环境因素对琥珀酸放线杆菌Actinobacillus succinogenes CGMCC 1593发酵生产丁二酸的影响

琥珀酸放线杆菌是发酵生产有应用前景的生物基原料-丁二酸的微生物。本研究室从牛瘤胃中筛选获得一株琥珀酸放线杆菌Actinobacillus succinogenes CGMCC 1593, 分析了环境气体、pH、氧化还原电位(ORP)环境因素对琥珀酸放线杆菌A. succinogenes CGMCC 1593发酵生产丁二酸的影响。结果表明: CO2不仅提供了A. succinogenes CGMCC 1593发酵生产丁二酸的最佳气体环境, 也是发酵生产丁二酸的底物之一; MgCO3是A. succinogenes CGMCC 1593发酵过程较好的pH调节剂, 发酵过程维持pH7.1~6.2, 可满足菌体生长与产酸的要求; 发酵液初始ORP过低, 不利于菌体生长, ORP在-270 mV时对丁二酸产生有利。在菌体对数生长期结束时, 通过Na2S·9H2O降低发酵液ORP到-270 mV, 发酵48 h时可产丁二酸37 g/L, 摩尔产率达到129%。这对深入研究A. succinogenes CGMCC 1593发酵生产丁二酸具有参考价值。     

环境因素对琥珀酸放线杆菌Actinobacillus succinogenes CGMCC 1593发酵生产丁二酸的影响

琥珀酸放线杆菌是发酵生产有应用前景的生物基原料-丁二酸的微生物。本研究室从牛瘤胃中筛选获得一株琥珀酸放线杆菌Actinobacillus succinogenes CGMCC 1593, 分析了环境气体、pH、氧化还原电位(ORP)环境因素对琥珀酸放线杆菌A. succinogenes CGMCC 1593发酵生产丁二酸的影响。结果表明: CO2不仅提供了A. succinogenes CGMCC 1593发酵生产丁二酸的最佳气体环境, 也是发酵生产丁二酸的底物之一; MgCO3是A. succinogenes CGMCC 1593发酵过程较好的pH调节剂, 发酵过程维持pH7.1~6.2, 可满足菌体生长与产酸的要求; 发酵液初始ORP过低, 不利于菌体生长, ORP在-270 mV时对丁二酸产生有利。在菌体对数生长期结束时, 通过Na2S·9H2O降低发酵液ORP到-270 mV, 发酵48 h时可产丁二酸37 g/L, 摩尔产率达到129%。这对深入研究A. succinogenes CGMCC 1593发酵生产丁二酸具有参考价值。     

利用氧化还原电位调控乳酸发酵

研究了控制不同氧化还原电位（oxidation—reduction potential,ORP）对乳酸发酵过程的影响。通过5L发酵罐分批发酵实验发现ORP水平控制在-170mV时最有利于乳酸生成,乳酸最高质量浓度达176g／L,糖酸转化率为94％,其平均乳酸产率3．7g/（L·h）,比ORP控制在-220mV和-120mV时分别高19％,37％;通过对发酵过程胞外有机酸浓度及代谢流分析发现氧化还原电位是通过影响细胞内代谢流分布来影响乳酸合成的。     

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%。     

烟酸转磷酸核糖激酶和丙酮酸羧化酶共表达对大肠杆菌BA002产丁二酸的影响

大肠杆菌BA002是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。pncB是烟酸转磷酸核糖激酶 (NAPRTase) 的编码基因,通过过量表达pncB基因能够提高NAD(H)总量与维持合适的NADH/NAD+,从而恢复了厌氧条件下重组菌E. coli BA014 (BA002/pTrc99a-pncB) 的生长和产丁二酸的性能。然而,BA014在厌氧发酵过程中有大量丙酮酸积累,为进一步提高菌株的丁二酸生产能力,减少副产物丙酮酸的生成,共表达NAPRTase和来自于乳酸乳球菌 NZ9000中丙酮酸羧化酶 (PYC) 的编码基因pyc,构建了重组菌E. coli BA016 (BA002/pTrc99a-pncB-pyc)。3 L发酵罐结果表明,BA016发酵112 h后,共消耗了35.00 g/L的葡萄糖。发酵结束时,菌体OD600为4.64,产生了25.09 g/L丁二酸。通过共表达pncB和pyc基因,使BA016的丙酮酸积累进一步降低,丁二酸产量进一步提高。     

