基于代谢通量分析的琥珀酸放线杆菌高产选育研究

在对产琥珀酸放线杆菌代谢分析的基础上选育出高产突变株对琥珀酸的工业生物转化有重要意义.在矩阵分析代谢通量基础上,围绕柔性节点下的副产物乙酸及乙醇的降低分别实施软X诱变及定点突变选育,并对比分析了突变株与出发株相关酶活及基因序列变化.针对出发株的流量分析显示产物琥珀酸的代谢通量为1.78(mmol/g/h),主要副产物乙酸与乙醇的代谢通量分别为(0.60mmol/g/h)和(1.04 mmol/g/h),并发现乙醇代谢加剧了琥珀酸合成中的H电子供体的不足;筛选出的氟乙酸抗性突变株S.JST1的乙酸代谢通量降低了96%,为0.024(mmol/g/h),酶活检测表明磷酸乙酰转移酶(Pta)的酶比活力从602降低到74,进一步的序列对比分析发现pta突变基因中产生了一个突变位点:adh定点复合突变株S.JST2的乙醇代谢通量降低了98%,为0.020(mmol/g/h),酶活检测表明Adh的酶比活力从585降低到62.最终突变株S.JST2琥珀酸累积产量达65.7 g/L.围绕产琥珀酸放线杆菌Pta及Adh酶活的降低实施定向选育,在降低副产物流量的同时,有助于改善细胞H供体代谢平衡进而提高琥珀酸的流量.所获突变株具有工业应用潜力.     

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.     

High levels of malic acid production by the bioconversion of corn straw hydrolyte using an isolated Rhizopus delemar strain

The microbial fermentation of malic acid, which is one of the most important organic acid platforms used widely in food and chemical engineering, has attracted considerable interest. A malate production strain was isolated, a mutation was induced, and regulation of the metabolic network was then conducted. The identification results showed that the malic acid production strain, HF- 119, belonged to Rhizopus delemar. An analysis of the metabolic pathway showed that the malic acid flux of this strain occurred through three main pathways, and many byproducts, such as succinic acid, fumaric acid and ethanol, were produced. Although corn straw hydrolyte was used, the metabolism of xylose was not as rapid as that of glucose. Subsequently, breeding of the strains and regulation of the metabolic network resulted in an increase in malate yield, and the strain HF-121 produced more than 120 g/L malic acid within 60 h. The ability to produce malic acid from biomass hydrolyte highlights the industrial development potential of this strain.     

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。     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

Enhanced production of succinic acid by metabolically engineered<Emphasis Type="Italic">Escherichia coli</Emphasis> with amplified activities of malic enzyme and fumarase

Apfl ldhA double mutantEscherichia coli strain NZN111 was used to produce succinic acid by overexpressing theE. coli malic enzyme gene (sfcA). This strain, however, produced a large amount of malic acid as well as succinic acid. After the analyses of the metabolic pathways, thefumB gene encoding the anaerobic fumarase ofE. coli was co-amplified to solve the problem of malic acid accumulation. A plasmid, pTrcMLFu, was constructed, which contains an artificial operon (sfcA-fumB) under the control of the inducibletrc promoter. From the batch culture of recombinantE. coli NZN111 harboring pTrcMLFu, 7 g/L of succinic acid was produced from 20 g/L of glucose, with no accumulation of malic acid. From the metabolic flux analysis the strain was found under reducing power limiting conditions by severe reorientation of metabolic fluxes.     

Differential labeling of the subunits of respiratory Complex III with [3H]succinic anhydride, [14C]succinic anhydride,andp-diazobenzene-[35S]sulfonate

Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [³H]succinic anhydride plus [³⁵S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [³H]succinic anhydride. In contrast, relative labeling by [³⁵S]DABS was similar to [³H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [³H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by¹⁴C- and³H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of¹⁴C/³H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in¹⁴C/³H labeling ratios of core proteins I and II, cytochromec ₁, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein.     

丙酮丁醇梭菌硫氧还蛋白系统在溶剂生产过程中的功能

[目的]探究丙酮丁醇梭菌硫氧还蛋白系统在生长和代谢过程中的功能.[方法]使用ClosTron系统对硫氧还蛋白系统中的硫氧还蛋白还原酶基因(trxB)进行插入失活,得到突变株,通过Southern杂交方法验证插入内含子的拷贝数;在基本培养基中进行分批发酵,比较并分析突变株的生长特点;通过pH控制,利用限磷的连续发酵方法使...     

From genome sequence to integrated bioprocess for succinic acid production by <Emphasis Type="Italic">Mannheimia succiniciproducens</Emphasis>

Mannheimia succiniciproducens is a capnophilic gram-negative bacterium isolated from bovine rumen. Wild-type M. succiniciproducens can produce succinic acid as a major fermentation product with acetic, formic, and lactic acids as byproducts during the anaerobic cultivation using several different carbon sources. Succinic acid is an important C4 building block chemical for many applications. Here, we review the progress made with M. succiniciproducens for efficient succinic acid production; the approaches taken towards the development of an integrated process for succinic acid production are described, which include strain isolation and characterization, complete genome sequencing and annotation, development of genetic tools for metabolic engineering, strain development by systems approach of integrating omics and in silico metabolic analysis, and development of fermentation and recovery processes. We also describe our current effort on further improving the performance of M. succiniciproducens and optimizing the mid- and downstream processes. Finally, we finish this mini-review by discussing the issues that need to be addressed to make this process of fermentative succinic acid production employing M. succiniciproducens to reach the industrial-scale process.     

Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol

Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H⁺ conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l^?1 of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli.     

Succinic acid production from corn stalk hydrolysate in an E. coli mutant generated by atmospheric and room-temperature plasmas and metabolic evolution strategies

AFP111 is a spontaneous mutant of Escherichia coli with mutations in the glucose-specific phosphotransferase system, pyruvate formate lyase system, and fermentative lactate dehydrogenase system, created to reduce byproduct formation and increase succinic acid accumulation. In AFP111, conversion of xylose to succinic acid only generates 1.67 ATP per xylose, but requires 2.67 ATP for xylose metabolism. Therefore, the ATP produced is not adequate to accomplish the conversion of xylose to succinic acid in chemically defined medium. An E. coli mutant was obtained by atmospheric and room-temperature plasmas and metabolic evolution strategies, which had the ability to use xylose and improve the capacity of cell growth. The concentration of ATP in the mutant was 1.33-fold higher than that in AFP111 during xylose fermentation. In addition, under anaerobic fermentation with almost 80 % xylose from corn stalk hydrolysate, a succinic acid concentration of 21.1 g l^?1 was obtained, with a corresponding yield of 76 %.     

肌醇、唾液酸及岩藻糖代谢途径对嗜水气单胞菌致病性的影响

【目的】调查肌醇、唾液酸以及岩藻糖代谢途径在嗜水气单胞菌感染宿主过程中对细菌致病性的影响。【方法】采用同源重组技术分别缺失嗜水气单胞菌NJ-35株的肌醇代谢相关基因iolC、唾液酸代谢相关基因nanA和岩藻糖代谢相关基因fucK,测定各缺失株对斑马鱼的半数致死量(LD_(50));将野生株与缺失株共感染鲫鱼,统计野生株和缺失株在不同组织中的细菌载量。【结果】各代谢相关基因的缺失均成功阻断了菌株对相应底物的降解能力。iolC的缺失导致菌株对斑马鱼的LD50升高近12倍,而nanA和fucK的缺失对LD50没有明显影响。野生株与iolC缺失株共感染鲫鱼,肝脏、脾脏和肾脏中野生株的载量显著高于缺失株,表现出明显的生长优势;nanA和fucK缺失株与野生株共感染鲫鱼,野生株和缺失株载量在各组织中均无明显差异。【结论】肌醇代谢途径在嗜水气单胞菌感染致病过程中发挥重要作用,而唾液酸和岩藻糖代谢途径对细菌无明显影响。     

调控大肠杆菌胞内ATP和NADH水平促进琥珀酸生产

琥珀酸作为一种重要的C4平台化合物,广泛应用于食品、化学、医药等领域。利用大肠杆菌(Escherichia coli)发酵生产琥珀酸受胞内辅因子不平衡的影响,存在产率低、生产强度低、副产物多等问题。为此,对不同氧气条件下琥珀酸产量和化学计量学分析发现,微厌氧条件下E.coli FMME-N-26高效积累琥珀酸需要借助三羧酸循环(tricarboxylic acid cycle,TCA)为还原性三羧酸途径(reductive tricarboxylic acid pathway,r-TCA)提供足够的ATP和NADH。通过减少ATP消耗、强化ATP合成、阻断NADH竞争途径和构建NADH回补路径等代谢工程策略,组合调控胞内ATP与NADH含量,获得工程菌株E.coli FW-17。通过发酵条件优化,菌株E.coli FW-17在5 L发酵罐能积累139.52 g/L琥珀酸,比出发菌株提高了17.81%,乙酸浓度为1.40 g/L,降低了67.59%。进一步在1000 L发酵罐中进行放大实验,琥珀酸产量和乙酸浓度分别为140.2 g/L和1.38 g/L。     

Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which had previously been identified by using genome-scale stoichiometric metabolic model simulations. The acl gene was deleted using the bipartite gene-targeting method, and the mutant was characterized in batch cultivation. It was found that the succinic acid yield was increased threefold by deleting the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger.     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

