Glutamate Fermentation Research Articles

Coupling fermentation of glutamic acid and γ-polyglutamic acid and preparation of poly(amino acid) superabsorbent polymers

γ-polyglutamic acid (γ-PGA) is a biomarker that can be directly obtained by microbial fermentation. Poly(amino acid) superabsorbent polymers (SAPs) were prepared with purified γ-PGA as raw material and ethylene glycol diglycidyl ether (EGDGE) as a cross-linking agent. However, γ-PGA fermentation broth has a high viscosity, requires complex extraction and separation processes, and entails high energy consumption, resulting in the high cost of poly (amino acid) SAPs. Therefore, the coupling fermentation processes of glutamate polyglutamic acid, the process of using glutamate fermentation broth instead of pure glutamate powder for fermentation, and the process of treating the fermentation broth under conditions of centrifugation, UV irradiation, and high temperature, were studied. The results showed that the yield of γ-PGA after centrifugation decreased by 5%, but it did not affect the synthesis of hydrogels, and the addition of γ-PGA fermentation broth had a significant effect on the performance of γ-PGA-co-PASP SAPs. The proposed method not only helps avoid the separation of complex γ-PGA fermentation broth and reduces the cost, but it also helps improve the performance of the super-absorbent resin, which has great application potential.

Intermittent fasting modulates the intestinal microbiota and improves obesity and host energy metabolism

Intermittent fasting (IF) is a promising paradigm for weight loss which has been shown to modulate the gut microbiota based on 16S rRNA gene amplicon sequencing. Here, 72 Chinese volunteers with a wide range of body mass index (BMI) participated in a three-week IF program during which an average loss of 3.67 kg body weight accompanied with improved clinical parameters was observed irrespective of initial anthropometric and gut microbiota status. Fecal samples were collected before and after the intervention and subjected to shotgun metagenomic sequencing. De novo assembly yielded 2934 metagenome-assembled genomes (MAGs). Profiling revealed significant enrichment of Parabacteroides distasonis and Bacteroides thetaiotaomicron after the intervention, with inverse correlations between their relative abundances and parameters related to obesity and atherosclerotic cardiovascular diseases (ASCVD). MAGs enriched after the intervention showed high richness and diversity of carbohydrate-active enzymes, with an increased relative abundances of genes related to succinate production and glutamate fermentation.

An interest-oriented laboratory microbial engineering course is helpful for the cultivation of scientific literacy

ABSTRACT Course-based undergraduate research experience (CURE) exposes an entire class of students to scientific research and is thought to increase students’ knowledge retention in science, technology, engineering and mathematics education. Microbial engineering is an interdisciplinary subject that combines science and engineering. We transformed a microbial engineering course into a CURE-type course by adopting an interest-oriented case teaching approach. Based on an investigation of the interest of students, which showed that the topics related to food and health were students’ favourites, we selected beer brewing, glutamate fermentation and Monascus cultivation as teaching topics. To simulate the reality of industrial fermentation, pilot-scale fermentation equipment was selected and installed. The course focused on fermentation research; most of the time was spent on performing experiments, and theoretical lectures were held at the intervals. To complete the course in 10 days, three series of experiments were alternatively carried out, and students took turns to conduct fermentation control and process analyses in groups. A questionnaire survey showed that students were satisfied with the CURE-type course, their fermentation knowledge and engineering skills were improved, their research abilities were developed, and their scientific attitudes were cultivated. CURE-type courses are worth popularising in biology teaching.

A biosensor based on oriented immobilization of an engineered l-glutamate oxidase on a screen-printed microchip for detection of l-glutamate in fermentation processes

This paper describes an amperometric biosensor utilizing an engineered l-glutamate oxidase for glutamate monitoring in microbial fermentation processes. We designed a general immobilization strategy that utilized a chitin-binding domain (ChBD-tag) as a biotether to further immobilize l-glutamate oxidase (GLOX) in an oriented manner on a screen-printed Prussian blue nanocube microchip (PB/SPC) with the biopolymer chitosan. The improved l-glutamate biosensor exhibited an enhanced sensitivity of 53.4 µA L mmol−1 cm−2 and a linear range from 25 µmol/L to 300 µmol/L with a detection limit of 9 µmol/L, and retained 95 % of its initial activity after two weeks of usage. In addition, the as-prepared biosensor was applied for real-time monitoring of food ingredient l-glutamate concentration during the fermentation process, which was in good agreement with that of high-performance liquid chromatography (HPLC). Above all, the l-glutamate biosensor prepared by this method had high analytical performance, and could fully realize real-time and high-efficiency monitoring in glutamate fermentation.

Potassium ion leakage impairs thermotolerance in Corynebacterium glutamicum

Corynebacterium glutamicum, a gram-positive bacterium, can produce amino acids such as glutamic acid and lysine. The heat generated during cell growth and/or glutamate fermentation disturbs both the cell growth and fermentation. To overcome such a negative effect of the fermentation heat, we have tried to establish a high temperature fermentation. One of the approach is to create a thermotolerant strains, while the other is to create an optimum culture conditions able for the strain to grow at higher temperatures. In this study, we focused on the latter approach, where we examined the effect of potassium ion on cell growth at high growth temperatures of C.glutamicum. The supplementation of high concentrations of potassium chloride (300mM) (or sorbitol, an osmolyte) mitigated the repressed cell growth induced by high temperature at 39°C or 40°C. The intracellular potassium concentration declines from 300mM to ∼150mM by increasing the growth temperature but not by supplementing potassium chloride or sorbitol. Furthermore, invitro experiments revealed that the potassium ion leakage occurs at high temperatures, which was mitigated in the presence of high concentrations of extracellular potassium chloride. This suggested that the presence of high osmolyte in the culture medium could inhibit the potassium ion leakage induced by high temperature and subsequently support cell growth at high temperatures.

Glutamate Fermentation Process Model Based on Gams with Double Penalty Approach

There are many fermentation variables which can influence fermentation product during fermentation process, including fermentation time, temperature, pH, oxygen uptake rate (OUR), carbon dioxide evolution rate (CER), dissolved oxygen (DO) and stirring speed. For understanding the relationship between fermentation product and associated fermentation variables, one current interest is the investigation of significant fermentation variables to construct an interpretable and stable fermentation model. In this study, the significant variables in the fermentation process of glutamate were selected based on the generalized additive models through a shrinkage approach. The GAM2 which is based on less fermentation variables after variable selection has the same value of adjusted Rsquare (0.972) and deviance explained (97.6%) as GAM1 which includes all the measuring fermentation variables. Results showed that the proposed approach not only can identify the significant variables in the fermentation process of glutamate, but also can improve the performance of the fermentation model.

Cofactor recycling for co-production of 1,3-propanediol and glutamate by metabolically engineered Corynebacterium glutamicum

Production of 1,3-propanediol (1,3-PDO) from glycerol is a promising route toward glycerol biorefinery. However, the yield of 1,3-PDO is limited due to the requirement of NADH regeneration via glycerol oxidation process, which generates large amounts of undesired byproducts. Glutamate fermentation by Corynebacterium glutamicum is an important oxidation process generating excess NADH. In this study, we proposed a novel strategy to couple the process of 1,3-PDO synthesis with glutamate production for cofactor regeneration. With the optimization of 1,3-PDO synthesis route, C. glutamicum can efficiently convert glycerol into 1,3-PDO with the yield of ~ 1.0 mol/mol glycerol. Co-production of 1,3-PDO and glutamate was also achieved which increased the yield of glutamate by 18% as compared to the control. Since 1,3-PDO and glutamate can be easily separated in downstream process, this study provides a potential green route for coupled production of 1,3-PDO and glutamate to enhance the economic viability of biorefinery process.

