Corynebacterium Glutamicum Strain Research Articles

Особенности получения кормовых аминокислот при культивировании коринебактерий на соевой мелассе

Currently, soybeans and soybean derivatives are widely used for food and animal feed. A significant content of carbohydrates in soy molasses makes it possible to use it as a component of a nutrient medium for cultivating microorganisms that produce feed amino acids. The aim of this work was to study the process of biosynthesis of feed amino acids on a soy molasses medium using bacterial strains of the genus Corynebacterium glutamicum. The following research methods were used: microscopy, spectrometry, refractometry, pH-metry and high-performance liquid chromatography. It has been established that when cultivating on soy molasses, the strains C. glutamicum B-1002 and C. glutamicum B-1722 have the greatest ability to assimilate the components of the medium. The addition of such growth components as nicotinic acid and NaCl had no significant effect on the accumulation of C. glutamicum B-1002 and C. glutamicum B-1722 biomass. It has been proven that the best medium for cultivation of C. glutamicum is molasse that has gone through the entire technological process and contains the maximum amount of components necessary for the cultivation of C. glutamicum and the production of feed amino acids. The production of feed amino acids by C. glutamicum is influenced by such factors as dry matter content and active acidity of the medium. It was found that the best producers of feed acids are C. glutamicum B-1002 and C. glutamicum B-1722 strains cultivated on media composed of soy molasses and distilled water at a dilution of 1 : 9.

Enhanced production of \u03b3-amino acid 3-amino-4-hydroxybenzoic acid by recombinant Corynebacterium glutamicum under oxygen limitation

BackgroundBio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics.ResultsUnder aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products. The specific productivity of 3,4-AHBA increased with decreasing levels of dissolved oxygen (DO) and was eightfold higher under oxygen limitation (DO = 0 ppm) than under aerobic conditions (DO ≥ 2.6 ppm). Metabolic profiles during 3,4-AHBA production were compared at three different DO levels (0, 2.6, and 5.3 ppm) using the DO-stat method. Results of the metabolome analysis revealed metabolic shifts in both the central metabolic pathway and amino acid metabolism at a DO of < 33% saturated oxygen. Based on this metabolome analysis, metabolic pathways were rationally designed for oxygen limitation. An ldh deletion mutant, with the loss of lactate dehydrogenase, exhibited 3.7-fold higher specific productivity of 3,4-AHBA at DO = 0 ppm as compared to the parent strain KT01 and produced 5.6 g/L 3,4-AHBA in a glucose fed-batch culture.ConclusionsOur results revealed changes in the metabolic state in response to DO concentration and provided insights into oxygen supply during fermentation and the rational design of metabolic pathways for improved production of related amino acids and their derivatives.Graphical

Enhancing β-alanine production from glucose in genetically modified Corynebacterium glutamicum by metabolic pathway engineering.

To directly produce β-alanine from glucose by microbial fermentation, a recombinant Corynebacterium glutamicum strain with high efficiency of β-alanine production was constructed in this study. To do this, the biosynthetic pathway of β-alanine in an L-lysine-producing strain XQ-5 was modified by enhancing carbon flux in biosynthetic pathway and limiting carbon flux in competitive pathway. This study showed that replacement of L-aspartate kinase (AK) with wild-type AK and disruption of lactate dehydrogenase and alanine/valine aminotransferases increase β-alanine production because of decreasing the by-products accumulation. Moreover, L-aspartate-α-decarboxylase (ADC) from Bacillus subtilis was designed as the best enzyme for increasing β-alanine production, and its variant (BsADCE56S/I88M) showed the highest activity for catalyzing L-aspartate to generate β-alanine. To further increase β-alanine production, expression level of BsADCE56S/I88M was controlled by optimizing promoter and RBS, indicating that Pgro plus ThirRBS is the best combination for BsADCE56S/I88M expression and β-alanine production. The resultant strain XQ-5.5 produced 30.7 ± 2.3g/L of β-alanine with a low accumulation of lactate (from 5.2 ± 0.14 to 0.2 ± 0.09g/L) and L-alanine (from 7.6 ± 0.22 to 3.8 ± 0. 32g/L) in shake-flask fermentation and produced 56.5 ± 3.2g/L of β-alanine with a productivity of 0.79g/(L·h) and the glucose conversion efficiency (α) of 39.5% in feed-batch fermentation. This is the first report of genetically modifying the biosynthetic pathway of β-alanine that improves the efficiency of β-alanine production in an L-lysine-producing strain, and these results give us a new insight for constructing the other valuable biochemical. KEY POINTS: • Optimization and overexpression of the key enzyme BsADC increased the accumulation of β-alanine. • The AK was replaced with wild-type AK to increase the conversion of aspartic acid to β-alanine. • A 56.5-g/L β-alanine production in fed-batch fermentation was achieved.

