过量表达Bacillus subtilis磷酸烯醇式丙酮酸羧化激酶对大肠杆菌产琥珀酸的影响

在大肠杆菌厌氧混合酸发酵途径中,磷酸烯醇式丙酮酸羧化酶(PPC)和磷酸烯醇式丙酮酸羧化激酶(PCK)皆可催化由磷酸烯醇式丙酮酸(PEP)到草酰乙酸(OAA)的反应。鉴于经由PCK催化的反应伴有ATP的生成,理论上更有利于菌体生长和产酸,本研究以大肠杆菌W3110(△pfl,△ldh)为出发菌株,利用λ-Red同源重组系统构建了其ppc缺陷菌株并在此基础上过量表达了Bacillus subtilispck基因。初步的厌氧发酵实验表明:过量表达pck可在一定程度上恢复初始菌株厌氧代谢葡萄糖的能力。其中又以ppc缺陷株更为明显,其耗糖能力和产酸能力分别为对照菌株的4.2和15.3倍。     

Activating Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Combination for Improvement of Succinate Production

Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production.     

Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.     

高等植物中的磷酸烯醇式丙酮酸羧激酶

简要介绍了近年来有关高等植物中磷酸烯醇式丙酮酸羧激酶（ＰＥＰＣＫ）的研究进展,并讨论了此酶的结构、功能和调节等方面的问题。     

植物磷酸烯醇式丙酮酸羧激酶(PEPCK)研究进展

磷酸烯醇式丙酮酸羧激酶(PEPCK)是一个广泛存在于开花植物中的酶, 在植物体内仅存在于特定的组织和细胞中, 其活性受自身磷酸化和一些相关代谢产物的调节。PEPCK的磷酸化在多种植物体内受光调控。ATP存在时, PEPCK催化OAA生成PEP, 而PEP是多种反应的前体物质。通过不同的代谢途径, PEPCK间接地参与贮油植物种子萌发和植物果实成熟的糖异生过程, C₄和CAM(景天科代谢)植物光合作用中的CO₂浓缩过程, 细胞内pH值平衡和植物体内氮代谢过程等, 从而调节植物的生长发育。该文综述了植物中已发现的PEPCK及其在植物生命活动过程中的自身活性调节和生理功能。     

Effect of Growth Phase Feeding Strategies on Succinate Production by Metabolically Engineered Escherichia coli

Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by Escherichia coli AFP111 with knockouts in rpoS, pflAB, ldhA, and ptsG genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.Succinic acid is valued as one of the key basic chemicals used in the preparation of biodegradable polymers or as raw material for chemicals of the C₄ family (8, 19). The fermentative production of succinic acid from renewable resources is environmentally acceptable and sustainable (3). A breakthrough in genetically engineering Escherichia coli (6, 7, 11, 18) for succinate production was the isolation of strain AFP111 (1, 4), a mutant of NZN111 with a spontaneous ptsG mutation (pflAB ldhA double mutant). The process involves a two-stage fermentation, with aerobic cell growth followed by anaerobic conditions for succinate production (16, 21, 22). The aerobically induced enzymes can maintain their activity during the anaerobic phase and significantly affect succinate fermentation (22, 23). Using the best transition time based on the activities of the key enzymes and other physiological states, a two-stage fermentation using the recombinant AFP111 strain harboring pTrc99A-pyc achieved a final succinic acid concentration and productivity of 99.2 g·liter⁻¹ and 1.3 g·liter⁻¹·h⁻¹, respectively (21).Aerobic cell growth is essential for the subsequent anaerobic fermentation. However, few studies have focused on the regulation of aerobic cell growth. As a regulation method, gluconeogenic carbon sources were used instead of glucose for the aerobic growth of Escherichia coli NZN111 and the activities of enzymes that are favorable for the anaerobic synthesis of succinate were enhanced (23, 24). Unfortunately, a gluconeogenic carbon source (e.g., sodium acetate) might increase the osmotic pressure of culture media, which would be detrimental to succinate production (23). As another regulation method, a glucose feeding strategy controlling the glucose concentration at about 0.5 g·liter⁻¹ up to 1 g·liter⁻¹ was reported to prevent excessive formation of acetic acid (16).In this study, we investigated different glucose feeding strategies for the aerobic growth phase of the two-phase process for succinate production by E. coli AFP111. Specifically, we compared several growth rates by using glucose limitation in addition to maximum growth under conditions of excess glucose.E. coli AFP111 [F⁺ λ⁻ rpoS396(Am) rph-1 ΔpflAB::Cam ldhA::Kan ptsG] (4, 16), which was a kind gift from D. P. Clark (Southern Illinois University), was the only strain used in this study. Luria-Bertani (LB) medium (60 ml) was used for inoculum culture in 1,000-ml flasks, and 3 liters of chemically defined medium (13, 14) was used for two-stage culture in a 7-liter fermentor. Two-stage fermentations were divided into three types, based on the glucose feeding strategy used during the aerobic stage. For type I culture, the glucose concentration was maintained at about 20 g·liter⁻¹ during aerobic cell growth. Type II and III cultures comprised a batch process and subsequent glucose-limited fed-batch process (Fig. ​(Fig.1).1). The batch process initially contained 13 g/liter of glucose. The fed-batch process began when the dry cell weight (DCW) reached about 6 g/liter, with type II and type III cultures using a 600 g/liter glucose feed to achieve cell growth rates of 0.15 h⁻¹ and 0.07 h⁻¹, respectively (10). When the DCW reached 12 g·liter⁻¹, the aerobically grown cells were directly transferred to anaerobic conditions (Fig. ​(Fig.1).1). For the anaerobic process, oxygen-free CO₂ was sparged at 0.5 liter·min⁻¹, the pH was controlled between 6.4 and 6.8 with intermittent supplementation of solid magnesium carbonate hydroxide, and the glucose concentration was maintained at about 20 g·liter⁻¹ by supplying glucose in an 800-g·liter⁻¹ solution.Open in a separate windowFIG. 1.Concentrations of glucose (circles), DCW (triangles), and succinic acid (squares) in the three types of two-stage fermentation by AFP111. μ, growth rate.The optical density at 600 nm was used to monitor cell growth, and this value was correlated to DCW. The concentration of glucose was assayed with an enzyme electrode analyzer, and organic acids were quantified by high-performance liquid chromatography (HPLC). The intracellular concentrations of NADH and NAD⁺ were assayed with a cycling method (12). The activities of isocitrate lyase (ICL) (20), pyruvate kinase (PYK) (17), phosphoenolpyruvate (PEP) carboxykinase (PCK) (20, 23), PEP carboxylase (PPC) (23), and malate dehydrogenase (MDH) (23) were measured spectrophotometrically at the end of the aerobic phase and 12 h after the onset of the anaerobic phase.All three types of fermentations were terminated when the succinate concentration increased less than 1 g·liter⁻¹ in 5 h. Type III fermentation was terminated at a final succinic acid concentration of 101.2 g·liter⁻¹ and an anaerobic-phase productivity of 1.89 g·liter⁻¹·h⁻¹ (Fig. ​(Fig.1).1). Trace amounts of by-products (such as acetate, ethanol, and pyruvate) accumulated and did not follow any trend in the anaerobic phase (data not shown).At the end of the aerobic culture phase, the specific enzyme activities of PCK, PYK, and ICL in type III culture were 2.9, 2.5, and 11.4 times higher, respectively, than the activities in type I culture (Table ​(Table1)1) . This phenomenon is consistent with published reports that suggest that the expression of enzymes involved in anaplerotic metabolism and the glyoxylate shunt (5, 15) is elevated in E. coli grown under glucose-limited conditions. These enzymes maintained their activities in the subsequent anaerobic phase (Table ​(Table1)1) and would be central to succinate production (22, 23). The elevated levels of PCK and PPC would provide the reductive branch of the tricarboxylic acid (TCA) cycle with oxaloacetate (OAA) at a higher rate (9), thereby supplying both malate and citrate (Table ​(Table11).

TABLE 1.

