In this study, the potential of oil palm trunk (OPT) sap as a sole substrate for succinic acid (SA) production was evaluated using Actinobacillus succinogenes 130Z. After OPT sap was characterised, the effects of adding carbonate, yeast extract (YE) and minerals to this medium were investigated in an attempt to develop a low-cost fermentation medium. The OPT sap alone, gave comparable SA yield and productivity (0.54 g/g and 0.35 g/L/h) to those supplemented with YE (0.50 g/g and 0.36 g/L/h) and minerals (0.55 g/g and 0.40 g/L/h). The findings showed that OPT sap has sufficient amount of nutrients for SA biosynthesis by A. succinogenes 130Z and could potentially reduce cost without requiring expensive nutrients supplementation.
Production of succinic acid via separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) are alternatives and are environmentally friendly processes. These processes have attained considerable positions in the industry with their own share of challenges and problems. The high-value succinic acid is extensively used in chemical, food, pharmaceutical, leather and textile industries and can be efficiently produced via several methods. Previously, succinic acid production via chemical synthesis from petrochemical or refined sugar has been the focus of interest of most reviewers. However, these expensive substrates have been recently replaced by alternative sustainable raw materials such as lignocellulosic biomass, which is cheap and abundantly available. Thus, this review focuses on succinic acid production utilizing lignocellulosic material as a potential substrate for SSF and SHF. SSF is an economical single-step process which can be a substitute for SHF - a two-step process where biomass is hydrolyzed in the first step and fermented in the second step. SSF of lignocellulosic biomass under optimum temperature and pH conditions results in the controlled release of sugar and simultaneous conversion into succinic acid by specific microorganisms, reducing reaction time and costs and increasing productivity. In addition, main process parameters which influence SHF and SSF processes such as batch and fed-batch fermentation conditions using different microbial strains are discussed in detail.
In the title mol-ecular salt, 2C6H10N3O+·C8H4O42-, the N atom of each of the two 2-amino-4-meth-oxy-6-methyl-pyrimidine mol-ecules lying between the amine and methyl groups has been protonated. The dihedral angles between the pyrimidine rings of the cations and the benzene ring of the succinate dianion are 5.04 (8) and 7.95 (8)°. Each of the cations is linked to the anion through a pair of N-H⋯O(carboxyl-ate) hydrogen bonds, forming cyclic R22(8) ring motifs which are then linked through inversion-related N-H⋯O hydrogen bonds, giving a central R24(8) motif. Peripheral amine N-H⋯O hydrogen-bonding inter-actions on either side of the succinate anion, also through centrosymmetric R22(8) extensions, form one-dimensional ribbons extending along [211]. The crystal structure also features π-π stacking inter-actions between the aromatic rings of the pyrimidine cations [minimum ring centroid separation = 3.6337 (9) Å]. The inter-molecular inter-actions were also investigated using Hirshfeld surface studies and two-dimensional fingerprint images.
The metabolic network is the reconstruction of the metabolic pathway of an organism that is used to represent the interaction between enzymes and metabolites in genome level. Meanwhile, metabolic engineering is a process that modifies the metabolic network of a cell to increase the production of metabolites. However, the metabolic networks are too complex that cause problem in identifying near-optimal knockout genes/reactions for maximizing the metabolite's production. Therefore, through constraint-based modelling, various metaheuristic algorithms have been improvised to optimize the desired phenotypes. In this paper, PSOMOMA was compared with CSMOMA and ABCMOMA for maximizing the production of succinic acid in E. coli. Furthermore, the results obtained from PSOMOMA were validated with results from the wet lab experiment.
The aim of this study was to establish an improved pretreatment and fermentation method i.e. immobilized cells for high recovery of fermentable sugars from palm kernel cake (PKC) and its effects on fermentability performance by Actinobacillus succinogenes 130Z in the conversion of the fermentable sugar to lactic acid. The effects of oxalic acid concentrations (1-6% w/v) and residence times (1-5 h) on the sugar recovery were initially investigated and it was found that the highest mannose concentration was 25.1 g/L at the optimum hydrolysis conditions of 4 h and 3% (w/v) oxalic acid. The subsequent enzymatic saccharification of the pretreated PKC afforded the highest enzymatic digestibility with the recovered sugars amounting to 25.18 g/L and 9.14 g/L of mannose and glucose, respectively. Subsequently, the fermentability performance of PKC hydrolysate was evaluated and compared in terms of cultivation phases (i.e. mono and dual-phases), carbonate loadings (i.e. magnesium and sodium carbonates), and types of sugars (i.e. glucose and mannose). The highest titer of 19.4 g/L lactic acid was obtained from the fermentation involving A. succinogenes 130Z in dual-phase cultivation supplemented with 30 g/L of magnesium carbonate. Lactic acid production was further enhanced by using immobilized cells with coconut shell-activated carbon (CSAC) of different sizes (A, B, C, and D) in the repeated batch cultivation of dual-phase fermentation producing 31.64 g/L of lactic acid. This work sheds light on the possibilities to enhance the utilization of PKC for lactic acid production via immobilized A. succinogenes 130Z.