过量表达烟酸单核苷酸腺苷酰转移酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因(ldhA)和丙酮酸-甲酸裂解酶的编码基因(pflB)的发酵生产丁二酸的潜力菌株。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。nadD为催化NAD(H)合成途径中烟酸单核苷酸(NaMN)生成烟酸腺嘌呤二核苷酸(NaAD)的烟酸单核苷酸腺苷酰转移酶(Nicotinic acid mononucleotide adenylyltransferase,NAMNAT)的编码基因,通过过量表达nadD基因能够提高NAD(H)总量与维持合适的NADH/NAD+比例。文中构建了重组菌E.coli NZN111/pTrc99a-nadD,在厌氧摇瓶发酵过程中通过添加终浓度为1.0 mmol/L的IPTG诱导表达,重组菌E.coli NZN111/pTrc99a-nadD中NAD+和NADH的浓度分别比宿主菌E.coli NZN111提高了3.21倍和1.67倍,NAD(H)总量提高了2.63倍,NADH/NAD+从0.64降低为0.41,使重组菌株恢复了厌氧条件下生长和代谢葡萄糖的能力。重组菌与对照菌相比,72 h内可以消耗14.0 g/L的葡萄糖产6.23 g/L的丁二酸,丁二酸产量增加了19倍。     

环境条件对丙酮酸分批发酵的影响

考察了搅拌转速、pH和温度对丙酮酸分批发酵的影响。高转速（500r／min）下,丙酮酸产率较高（71％）,但葡萄糖消耗速度较慢（1．23g／（L·h））;低转速（300r／min）下,细胞消耗葡萄糖的速度加快（1．95g/（L·h））,而丙酮酸产率（0．48％）却明显下降。将搅拌转速恒定在400r／min可在一定程度上获得较高的丙酮酸产率（0．62％）和葡萄糖消耗速度（1．66g／（L·h））。CaCO3调节pH时,较多碳流从丙酮酸节点转向α-酮戊二酸节点和细胞生长,最终丙酮酸产量比NaOH调节pH时的发酵结果低38．7％;NH3·H2O调节pH时最终细胞浓度和丙酮酸产量仅为NaOH调节时的77．8％和90．9％。pH5．5时最利于丙酮酸的合成。较高的发酵温度加速T.glabrata积累丙酮酸,但同时会导致α-酮戊二酸的提前积累;而较低的温度下甘油和α-酮戊二酸积累较少,丙酮酸发酵的最适温度为28～30℃。     

过量表达烟酸转磷酸核糖激酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111厌氧发酵的主要产物为丁二酸,是发酵生产丁二酸的潜力菌株。但是由于敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸甲酸裂解酶的编码基因 (pflB),导致辅酶NADH/NAD+不平衡,厌氧条件下不能利用葡萄糖生长代谢。构建烟酸转磷酸核糖激酶的重组菌Escherichia coli NZN111/pTrc99a-pncB,在厌氧摇瓶发酵过程中通过添加0.5 mmol/L的烟酸、0.3 mmol/L的IPTG诱导后重组菌的烟酸转磷酸核糖激酶 (Nicotinic acid phosphor     

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%。     

最新专利文摘

CN1814747:一种微生物发酵生产丁二酸的菌种和方法本专利具体涉及一种发酵糖质原料产生丁二酸的微生物琥珀酸放线杆菌(Actinobacillus succinogenes)SW 0580,保藏号CGMCC No·1593,以及利用该微生物发酵生产丁二酸的方法。该微生物是从瘤胃中分离并鉴定的琥珀酸放线杆菌(Actino     

