Role of tktA gene in pentose phosphate pathway on odd-ball biosynthesis of poly-beta-hydroxybutyrate in transformant Escherichia coli harboring phbCAB operon

A cloned tktA gene encoding transketolase (TK), the most critical enzyme in the nonoxidative pentose phosphate (PP) pathway, was reinforced into the parent Escherichia coli for metabolic flux control of carbohydrate. It was also transformed into the transformant E. coli harboring phbCAB operon for the odd-ball biosynthesis of PHB. The biosynthesis of PHB significantly increased up to 1.7-fold after the cotransformation of the phbCAB operon and tktA gene, due to the active supplementation of the precursor molecules, acetyl-CoA and NADPH.     

Utilization of oxidative pressure for enhanced production of poly-beta-hydroxybutyrate and poly(3-hydroxybutyrate-3-hydroxyvalerate) in Ralstonia eutropha

Ralstonia eutropha was cultivated under oxidative conditions in the presence of hydrogen peroxide and methyl viologen to stimulate the flux of NADPH to the PHA biosynthesis pathway. The effects of oxidants on the biosynthesis of poly-beta-hydroxybutyrate(PHB) and poly(3-hydroxybutyrate-3-hydroxyvalerate)[P(3HB-3HV)] were investigated. The biosynthesis rate and concentrations of PHB and its copolymer P(3HB-3HV) increased significantly under the oxidative pressure of methyl viologen, meanwhile, the molar fraction of 3HV in P(3HB-3HV) remained constant. The effect of methyl viologen on the flux of the intermediate compounds was investigated by measuring the activity of enzymes related to PHB biosynthesis, glucose 6-phosphate dehydrogenase (G6PD), and NADPH level. The oxidant significantly activated the G6PD of R. eutropha, increasing the NADPH level used for the reduction reaction of acetoacetyl-CoA to beta-hydroxybutyryl-CoA, thereby enhancing the biosynthesis of PHB and P(3HB-3HV).     

Modulation of talA gene in pentose phosphate pathway for overproduction of poly-beta-hydroxybutyrate in transformant Escherichia coli harboring phbCAB operon

The talA gene encoding transaldolase, the key enzyme in the nonoxidative pentose phosphate pathway, was amplified in a transformant Escherichia coli harboring the phbCAB operon to shift the metabolic flux of the hexose mono-phosphate shunt to the odd-ball biosynthesis pathway for poly-beta-hydroxybutyrate overproduction. The PHB content in the transformant E. coli coharboring the phbCAB operon and talA gene increased from 28.2% to 52.3%, and the retarded cell growth was overcome. This increase seems to be mainly due to the concomitant supplies of the intermediates NADPH and acetyl-CoA, which are from the activated pentose phosphate pathway through the modulation of the talA gene and from the Embden-Meyerhof pathway.     

葡萄糖-6-磷酸脱氢酶基因的Candida tropicalis过量表达及其对木糖醇合成代谢的影响

研究葡萄糖-6-磷酸脱氢酶基因g6pd过量表达对Candida tropicalis木糖醇生物合成代谢的影响。克隆Candidatropicalis CT16的g6pd基因,并将其与表达载体pYES-pgk重组连接,构建重组载体pYES-pgk-g6pd,LiAc/ssDNA/PEG方法转化导入C. tropicalis CT16,筛选阳性转化子,实现g6pd基因的过量表达。结果表明：发酵62 h,阳性转化子C. tropicalis SYG5的葡萄糖-6-磷酸脱氢酶活力提高了300%,发酵液中木糖醇质量浓度达到79.90 g/L,较野生型对照菌株的木糖醇产量提高了12.41%,木糖醇产率提高了44.94%。因此,葡萄糖-6-磷酸脱氢酶在C. tropicalis木糖醇的合成代谢途径中发挥重要作用,增强g6pd基因的表达,可以明显提高菌体NADPH供应量和还原力,有利于木糖醇的生物合成。     

Effect of modifying metabolic network on poly-3-hydroxybutyrate biosynthesis in recombinant Escherichia coli