Activities of enzymes at the end of the aerobic culture phase and 12 h after the onset of the anaerobic phase

好氧发酵生产琥珀酸工程菌株的构建

通过分析大肠杆菌的碳源代谢途径, 利用基因敲除手段, 以Escherichia coli MG1655为出发菌株, 成功构建了琥珀酸好氧发酵生产工程菌E. coli QZ1111 (MG1655?ptsG?poxB?pta?iclR?sdhA)。检测结果表明该菌株能以葡萄糖为碳源, 在好氧发酵且不表达任何异源基因的条件下大量积累琥珀酸。摇瓶试验证明, 琥珀酸发酵产量达到26.4 g/L, 乙酸盐作为唯一检测到的副产物产量为2.3 g/L。二者浓度比达到11.5:1。     

大肠杆菌突变株QQS101发酵生产琥珀酸初步研究

琥珀酸是一种具有重要应用价值的生物基平台化合物。对大肠杆菌focA-pflB ldhA突变株QQS101在严格厌氧条件下生长和葡萄糖代谢能力进行了考察,比较分析了葡萄糖与大肠杆菌混合酸发酵产物的单位碳的还原程度,认为非严格厌氧条件有利于QQS101发酵葡萄糖积累琥珀酸,进一步对有氧生长碳源进行了对比试验的结果表明,以木糖支持有氧生长,QQS101摇瓶发酵39 h消耗葡萄糖37.6 g/L,琥珀酸的产量达到31.01 g/L,摩尔产率为1.258 mol Succinate/mol Glucose。发酵过程中,丙氨酸的添加能够提高琥珀酸的摩尔产率。     

Reengineering Escherichia coli for Succinate Production in Mineral Salts Medium

The fermentative metabolism of glucose was redirected to succinate as the primary product without mutating any genes encoding the native mixed-acid fermentation pathway or redox reactions. Two changes in peripheral pathways were together found to increase succinate yield fivefold: (i) increased expression of phosphoenolpyruvate carboxykinase and (ii) inactivation of the glucose phosphoenolpyruvate-dependent phosphotransferase system. These two changes increased net ATP production, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production. Modest further improvements in succinate yield were made by inactivating the pflB gene, encoding pyruvate formate lyase, resulting in an Escherichia coli pathway that is functionally similar to the native pathway in Actinobacillus succinogenes and other succinate-producing rumen bacteria.Succinic acid is used as a specialty chemical in the agricultural, food, and pharmaceutical industries (17, 32). It has also been identified by the U.S. Department of Energy as one of the top 12 building block chemicals (30), because it can be converted into a variety of products, including green solvents, pharmaceutical products, and biodegradable plastics (17, 32). Although succinic acid is currently produced from petroleum-derived maleic anhydride, considerable interest in the fermentative production of succinate from sugars has emerged during the past decade (9, 10, 17).Several natural succinate-producing rumen bacteria that have high rates of succinate production and high succinate yields, such as Anaerobiospirillum succiniciproducens (22), Actinobacillus succinogenes (13, 28), and “Mannheimia succiniciproducens” (15, 16), have been isolated. However, these strains require complex organic nutrients that increase the costs associated with production, purification, and waste disposal (15, 22, 28). Low levels of succinate are produced by native strains of Escherichia coli in complex and mineral salts media (1, 4). Most mutant strains of E. coli that have been described previously as succinate producers also require complex organic nutrients (18, 23-26, 29, 31). Many involve two-step aerobic and anaerobic processes (3, 23-25, 29) and the addition of foreign genes (5, 6, 23-26, 29, 31).Novel E. coli biocatalysts (KJ060, KJ071, and KJ073) for the anaerobic production of succinate in mineral salts medium have been developed recently without the use of foreign genes or resident plasmids (9, 10). These biocatalysts were developed by combining constructed mutations to eliminate alternative routes of NADH oxidation in the mixed-acid pathway with growth-based selection (metabolic evolution). In subsequent studies (33), these strains were found to have recruited the glucose-repressed (7), gluconeogenic pck gene (11, 12, 19, 21, 27), encoding phosphoenolpyruvate carboxykinase (PCK) (derepressed via a point mutation in the promoter region), to replace the native phosphoenolpyruvate carboxylase (ppc) and serve as the primary route for CO₂ fixation (Fig. ​(Fig.1).1). A second acquired mutation was also identified as a frameshift mutation in the carboxy terminus of ptsI, inactivating the phosphoenolpyruvate-dependent phosphotransferase system (33). Glucose uptake by the phosphotransferase system was functionally replaced by galactose permease (galP) and glucokinase (glk).Open in a separate windowFIG. 1.Anaerobic metabolism of E. coli using the mixed-acid fermentation pathway (data from reference 1). The native phosphotransferase system pathway for glucose uptake and the mixed-acid pathway for fermentation are shown with black arrows. Peripheral reactions for glucose uptake, carboxylation, and acetyl-CoA synthesis are shown as dotted green arrows and represent new metabolic functions that have been recruited for succinate production from glucose. Reactions that have been blocked by gene deletions or point mutations are marked with an X. pck* indicates a novel mutation that derepressed pck, allowing the enzyme to serve as the primary route for oxaloacetate production. Pyruvate (boxed) appears at two sites but is presumed to exist as a single intracellular pool.Based on these previous studies, we have now determined the core mutations needed to direct carbon flow from glucose to succinate in E. coli and have constructed new succinate-producing strains with a minimum of genetic change.     

Succinate production in Escherichia coli

Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for the production of four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve an optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon channeled into the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose and other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, the requirement for efficient recovery of succinate, and the reliability of the performance under scaleup are important in the overall process. The costs of the overall biorefinery-compatible process will determine the economic commercialization of succinate and its impact in larger chemical markets.     

产琥珀酸重组大肠杆菌的发酵性能研究

研究了重组大肠杆菌JM001(△ppc)/pTrc99a-pck发酵产琥珀酸的性能,结果表明厌氧条件下其耗糖能力和产酸能力分别为对照菌株JM001的4.2倍和15.3倍。进一步优化发酵条件表明：采用接入菌泥的发酵方式比按照10%接种量转接厌氧发酵的效果要好,琥珀酸的对葡萄糖的质量收率提高了约10%,且副产物乙酸的量进一步降低。初始葡萄糖浓度高于60g/L时会对菌株的生长和产酸产生抑制,且浓度越高,抑制作用越明显。7L发酵罐放大实验中,整个厌氧发酵阶段葡萄糖的消耗速率为0.42g/(L.h),琥珀酸对葡萄糖的质量收率为67.75%,琥珀酸的生产强度为0.28g/(L.h)。     

Characterization of Phosphoenolpyruvate Carboxykinase from Panicum maximum

Phosphoenolpyruvate carboxykinase, EC 4.1.1.32 (PEPCK), was purified 43-fold from the grass Panicum maximum. Michaelis constants (Km) were determined for the exchange reaction, the carboxylation reaction, and the decarboxylation reaction. The Km values for oxaloacetate and ATP in the decarboxylation reaction were found to be lower than the Km values for the substrates used in the exchange reaction and in the carboxylation reaction. Phosphoenolpyruvate carboxylase was not detectable in the purified PEPCK preparation.     

Succinate Transport in Escherichia coli Mutants Defective in Succinate Metabolism

To investigate the operation of a succinate transport system in Escherichia coli, mutants defective in succinate metabolism were isolated. Although the metabolic blocks in the mutant cells were not complete, the succinate transport assays became possible.

Pyruvate, lactate or many other carbon sources stimulated succinate uptake, and the uptake was strongly inhibited by some electron transport inhibitors, uncouplers of oxidative phosphorylation and sulfhydryl reagents. The mutant strains accumulated succinate into the cells against a concentration gradient when suitable energy sources were supplied.

Presence of glucose in the medium strongly repressed the formation of the succinate transport system. The optimum pH for the succinate uptake was between 7.8 and 8.0.     

碳源对琥珀酸放线杆菌发酵产丁二酸的影响及其代谢流量分析

在厌氧条件下, Actinobacillus succinogenes能够利用单糖、双糖和糖醇等碳水化合物发酵生成丁二酸, 其中以山梨醇为碳源时丁二酸的产量最高。代谢流量分析结果表明: 与葡萄糖发酵相比较, 由于代谢系统中积累了更多的NADH, 使得代谢网络关键节点PYR和AcCoA处的代谢流量分配有了较大的变化, 导致更多的碳源流向丁二酸和乙醇, 而乙酸和甲酸的分泌相对减少。     

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%。