Economical succinic acid production from cane molasses by Actinobacillus succinogenes

In this work, production of succinic acid by Actinobacillus succinogenes CGMCC1593 using cane molasses as a low cost carbon source was developed. In anaerobic bottles fermentation, succinic acid concentration of 50.6+/-0.9 g l(-1) was attained at 60 h using an optimum medium containing molasses pretreated with sulfuric acid, resulting in a succinic acid yield of 79.5+/-1.1% and sugar utilization of 97.1+/-0.6%. When batch fermentation was carried out in a 5-l stirred bioreactor with pretreated molasses, 46.4 g l(-1) of succinic acid was attained at 48 h and faster cells growth was also observed. Fed batch fermentation was performed to minimize the substrate (sugar) inhibition effect, giving 55.2 g l(-1) of succinic acid and 1.15 g l(-1)h(-1) of productivity at 48 h. The present study suggests that the inexpensive cane molasses could be utilized for the economical and efficient production of succinic acid by A. succinogenes.     

一株琥珀酸产生菌的筛选及鉴定

从牛的瘤胃中筛选获得一株能发酵生产琥珀酸的兼性厌氧菌。对其进行生理生化特性鉴定及16S rRNA基因分析。该菌株短杆状,无鞭毛,革兰氏染色阴性,V-P反应阴性,能发酵多种糖类产酸;其16S rRNA基因与琥珀酸放线杆菌的同源性高达99．8%,认为属于琥珀酸放线杆菌(Actinobacillus succinogenes),并将其命名为琥珀酸放线杆菌(Actinobacillus succinogenes)SW0580,保藏号CGMCC 1593。初步发酵试验表明该菌能发酵60g/L葡萄糖产生25．8g/L的丁二酸。     

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.     

基因组改组技术选育耐酸性琥珀酸放线杆菌

以琥珀酸产生菌Actinobacillus succinogenes CGMCC 1593为出发菌,分别经过紫外线-甲基磺酸乙酯(UV-EMS)和紫外线-硫酸二乙酯(UV-DES)诱变处理,得到7株耐酸性有所提高的突变株.以此作为候选菌库,经3轮原生质体递进融合,筛选获得4株可以在pH 5.6下生长的改组菌株.其中改组菌株F3-21在pH 5.6的完全液体培养基中生长的OD值是原始菌的7倍,在pH 5.2条件下仍能生长;其摇瓶发酵48h琥珀酸产量较原始菌株提高48%.在5L发酵罐中进行分批发酵,当控制pH在较低值(5.6～6.0)时,F3-21厌氧发酵48h积累琥珀酸38.1g/L,较出发菌株提高了45%;当控制pH在6.5～7.0时,F3-21厌氧发酵32h积累琥珀酸40.7g/L.F3-21在5L发酵罐中进行补料分批发酵,厌氧发酵72h,产琥珀酸达67.4g/L.结果说明基因组改组技术能够改进琥珀酸放线菌的耐酸性能及其琥珀酸的产量.     

琥珀酸放线杆菌发酵培养基的优化

对琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC1593发酵产生琥珀酸培养基的主要成分,及其含量进行优化。通过单因素试验,得出发酵培养基中葡萄糖、酵母膏和玉米浆的含量对产生琥珀酸有显著影响;采用响应面法(RSM),得出多元二次回归方程拟合的三种因素与琥珀酸含量间的函数关系,并根据优化结果与实验,CGMCC1593产琥珀酸达到41.69g/L。     