The regulatory mechanism for poly-3-hydroxybutyrate (PHB) biosynthesis in recombinant Escherichia coli is markedly different from that of Ralstonia eutropha (formerly, Alcaligenes eutrophus) since the former efficiently synthesizes PHB during growth without any nutrient limitation. To analyze how the central metabolic pathways should be balanced with pathways necessary for cell growth and PHB formation, a stoichiometric model was developed to predict the theoretical maximum PHB production capacity for different metabolic variants. Flux analysis results illustrated the importance of the availability of acetyl-CoA and NADPH for achieving the maximum yield of PHB. In order to examine whether the increased availability of the above substances can enhance PHB synthesis in recombinant E. coli, both genetic and environmental perturbations were attempted. Several E. coli K12 derivatives, namely, HMS174, TA3516 (pta-/ack-), and DF11 (pgi-), were transformed with a plasmid which contains the native phb operon. The fermentation characteristics of these recombinant strains were studied and compared. In this study we examined the effects of intracellular acetyl-CoA accumulation, which may promote PHB synthesis in vivo, by perturbations induced from attenuation of acetate kinase and phosphotransacetylase (TA3516, blocked in the acetate pathway) and by cultivation of E. coli HMS174 on gluconate; it can convert gluconate to acetyl-CoA at a higher rate. The effects of intracellular accumulation of NADPH were investigated by introducing a perturbation induced from attenuation of phosphoglucose isomerase, which redirects the carbon flow to the pentose-phosphate (PP) pathway. Results from the analyses of these perturbations indicate that intracellular buildup of acetyl-CoA may not be able to promote PHB synthesis in vivo. On the other hand, since the biosynthesis of PHB in the pgi- mutant strain can utilize the NADPH overproduced through the PP pathway, the growth of the pgi- mutant on glucose was recovered, indicating that the overproduction of NADPH might be able to enhance PHB synthesis.     

Respiratory Enzyme Activity in Low Temperature Sweetening of Susceptible and Resistant Potatoes

During storage at 4°C and 12°C, a potato cultivar susceptible to chill-sweetening (Norchip) accumulated significantly (P≤0.05) higher levels of sucrose, fructose and glucose than a potato selection resistant to chill-sweetening (ND 860-2). ND 860-2 tubers exhibited a significantly (P≤0.05) higher respiration rate throughout storage, reflected in higher activities of phosphofructokinase (PFK), glucose-6-phos-phate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Storage significantly (P≤0.05) reduced respiration rate for both cultivars. G6PDH showed no significant (P>0.05) difference in specific activity or V_max between 4°C and 12°C for either cultivar. However, Km decreased at 4°C for both cultivars, possibly due to build up of substrate.     

The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains

Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H(2)O(2) than the control strain TMB3001.     

温度对Zymomonas mobilis糖代谢关键酶活力和代谢流量的影响

以运动发酵单胞菌(Zynwmonas mobilis)ATCC31821为模式菌株,研究不同温度条件对其葡萄糖代谢关键酶活力的影响.采用全自动发酵罐,在整个发酵过程中通过充入氮气调节发酵液的溶氧量(DO)=0%,添加0.5mol/LNaOH溶液控制pH=5.5,发酵温度分别控制为25、30、35、40℃,发酵24h,测定其糖代谢网络中ED、HMP、TCA等途径的关键酶活力和代谢物成分.结果表明,在发酵温度为30～35℃时,乙醇脱氢酶(ADH)、葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC)、葡萄糖激酶(GK)、丙酮酸激酶(PK)和甘油醛-3-磷酸脱氢酶(GD-3-PDH)的活力较高,菌体的ED途径代谢活跃,碳素流量增加,乙醇生产量和糖转化率较高,而TCA途径的苹果酸脱氢酶(MDH)和异柠檬酸脱氢酶(ICDH)等活力较低,进入TCA途径的碳素流量明显减少;发酵温度为25、40℃时,TCA途径的酶活力升高,ED途径的酶活力减弱,生成乙醇的代谢流量减少,因此温度是z.mobilis发酵过程中调控菌体细胞生长和糖代谢的一个重要因素. 、葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC) 葡萄糖激酶(GK)、丙酮酸激酶(PK)和甘油醛-3-磷酸脱氢酶(GD-3-PDH)的活力较高,菌体的ED途径代谢活跃,碳素流量增加,乙醇生产量和糖转化率较高,而TCA途径的苹果酸脱氢酶(MDH)和异柠檬酸脱氢酶(ICDH)等活力较低,进入TCA途径的碳素流量明显减少;发酵温度为25、40℃时,TCA途径的酶活力升高,ED途径的酶活力减弱,生成乙醇的代谢流量减少,因此温度是z.mobilis发酵过程中调控菌体细胞生长和糖代谢的一个重要因素.葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC) 葡萄糖激酶(GK)、     

Proteome analysis of a temperature-inducible recombinant Escherichia coli for poly-beta-hydroxybutyrate production

A recombinant Escherichia coli strain harboring the lambdap(R)-p(L) promoter and heterologous poly-beta-hydroxybutyrate (PHB) biosynthesis genes was shown to accumulate PHB when the incubation temperature was changed from 34 degrees C to temperatures higher than 37 degrees C. In the present research, total gene expression patterns of the recombinant E. coli before and after induction were investigated by two-dimensional gel electrophoresis. Proteins encoded by serS, sucC, trpA, and alaS were found to be expressed before induction of phb genes at a culture temperature of 34 degrees C. On the other hand, proteins encoded by metG, rplI, and carA were found to be expressed after induction achieved by increasing the temperature to 40 degrees C. In the case of plasmid-free cells, all the selected genes have been shown to be expressed except metG, and ibpA and ibpB among the heat-shock proteins. The heat-shock proteins were found to be upregulated upon induction of phb genes, which may be due to the stress caused by the accumulation of PHB granules as well as by the temperature upshift. The changes in the expression of some of the metabolic pathway-related proteins before and after induction were interpreted in relation to the consumption of NADPH and acetyl-CoA for PHB synthesis.     