Multi-Phase Support Vector Regression Soft Sensor for Online Product Quality Prediction in Glutamate Fermentation Process

Glutamate fermentation is inherently nonlinear, multi-phase and an aerobic fermentation process. As long measurement delays and expensive apparatus cost, on-line measurement of the product concentration is not necessarily available. The present fermentation process monitoring and quality prediction involve manual interpretation of highly informative, however, the concentrations of substrates, biomass and products are only low frequency off-line measurements. In this paper, we propose a novel Multi-Phase Support Vector Regression (MPSVR) based soft sensor model for online quality prediction of glutamate concentration. The glutamate fermentation process can be divided into a sequence of five phases by detecting the trend variation events (also termed as singular points or inflection point) of online measured O2 in the exhaust gas, the Inflection Point (IP) are easily identified through combining Moving Window (MW) with Pearson Correlation Coefficient (PCC). For each estimation phase, SVR soft sensor model are constructed and their performance is evaluated against fermentation data in a 5 L fermenter. The efficiency of the proposed soft sensor model for online product quality prediction has been demonstrated to be superior compared to that of reported techniques in a 5 L glutamate fermentation process.

An on-line adaptive glucose feeding system incorporating patterns recognition for glucose concentration control in glutamate fermentations

In glutamate fermentation, intermittent feeding is the most widely used glucose feed strategy. This feeding strategy causes severe fluctuations of glucose concentration and osmotic pressure in fermentation broth, which deteriorates the viability of the cell and reduces glutamate production in turn. In order to maintain glucose concentration at stable and constant levels, an on-line prediction and feedback control system based an empiric mass balance model was developed. However, the control system did not work properly and sometimes glucose concentration could even decline to 0 level (glucose exhaustion), as the model parameter varies in different runs. As a result, a novel model-based adaptive feedback control system incoporating with an artificial neural network (ANN) based pattern reconition unit for on-line diagnosizing the fault of glucose exhaustion was proposed and applied for glutamate fermentation. This adaptive control system could accurately detect glucose exhaustion when it occurs, and then immediately updates the control parameter based on some pre-defined rule. With the proposed control system, glucose was automatically fed, and its concentration could be maintained at desired levels constantly. As a result, glutamate concentration was 17 ~ 30% higher than that of the traditional fermentations using the intermittent glucose feed strategy.

Soft Sensor Modeling of Product Concentration in Glutamate Fermentation using Gaussian Process Regression

The on-line control of glutamate fermentation process is difficult, owing to the typical uncertainties of biochemical process and the lack of suitable on-line sensors for primary process variables. A prediction model based on Gaussian Process Regression (GPR) is presented to predict glutamate concentration online. First, Partial Least Squares (PLS) is applied to extract the features of the input secondary variables to reduce the number of the variables dimension and eliminate the correlation, through variables selection to reduce model complexity and improve model tracking performance. Validation was carried out in a 5 L fermentation tank for 10 batches glutamate fermentation process. Simulation results show that the proposed model outperforms the PLS and Support Vector Machine (SVM) model and the Root Mean Square Error (RMSE) are 1.59, 7.98 and 1.95, respectively. It can provide effective operation guidance for control and optimization of the glutamate fermentation process.

Lysine Fermentation: History and Genome Breeding.