Actinobacillus succinogenes抗氟乙酸突变株的选育及其代谢流量分析

琥珀酸是一种用于合成树脂、可降解塑料及许多化学中间体的重要绿色化工原料。为了提高琥珀酸的发酵产率, 基于Actinobacillus succinogenes的代谢流量分布情况对其育种机制进行了研究。以Actinobacillus succinogenes CGMCC1593为原始菌株进行NTG诱变, 挑选在含有50~100 mmol/L氟乙酸平板生长较快的菌落, 经过初筛和复筛, 发现SF-9菌株产生更多琥珀酸且积累乙酸较少。以50 g/L的葡萄糖为碳源, 在5 L发酵罐上进行分批发酵, 该菌株发酵32 h时琥珀酸产量(34.8 g/L)提高了23.4%, 琥珀酸/乙酸比率为9:1, 副产物乙酸量比原始菌株降低了约50%。代谢流量分析(MFA)结果表明, PEP是影响琥珀酸合成的关键节点, PYR是影响乙酸等杂酸生成比例的关键节点, 并且这两个节点均非刚性节点。通过氟乙酸抗性诱变, 成功地筛选出了流向乙酸、甲酸和乳酸等杂酸的流量相对减少, 而流向琥珀酸的流量明显增强的突变菌株SF-9。     

Actinobacillus succinogenes抗氟乙酸突变株的选育及其代谢流量分析

琥珀酸是一种用于合成树脂、可降解塑料及许多化学中间体的重要绿色化工原料。为了提高琥珀酸的发酵产率, 基于Actinobacillus succinogenes的代谢流量分布情况对其育种机制进行了研究。以Actinobacillus succinogenes CGMCC1593为原始菌株进行NTG诱变, 挑选在含有50~100 mmol/L氟乙酸平板生长较快的菌落, 经过初筛和复筛, 发现SF-9菌株产生更多琥珀酸且积累乙酸较少。以50 g/L的葡萄糖为碳源, 在5 L发酵罐上进行分批发酵, 该菌株发酵32 h时琥珀酸产量(34.8 g/L)提高了23.4%, 琥珀酸/乙酸比率为9:1, 副产物乙酸量比原始菌株降低了约50%。代谢流量分析(MFA)结果表明, PEP是影响琥珀酸合成的关键节点, PYR是影响乙酸等杂酸生成比例的关键节点, 并且这两个节点均非刚性节点。通过氟乙酸抗性诱变, 成功地筛选出了流向乙酸、甲酸和乳酸等杂酸的流量相对减少, 而流向琥珀酸的流量明显增强的突变菌株SF-9。     

Effects of glucose, vitamins, and DO concentrations on pyruvate fermentation using Torulopsis glabrata IFO 0005 with metabolic flux analysis

The effects of glucose, vitamins, and DO concentrations on efficient pyruvic acid fermentation were investigated using Torulopsis glabrata IFO 0005, and a novel biphasic culture method was developed on the basis of the metabolic flux analysis. T. glabrata requires the four vitamins nicotinic acid (NA), thiamine hydrochloride (B(1)), pyridoxine hydrochloride, and biotin for cell growth. The deficiency of these vitamins plays an essential role in pyruvate fermentation. In the present study, we considered the effects of the first two vitamins on the pyruvate fermentation. On the basis of several batch and fed-batch experiments, it was found that, as a result of glucose inhibition of cell growth, the initial glucose concentration should be around 30-40 g/L, and the glucose concentration during fermentation should be controlled at high level around 30 g/L. On the basis of an analysis of carbon flux distribution, a biphasic fermentation method was developed where the cultivation started with a high DO (at 40-50% of air saturation) for efficient cell growth and then was reduced to 5-10% for efficient pyruvate production. Since a fair amount of ethanol was formed when the DO concentration was decreased, the addition of NA turned out to be effective in reducing the ethanol formation. This may be due to a relaxing of the requirement for NADH oxidation by the alcohol dehydrogenase pathway. Since B(1) affects both the pyruvate dehydrogenase complex and pyruvate decarboxylase, its initial concentration must be carefully determined by considering both the cell growth and pyruvate production phases.