高产琥珀酸菌株的通量筛选

以琥珀酸放线杆菌Actinobacillus succinogenes F3—21为出发菌株,分别用吖啶黄、紫外线、紫外线．硫酸二乙酯和亚硝基胍进行诱变,产生突变菌库。用“96孔板培养－HPLC浓缩检测－厌氧瓶复筛”的模式筛选高产突变株。从1056株突变株中,筛选到一株高产菌株Ⅵ-10-C。连续传代10次,产酸水平不变。在5L发酵罐中补料分批发酵72h,Ⅵ－10－C产琥珀酸87．6g／L,生产强度1．22g/（L·h）,糖酸转化率0．66g／g;琥珀酸产量比出发菌提高了30％。代谢通量与关键酶活性分析表明：相比于F3-21,Ⅵ-10-C发酵过程中从磷酸烯醇式丙酮酸节点处流向草酰乙酸的代谢流量增加了28．9％,相对应的磷酸烯醇式丙酮酸羧化激酶（PEPCK）酶活提高了23．5％。结果表明用“96孔板培养-HPLC浓缩检测-厌氧瓶复筛”的模式能快速有效筛选高产琥珀酸菌株。     

Metabolic flux distribution for γ-linolenic acid synthetic pathways in<Emphasis Type="Italic">Spirulina platensis</Emphasis>

Spirulina produces γ-linolenic acid (GLA), an important pharmaceutical substance, in a relatively low level compared with fungi and plants, prompting more research to improve its GLA yield. In this study, metabolic flux analysis was applied to determine the cellular metabolic flux distributions in the GLA synthetic pathways of twoSpirulina strains, wild type BP and a high-GLA producing mutant Z19/2. Simplified pathways involving the GLA synthesis ofS. platensis formulated comprise of photosynthesis, gluconeogenesis, the pentose phosphate pathway, the anaplerotic pathway, the tricarboxylic cycle, the GLA synthesis pathway, and the biomass synthesis pathway. A stoichiometric model reflecting these pathways contains 17 intermediates and 22 reactions. Three fluxes—the bicarbonate (C-source) uptake rate, the specific growth rate, and the GLA synthesis rate—were measured and the remaining fluxes were calculated using linear optimization. The calculation showed that the flux through the reaction converting acetyl-CoA into malonyl-CoA in the mutant strain was nearly three times higher than that in the wild-type strain. This finding implies that this reaction is rate controlling. This suggestion was supported by experiments, in which the stimulating factors for this reaction (NADPH and MgCl₂) were added into the culture medium, resulting in an increased GLA-synthesis rate in the wild type strain.     

Metabolism associated with raised metabolic flux to sugar nucleotide precursors of exopolysaccharides in Lactobacillus delbrueckii subsp. bulgaricus

Exopolysaccharide (EPS) metabolism was studied in a galactose-negative strain of Lactobacillus delbrueckii subsp. bulgaricus, using two different approaches. Firstly, using both the parent strain and a chemically induced mutant with higher yield and specific productivity of EPS than the parent, comparative information was obtained relating to enzyme activities and metabolite levels associated with EPS formation when grown on lactose. Under continuous culture conditions (D=0.10 h⁻¹), the higher metabolic flux towards EPS formation in the mutant strain relative to the parent appeared to be mediated by raised levels of UDP-glucose pyrophosphorylase (UGP). Marginally raised UDP-galactose 4-epimerase (UGE) activity in the mutant strain suggested that this enzyme could also play a role in EPS overproduction. The second approach involved investigating the effect of growth rate on sugar nucleotide metabolism in the parent, as it is known that EPS production is growth-associated in this strain. UGE activity in the parent strain appeared to increase when the growth rate was elevated from 0.05 to 0.10 h⁻¹, and further to 0.35 h⁻¹, conditions that can be associated with higher levels of metabolic flux to EPS formation. Concurrent with these increments, intracellular ATP levels in the cell were raised. In both investigations glucose-6-phosphate accumulated pointing to a constriction at this branch-point, and a limitation in the flow of carbon towards fructose-6-phosphate or glucose-1-phosphate. The changes in metabolism associated with enhanced flux to EPS provide guidance as to how the yield of Lactobacillus delbrueckii subsp. bulgaricus EPS can be improved.     

耐铵型产琥珀酸放线杆菌的选育及铵离子对其生长代谢的影响

经硫酸二乙酯(DES)诱变,在含61～242mmol/LNH4+梯度平板中,筛选到一株耐铵型突变株YZ25,该菌株在含121mmol/LNH4+发酵培养基中,琥珀酸产量达32.68g/L,转化率为65.4%,比出发菌提高了180.5%。进一步考察了不同形态铵盐对YZ25生长的影响,结果表明添加少量铵盐能够提高突变菌的生长速率,但当超过一定量后菌株生长受到抑制,不同铵盐对菌株的抑制程度不同,硫酸铵、碳酸氢铵、氯化铵和硝酸铵对突变株YZ25的半抑制浓度分别为:215mmol/L、265mmol/L、235mmol/L、210mmol/L。为了考察铵离子对YZ25发酵产琥珀酸的影响过程,在3.0L发酵罐以氨水作为pH的调控剂发酵,结果表明在稳定期前菌株生长基本不受铵离子抑制,生物量能够达到正常水平,但是进入稳定期后铵离子抑制作用越来越明显,导致菌株生长提前结束,耗糖不完全,产酸受阻。最后结合产琥珀酸放线杆菌Actinobacillus succinogenes代谢途径分析了铵离子对菌株抑制作用的机理。