腺嘌呤对大肠杆菌DH5α和其耐乙酸突变株DA19 代谢流分布的影响

【目的】本文通过分析在基本培养基中添加腺嘌呤对大肠杆菌DH5α和其耐乙酸突变株DA19代谢流分布的影响,从而进一步了解二者在代谢调控方面的差异。【方法】对2个菌株分别在氮源限制基本培养基及添加腺嘌呤的氮源限制基本培养基中进行连续培养,分析两者代谢流变化差异,并与酶活测定结果进行比较。【结果】添加腺嘌呤降低了DH5α的葡萄糖比消耗速率和乙酸的比生成速率,提高了菌体关于葡萄糖的得率,而丙酮酸比生成速率变化不明显。与MN培养基相比,添加腺嘌呤后DH5α降低了乙酸分流比,提高了分泌丙酮酸和三羧酸循环分流比,同时明显改变了磷酸果糖激酶、6-磷酸葡萄糖脱氢酶和乙酸激酶酶活。与DH5α不同,添加腺嘌呤使得DA19的丙酮酸比生成速率增加了近57%,而其它参数无明显改变。与MN培养基相比,DA19在添加腺嘌呤后降低了三羧酸循环分流比,大大提高了分泌丙酮酸分流比,而关键酶活未发生明显改变。酶活变化与代谢流结果基本一致。【结论】由于大肠杆菌DH5α和DA19嘌呤核苷酸从头合成途径能力存在差异,因此添加腺嘌呤对两个菌株的代谢流分布产生了完全不同的影响。     

大肠杆菌DH5α及其耐乙酸突变株DA19在氮源限制下的代谢和关键酶特性研究

在氮源限制的基本培养基中对大肠杆菌DH5α及其耐乙酸突变株DA19进行连续培养,通过测定中心代谢途径关键酶的活性分析二者代谢差异。结果表明,DA19的6-磷酸葡萄糖脱氢酶(G6PDH)和异柠檬酸脱氢酶(ICDH)活性高于DH5α,而磷酸果糖激酶(PFK)和乙酸激酶(ACK)活性低于DH5α,反映了DA19进入磷酸戊糖途径(PPP)的碳流增加,而进入酵解和乙酸产生(Ack-Pta)途径的碳流减少。因此,关键酶活差异与DA19菌体关于葡萄糖得率提高、副产物乙酸和丙酮酸的生成减少相一致。添加腺嘌呤后,DH5α的G6PDH和ICDH活性增加,PFK和ACK活性降低,而DA19各酶活变化不明显。乙酸钠的添加导致除PFK外的其他酶活性均降低,尤其是DH5α的ICDH明显降低,这些结果反映的中心代谢途径流量变化也与二者生长和代谢副产物积累的变化一致。     

TCA循环中间产物对酿酒酵母胞内代谢关键酶活性的影响

对酿酒酵母在添加苹果酸、柠檬酸和琥珀酸的混合培养基与其在YEPD培养基中胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活力差异进行了对比分析。结果表明:添加苹果酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.82%、57.23%、39.15%、12.10%;添加柠檬酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶的酶活分别下降50.17%、42.20%、48.40%;添加琥珀酸使胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.16%、34.16%、50.87%、50.87%、12.37%。丙酮酸激酶、异柠檬酸脱氢酶和苹果酸脱氢酶对3种有机酸的耐受性较差,葡萄糖-6-磷酸脱氢酶、乙醇脱氢酶对3种有机酸的耐受具有选择性。     

碳源对重组大肠杆菌两阶段发酵产丁二酸的影响

研究了在好氧培养基中分别添加不同碳源对两阶段发酵菌体生长、酶活及代谢产物分布的影响,结果表明添加4mmol/L葡萄糖和12,54,80mmol/L乙酸钠均可以提高好氧阶段的菌体密度和相关酶活。将不同条件下培养的菌体转接厌氧发酵后,厌氧阶段的酶活和代谢产物分布也发生改变。进一步对酶活及代谢产物分析表明：Escherichia coli NZN111(sfcA)厌氧发酵过程中,磷酸烯醇式丙酮酸羧化激酶(PCK)是产丁二酸的关键酶,丙酮酸激酶(PYK)主要和副产物丙酮酸的积累有关,异柠檬酸裂解酶(ICL)对丁二酸产量也有一定影响。好氧培养基中添加80mmol/L乙酸钠,厌氧发酵结束时丁二酸的质量收率可达89.0%,相比对照提高了16.6%。     

外源添加代谢中间体对产琥珀酸放线杆菌厌氧发酵制备丁二酸的影响

考察了外源添加中间代谢产物对菌体生长及发酵产酸的影响,结果表明添加0.5g/L磷酸烯醇式丙酮酸(PEP)时丁二酸产量最高。围绕产琥珀酸放线杆菌NJ113厌氧发酵产丁二酸的代谢网络进行代谢通量分析,发现添加PEP后己糖磷酸途径(HMP)与糖酵解途径(EMP)的通量比由39.4∶60.3提高至76.8∶22.6,解决了丁二酸合成过程中还原力不足的矛盾,导致PEP生成草酰乙酸的通量提高了23.8%,丁二酸代谢通量从99.8mmol/(gDCW·h)增至124.4mmol/(gDCW·h),而副产物乙酸及甲酸的代谢通量分别降低了22.9%、15.4%;关键酶活分析结果表明,添加0.5g/LPEP后PEP羧化激酶比酶活达到1910U/mg,与对照相比提高了74.7%,而丙酮酸激酶的比酶活降低了67.5%。最终丁二酸浓度为29.1g/L,收率达到76.2%,比未添加PEP时提高了11.0%。     

大肠杆菌DH5α耐乙酸突变株的选育及其代谢特性研究

大肠杆菌DH5α是基因工程常用的宿主菌之一,但由于对代谢副产物乙酸十分敏感,影响外源基因的表达效率。为了提高E. coli DH5α乙酸耐受力,采用60Co诱变结合连续培养,逐步提高稀释率和乙酸钠选择压力,于含乙酸钠平板进一步筛选,得到5株对乙酸耐受能力显著增强的突变菌株,具有良好的遗传稳定性,其中DA19显示最强的耐受性能。DA19与DH5α相比,在复合培养基YPS和YPS2G中菌体浓度分别提高17％和5％,最大比生长速率分别提高8％和27％,产乙酸分别减少为6％和59％;在基本培养基中的细胞浓度提高24倍,在含10g/L乙酸钠培养基中达到的细胞浓度与不加乙酸钠DH5α的细胞浓度相当。     

基于代谢流量分布信息理解大肠杆菌中异柠檬酸裂解酶调节因子的代谢调控作用

基因的表达受不同的转录调节因子调节。大肠杆菌中的异柠檬酸裂解酶调节因子(IclR)能够抑制编码乙醛酸支路酶的aceBAK操纵子的表达。本研究基于代谢物的13C同位体物质分布来定量解析代谢反应,主要研究了iclR基因在大肠杆菌生理和代谢中的作用。大肠杆菌iclR基因缺失突变株的生长速率、糖耗速率和乙酸的产量相对于原始菌株都有所降低,但菌体得率略有增加。通过代谢途径的流量比率分析发现基因缺失株的乙醛酸支路得到了激活,33%的异柠檬酸流经了乙醛酸支路;戊糖磷酸途径的流量变小,使得CO2的生成量减少。同时,乙醛酸支路激活,但草酰乙酸形成磷酸烯醇式丙酮酸的流量基本不变,说明磷酸烯醇式丙酮酸-乙醛酸循环没有激活,没有过多的碳原子在磷酸烯醇式丙酮酸羧化激酶反应中以CO2形式排出,从而确保了菌体得率。葡萄糖利用速率的降低、乙酰辅酶A的代谢效率提高等使得iclR基因敲除菌的乙酸分泌较原始菌株有所降低。     

溶解氧对γ-聚谷氨酸合成的影响

在5L发酵罐上研究了溶解氧(DO)对地衣芽孢杆菌分批发酵生产γ-聚谷氨酸(γ-PGA)的影响并考察在8h、32h、56h时,葡萄糖激酶、6-磷酸葡萄糖脱氢酶、丙酮酸脱氢酶、异柠檬酸脱氢酶和谷氨酸脱氢酶的活性及对应时间点上γ-PGA的生产速率。通过溶解氧电极和搅拌转速的串联控制发酵过程中溶解氧水平,发现高溶解氧(60%)水平和低溶解氧(10%)水平均不能高效积累γ-PGA。6-磷酸葡萄糖脱氢酶活性的提高对产物的积累有抑制作用,葡萄糖激酶和谷氨酸脱氢酶的酶活提高对产物积累有促进作用,过高的丙酮酸脱氢酶和异柠檬酸脱氢酶的活性在一定程度上可以促进菌体生长但不利于产物的积累。此外,通过对三种不同DO水平下γ-PGA生物合成途径中相关代谢流量的计算表明,在p H 6.5的条件下,对于谷氨酸依赖型生产菌株,提高外源谷氨酸利用率可以促进γ-PGA的生物合成。     

外源添加代谢中间产物对米根霉生产富马酸的影响

通过外源添加代谢中间产物(苹果酸、草酰乙酸和丙酮酸),研究其对米根霉积累富马酸及代谢关键酶的影响。结果表明,添加4.0 g/L苹果酸时,富马酸质量浓度可达34.0 g/L,与对照相比提高了24.5%,同时提高富马酸酶(FUMR)比酶活,抑制乙醇脱氢酶(ADH)比酶活。添加少于1.0 g/L草酰乙酸可提高FUMR比酶活,降低ADH比酶活,促进富马酸积累;但添加高于1.0 g/L草酰乙酸抑制了FUMR和ADH比酶活,导致富马酸和乙醇产量下降。添加1.5 g/L丙酮酸,使富马酸的质量浓度从27.3 g/L增加到38.7 g/L,提高了41.8%,但FUMR比酶活下降34.7%,而ADH比酶活保持稳定。     

嗜乙酰乙酸棒杆菌Corynebacterium acetoacidophilum-Δldh缺氧条件下代谢葡萄糖途径的变化

缺氧条件下嗜乙酰乙酸棒杆菌Corynebacterium acetoacidophilum ATCC13870生长停滞,却能够代谢葡萄糖产生以乳酸和琥珀酸为主的有机酸。采用以sacB基因为反向筛选标记的同源重组染色体基因敲除系统,敲除嗜乙酰乙酸棒杆菌的乳酸脱氢酶基因,得到的Δldh菌株CCTCC NO.M20122041在缺氧条件下不产乳酸,葡萄糖消耗速率降低了29.3%,产琥珀酸和乙酸浓度分别提高45.6%和182%;NADH/NAD+值小于1(约0.7);磷酸烯醇式丙酮酸羧化酶和乙酸激酶的比酶活分别提高84%和12倍。说明嗜乙酰乙酸棒杆菌中乳酸合成途径的阻断驱使了琥珀酸和乙酸代谢途径加强,推测加强NADH供给和阻断乙酸产生支路可能是提高C.acetoacidophilum菌株产琥珀酸产量的有效途径。     

Acetate scavenging activity in <Emphasis Type="Italic">Escherichia coli</Emphasis>: interplay of acetyl–CoA synthetase and the PEP–glyoxylate cycle in chemostat cultures

Impairment of acetate production in Escherichia coli is crucial for the performance of many biotechnological processes. Aerobic production of acetate (or acetate overflow) results from changes in the expression of central metabolism genes. Acetyl−CoA synthetase scavenges extracellular acetate in glucose-limited cultures. Once converted to acetyl−CoA, it can be catabolized by the tricarboxylic acid cycle or the glyoxylate pathway. In this work, we assessed the significance of these pathways on acetate overflow during glucose excess and limitation. Gene expression, enzyme activities, and metabolic fluxes were studied in E. coli knock-out mutants related to the glyoxylate pathway operon and its regulators. The relevance of post-translational regulation by AceK-mediated phosphorylation of isocitrate dehydrogenase for pathway functionality was underlined. In chemostat cultures performed at increasing dilution rates, acetate overflow occurs when growing over a threshold glucose uptake rate. This threshold was not affected in a glyoxylate-pathway-deficient strain (lacking isocitrate lyase, the first enzyme of the pathway), indicating that it is not relevant for acetate overflow. In carbon-limited chemostat cultures, gluconeogenesis (maeB, sfcA, and pck), the glyoxylate operon and, especially, acetyl−CoA synthetase are upregulated. A mutant in acs (encoding acetyl−CoA synthetase) produced acetate at all dilution rates. This work demonstrates that, in E. coli, acetate production occurs at all dilution rates and that overflow is the result of unbalanced synthesis and scavenging activities. The over-expression of acetyl−CoA synthetase by cAMP−CRP-dependent induction limits this phenomenon in cultures consuming glucose at low rate, ensuring the recycling of the acetyl−CoA and acetyl−phosphate pools, although establishing an energy-dissipating substrate cycle.     

Metabolic regulation analysis of <Emphasis Type="Italic">icd</Emphasis>-gene knockout <Emphasis Type="Italic">Escherichia coli</Emphasis> based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements

An integrated study of cell growth characteristics, enzyme activities and protein expression patterns was carried out to investigate how the central metabolism of Escherichia coli changes upon knockout of the isocitrate dehydrogenase (ICDH) gene (icd) in the tricarboxylic acid cycle. Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption rate. The reduced specific growth rate in the icd mutant was due mainly to the lower intracellular ATP/ADP ratio as well as to the lower NADPH/NADP⁺ ratio compared with those in the parent strain. However, the specific carbon dioxide evolution rate was found to be higher in the icd mutant strain compared to the parent E. coli. This may be due to the higher activity of 6-phosphogluconate dehydrogenase, phosphoenol pyruvate carboxykinase and NADP⁺-dependent malic enzymes. The glyoxylate pathway was also utilized, as evidenced by the significant upregulation of isocitrate lyase and malate synthase activity in the icd mutant E. coli. The appearance of the glyoxylate pathway caused lower acetate production. Of 21 proteins showing altered expression levels, 17 were successfully identified with the aid of MALDI-TOF mass spectrometry. The results showed that the abolition of ICDH activity significantly affected the respiratory system and electron transport chain, as evidenced by the significant downregulation of proteins encoded by the genes nuoE, nuoH, cydA and cyoA in icd mutant E. coli compared to the parent.     

Acetate assimilation pathway of Methanosarcina barkeri.

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.     

Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase.

Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : I. Oxidative Activities with Soluble Substrates

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.     

Chemostat studies on the regulation of glucose metabolism in Pseudomonas aeruginosa by citrate

The effect of the relative concentrations of citrate and glucose on the regulation of key enzymes of the direct oxidative, phosphorylative, Entner-Doudoroff and pentose-cycle pathways of glucose metabolism in Pseudomonas aeruginosa has been investigated in continuous culture under conditions of NH(4) (+)-limitation. For comparison isocitrate dehydrogenase and aconitase were also assayed. Measurements were made for steady-state and transient conditions and the effect of growth rate was also studied. When cells grew on 75mm-citrate the glucose concentration had to attain 6-8mm before significant induction of enzymes of glucose metabolism occurred; the specific activities increased further as the result of both raising the glucose concentration to 30mm and then subsequently lowering the citrate to 60mm and then to 45mm. The specific activities of the glucose enzymes increased immediately during the transient period between the steady states characteristic of growth on 6mm- and 8mm-glucose, the increase continuing for about two doubling times. The converse experiment of adding increasing citrate concentrations to 45mm-glucose medium revealed an immediate induction of the citrate-transport system, oxidation of citrate following the increase in citrate concentration up to 8mm. Between 8mm- and 16mm-citrate a marked repression of gluconate, glucose 6-phosphate and 6-phosphogluconate dehydrogenases and the Entner-Doudoroff enzymes occurred. Increased growth rate in citrate medium resulted in decreased specific activities of glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase. Increased growth rate in citrate-glucose medium gave decreased specific activities of isocitrate dehydrogenase and aconitase whereas the activities of some of the glucose enzymes decreased initially but then increased at the highest growth rate (0.5h(-1)), at which a marked increase in glucose utilization occurred. These observations accord with the regulation of glucose enzymes by induction with glucose or its metabolites and repression by citrate or its metabolic products.     

Metabolic roles of peptone and yeast extract for the culture of a recombinant strain ofEscherichia coli

Summary The influence of complex compounds on the growth of a recombinant strain ofEscherichia coli containing the gene encoding glyceraldehyde 3-phosphate dehydrogenase, as well as the production of this enzyme have been studied. Batchwise cultures led to an accumulation of acetate, which was not utilized in a yeast extract-free medium. After glucose exhaustion, growth stopped and enzyme activity decreased. Whereas yeast extract allowed acetate assimilation and growth, peptone stabilized the enzymatic activity. The addition of both compounds resulted in optimal performances for enzyme production.     

Comparative Intermediary Metabolism of Vegetative Cells and Microcysts of Myxococcus xanthus

Crude extracts of both vegetative cells and glycerol-induced microcysts of Myxococcus xanthus contained the following enzyme activities: phosphofructokinase, phosphoglucoisomerase, fructose-1,6-diphosphatase, fructosediphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate carboxylase, citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and uridine diphosphate glucose pyrophosphorylase. With the exception of isocitrate dehydrogenase, which was present at a fivefold higher concentration in microcysts, all activities in extracts from both types of cells were essentially equal. Hexokinase and pyruvate kinase could not be detected in extracts from either type of cell. Microcysts metabolized acetate at a lower rate than did vegetative cells. Most of this decrease was reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO(2). In addition, microcysts and vegetative cells showed a different distribution of (14)C-label from incorporated acetate.