Lysine fermentation by Corynebacterium glutamicum was developed in 1958 by Kyowa Hakko Kogyo Co. Ltd. (current Kyowa Hakko Bio Co. Ltd.) and is the second oldest amino acid fermentation process after glutamate fermentation. The fundamental mechanism of lysine production, discovered in the early stages of the process's history, gave birth to the concept known as "metabolic regulatory fermentation," which is now widely applied to metabolite production. After the development of rational metabolic engineering, research on lysine production first highlighted the need for engineering of the central metabolism from the viewpoints of precursor supply and NADPH regeneration. Furthermore, the existence of active export systems for amino acids was first demonstrated for lysine in C. glutamicum, and this discovery has resulted in the current recognition of such exporters as an important consideration in metabolite production. Lysine fermentation is also notable as the first process to which genomics was successfully applied to improve amino acid production. The first global "genome breeding" strategy was developed using a lysine producer as a model; this has since led to new lysine producers that are more efficient than classical industrial producers. These advances in strain development technology, combined with recent systems-level approaches, have almost achieved the optimization of entire cellular systems as cell factories for lysine production. In parallel, the continuous improvement of the process has resulted not only in fermentation processes with reduced load on downstream processing but also in commercialization of various product forms according to their intended uses. Nowadays lysine fermentation underpins a giant lysine demand of more than 2 million metric tons per year.

Discovery and History of Amino Acid Fermentation.

There has been a strong demand in Japan and East Asia for L-glutamic acid as a seasoning since monosodium glutamate was found to present umami taste in 1907. The discovery of glutamate fermentation by Corynebacterium glutamicum in 1956 enabled abundant and low-cost production of the amino acid, creating a large market. The discovery also prompted researchers to develop fermentative production processes for other L-amino acids, such as lysine. Currently, the amino acid fermentation industry is so huge that more than 5 million metric tons of amino acids are manufactured annually all over the world, and this number continues to grow. Research on amino acid fermentation fostered the notion and skills of metabolic engineering which has been applied for the production of other compounds from renewable resources. The discovery of glutamate fermentation has had revolutionary impacts on both the industry and science. In this chapter, the history and development of glutamate fermentation, including the very early stage of fermentation of other amino acids, are reviewed.

Present Global Situation of Amino Acids in Industry.

At present, amino acids are widely produced and utilized industrially. Initially, monosodium glutamate (MSG) was produced by extraction from a gluten hydrolysate. The amino acid industry started using the residual of the lysate. The discovery of the functions of amino acids has led to the expansion of their field of use. In addition to seasoning and other food use, amino acids are used in many fields such as animal nutrients, pharmaceuticals, and cosmetics. On the other hand, the invention of the glutamate fermentation process, followed by the development of fermentation methods for many other amino acids, is no less important. The supply of these amino acids at a low price is very essential for their industrial use. Most amino acids are now produced by fermentation. The consumption of many amino acids such as MSG or feed-use amino acids is still rapidly increasing.

Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB.

Mesaconase catalyzes the hydration of mesaconate (methylfumarate) to (S)-citramalate. The enzyme participates in the methylaspartate pathway of glutamate fermentation as well as in the metabolism of various C5-dicarboxylic acids such as mesaconate or L-threo-β-methylmalate. We have recently shown that Burkholderia xenovorans uses a promiscuous class I fumarase to catalyze this reaction in the course of mesaconate utilization. Here we show that classical Escherichia coli class I fumarases A and B (FumA and FumB) are capable of hydrating mesaconate with 4% (FumA) and 19% (FumB) of the catalytic efficiency k cat/K m, compared to the physiological substrate fumarate. Furthermore, the genomes of 14.8% of sequenced Enterobacteriaceae (26.5% of E. coli, 90.6% of E. coli O157:H7 strains) possess an additional class I fumarase homologue which we designated as fumarase D (FumD). All these organisms are (opportunistic) pathogens. fumD is clustered with the key genes for two enzymes of the methylaspartate pathway of glutamate fermentation, glutamate mutase and methylaspartate ammonia lyase, converting glutamate to mesaconate. Heterologously produced FumD was a promiscuous mesaconase/fumarase with a 2- to 3-fold preference for mesaconate over fumarate. Therefore, these bacteria have the genetic potential to convert glutamate to (S)-citramalate, but the further fate of citramalate is still unclear. Our bioinformatic analysis identified several other putative mesaconase genes and revealed that mesaconases probably evolved several times from various class I fumarases independently. Most, if not all iron-dependent fumarases, are capable to catalyze mesaconate hydration.

