Increased Succinate Production Research Articles

Genetic optimization of the human gut bacterium Phocaeicola vulgatus for enhanced succinate production

The demand for sustainably produced bulk chemicals is constantly rising. Succinate serves as a fundamental component in various food, chemical, and pharmaceutical products. Succinate can be produced from sustainable raw materials using microbial fermentation and enzyme-based technologies. Bacteroides and Phocaeicola species, widely distributed and prevalent gut commensals, possess enzyme sets for the metabolization of complex plant polysaccharides and synthesize succinate as a fermentative end product. This study employed novel molecular techniques to enhance succinate yields in the natural succinate producer Phocaeicola vulgatus by directing the metabolic carbon flow toward succinate formation. The deletion of the gene encoding the methylmalonyl-CoA mutase (Δmcm, bvu_0309-0310) resulted in a 95% increase in succinate production, as metabolization to propionate was effectively blocked. Furthermore, deletion of genes encoding the lactate dehydrogenase (Δldh, bvu_2499) and the pyruvate:formate lyase (Δpfl, bvu_2880) eliminated the formation of fermentative end products lactate and formate. By overproducing the transketolase (TKT, BVU_2318) in the triple deletion mutant, succinate production increased from 3.9 mmol/g dry weight in the wild type to 10.9 mmol/g dry weight. Overall, succinate yield increased by 180% in the new mutant strain P. vulgatus Δmcm Δldh Δpfl pG106_tkt relative to the parent strain. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus, and forms the basis for targeted genetic optimization. The increase of efficiency highlights the huge potential of P. vulgatus as a succinate producer with applications in sustainable bioproduction processes.Key points• Deleting methylmalonyl-CoA mutase gene in P. vulgatus doubled succinate production• Triple deletion mutant with transketolase overexpression increased succinate yield by 180%• P. vulgatus shows high potential for sustainable bulk chemical production via genetic optimization

Bioupcycling Methane and CO2 for Succinate Production by an Engineered Type I Methanotrophic Bacterium.

Methane, a byproduct of agricultural activities, has shown potential as a nonedible substrate for biomanufacturing. The production of succinate by a methanotrophic bacterium utilizing methane presents an innovative route for the sustainable synthesis of chemicals. In this study, Methylotuvimicrobium buryatense 5GB1S was genetically modified through the reconstruction of an artificial serine cycle to enable the bioconversion of both methane and CO2 into succinate. The 13C labeling analysis confirmed the CO2 fixing in M. buryatense 5GB1S, leading to a 46% improvement in carbon conversion efficiency and a 107% increase in succinate production compared to the wild-type strain. The transcriptome data on carbon metabolisms was assessed to guide future optimizations for strengthening the overall carbon flux from methane to succinate. Finally, the maximum succinate titer of 299.36 mg/L was achieved under oxygen-limited conditions in 3 L bioreactors, which resulted in the volumetric productivity of 199.60 mg/L/day, representing a 23-fold enhancement compared to the wild-type strain. This study offers a new strategy for upcycling greenhouse gases into succinate in a sustainable manner through methanotrophic-based biomanufacturing.

HIF1α/ATF3 partake in PGK1 K191/K192 succinylation by modulating P4HA1/succinate signaling in glioblastoma.

Hypoxia is a pathological hallmark in most cancers, including glioblastoma (GBM). Hypoxic signaling activation and post-translational modification (PTM) of oncogenic proteins are well-studied in cancers. Accumulating studies indicate glycolytic enzyme PGK1 plays a crucial role in tumorigenesis, yet the underlying mechanisms remain unknown. We first used ChIP assays to uncover the crosstalk between HIF1α and ATF3 and their roles in P4HA1 regulation. Protein degradation analysis, LC-MS/MS, and in vitro succinate production assays were performed to examine the effect of protein succinylation on GBM pathology. Seahorse assay measured the effects of PGK1 succinylation at K191/K192 or its mutants on glucose metabolism. We utilized an in vivo intracranial mouse model for biochemical studies to elucidate the impact of ATF3 and P4HA1 on aerobic glycolysis and the tumor immune microenvironment. We demonstrated that HIF1α and ATF3 positively and negatively regulate the transcription of P4HA1, respectively, leading to an increased succinate production and increased activation of HIF1α signaling. P4HA1 expression elevated the succinate concentration, resulting in the enhanced succinylation of PGK1 at the K191 and K192 sites. Inhibition of proteasomal degradation of PGK1 by succinylation significantly increased aerobic glycolysis to generate lactate. Furthermore, ATF3 overexpression and P4HA1 knockdown reduced succinate and lactate levels in GBM cells, inhibiting immune responses and tumor growth. Together, our study demonstrates that HIF1α/ATF3 participated in P4HA1/succinate signaling, which is the major regulator of succinate biosynthesis and PGK1 succinylation at K191 and K192 sites in GBM. The P4HA1/succinate pathway might be a novel and promising target for aerobic glycolysis in GBM.