This paper presents a study on gene knockout strategies to identify candidate genes to be knocked out for improving the production of succinic acid in Escherichia coli. Succinic acid is widely used as a precursor for many chemicals, for example production of antibiotics, therapeutic proteins and food. However, the chemical syntheses of succinic acid using the traditional methods usually result in the production that is far below their theoretical maximums. In silico gene knockout strategies are commonly implemented to delete the gene in E. coli to overcome this problem. In this paper, a hybrid of Ant Colony Optimization (ACO) and Minimization of Metabolic Adjustment (MoMA) is proposed to identify gene knockout strategies to improve the production of succinic acid in E. coli. As a result, the hybrid algorithm generated a list of knockout genes, succinic acid production rate and growth rate for E. coli after gene knockout. The results of the hybrid algorithm were compared with the previous methods, OptKnock and MOMAKnock. It was found that the hybrid algorithm performed better than OptKnock and MOMAKnock in terms of the production rate. The information from the results produced from the hybrid algorithm can be used in wet laboratory experiments to increase the production of succinic acid in E. coli.
Cancer is the second leading cause of death-1 in 6 deaths globally is due to cancer. Cancer metabolism is a complex and one of the most actively researched area in cancer biology. Metabolic reprogramming in cancer cells entails activities that involve several enzymes and metabolites to convert nutrient into building blocks that alter energy metabolism to fuel rapid cell division. Metabolic dependencies in cancer generate signature metabolites that have key regulatory roles in tumorigenesis. In this minireview, we highlight recent advances in the popular methods ingrained in biochemistry research such as stable and flux isotope analysis, as well as radioisotope labeling, which are valuable in elucidating cancer metabolites. These methods together with analytical tools such as chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry have helped to bring about exploratory work in understanding the role of important as well as obscure metabolites in cancer cells. Information obtained from these analyses significantly contribute in the diagnosis and prognosis of tumors leading to potential therapeutic targets for cancer therapy.
Organostannum(IV) complexes derived from dicarboxylic acids have been successfully synthesised from triphenylstannum(IV) hydroxide (Ph3SnOH). The complexes were obtained by refluxing Ph3SnOH and the respective dicarboxylic acids (malonic, succinic, glutaric and adipic acids) using a Dean and Stark apparatus in the appropriate solvent. The free ligands and complexes were characterised by C, Hand Sn elemental analysis and spectroscopic methods such as infrared and nuclear magnetic resonance (1H, 13C NMR). Structure determination of triphenylstannum(IV) succinate, 2, showed that each dicarboxylate anion was bonded monodentatically to two triphenylstannum(IV) moieties. The geometry surrounding each stannum atom is close to a tetrahedron. Spectroscopic data obtained for all complexes were found to be similar and thus it may be concluded that all complexes have the same structure as 2.
Using 100-fold molar excess of succinic anhydride, about 99% of lysine residues of hen egg white lysozyme (HEWL) were modified. Succinylated (S(99)) HEWL showed both charge and size homogeneity as judged by PAGE and gel filtration, respectively. Hydrodynamic parameters such as Stokes radius and frictional ratio (f/f(o)) showed more expanded conformation of S(99) HEWL compared to native HEWL as evident from the increase in Stokes radius (from 1.36 to 1.86 nm) and f/f(o) (from 0.86 to 1.15) values. Guanidine hydrochloride (GdnHCl) denaturation studies using fluorescence spectroscopy connoted a marked decrease in conformational stability of HEWL upon succinylation. Complete denaturation of S(99) HEWL was achieved at lower GdnHCl concentration ( approximately 3.8 M) compared to native HEWL ( approximately 5 M). Furthermore, free energy of stabilization (DeltaG(D)(H(2)O)) value also showed a notable decrease from 8,559 and 7,956 cal/mol (for native HEWL) to 4,404 and 4,669 cal/mol (for succinylated HEWL) using excitation at 280 and 295 nm, respectively. Both expanded conformation and decreased DeltaG(D)(H(2)O) can be attributed to the increase in the net negative charge on the protein upon succinylation. All these results manifested the importance of positively charged lysine residues in maintaining the conformational stability of HEWL through electrostatic interactions.