Improving the stability of glutamate fermentation by Corynebacterium glutamicum via supplementing sorbitol or glycerol

Corynebacterium glutamicum is widely used in glutamate fermentation. The fermentation feature of the strain varies sometimes. These variations may lead to the reduction in the ability of the strain to resist environmental changes and to synthesize glutamate, resulting in abnormal glutamate fermentations. In the abnormal glutamate fermentations, glutamate accumulation stopped after glucose feeding and the final glutamate concentration was at a lower level (50 to 60 g/L). The r NAD +/r NADH ratio was lower than that in normal batch which was reflected by lower oxidation-reduction potential (ORP) value. The abnormal fermentation performance was improved when glucose was co-fed with sorbitol/glycerol at a weight ratio of 5:1 or adding 10 to 15 g/L of sorbitol/glycerol in the initial medium. Under these conditions, glutamate synthesis continued after substrate(s) feeding and final glutamate concentration was restored to normal levels (≥72 g/L). r NAD +/rNADH ratio, ORP, and pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH), and cytochrome c oxidase (CcO) activities were maintained at higher levels. Sorbitol and glycerol were not used as carbon sources for the fermentation. They were considered as effective protective agents to increase cells' resistance ability against environmental changes and maintain key enzymes activities.

Parameters Optimization and Application to Glutamate Fermentation Model Using SVM

Aimed at the parameters optimization in support vector machine (SVM) for glutamate fermentation modelling, a new method is developed. It optimizes the SVM parameters via an improved particle swarm optimization (IPSO) algorithm which has better global searching ability. The algorithm includes detecting and handling the local convergence and exhibits strong ability to avoid being trapped in local minima. The material step of the method was shown. Simulation experiments demonstrate the effectiveness of the proposed algorithm.

Peptidoglycan from fermentation by-product triggers defense responses in grapevine.

Plants are constantly under attack from a variety of microorganisms, and rely on a series of complex detection and response systems to protect themselves from infection. Here, we found that a by-product of glutamate fermentation triggered defense responses in grapevine, increasing the expression of defense response genes in cultured cells, foliar chitinase activity, and resistance to infection by downy mildew in leaf explants. To identify the molecule that triggered this innate immunity, we fractionated and purified candidates extracted from Corynebacterium glutamicum, a bacterium used in the production of amino acids by fermentation. Using hydrolysis by lysozyme, a silkworm larva plasma detection system, and gel filtration analysis, we identified peptidoglycan as inducing the defense responses. Peptidoglycans of Escherichia coli, Bacillus subtilis, and Staphylococcus aureus also generated similar defensive responses.

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum

Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production.

A novel metabolic model incorporating directed signal flow diagram with enzymatic activities data for evaluating glutamate yield in glutamate fermentation

Glutamate conversion yield is one of the most important performance indexes in glutamate fermentation. The experimental results showed that anaplerotic reaction could be enhanced by jointly manipulating pH regulation and NaHCO3 supplement during fermentations by Corynebacterium glutamicum, leading to a 36% increase in the yield and comparably high glutamate productivity. A novel metabolic model incorporating directed signal flow diagram and enzymatic activities data was proposed to interpret the yield enhancement. The simulation and experimental results revealed that singly regulating each individual enzyme could not increase the yield, and glutamate yield could be enhanced only when six key enzymes of pyruvate carboxylase (PC), pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH), isocitrate lyase (ICL), glutamate dehydrogenase (GDH) and α-oxoglutarate dehydrogenase (ODHC) works in a coordinated way. Namely, relative activities ratios of enzymatic pairs of PC/PDH should be controlled at moderate level of 6:4, while those of ICDH/ICL and GDH/ODHC at higher level of 8:2 simultaneously. The model could cluster data pairs of glutamate yields and enzymatic activities obtained under different operation conditions into different categories, indicating its abilities in guiding optimal enzyme regulation ways for fermentations characterized with multiple enzymatic reactions and closed reaction loops.