Fed-batch culture of a metabolically engineered Escherichia coli strain designed for high-level succinate production and yield under aerobic conditions

An aerobic succinate production system developed by Lin et al. (Metab Eng, in press) is capable of achieving the maximum theoretical succinate yield of 1.0 mol/mol glucose for aerobic conditions. It also exhibits high succinate productivity. This succinate production system is a mutant E. coli strain with five pathways inactivated: DeltasdhAB, Delta(ackA-pta), DeltapoxB, DeltaiclR, and DeltaptsG. The mutant strain also overexpresses Sorghum vulgare pepc. This mutant strain is designated HL27659k(pKK313). Fed-batch reactor experiments were performed for the strain HL27659k(pKK313) under aerobic conditions to determine and demonstrate its capacity for high-level succinate production. Results showed that it could produce 58.3 g/l of succinate in 59 h under complete aerobic conditions. Throughout the entire fermentation the average succinate yield was 0.94+/-0.07 mol/mol glucose, the average productivity was 1.08+/-0.06 g/l-h, and the average specific productivity was 89.77+/-3.40 mg/g-h. Strain HL27659k (pKK313) is, thus, capable of large-scale succinate production under aerobic conditions. The results also showed that the aerobic succinate production system using the designed strain HL27659k(pKK313) is more practical than conventional anaerobic succinate production systems. It has remarkable potential for industrial-scale succinate production and process optimization.     

Succinate production from sucrose by metabolic engineered Escherichia coli strains under aerobic conditions

Two metabolically engineered E. coli strains HL2765k and HL27659k, while capable of producing succinate from glucose with high yields, are not able to grow and produce succinate on sucrose. Consequently, the pUR400 plasmid containing scrK, Y, A, B, and R genes was introduced into HL2765k and HL27659k, respectively. Shake flask culture studies showed that the resulting strains can utilize sucrose; the strain HL2765k pUR400 and HL27659k pUR400 can produce succinate aerobically with a molar yield of 0.78 ± 0.02 mol/mol and 1.35 ± 0.13 mol/mol, respectively. On introduction of the plasmid pHL413, which encodes the heterologous pyruvate carboxylase (PYC) from Lactococcus lactis, the molar succinate yield increased to 1.60 ± 0.01 mol of succinate per mole of sucrose by the HL2765k pUR400 pHL413 strain and to 1.84 ± 0.10 by the HL27659k pUR400 pHL413 strain. In aerobic batch bioreactor studies, the succinate production rate was faster, and succinate production reached 101.83 mM with a yield of 1.90 when dissolved oxygen (DO) was controlled at 40 ± 7%. In addition, the results showed that DO had an important effect on succinate production by influencing PYC activity. This work demonstrates the possibility of producing succinate aerobically using sucrose as the carbon source.     

大肠杆菌木糖生物合成琥珀酸的代谢工程研究

目的:对大肠杆菌进行代谢网络改造,考察木糖好氧发酵生产琥珀酸的可行性。方法:以有氧条件下大肠杆菌木糖生物合成琥珀酸的代谢途径分析为基础,以大肠杆菌BL21为出发菌株,通过P1噬菌体一步敲除法敲除琥珀酸脱氢酶基因(sdhA)、磷酸转乙酰基酶基因(pta)、丙酮酸脱氢酶基因(poxB)及异柠檬酸裂解酶阻遏物基因(iclR),构建木糖好氧发酵生产琥珀酸的大肠杆菌工程菌JLS400(△poxB△pta△iclR△sdhA)。将携带磷酸烯醇式丙酮酸羧化酶基因的质粒pJW225转化到JLS400中。结果:摇瓶发酵结果表明,构建的工程菌能以木糖为碳源,在好氧发酵条件下琥珀酸产率较高,副产物仅有少量乙酸和丙酮酸。结论:基因工程大肠杆菌JLS400pJW225的构建,为有氧条件下以木糖为原料生产琥珀酸的进一步研究奠定了基础。     

Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield

The potential to produce succinate aerobically in Escherichia coli would offer great advantages over anaerobic fermentation in terms of faster biomass generation, carbon throughput, and product formation. Genetic manipulations were performed on two aerobic succinate production systems to increase their succinate yield and productivity. One of the aerobic succinate production systems developed earlier (Biotechnol, Bioeng., 2004, accepted) was constructed with five mutations (DeltasdhAB, Deltaicd, DeltaiclR, DeltapoxB, and Delta(ackA-pta)), which created a highly active glyoxylate cycle. In this study, a second production system was constructed with four of the five above mutations (DeltasdhAB, DeltaiclR, DeltapoxB, and Delta(ackA-pta)). This system has two routes in the aerobic central metabolism for succinate production. One is the glyoxylate cycle and the other is the oxidative branch of the TCA cycle. Inactivation of ptsG and overexpression of a mutant Sorghum pepc in these two production systems showed that the maximum theoretical succinate yield of 1.0 mol/mol glucose consumed could be achieved. Furthermore, the two-route production system with ptsG inactivation and pepc overexpression demonstrated substantially higher succinate productivity than the previous system, a level unsurpassed for aerobic succinate production. This optimized system showed remarkable potential for large-scale aerobic succinate production and process optimization.     

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。     

Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate

Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes. This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth-based selection. After deletion of the central anaerobic fermentation genes (ldhA, adhE, ackA), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co-selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (focA, pflB, poxB, mgsA). The best succinate biocatalysts, strains KJ060(ldhA, adhE, ackA, focA, pflB) and KJ073(ldhA, adhE, ackA, focA, pflB, mgsA, poxB), produce 622-733 mM of succinate with molar yields of 1.2-1.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(ldhA, adhE, ackA, focA, pflB, mgsA), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized.     

Effect of culture operating conditions on succinate production in a multiphase fed-batch bioreactor using an engineered <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> strain

A metabolically engineered Escherichia coli strain SBS550MG (pHL413) was used in this study to investigate the impact of various culture operating conditions for improving the specific succinate production rate for better final titer while maintaining the theoretical succinate yield on glucose in multiphase fed-batch cultures. Previously, we reported that changes in the level of aeration during the cell growth phase significantly modified gene expression profiles and metabolic fluxes in this system (Martinez et al. 2010). Based on these observations, the examination of culture conditions was mainly focused on the aerobic growth phase. It was found that 2–5 h of low dissolved oxygen culture during the aerobic phase improves cell productivity, but pH control during the aerobic phase was not favorable for the system. Cell viability has been identified as a major limiting factor for succinate production. Supplementing LB medium and betaine, an anti-osmotic stress reagent, did not improve cell activity. A higher succinate titer (537.8 mM) using the current metabolic engineering E. coli strain was achieved, which can potentially be improved further by increasing cell viability.     

Chemostat culture characterization of Escherichia coli mutant strains metabolically engineered for aerobic succinate production: a study of the modified metabolic network based on metabolite profile, enzyme activity, and gene expression profile

Various Escherichia coli mutant strains designed for succinate production under aerobic conditions were characterized in chemostat. The metabolite profiles, enzyme activities, and gene expression profiles were studied to better understand the metabolic network operating in these mutant strains. The most efficient succinate producing mutant strain HL27659k was able to achieve a succinate yield of 0.91 mol/mol glucose at a dilution rate of 0.1/h. This strain has the five following mutations: sdhAB, (ackA-pta), poxB, iclR, and ptsG. Four other strains involved in this study were HL2765k, HL276k, HL2761k, and HL51276k. Strain HL2765k has mutations in sdhAB, (ackA-pta), poxB and iclR, strain HL276k has mutations in sdhAB, (ackA-pta) and poxB, strain HL2761k has mutations in sdhAB, (ackA-pta), poxB and icd, and strain HL51276k has mutations in iclR, icd, sdhAB, (ackA-pta) and poxB. Enzyme activity data showed strain HL27659k has substantially higher citrate synthase and malate dehydrogenase activities than the other four strains. The data also showed that only iclR mutation strains exhibited isocitrate lyase and malate synthase activities. Gene expression profiles also complemented the studies of enzyme activity and metabolites from chemostat cultures. The results showed that the succinate synthesis pathways engineered in strain HL27659k were highly efficient, yielding succinate as the only major product produced under aerobic conditions. Strain HL27659k was the only strain without pyruvate accumulation, and its acetate production was the least among all the mutant strains examined.     

Synergy between (13)C-metabolic flux analysis and flux balance analysis for understanding metabolic adaptation to anaerobiosis in E. coli

Genome-based Flux Balance Analysis (FBA) and steady-state isotopic-labeling-based Metabolic Flux Analysis (MFA) are complimentary approaches to predicting and measuring the operation and regulation of metabolic networks. Here, genome-derived models of Escherichia coli (E. coli) metabolism were used for FBA and 13C-MFA analyses of aerobic and anaerobic growths of wild-type E. coli (K-12 MG1655) cells. Validated MFA flux maps reveal that the fraction of maintenance ATP consumption in total ATP production is about 14% higher under anaerobic (51.1%) than aerobic conditions (37.2%). FBA revealed that an increased ATP utilization is consumed by ATP synthase to secrete protons from fermentation. The TCA cycle is shown to be incomplete in aerobically growing cells and submaximal growth is due to limited oxidative phosphorylation. An FBA was successful in predicting product secretion rates in aerobic culture if both glucose and oxygen uptake measurement were constrained, but the most-frequently predicted values of internal fluxes yielded from sampling the feasible space differ substantially from MFA-derived fluxes.     

Quantification and analysis of metabolic characteristics of aerobic succinate-producing Escherichia coli under different aeration conditions

The production of succinate by engineered Escherichia coli strains has been widely investigated. In this study, quantitative comparison of metabolic fluxes was carried out for the wild-type E. coli strain and a quintuple mutant strain QZ1111 that was designed for the production of succinate aerobically by knocking out five genes (ptsG, poxB, pta, sdhA, iclR) of the wild-type E. coli MG1655. Metabolic flux distributions of both strains were quantified by ¹³C-labeling experiments, together with the determination of physiological parameters and the expression level of key genes. The experimental results indicated that under the same aeration condition the fraction of oxaloacetate molecules originating from phosphoenolpyruvate was increased in E. coli QZ1111 compared to that in the wild-type E. coli MG1655. The glyoxylate shunt was likely activated in E. coli QZ1111 only under high aeration condition but repressed in other conditions, indicating that the deletion of the iclR gene could not completely remove the repression of the glyoxylate shunt with limited oxygen supply. Our results also suggested further genetic manipulation strategies to enhance the production yield of succinate.     

Metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems

Glycogen-accumulating organisms (GAO) have the potential to directly compete with polyphosphate-accumulating organisms (PAO) in EBPR systems as both are able to take up VFA anaerobically and grow on the intracellular storage products aerobically. Under anaerobic conditions GAO hydrolyse glycogen to gain energy and reducing equivalents to take up VFA and to synthesise polyhydroxyalkanoate (PHA). In the subsequent aerobic stage, PHA is being oxidised to gain energy for glycogen replenishment (from PHA) and for cell growth. This article describes a complete anaerobic and aerobic model for GAO based on the understanding of their metabolic pathways. The anaerobic model has been developed and reported previously, while the aerobic metabolic model was developed in this study. It is based on the assumption that acetyl-CoA and propionyl-CoA go through the catabolic and anabolic processes independently. Experimental validation shows that the integrated model can predict the anaerobic and aerobic results very well. It was found in this study that at pH 7 the maximum acetate uptake rate of GAO was slower than that reported for PAO in the anaerobic stage. On the other hand, the net biomass production per C-mol acetate added is about 9% higher for GAO than for PAO. This would indicate that PAO and GAO each have certain competitive advantages during different parts of the anaerobic/aerobic process cycle.     

基于代谢流量分布信息理解大肠杆菌中异柠檬酸裂解酶调节因子的代谢调控作用

基因的表达受不同的转录调节因子调节。大肠杆菌中的异柠檬酸裂解酶调节因子(IclR)能够抑制编码乙醛酸支路酶的aceBAK操纵子的表达。本研究基于代谢物的13C同位体物质分布来定量解析代谢反应,主要研究了iclR基因在大肠杆菌生理和代谢中的作用。大肠杆菌iclR基因缺失突变株的生长速率、糖耗速率和乙酸的产量相对于原始菌株都有所降低,但菌体得率略有增加。通过代谢途径的流量比率分析发现基因缺失株的乙醛酸支路得到了激活,33%的异柠檬酸流经了乙醛酸支路;戊糖磷酸途径的流量变小,使得CO2的生成量减少。同时,乙醛酸支路激活,但草酰乙酸形成磷酸烯醇式丙酮酸的流量基本不变,说明磷酸烯醇式丙酮酸-乙醛酸循环没有激活,没有过多的碳原子在磷酸烯醇式丙酮酸羧化激酶反应中以CO2形式排出,从而确保了菌体得率。葡萄糖利用速率的降低、乙酰辅酶A的代谢效率提高等使得iclR基因敲除菌的乙酸分泌较原始菌株有所降低。     

A metabolic model of cellular energetics and carbon flux during aerobic Escherichia coli fermentation

An integrated metabolic model for the production of acetate by Escherichia coli growing on glucose under aerobic conditions was presented previously (Ko et al., 1993). The resulting model equations can be used to explain phenomena often observed with industrial fermentations, i.e., increased acetate production which follows from high glucose uptake rate, a low dissolved oxygen concentration, a high specific growth rate, or a combination of these conditions. However, several questions still need to be addressed. First, cell composition is growth rate and media dependent. Second, the macromolecular composition varied between E. coli strains. And finally, a model that represents the carbon fluxes between the Embden-Meyerhof-Parnas (EMP) and the hexose monophosphate (HMP) pathways when cells are subject to internal and/or external stresses is still not well defined. In the present work, we have made an effort to account for these effects, and the resulting model equations show good agreement for wild-type and recombinant E. coli experimental data for the acetate concentration, the onset of acetate secretion, and cell yield based on glucose. These results are useful for optimizing aerobic E. coli fermentation processes. More specifically, we have determined the EMP pathway carbon flux profiles required by the integrated metabolic model for an accurate fit of the acetic acid profile data from a wild-type E. coli strain ML308. These EMP carbon flux profiles were correlated with a dimensionless measurement of biomass and then used to predict the acetic acid profiles for E. coli strain F-122 expressing human immunodeficiency virus-(HIV(528)) beta-galactosidase fusion protein. The effect of different macromolecular compositions and growth rates between these two E. coli strains required a constant scaling factor for improved quantitative predictions.     

Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum

Corynebacterium glutamicum, an established industrial amino acid producer, has been genetically modified for efficient succinate production from the renewable carbon source glucose under fully aerobic conditions in minimal medium. The initial deletion of the succinate dehydrogenase genes (sdhCAB) led to an accumulation of 4.7 g l^?1 (40 mM) succinate as well as high amounts of acetate (125 mM) as by‐product. By deleting genes for all known acetate‐producing pathways (pta‐ackA, pqo and cat) acetate production could be strongly reduced by 83% and succinate production increased up to 7.8 g l^?1 (66 mM). Whereas overexpression of the glyoxylate shunt genes (aceA and aceB) or overproduction of the anaplerotic enzyme pyruvate carboxylase (PCx) had only minor effects on succinate production, simultaneous overproduction of pyruvate carboxylase and PEP carboxylase resulted in a strain that produced 9.7 g l^?1 (82 mM) succinate with a specific productivity of 1.60 mmol g (cdw)^?1 h^?1. This value represents the highest productivity among currently described aerobic bacterial succinate producers. Optimization of the production conditions by decoupling succinate production from cell growth using the most advanced producer strain (C. glutamicumΔpqoΔpta‐ackAΔsdhCABΔcat/pAN6‐pyc^P458Sppc) led to an additional increase of the product yield to 0.45 mol succinate mol^?1 glucose and a titre of 10.6 g l^?1 (90 mM) succinate.     

Three classes of Escherichia coli mutants selected for aerobic expression of fumarate reductase

Fumarate reductase (encoded by frd) and succinate dehydrogenase (encoded by sdh) of Escherichia coli are both known to catalyze the interconversion of fumarate and succinate. Fumarate reductase, however, is not inducible aerobically and therefore cannot participate in the dehydrogenation of succinate. Three classes of suppressor mutants, classified as frd oxygen-resistant [frd(Oxr)], constitutive [frd(Con)], and gene amplification [frd(Amp)] mutants, were selected from an sdh strain as pseudorevertants that regained the partial ability to grow aerobically on succinate. All contained increased aerobic levels of fumarate reductase activity. In frd(Oxr) mutants expression of the operon showed increased resistance to aerobic repression. Under anaerobic conditions expression of the operon became less dependent on the fnr+ gene product, a pleiotropic activator protein for genes encoding anaerobic respiratory enzymes. Exogenous fumarate, however, was still required for full induction, and repression by nitrate was undiminished. Thus, aerobic repression and anaerobic nitrate repression appear to involve separate mechanisms. In frd(Con) mutants expression of the operon became highly resistant to aerobic repression. Under anaerobic conditions expression of the operon no longer required the fnr+ gene product or exogenous fumarate and became immune to nitrate repression. In partial diploids bearing an frd(Oxr) or an frd(Con) allele and phi(frd+-lac) there was no mutual regulatory influence between the two genetic loci. Thus, the frd mutations act in cis and hence are probably in the promoter region. In frd(Amp) mutants the frd locus was amplified without significant alteration in the pattern of regulation.     

Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.     

Glycerol as a substrate for aerobic succinate production in minimal medium with Corynebacterium glutamicum

Corynebacterium glutamicum, an established microbial cell factory for the biotechnological production of amino acids, was recently genetically engineered for aerobic succinate production from glucose in minimal medium. In this work, the corresponding strains were transformed with plasmid pVWEx1-glpFKD coding for glycerol utilization genes from Escherichia coli. This plasmid had previously been shown to allow growth of C. glutamicum with glycerol as sole carbon source. The resulting strains were tested in minimal medium for aerobic succinate production from glycerol, which is a by-product in biodiesel synthesis. The best strain BL-1/pVWEx1-glpFKD formed 79 mM (9.3 g l⁻¹) succinate from 375 mM glycerol, representing 42% of the maximal theoretical yield under aerobic conditions. A specific succinate production rate of 1.55 mmol g⁻¹ (cdw) h⁻¹ and a volumetric productivity of 3.59 mM h⁻¹ were obtained, the latter value representing the highest one currently described in literature. The results demonstrate that metabolically engineered strains of C. glutamicum are well suited for aerobic succinate production from glycerol.     

Studies of regulatory metabolism in Moniezia expansa: general considerations.

The activities of selected enzymes in the branched metabolic pathway to succinate or lactate were determined in cytosol and mitochondrial fractions. The enzymes of lowest activity in the cytosol, and thus possibly regulatory, are phosphofructokinase and pyruvate kinase. Malic enzyme activity could scarcely be detected in either compartment; phosphoenolpyruvate carboxykinase and malate dehydrogenase occur in both. The end products of metabolism are succinate and lactate; under anaerobic conditions lactate production increases whereas succinate production shows a small decrease. The presence of glucose in the medium does not influence the change, but causes an increase in total endproduct accumulation. Levels of metabolic intermediates in worms incubated aerobically and anaerobically are presented, and ‘cross-over’ plots and calculations of apparent equilibrium constants identify hexokinase, phosphofructokinase and pyruvate kinase as regulatory. Under aerobic conditions a large increase in the size of the malate pool is observed suggesting that the depression of lactate production is produced by its inhibitory effect on pyruvate kinase. Adenine nucleotide levels are maintained whether or not the worm is incubated under anaerobic conditions.     

Anaerobic Expression of Escherichia coli Succinate Dehydrogenase: Functional Replacement of Fumarate Reductase in the Respiratory Chain during Anaerobic Growth

Succinate-ubiquinone oxidoreductase (SQR) from Escherichia coli is expressed maximally during aerobic growth, when it catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and reduces ubiquinone in the membrane. The enzyme is similar in structure and function to fumarate reductase (menaquinol-fumarate oxidoreductase [QFR]), which participates in anaerobic respiration by E. coli. Fumarate reductase, which is proficient in succinate oxidation, is able to functionally replace SQR in aerobic respiration when conditions are used to allow the expression of the frdABCD operon aerobically. SQR has not previously been shown to be capable of supporting anaerobic growth of E. coli because expression of the enzyme complex is largely repressed by anaerobic conditions. In order to obtain expression of SQR anaerobically, plasmids which utilize the P_FRD promoter of the frdABCD operon fused to the sdhCDAB genes to drive expression were constructed. It was found that, under anaerobic growth conditions where fumarate is utilized as the terminal electron acceptor, SQR would function to support anaerobic growth of E. coli. The levels of amplification of SQR and QFR were similar under anaerobic growth conditions. The catalytic properties of SQR isolated from anaerobically grown cells were measured and found to be identical to those of enzyme produced aerobically. The anaerobic expression of SQR gave a greater yield of enzyme complex than was found in the membrane from aerobically grown cells under the conditions tested. In addition, it was found that anaerobic expression of SQR could saturate the capacity of the membrane for incorporation of enzyme complex. As has been seen with the amplified QFR complex, E. coli accommodates the excess SQR produced by increasing the amount of membrane. The excess membrane was found in tubular structures that could be seen in thin-section electron micrographs.