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.
This study aimed to enhance the crystallizability of bio-based succinic acid for its efficient recovery while maintaining the end product at the highest purity. Immobilization of Actinobacillus succinogenes was initially evaluated based on three different carriers: volcanic glass, clay pebbles, and silica particles. The adsorption capacity of metabolites with a low concentration (10 g/L) and a high concentration (40 g/L) was investigated. It was demonstrated that clay pebbles adsorbed the least succinic acid (acid with an average concentration of up to 36.3 g/L with a metabolite-production ratio of 3:1 (succinic acid to by-products) could be attained within 130 h. Subsequently, the purification of succinic acid through crystallization was assessed in terms of pH, temperature, crystallization time, initial succinic acid concentration and multiple recrystallization processes. Increasing the crystallization time from 6 h to 9 h afforded an improvement of 17% in the recovery of succinic acid crystals. Moreover, a fourfold concentration coefficient of the broth yielded the highest purity percentage (99.9%). The crystallization in three consecutive stages at 9 h (with a fourfold concentration coefficient) successfully improved the total recovery percentage of succinic acid from 55.0 to 84.8%.
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.
Despite the advantages of continuous fermentation whereby ethanol is selectively removed from the fermenting broth to reduce the end-product inhibition, this process can concentrate minor secondary products to the point where they become toxic to the yeast. This study aims to develop a new mathematical model do describe the inhibitory effect of byproducts on alcoholic fermentation including glycerol, lactic acid, acetic acid, and succinic acid, which were reported as major byproducts during batch alcoholic fermentation. The accumulation of these byproducts during the different stages of batch fermentation has been quantified. The yields of total byproducts, glycerol, acetic acid, and succinic acid per gram of glucose were 0.0442, 0.023, 0.0155, and 0.0054, respectively. It was found that the concentration of these byproducts linearly increases with the increase in glucose concentration in the range of 25-250 g/L. The results have also showed that byproduct concentration has a significant inhibitory effect on specific growth coefficient (μ) whereas no effect was observed on the half-velocity constant (Ks). A new mathematical model of alcoholic fermentation was developed considering the byproduct inhibitory effect, which showed a good performance and more accuracy compared to the classical Monod model.
The aim of this study was to examine the variation in metabolite constituents of five commercial varieties of date fruits; Ajwa, Safawi and Ambar which originated from Madinah, the Iranian Bam and Tunisian Deglet Noor. The differences of metabolome were investigated using proton nuclear magnetic resonance (1H NMR) spectroscopy combined with multivariate data analysis (MVDA). Principal Component Analysis (PCA) revealed clear separation between the date varieties. The Tunisian Deglet Noor demonstrated distinct cluster from the rest of the palm date samples based on the metabolite composition as shown by the pattern observed in Hierarchical Clustering Analysis (HCA) and PCA. Deglet Noor exhibited a significant higher level of sucrose (δ 5.40) and fructose (δ 4.16) in comparison with the other four varieties which can be associated with the distinctive sweet taste of this variety. Dates originated from Madinah and Tunisia exhibited a contrast manner in the amount of xylose and moisture content. These two aspects may contribute towards the soft texture of Tunisian dates. All Madinah dates were found to contain phenolic compounds which were well established as great antioxidant and anti-inflammatory agent. Ajwa dates exerted greater effect in inhibiting the generation of nitric oxide (NO) from the stimulated RAW264.7 cells at 95.37% inhibition. Succinic acid was suggested to have the most significant correlation with the trend of NO inhibitory shown by the selected date palm varieties.
The increase in the price of commercial succinic acid has necessitated the need for its
synthesis from waste materials such as glycerol. Glycerol residue is a waste product of
Oleochemical production which is cheaply available and a very good source of carbon.
The use of immobilized cells can further reduce the overall cost of the production process.
