谷氨酸棒状杆菌厌氧产丁二酸的发酵条件

考察谷氨酸棒状杆菌ATCC13032Δldh厌氧产丁二酸的发酵条件。结果发现:补加NaHCO3的效果最好,并且考察了NaHCO3浓度对葡萄糖转化速率及丁二酸生成速率的影响。运用代谢流分析方法分析了乳酸脱氢酶基因敲除对谷氨酸棒状杆菌厌氧代谢的影响,发现乳酸脱氢酶基因敲除导致磷酸烯醇式丙酮酸生成丁二酸的流量提高了214.3%,流向乳酸的流量变为0;分批厌氧转化36 h生成41.2 g/L丁二酸,产率45.0%。     

一株丁二酸产生菌的选育及代谢分析

[目的]了解细胞代谢过程,并最终选育出高产突变株,对丁二酸的工业生物转化有重要意义.[方法]在菌株生化及分子鉴定基础上,讨论了菌株的代谢途径,便于实施有针对性的诱变选育,利用矩阵计算了流量分布以及用扰动法分析了代谢节点.[结果]菌株S.JST经鉴定为产丁二酸放线杆菌,酶活检测表明磷酸烯醇式丙酮酸羧激酶、苹果酸脱氢酶在丁二酸代谢过程中具有较高酶活,出发株的流量分布显示副产物乙醇的流量仅次于丁二酸的流量,选育获得的突变株乙醇脱氧酶酶活显著降低,丁二酸与乙醇的流量分别有34%升高与93%的降低,序列分析发现突变株的乙醇脱氢酶酶基因中存在一个突变位点,且生物信息学表明该位点编码的氨基酸序列与该酶的NADH连接活性有联系.[结论]对产丁二酸放线杆菌采用定向选育的方法能够有效改善细胞代谢,并最终提高丁二酸产量.     

重组大肠杆菌利用蔗糖及糖蜜发酵生产丁二酸

富含蔗糖的甘蔗糖蜜可作为制备丁二酸的廉价原料。然而生产丁二酸的潜力菌株大肠杆菌Escherichia coli AFP111不能代谢蔗糖。为了使其具有蔗糖代谢能力,将E.coli W中非PTS蔗糖利用系统蔗糖通透酶的编码基因csc B,果糖激酶的编码基因csc K和蔗糖水解酶的编码基因csc A克隆并表达到AFP111中,获得重组菌株AFP111/p MD19T-csc BKA。经厌氧发酵验证,重组菌株72 h消耗20 g/L蔗糖,丁二酸产量达到12 g/L。在3L发酵罐中采用有氧阶段培养菌体、厌氧阶段发酵的两阶段发酵方式,厌氧发酵30 h,重组菌株以蔗糖和糖蜜为碳源丁二酸产量分别为34 g/L和30 g/L。结果表明,通过外源引入非PTS蔗糖利用系统,重组菌株具有较强的代谢蔗糖生长及合成丁二酸的能力,并且能够利用廉价糖蜜发酵制备丁二酸。     

氧化还原电位对Actinobacillus succinogenes厌氧发酵生产丁二酸的影响

为提高琥珀酸放线菌Actinobacillus succinogenes CGMCC1593厌氧发酵产丁二酸的水平。研究了以葡萄糖为C源,发酵液中不同氧化还原电位（VORP）对A．succirtogenes CGMCC1593生长和代谢产物分布的影响。结果表明：菌体生长和丁二酸积累的较佳VORP分别为-220mV和-270mV;利用代谢流分析法,比较VORP在-220mV和-270mV时发酵对数生长期（8h）和稳定期（20h）的代谢通量分布,以及发酵过程中磷酸烯醇式丙酮酸（PEP）、丙酮酸（Pyr）节点,NADH通量分配的变化,由此得出在VORP为-270mV时,NADH总通量和丁二酸方向代谢通量增幅明显。在发酵过程中,通过降低VORP至-270mV,使丁二酸的产率从70％提高到85％。     

不同pH调节剂对产琥珀酸放线杆菌NJ113发酵产丁二酸的影响

在3L发酵罐中分别采用不同的碱性物质作为pH调节剂,考察其对产琥珀酸放线杆菌Actinobacillus succinogenes NJ113厌氧发酵制备丁二酸的影响。结果表明:Ca2+、NH4+调节剂对菌体生长代谢有较大阻碍作用,丁二酸产量较低;采用含Na+调节剂,在发酵中后期菌体出现絮凝现象严重,且产丁二酸能力骤降;采用含Mg2+调节剂,整个发酵过程菌体代谢旺盛,发酵效果较佳。根据各碱性物质的调节能力以及对菌体生长代谢的影响,选择NaOH、Mg(OH)2和Na2CO3、Mg(OH)2分别作为混合碱组分调节pH,并对两组混合碱中各物质的质量比例进行优化。结果表明,以NaOH、Mg(OH)2混合,两者质量比为1:1时,发酵效果最好,丁二酸质量浓度高达到69.8g/L,质量收率74.5%。该种混合碱配比可有效替代碱式MgCO3调节pH,既达到高产丁二酸的目的,又可降低生物制备丁二酸的成本。     

微生物发酵生产丁二酸研究进展

丁二酸是微生物三羧酸循环中重要的代谢中间产物,广泛用于生物高分子、食品与医药等行业,市场潜在需求量巨大。文中从3个方面归纳了国内外生物基丁二酸研究进展：能够过量积累丁二酸的微生物的发现和筛选,产丁二酸工程菌构建中所采用的基因工程策略及代谢工程技术,丁二酸发酵过程控制与优化。最后,讨论了微生物法生产丁二酸今后的研究方向。     

常压室温等离子体诱变高效利用木糖产丁二酸菌株

大肠杆菌Escherichia coli AFP111是E. coli NZN111 (△pflAB△ldhA) 的ptsG自发突变株,其转化1 mol的木糖合成丁二酸的过程中净产生1.67 mol ATP,但是转化1 mol的木糖合成丁二酸的过程中实际需要2.67 mol ATP,因此在厌氧条件下,ATP的供给不足导致E. coli AFP111不能代谢木糖。采用常压室温等离子体射流诱变产丁二酸大肠杆菌菌株,在厌氧条件下,利用以木糖为碳源的M9培养基,筛选得到一株可以代谢木糖并积累丁二酸的突变株DC111。该突变菌株在发酵培养基中,72 h内可以消耗10.52 g/L木糖产6.46 g/L的丁二酸,丁二酸的得率达到了0.78 mol/mol。而且突变株中伴有ATP产生的磷酸烯醇式丙酮酸羧激酶 (PCK) 途径得到加强,PCK的比酶活相对于出发菌株提高了19.33倍,使得其在厌氧条件下能够有足够的ATP供给来代谢木糖发酵产丁二酸。     

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%。     

采用玉米秸秆水解糖和玉米浆发酵生产丁二酸

研究了以玉米秸秆水解糖为碳源,不同氮源条件下琥珀酸放线杆菌Actinobacillus succinogenesSF-9的丁二酸发酵产酸能力。结果表明玉米浆可以替代酵母膏作为丁二酸发酵的廉价氮源。厌氧摇瓶丁二酸发酵单因素试验,得到在初糖浓度50 g/L时,玉米浆的较佳用量为20 g/L。在5 L搅拌罐上,考察了不同初始玉米秸秆水解糖浓度对A.succinogenes SF-9发酵生产丁二酸的影响,结果显示高初始秸秆糖浓度对琥珀酸放线杆菌的生长有抑制作用。采用补料分批发酵,发酵60 h丁二酸的产量达到42.7g/L,丁二酸产率82.7%,生产强度0.81 g/(L·h)。丁二酸的产量和生产强度较分批发酵有明显提高。     

大肠杆菌AFP111菌体回收连续转化生产丁二酸

在利用大肠杆菌AFP111厌氧发酵生产丁二酸过程中,随着产物丁二酸的不断积累,菌体活力和产酸能力逐渐降低,而通过回收菌体在新鲜培养基中重复发酵,可延长厌氧发酵时间,但是丁二酸生产效率较低。为了提高菌体回收丁二酸的转化效率,通过在回收菌体时有氧诱导 3 h,以纯水为培养基,进行丁二酸转化发酵。在连续进行 3 批次的发酵后,丁二酸的总产量和最终收率分别为 56.50 g/L和90%,生产速率达到了 0.81 g/(L·h),比未诱导情况下的生产速率提高了13%。     

Effect of carbon sources differing in oxidation state and transport route on succinate production in metabolically engineered Escherichia coli

In mixed-acid fermentation, succinate synthesis requires one mole of phosphoenolpyruvate (PEP), one mole of CO₂, and two moles of NADH for every mole of succinate to be formed. Different carbon sources with different properties were used to address these requirements. Sorbitol generates one more mole of NADH than glucose. Fermentation of sorbitol was shown in this study (and by others) to produce significantly more succinate than fermentation of glucose, due to increased NADH availability. Xylose fermentation conserves the intracellular PEP pool, since its transport does not require the phosphotransferase system normally used for glucose transport. The extra PEP can then be assimilated in the succinate pathway to improve production. In this study, fermentation of xylose did yield higher succinate production than glucose fermentation. Subsequent inactivation of the acetate and lactate pathways was performed to study metabolite redistribution and the effect on succinate production. With the acetate pathway inactivated, significant carbon flux shifted toward lactate rather than succinate. When both acetate and lactate pathways were inactivated, succinate yield ultimately increased with a concomitant increase in ethanol yield.     

Anaerobic fermentation balance of Escherichia coli as observed by in vivo nuclear magnetic resonance spectroscopy.

Fermenting anaerobic cultures of Escherichia coli were observed by the nonintrusive technique of in vivo, whole-culture nuclear magnetic resonance. Fermentation balances were calculated for hexoses, pentoses, sugar alcohols, and sugar acids. Substrates more reduced than glucose yielded more of the highly reduced fermentation product ethanol, whereas more-oxidized substrates produced more of the less-reduced fermentation product acetate. These relationships were made more obvious by the introduction of ldhA mutations, which abolished lactate production, and delta frd mutations, which eliminated succinate. When grown anaerobically on sugar alcohols such as sorbitol, E. coli produced ethanol in excess of the amount calculated by the standard fermentation pathways. Reducing equivalents must be recycled from formate to account for this excess of ethanol. In mutants deficient in hydrogenase (hydB), ethanol production from sorbitol was greatly decreased, implying that hydrogen gas released from formate by the formate-hydrogen lyase system may be partially recycled, in the wild type, to increase the yield of the highly reduced fermentation product ethanol.     

Use of carbon and energy balances in the study of the anaerobic metabolism of Enterobacter aerogenes at variable starting glucose concentrations

The anaerobic metabolism of Enterobacter aerogenes was studied in batch culture at increasing initial glucose levels (9.0< S(o) <72 g l(-1)). The ultimate concentrations of fermentation products were utilized to check a metabolic flux analysis based on simple carbon mass and energy balances that promise to be suitable for the study of different fermentation processes, either under aerobic or anaerobic conditions. The stoichiometric coefficients of products collected at increasing starting glucose concentrations under anaerobic conditions suggest: (a) little influence of starting glucose level on the formation of the main fermentation products (2,3-butanediol and ethanol); (b) possible inhibition of 2,3-butanediol and lactate formations by increased ethanol concentration; (c) consequent increase in carbon flux through the remaining metabolic pathways with increased molar productions of succinate, acetate and hydrogen; (d) relative constancy of the molar production of ATP and CO(2).     

Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers

Actinobacillus succinogenes is a promising candidate for industrial succinate production. However, in addition to producing succinate, it also produces formate and acetate. To understand carbon flux distribution to succinate and alternative products we fed A. succinogenes [1-(13)C]glucose and analyzed the resulting isotopomers of excreted organic acids, proteinaceous amino acids, and glycogen monomers by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The isotopomer data, together with the glucose consumption and product formation rates and the A. succinogenes biomass composition, were supplied to a metabolic flux model. Oxidative pentose phosphate pathway flux supplied, at most, 20% of the estimated NADPH requirement for cell growth. The model indicated that NADPH was instead produced primarily by the conversion of NADH to NADPH by transhydrogenase and/or by NADP-dependent malic enzyme. Transhydrogenase activity was detected in A. succinogenes cell extracts, as were formate and pyruvate dehydrogenases, which the model suggested were contributing to NADH production. Malic enzyme activity was also detected in cell extracts, consistent with the flux analysis results. Labeling patterns in amino acids and organic acids showed that oxaloacetate and malate were being decarboxylated to pyruvate. These are the first in vivo experiments to show that the partitioning of flux between succinate and alternative fermentation products can occur at multiple nodes in A. succinogenes. The implications for designing effective metabolic engineering strategies to increase A. succinogenes succinate production are discussed.     

Formation of sorbitol by Zymomonas mobilis

Summary Sorbitol is formed as the major by-product in ethanol fermentations by Zymomonas mobilis when both glucose and fructose are present in the fermentation medium. The amount of sorbitol produced was equivalent to as much as 11% of the original carbon source, decreasing the ethanol yield correspondingly. Only minor amounts of sorbitol were formed from glucose or fructose alone. The formation of sorbitol is apparently a consequence of the inhibition of fructokinase by glucose.     

13C-metabolic flux analysis of Actinobacillus succinogenes fermentative metabolism at different NaHCO3 and H2 concentrations

Actinobacillus succinogenes naturally produces high concentrations of succinate, a potential intermediary feedstock for bulk chemical productions. A. succinogenes responds to high CO(2) and H(2) concentrations by producing more succinate and by producing less formate, acetate, and ethanol. To determine how intermediary fluxes in A. succinogenes respond to CO(2) and H(2) perturbations, (13)C-metabolic flux analysis was performed in batch cultures at two different NaHCO(3) concentrations, with and without H(2), using a substrate mixture of [1-(13)C]glucose, [U-(13)C]glucose, and unlabeled NaHCO(3). The resulting amino acid, organic acid, and glycogen isotopomers were analyzed by gas chromatography-mass spectrometry and NMR. In all conditions, exchange flux was observed through malic enzyme and/or oxaloacetate decarboxylase. The presence of an exchange flux between oxaloacetate, malate, and pyruvate indicates that, in addition to phosphoenolpyruvate, oxaloacetate, and malate, pyruvate is a fourth node for flux distribution between succinate and alternative fermentation products. High NaHCO(3) concentrations decreased the amount of flux shunted by C(4)-decarboxylating activities from the succinate-producing C(4) pathway to the formate-, acetate-, and ethanol-producing C(3) pathway. In addition, pyruvate carboxylating flux increased in response to high NaHCO(3) concentrations. C(3)-pathway dehydrogenase fluxes increased or decreased appropriately in response to the different redox demands imposed by the different NaHCO(3) and H(2) concentrations. Overall, these metabolic flux changes allowed A. succinogenes to maintain a constant growth rate and biomass yield in all conditions. These results are discussed with respect to A. succinogenes' physiology and to metabolic engineering strategies to increase the flux to succinate.     

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

Summary The intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [¹⁴C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.     

Proton correlation nuclear magnetic resonance study of anaerobic metabolism of Escherichia coli.

Proton correlation nuclear magnetic resonance has been used to investigate anaerobic metabolism of glucose in Escherichia coli cells. The time course of the concentrations of six metabolites (ethanol, lactate, acetate, pyruvate, succinate, and formate) has been followed at the very early state of fermentation, and used to discuss dynamical aspects of the mixed-acid fermentation of glucose by E. coli.     

The effect of ethanol or sorbitol on glucose production from pyruvate in isolated hepatocytes from 48-hour fasted guinea-pigs

Hepatocytes isolated from 48-hour, fasted guinea-pigs were incubated with glucose precursors to compare relative rates of glucose production. Glucose production from lactate and pyruvate was similar (2.61 vs 3.18 mumol/hr per 100 mg wet weight). Glucose production from fructose was greater than that from sorbitol (4.68 vs 1.63 mumol/hr per 100 mg wet weight). When ethanol was added to pyruvate-containing buffer, the flux of pyruvate to glucose and lactate was synergistically enhanced (5.28 vs 3.76 and 7.51 vs 2.88 mumol/hr per 100 mg wet weight, respectively). When sorbitol was added to buffer containing pyruvate, glucose and lactate production were even greater than that seen with ethanol (8.32 vs 5.38 and 15.99 vs 7.51 mumol/hr per 100 mg wet weight, respectively).