Designing a periplasmic photosynthetic biohybrid system for succinate and electric energy production

The emergence of photosynthetic biohybrid systems provides an environmentally friendly and sustainable approach to produce fuels and chemicals in microbial cell factories. However, the low efficiency of electron transfer between semiconductors and microorganisms hinders production efficiency. In this study, a periplasmic photosynthetic biohybrid system (PPB) was developed in E. coli through biological self-synthesis and optimized through engineering the periplasmic cytochrome network and cell membrane fluidity. The PPB significantly increased succinate production to a titer of 121.8 g/L in a 5 L fermenter by improving the reducing equivalents utilization. Additionally, the PPB was employed to establish a glucose-utilizing microbial consortium that exhibited robust performance, producing consistent electric energy output for a minimum of 17 days and achieving a maximum power density of 225.3 mW/m2. These findings demonstrate the capability of the PPB to enhance chemical production and generate electric energy from renewable carbon sources, thereby driving advances in green chemistry and promoting environmentally friendly industrial processes.

Effect of Salt Stress on the Activity, Expression, and Promoter Methylation of Succinate Dehydrogenase and Succinic Semialdehyde Dehydrogenase in Maize (Zea mays L.) Leaves

The effect of salt stress on the expression of genes, the methylation of their promoters, and the enzymatic activity of succinate dehydrogenase (SDH) and succinic semialdehyde dehydrogenase (SSADH) was investigated in maize (Zea mays L.). The incubation of maize seedlings in a 150 mM NaCl solution for 24 h led to a several-fold increase in the activity of SSADH that peaked at 6 h of NaCl treatment, which was preceded by an increase in the Ssadh1 gene expression and a decrease in its promoter methylation observed at 3 h of salt stress. The increase in SDH activity and succinate oxidation by mitochondria was slower, developing by 24 h of NaCl treatment, which corresponded to the increase in expression of the genes Sdh1-2 and Sdh2-3 encoding SDH catalytic subunits and of the gene Sdh3-1 encoding the anchoring SDH subunit. The increase in the Sdh2-3 expression was accompanied by the decrease in promoter methylation. It is concluded that salt stress results in the rapid increase in succinate production via SSADH operating in the GABA shunt, which leads to the activation of SDH, the process partially regulated via epigenetic mechanisms. The role of succinate metabolism under the conditions of salt stress is discussed.

A shift towards succinate-producing Prevotella in the ruminal microbiome challenged with monensin.

The time-resolved impact of monensin on the active rumen microbiome was studied in a rumen-simulating technique (Rusitec) with metaproteomic and metabolomic approaches. Monensin treatment caused a decreased fibredegradation potential that was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Decreased proteolytic activities resulted in reduced amounts of ammonium as well as branched-chain fatty acids. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of the metabolism from acetate to succinate production. Prevotella species harbour a membrane-bound electron transfer complex, which drives the reduction of fumarate to succinate, which is the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin.

An Escherichia coli FdrA Variant Derived from Syntrophic Coculture with a Methanogen Increases Succinate Production Due to Changes in Allantoin Degradation.

ABSTRACTWild-type Escherichia coli was adapted to syntrophic growth with Methanobacterium formicicum for glycerol fermentation over 44 weeks. Succinate production by E. coli started to increase in the early stages of syntrophic growth. Genetic analysis of the cultured E. coli population by pooled sequencing at eight time points suggests that (i) rapid evolution occurred through repeated emergence of mutators that introduced a large number of nucleotide variants and (ii) many mutators increased to high frequencies but remained polymorphic throughout the continuous cultivation. The evolved E. coli populations exhibited gains both in fitness and succinate production, but only for growth under glycerol fermentation with M. formicicum (the condition for this laboratory evolution) and not under other growth conditions. The mutant alleles of the 69 single nucleotide polymorphisms (SNPs) identified in the adapted E. coli populations were constructed individually in the ancestral wild-type E. coli. We analyzed the phenotypic changes caused by 84 variants, including 15 nonsense variants, and found that FdrAD296Y was the most significant variant leading to increased succinate production. Transcription of fdrA was induced under anaerobic allantoin degradation conditions, and FdrA was shown to play a crucial role in oxamate production. The FdrAD296Y variant increased glyoxylate conversion to malate by accelerating oxamate production, which promotes carbon flow through the C4 branch, leading to increased succinate production.IMPORTANCE Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate. We found that the production of succinate by E. coli significantly increased during successive cocultivation. Genomic DNA sequencing, evaluation of relative fitness, and construction of SNPs were performed, from which FdrAD296Y was identified as the most significant variant to enable increased succinate production by E. coli. The function of FdrA is uncertain. In this study, experiments with gene expression assays and metabolic analysis showed for the first time that FdrA could be the “orphan enzyme” oxamate:carbamoyltransferase in anaerobic allantoin degradation. Furthermore, we demonstrate that the anaerobic allantoin degradation pathway is linked to succinate production via the glyoxylate pathway during glycerol fermentation.

Short-Term Adaptation Modulates Anaerobic Metabolic Flux to Succinate by Activating ExuT, a Novel D-Glucose Transporter in Escherichia coli.

The sugar phosphotransferase system (PTS) is an essential energy-saving mechanism, particularly under anaerobic conditions. Since the PTS consumes equimolar phosphoenolpyruvate to phosphorylate each molecule of internalized glucose in the process of pyruvate generation, its absence can adversely affect the mixed acid fermentation profile and cell growth under anaerobic conditions. In this study, we report that the ΔptsG mutant cells of Escherichia coli K-12 strain exhibited inefficient glucose utilization, produced a significant amount of succinate, and exhibited a low growth rate. However, cells adapted soon after and started to grow rapidly in the same batch culture. As a result, the adapted ΔptsG cells showed the same mixed acid fermentation profiles as the wild-type cells, which was attributed to the mutation of the mlc gene, a repressor of the D-mannose PTS, another transporter for D-glucose. Similar adaptations were observed in the cells with ΔptsGΔmanX and the cells with ΔptsI that resulted in the production of a substantial amount of succinate and fast growth rate. The genome sequencing showed the presence of null mutations in the exuR gene, which encodes a modulator of exuT-encoded non-PTS sugar transporter, in adapted ΔptsGΔmanX and ΔptsI strains. Results from the RT-qPCR analysis and genetic test confirmed that the enhanced expression of ExuT, a non-PTS sugar transporter, was responsible for the uptake of D-glucose, increased succinate production, and fast growth of adapted cells. In conclusion, our study showed that the regulatory network of sugar transporters can be modulated by short-term adaptation and that downstream metabolic flux could be significantly determined by the choice of sugar transporters.

Succinate induces synovial angiogenesis in rheumatoid arthritis through metabolic remodeling and HIF-1α/VEGF axis

Background and purposeIn response to hypoxic succinate accumulates in arthritis synovium, however, the implication is little known. This study aims to investigate whether succinate could act as a metabolic signal linking metabolic alternation with angiogenesis in arthritis synovium. Experimental approachThe interaction between elevated succinate and VEGF production was examined in endothelial cells. Succinate production, HIF-1α induction and angiogenesis in the hypoxic synovium of collagen-induced arthritis rats were also investigated. Key resultsIntracellular succinate promoted VEGF production and induced angiogenic response dependent on HIF-1α induction in endothelial cells. Luciferase reporter assay showed that succinate increased VEGF expression through gene promoter activation dependent on HIF-1α induction. Intracellular succinate released into intercellular space, where extracellular succinate activated succinate receptor G-protein-coupled receptor 91 (GPR91) and induced VEGF production, further exacerbating angiogenesis. In addition, TGF-β1 treatment increased succinate production due to the reversal of succinate dehydrogenase (SDH) activation, and consistently, SDH inhibitor dimethyl malonate reduced angiogenesis in the arthritis synovium. Conclusion and implicationsMore than an intermediate, succinate functioned as a signaling molecule to link metabolic reprograming with angiogenesis. Intracellular succinate induced angiogenesis through HIF-1α induction, while extracellular succinate acted on GPR91 activation, working together to disturb energy metabolism and exacerbate inflammation and angiogenesis in arthritis synovium. Our work suggested that suppression of SDH could prevent succinate accumulation and inhibit angiogenesis via blocking HIF-1α/VEGF axis. This finding not only provides a novel insight into angiogenesis, but also reveals a potential therapeutical strategy to attenuate revascularization in arthritis.

Escherichia coli genome-scale metabolic gene knockout of lactate dehydrogenase (ldhA), increases succinate production from glycerol

Genome-scale metabolic model (GEM) of Escherichia coli has been published with applications in predicting metabolic engineering capabilities on different carbon sources and directing biological discovery. The use of glycerol as an alternative carbon source is economically viable in biorefinery. The use of GEM for predicting metabolic gene deletion of lactate dehydrogenase (ldhA) for increasing succinate production in Escherichia coli from glycerol carbon source remained largely unexplored. Here, I hypothesized that metabolic gene knockout of ldhA in E. coli from glycerol could increase succinate production. A proof-of-principle strain was constructed and designated as E. coli BMS5 (ΔldhA), by predicting increased succinate production in E. coli GEM and confirmed the predicted outcomes using wet cell experiments. The mutant GEM (ΔldhA) predicted 11% increase in succinate production from glycerol compared to its wild-type model (iAF1260), and the E. coli BMS5 (ΔldhA) showed 1.05 g/l and its corresponding wild-type produced .05 g/l (23-fold increase). The proof-of-principle strain constructed in this study confirmed the aforementioned hypothesis and further elucidated the fact that E. coli GEM can prospectively and effectively predict metabolic engineering interventions using glycerol as substrate and could serve as platform for new strain design strategies and biological discovery.

Bio-based succinate from sucrose: High-resolution 13C metabolic flux analysis and metabolic engineering of the rumen bacterium Basfia succiniciproducens

Succinic acid is a platform chemical of recognized industrial value and accordingly faces a continuous challenge to enable manufacturing from most attractive raw materials. It is mainly produced from glucose, using microbial fermentation. Here, we explore and optimize succinate production from sucrose, a globally applied substrate in biotechnology, using the rumen bacterium Basfia succiniciproducens DD1. As basis of the strain optimization, the yet unknown sucrose metabolism of the microbe was studied, using 13C metabolic flux analyses. When grown in batch culture on sucrose, the bacterium exhibited a high succinate yield of 1molmol−1 and a by-product spectrum, which did not match the expected PTS-mediated sucrose catabolism. This led to the discovery of a fructokinase, involved in sucrose catabolism. The flux approach unraveled that the fructokinase and the fructose PTS both contribute to phosphorylation of the fructose part of sucrose. The contribution of the fructokinase reduces the undesired loss of the succinate precursor PEP into pyruvate and into pyruvate-derived by-products and enables increased succinate production, exclusively via the reductive TCA cycle branch. These findings were used to design superior producers. Mutants, which (i) overexpress the beneficial fructokinase, (II) lack the competing fructose PTS, and (iii) combine both traits, produce significantly more succinate. In a fed-batch process, B. succiniciproducens ΔfruA achieved a titer of 71gL−1 succinate and a yield of 2.5molmol−1 from sucrose.

Improvement of succinate production by release of end-product inhibition in Corynebacterium glutamicum

Succinate is a renewable-based platform chemical that may be used to produce a wide range of chemicals including 1,4-butanediol, tetrahydrofurane, and γ-butyrolactone. However, industrial fermentation of organic acids is often subject to end-product inhibition, which significantly retards cell growth and limits metabolic activities and final productivity. In this study, we report the development of metabolically engineered Corynebacterium glutamicum for high production of succinate by release of end-product inhibition coupled with an increase of key metabolic flux. It was found that the rates of glucose consumption and succinate production were significantly reduced by extracellular succinate in an engineered strain, S003. To understand the mechanism underlying the inhibition by succinate, comparative transcriptome analysis was performed. Among the downregulated genes, overexpression of the NCgl0275 gene was found to suppress the inhibition of glucose consumption and succinate production, resulting in a 37.7% increase in succinate production up to 55.4g/L in fed-batch fermentation. Further improvement was achieved by increasing the metabolic flux from PEP to OAA. The final engineered strain was able to produce 152.2g/L succinate, the highest production reported to date, with a yield of 1.1g/g glucose under anaerobic condition. These results suggest that the release of end-product inhibition coupled with an increase in key metabolic flux is a promising strategy for enhancing production of succinate.

Model-guided identification of novel gene amplification targets for improving succinate production in Escherichia coli NZN111.

Reconstruction and application of genome-scale metabolic models (GEMs) have facilitated metabolic engineering by providing a platform on which systematic computational analysis of metabolic networks can be performed. In this study, a GEM of Escherichia coli NZN111 was employed by the analysis of production and growth coupling (APGC) algorithm to identify genetic strategies for the overproduction of succinate. Through in silico simulation and reaction expression analysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), and phosphoenolpyruvate carboxylase (PPC), encoded by gapA, pgk, tpiA, and ppc, respectively, were selected for experimental overexpression. The results showed that overexpressing any of these could improve both growth and succinate production. Specifically, overexpression of GAPDH or PGK showed a significant effect with up to 24% increase in succinate production. These results indicate that the APGC algorithm can be effectively used to guide genetic manipulation for strain design by identifying genome-wide gene amplification targets.

Application of theoretical methods to increase succinate production in engineered strains

Computational methods have enabled the discovery of non-intuitive strategies to enhance the production of a variety of target molecules. In the case of succinate production, reviews covering the topic have not yet analyzed the impact and future potential that such methods may have. In this work, we review the application of computational methods to the production of succinic acid. We found that while a total of 26 theoretical studies were published between 2002 and 2016, only 10 studies reported the successful experimental implementation of any kind of theoretical knowledge. None of the experimental studies reported an exact application of the computational predictions. However, the combination of computational analysis with complementary strategies, such as directed evolution and comparative genome analysis, serves as a proof of concept and demonstrates that successful metabolic engineering can be guided by rational computational methods.

Systematic engineering of pentose phosphate pathway improves Escherichia coli succinate production.

BackgroundSuccinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production.ResultsAltering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge.ConclusionsIn summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0675-y) contains supplementary material, which is available to authorized users.

Combinatorial optimization of CO2 transport and fixation to improve succinate production by promoter engineering.

To balance the flux of an engineered metabolic pathway to achieve high yield of target product is a major challenge in metabolic engineering. In previous work, the collaborative regulation of CO2 transport and fixation was investigated with co-overexpressing exogenous genes regulating both CO2 transport (sbtA and bicA) and PEP carboxylation (phosphoenolpyruvate (PEP) carboxylase (ppc) and carboxykinase (pck)) under trc promoter in Escherichia coli for succinate biosynthesis. For balancing metabolic flux to maximize succinate titer, a combinatorial optimization strategy to fine-tuning CO2 transport and fixation process was implemented by promoter engineering in this study. Firstly, based on the energy matrix a synthetic promoter library containing 20 rationally designed promoters with strengths ranging from 0.8% to 100% compared with the widely used trc promoter was generated. Evaluations of rfp and cat reporter genes provided evidence that the synthetic promoters were stably and had certain applicability. Secondly, four designed promoters with different strengths were used for combinatorial assembly of single CO2 transport gene (sbtA or bicA) and single CO2 fixation gene (ppc or pck) expression. Three combinations, such as Tang1519 (P4 -bicA + pP19 -pck), Tang1522 (P4 -sbtA + P4 -ppc), Tang1523 (P4 -sbtA + P17 -ppc) with a more than 10% increase in succinate production were screened in bioreactor. Finally, based on the above results, co-expression of the four transport and fixation genes were further investigated. Co-expression of sbtA, bicA, and ppc with weak promoter P4 and pck with strong promoter P19 (AFP111/pT-P4 -bicA-P4 -sbtA + pACYC-P19 -pck-P4 -ppc) provided the best succinate production among all the combinations. The highest succinate production of 89.4 g/L was 37.5% higher than that obtained with empty vector control. This work significantly enhanced succinate production through combinatorial optimization of CO2 transport and fixation. The promoter engineering and combinatorial optimization strategies used herein represents a powerful approach to tailor-making metabolic pathways for the production of other industrially important chemicals. Biotechnol. Bioeng. 2016;113: 1531-1541. © 2016 Wiley Periodicals, Inc.

Model-assisted formate dehydrogenase-O (fdoH) gene knockout for enhanced succinate production in Escherichia coli from glucose and glycerol carbon sources

Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H+ conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l−1 (41.2-fold in 7 days) from glycerol and .39 g l−1 (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.

Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium.

Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique.

A two-stage process for succinate production using genetically engineered Corynebacterium acetoacidophilum

Corynebacterium acetoacidophilum, which is related to Corynebacterium glutamicum, was genetically engineered to produce succinate for the first time. The results of genetic experiments in C. acetoacidophilum ATCC13870 showed that disrupting the lactate and acetate formation pathways, as well as overexpressing the anaplerotic enzyme pyruvate carboxylase, could increase succinate production and decrease the formation of by-products. Among the six engineered C. acetoacidophilum strains, ΔldhA, ΔldhAΔpta-ackA, ΔldhAΔpta-ackAΔctfA, ΔldhAΔaceE, ΔldhA/pXMJ19-pyc, and ΔldhAΔaceE/pXMJ19-pyc, merely deleting the l-lactate dehydrogenase gene ldhA and overexpressing the pyruvate carboxylase gene pyc yielded the optimum production of succinate under oxygen deprivation. A two-stage bioprocess was conducted in a 3-L fermenter to produce succinate using strain ΔldhA/pXMJ19-pyc, in which cells were first grown aerobically, after which glucose and bicarbonate were directly added to the cultures for conversion under oxygen-deprived conditions. The succinate concentration reached 908mM, with an overall yield of 1.34mol (mol glucose)−1 and volumetric productivity of 9.5mMh−1, suggesting a new potential approach for the industrial production of succinate.

In silico deletion of PtsG gene in Escherichia coli genome-scale model predicts increased succinate production from glycerol

Systems metabolic engineering and in silico analyses are necessary to study gene knockout candidate for enhanced succinic acid production by Escherichia coli. Metabolically engineered E. coli has been reported to produce succinate from glucose and glycerol. However, investigation on in silico deletion of ptsG/b1101 gene in E. coli from glycerol using minimization of metabolic adjustment algorithm with the OptFlux software platform has not yet been elucidated. Herein we report what is to our knowledge the first direct predicted increase in succinate production following in silico deletion of the ptsG gene in E. coli GEM from glycerol with the OptFlux software platform. The result indicates that the deletion of this gene in E. coli GEM predicts increased succinate production that is 20% higher than the wild-type control model. Hence, the mutant model maintained a growth rate that is 77% of the wild-type parent model. It was established that knocking out of the ptsG/b1101 gene in E. coli using glucose as substrate enhanced succinate production, but the exact mechanism of this effect is still obscure. This study informs other studies that the deletion of ptsG/b1101 gene in E. coli GEM predicted increased succinate production, enabling a model-driven experimental inquiry and/or novel biological discovery on the underground metabolic role of this gene in E. coli central metabolism in relation to increasing succinate production when glycerol is the substrate.