This study primarily aims to produce succinic acid from glycerol residue through the use
of immobilized Escherichia coli in a batch fermentation process. The parameters which
affect bacterial fermentation process such as the mass substrate, temperature, inoculum
size and duration of fermentation were screened using One-Factor-At-a-Time (OFAT)
method. The result of the screening process shows that a substrate (glycerol) concentration
of 30 g, inoculum size 20% v/v, and time 4 h produced the maximum succinic acid
concentration of 117.99 g/L. The immobilized cells were found to be stable as well as
retain their fermentative ability up to the 6th cycle of recycling, thereby presenting as an
advantage over the free cell system. Therefore, conclude that using immobilized cells can
contribute immensely to the cost-effective production of succinic acid from glycerol
residue.
Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Because of its high recurrence rate and heterogeneity, effective treatment for advanced stage of HCC is currently lacking. There are accumulating evidences showing the therapeutic potential of pharmacologic vitamin C (VC) on HCC. However, the metabolic basis underlying the anticancer property of VC remains to be elucidated. In this study, we used a high-resolution proton nuclear magnetic resonance-based metabolomics technique to assess the global metabolic changes in HCC cells following VC treatment. In addition, the HCC cells were also treated with oxaliplatin (OXA) to explore the potential synergistic effect induced by the combined VC and OXA treatment. The current metabolomics data suggested different mechanisms of OXA and VC in modulating cell growth and metabolism. In general, VC treatment led to inhibition of energy metabolism via NAD+ depletion and amino acid deprivation. On the other hand, OXA caused significant perturbation in phospholipid biosynthesis and phosphatidylcholine biosynthesis pathways. The current results highlighted glutathione metabolism, and pathways related to succinate and choline may play central roles in conferring the combined effect between OXA and VC. Taken together, this study provided metabolic evidence of VC and OXA in treating HCC and may contribute toward the potential application of combined VC and OXA as complementary HCC therapies.
A semiconducting water-soluble core-shell quantum dots (QDs) system capped with thiolated ligand was used in this study for the sensitive detection of glucose in aqueous samples. The QDs selected are of CdSe-coated ZnS and were prepared in house based on a hot injection technique. The formation of ZnS shell at the outer surface of CdSe core was made via a specific process namely, SILAR (successive ionic layer adsorption and reaction). The distribution, morphology, and optical characteristics of the prepared core-shell QDs were assessed by transmission electron microscopy (TEM) and spectrofluorescence, respectively. From the analysis, the results show that the mean particle size of prepared QDs is in the range of 10-12 nm and that the optimum emission condition was displayed at 620 nm. Further, the prepared CdSe/ZnS core shell QDs were modified by means of a room temperature ligand-exchange method that involves six organic ligands, L-cysteine, L-histidine, thio-glycolic acid (TGA or mercapto-acetic acid, MAA), mercapto-propionic acid (MPA), mercapto-succinic acid (MSA), and mercapto-undecanoic acid (MUA). This process was chosen in order to maintain a very dense water solubilizing environment around the QDs surface. From the analysis, the results show that the CdSe/ZnS capped with TGA (CdSe/ZnS-TGA) exhibited the strongest fluorescence emission as compared to others; hence, it was tested further for the glucose detection after their treatment with glucose oxidase (GOx) and horseradish peroxidase (HRP) enzymes. Here in this study, the glucose detection is based on the fluorescence quenching effect of the QDs, which is correlated to the oxidative reactions occurred between the conjugated enzymes and glucose. From the analysis of results, it can be inferred that the resultant GOx:HRP/CdSe/ZnS-TGA QDs system can be a suitable platform for the fluorescence-based determination of glucose in the real samples.
Metabolic engineering is a research field that focuses on the design of models for metabolism, and uses computational procedures to suggest genetic manipulation. It aims to improve the yield of particular chemical or biochemical products. Several traditional metabolic engineering methods are commonly used to increase the production of a desired target, but the products are always far below their theoretical maximums. Using numeral optimisation algorithms to identify gene knockouts may stall at a local minimum in a multivariable function. This paper proposes a hybrid of the artificial bee colony (ABC) algorithm and the minimisation of metabolic adjustment (MOMA) to predict an optimal set of solutions in order to optimise the production rate of succinate and lactate. The dataset used in this work was from the iJO1366 Escherichia coli metabolic network. The experimental results include the production rate, growth rate and a list of knockout genes. From the comparative analysis, ABCMOMA produced better results compared to previous works, showing potential for solving genetic engineering problems.
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.
The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35gl(-1) and 1.40gl(-1) from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals.