株.首先通过下调thrB和thrC的转录弱化L-高丝氨酸的降解途径;并过表达thrA、ppc和pntAB以强化L-高丝氨酸合成代谢流、提高前体物以及辅酶供应;在此基础上过表达rht...     

利用代谢工程改造谷氨酸棒杆菌产丙酮酸研究

丙酮酸是一种重要的有机酸,可以作为前体物质,参与合成多种有机化合物,在生物体的能量代谢中发挥着重要作用。为提高丙酮酸产量,选择利用代谢工程改造谷氨酸棒杆菌(Corynebacterium glutamicum)生产丙酮酸。利用同源重组的方法,依次敲除谷氨酸棒杆菌中与丙酮酸代谢支流相关的5个关键基因(丙酮酸醌氧化还原酶基因pqo、丙酮酸羧化酶基因pyc、转氨酶基因alaT、缬氨酸-丙酮酸氨基转移酶基因avtA、丙酮酸脱氢酶基因aceE),摇瓶发酵72h后丙酮酸的产量达到14.64g/L。通过过表达编码转酮醇酶基因tkt、转醛酶基因tal、磷酸烯醇丙酮酸羧激酶基因pck,增加合成丙酮酸前体物质的供应。最终,复合培养基摇瓶发酵72h后,发酵液中丙酮酸的产量达到15.39g/L,与野生型菌株相比提高了28倍。研究旨在为利用微生物发酵生产丙酮酸提供一定的理论参考。