内源性氧化胁迫促进酿酒酵母合成谷胱甘肽的潜在机制分析

利用转录组学分析手段结合生理生化特性来研究酿酒酵母突变株高产谷胱甘肽的潜在机制。结果表明:突变株谷胱甘肽合成限速酶、抗氧化酶活力及其编码基因表达量、过氧化氢和还原型辅酶Ⅱ(nicotinamide adenine dinucleotide phosphate,NADPH)含量显著提高;丙酮酸激酶活力、丙酮酸、柠檬酸和琥珀酸含量显著降低;此外,三羧酸循环和磷酸戊糖途径的基因表达量分别显著下调和上调。因此,突变株可能在遭受内源性活性氧过氧化氢的胁迫下,通过调节谷胱甘肽合成限速酶活力加强了谷胱甘肽的合成,与抗氧化酶共同抵御氧化胁迫;丙酮酸激酶活力减弱降低了丙酮酸的合成,减少了三羧酸循环的通量,使得磷酸戊糖途径通量增加,从而提高了NADPH含量,为谷胱甘肽的合成提供了充足的还原力。     

Key role for transketolase activity in erythritol production by Trichosporonoides megachiliensis SN-G42

Erythritol is an important sugar alcohol industrially produced only by fermentation. The highly osmophilic yeast-like fungi, Trichosporonoides megachiliensis SN-G42, enables commercial production of erythritol with a high conversion from glucose to erythritol of more than 47%. However, the microbial production pathway of erythritol remains unclear. In the present study, the activities of enzymes in the pentose phosphate pathway of Trichosporonoides megachiliensis SN-G42 used for industrial erythritol production were measured under various culture conditions to examine the production mechanism and the key-enzymes.As a result, the various enzyme activities of this organism are revealed in the pentose phosphate pathway, i.e., those of hexokinase, glucose-6-phosphate dehydrogenase, gluconate dehydrogenase, transketolase, transaldolase, and erythrose reductase. In the cultures in which erythritol was produced after completion of cell growth, the enzyme activities of the pentose phosphate pathway were higher than those of the TCA cycle. In particular, transketolase activity was correlated with erythritol productivity under various production cultures with different agitation speeds and thiamine concentrations.These results suggest that erythritol may be produced mainly through the pentose phosphate pathway. In addition, the high activity of transketolase is required to produce abundant intermediates, which results in high erythritol productivity. As such, transketolase appears to be a key-enzyme for erythritol production in the organism studied.     

吐温对钝齿棒杆菌产L-精氨酸代谢的影响

目的:以课题组构建的钝齿棒杆菌(Corynebacterium crenatum)MT NAGK M4 △proBANcgl1221AdtsR1(CCM01)为出发菌株,探究吐温(Tween)对该菌产精氨酸代谢的影响.方法:优化精氨酸发酵及Tween质量浓度,测定菌体生长量、L-精氨酸产量、发酵液残糖量考察Tween ...     

The Influence of Barley Storage on Respiration and Glucose‐6‐Phosphate Dehydrogenase During Malting

Malt is produced by the controlled, but limited germination of barley. To produce good quality malt, the barley employed must be able to germinate rapidly and synchronously. Dormancy is a seed characteristic that can interfere with the rapid and uniform germination of barley, thereby reducing the resultant malt quality. Various studies have shown that post harvest storage can be used to remove dormancy and enhance barley germination characteristics and malt quality. Because of its complexity, the fundamental basis of dormancy induction, maintenance and termination remain unknown. Glucose‐6‐phosphate dehydrogenase (G6PDH) is the rate limiting enzyme of the pentose phosphate pathway and has been associated with dormancy decay and increased seed vigor of a variety of different seeds. The aim of this study was to determine if changes in barley germination vigour were associated with respiration and/or G6PDH changes during malting. Commercially grown barley (cv. Gairdner) was obtained from various states of Australia and stored at room temperature for up to 7 months. At 1, 3 and 7 months, samples were taken and stored at ?18°C. The germinative energy (GE) and germinative index (GI) of these samples were measured. Samples were micro‐malted and the α‐amylase activity, respiration rate, and G6PDH activity of the germinating grains were measured at various stages of malting. It was found that storage of barley for up to seven months significantly improved the germination characteristics and increased the α‐amylase activity during malting. However, these improvements were not associated with concomitant changes in respiration rate or G6PDH activity during malting.     

Fermentative production of (R)-(-)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Two systems, one using an (R)-(-)-3-hydroxybutyrate dehydrogenase (BDH) null mutant of Ralstonia eutropha and the other using a recombinant Escherichia coli strain containing a synthetic poly[(R)-(-)-3-hydroxybutyrate] (PHB) operon and an extracellular PHB depolymerase gene, were used for the fermentative production of (R)-(-)-3-hydroxybutyrate (3HB). The concentration of 3HB in the culture supernatant of the mutant R. eutropha system reached about 30 mM after 5 d under anaerobic conditions, although it was about 4-10 mM under aerobic conditions. On the other hand, the 3HB concentration in the culture supernatant of the recombinant E. coli system reached about 70 mM after 4 d, indicating that about 70% of the glucose added was converted to 3HB.     

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。     

吐温-80对钝齿棒杆菌产L-赖氨酸的影响

对吐温-80提高L-赖氨酸产量的分子机制进行探究。以钝齿棒杆菌MT-M4 ΔproB为出发菌株,在菌株生长至对数初期36 h时,添加5 mg/mL吐温-80进行摇瓶发酵,通过反转录实时荧光定量聚合酶链式反应（reversetranslate real-time fluorescence quantitative polymerase chain reaction,RT-qPCR）监控相关基因的转录水平、胞内的还原型烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate reduced form,NADPH）水平及α-酮戊二酸脱氢酶复合物（α-oxoglutarate dehydrogenase complex,ODHC）的活力。摇瓶发酵结果表明,添加吐温-80使L-赖氨酸产量提高了215%;RT-qPCR结果表明,编码ODHC的基因odhA、sucB和lpdA、参与ODHC调控的基因pknG、dtsR和ppp以及合成NADPH的磷酸戊糖途径基因zwf的转录水平均发生了变化,其中odhA、sucB、lpdA、pknG及dtsR1基因分别发生了上调,ppp基因下调,相应ODHC活力提高了32.9%;zwf基因上调了17.6 倍,相应胞内NADPH水平提高了137%。因此,在对数生长初期36 h添加吐温-80具有双重功效,既提高了ODHC的表达又使NADPH积累,从而诱导L-赖氨酸的高产。     

BTH处理对采后草莓果实还原势和抗病性的调控作用

为明确苯丙噻唑硫代乙酸甲酯(BTH)处理抑制草莓果实采后灰霉病的相关机理,本研究分析了BTH对果实在20 ℃贮藏期间还原势及抗病性的调控作用。结果显示,单一BTH处理在贮藏1 d后可有效诱导草莓果实磷酸戊糖代谢途径关键酶G6PDH(葡萄糖-6-磷酸脱氢酶)、6PGDH(6-磷酸葡萄糖酸脱氢酶)活性以及AsA-GSH(抗坏血酸-谷胱甘肽循环)循环关键酶MDHAR(单脱氢抗坏血酸还原酶)、DHAR(脱氢抗坏血酸还原酶)、GR(谷胱甘肽还原酶)和APX(抗坏血酸过氧化物酶)活性的上升,促进NADPH(还原型烟酰胺腺嘌呤二核苷酸磷酸)、GSH(还原型谷胱甘肽)和AsA(抗坏血酸)等还原性物质含量的上升,并抑制NADP+(烟酰胺腺嘌呤二核苷磷酸)、GSSG(氧化型谷胱甘肽)和DHA(脱氢抗坏血酸)等氧化产物的合成,从而提高果实内部还原势;伴随着还原势的升高,果实中FaPR1、FaPR5、FaCHI-1和Faβglu等PRs基因表达丰度出现明显的上升趋势,同时果实由Botrytis cinerea导致的灰霉病发病率显著(p<0.05)降低。另一方面,经BTH处理的果实在接种病原菌B. cinerea后,其果实内PPP(磷酸戊糖)途径和AsA-GSH循环关键酶活性以及PRs基因表达丰度在整个贮藏期间出现较单一BTH处理更为显著的提高(p<0.05),同时果实发病率和病斑直径也显著下降(p<0.05)。这些结果说明,0.1 mmol/L BTH可通过诱导Priming抗性,使草莓果实在受到B. cinerea侵染时产生强烈抗病性;同时,BTH处理可提高PPP途径和AsA-GSH循环关键酶活性,促进果实中还原性物质积累,从而为PRs基因表达提供高还原势的有利环境。