Improve gamma-aminobutyric acid production in Corynebacterium glutamicum by optimizing the metabolic flux

Gamma-aminobutyric acid is an important nonprotein amino acid and has been extensively applied in pharmaceuticals, livestock, food additives, and so on. It is important to develop Corynebacterium glutamicum strains that can efficiently produce gamma-aminobutyric acid from glucose. In this study, production of gamma-aminobutyric acid in C. glutamicum CGY700 was improved by construction of CO2 anaplerotic reaction and overexpression of citrate synthase. The co-expression of ppc encoding phosphoenolpyruvate carboxylase and gltA encoding citrate synthase was constructed and optimized in the chromosome to compensate carbon loss and conquer metabolic bottleneck. The expression of ppc and gltA were controlled by promoters Ptac and PtacM, and the optimal mode of PtacM-ppc-Ptac-gltA was determined. Simultaneously, the genes pknG encoding serine/threonine protein kinase G and ldh encoding l-lactate dehydrogenase were deleted, and glnA2 encoding glutamine synthase was overexpressed in the chromosome. The final strain CGY-PG-304 constructed in this study could produce 41.17 g/L gamma-aminobutyric acid in shake flask cultivation and 58.33 g/L gamma-aminobutyric acid via Fed-Batch fermentation with a yield of 0.30 g/g glucose. CGY-PG-304 was constructed by genome editing; therefore, it is stable and not necessary to add any antibiotics and inducer during fermentation.

Safety evaluation of the food enzyme d-psicose 3-epimerase from the genetically modified Corynebacterium glutamicum strain FIS002.

This assessment addresses a food enzyme preparation consisting of the immobilised intact but non‐viable cells of the genetically modified Corynebacterium glutamicum strain FIS002 by CJ‐Tereos Sweeteners Europe SAS. The production strain produces the food enzyme d‐fructose 3‐epimerase (d‐psicose 3‐epimerase; EC 5.1.3.30). The food enzyme preparation is used in processing fructose to produce a speciality carbohydrate d‐allulose (synonym d‐psicose). Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of d‐allulose, dietary exposure was not calculated. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 1,796 mg TOS/kg body weight (bw) per day, the highest dose tested. A search for similarity of the amino acid sequence of the enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood of such reactions to occur is low. The food enzyme preparation contains multiple copies of an antimicrobial resistance gene, which is considered a hazard. However, under the specific intended conditions of use described by the applicant, and based on the evidence showing the removal of TOS during the production of d‐allulose and the absence of recombinant DNA in the d‐allulose, the Panel concluded that the identified hazard associated with the food enzyme d‐psicose 3‐epimerase produced with the genetically modified C. glutamicum strain FIS002 will not result in a risk.

The PhoPR two-component system responds to oxygen deficiency and regulates the pathways for energy supply in Corynebacterium glutamicum.

The PhoPR two-component system, a highly conserved system in corynebacteria and mycobacteria, is involved in the cellular response to environmental stress. When analysing the transcriptomic data of Corynebacterium glutamicum strains under different dissolved oxygen (DO) levels, PhoPR was found to be the most responsive two-component system to DO changes. Here, we systematically investigated the expression of PhoPR in response to different DO levels and its impact on genes related to global regulation and energy metabolism. Using Green fluorescent protein as a reporter, we confirmed that PhoPR was significantly upregulated upon decrease of DO. Through real-time quantitative PCR and electrophoretic mobility shift assay, we found that the effector protein PhoP directly activated glxR (encoding a global regulator), pfk and gapA (encoding the glycolytic enzymes) and ctaD (encoding cytochrome c in the electron transport chain), while downregulated aceE and gltA (encoding the TCA cycle enzymes). Overexpression of phoP or phoR resulted in a decreased intracellular NAD+/NADH ratio and increased intracellular ATP level, consistent with the gene expression changes regulated by PhoP. These results reveal the PhoPR system respond to oxygen deficiency and is responsible for the regulation of pathways involved in the sustainability of the energy levels required under low oxygen conditions. Our findings in this study not only provide new insights into the adaptation pathways of C. glutamicum in response to low oxygen conditions but also identify new possible genetic targets for the construction of the new cell factories aimed toward industrial applications.

Metabolic and process engineering for microbial production of protocatechuate with Corynebacterium glutamicum.

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of high interest in the chemical industry and has wide potential for pharmaceutical applications. We designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of d-xylose for microbial production of PCA. Shake flask cultivation of the engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L-1 ) from d-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-mol PCA per C-mol d-xylose, which corresponds to 38% of the maximum theoretical yield. Under growth-decoupled bioreactor conditions, a comparable PCA titer and a total amount of 16.5 ± 1.1 g PCA could be achieved when d-glucose and d-xylose were combined as orthogonal carbon substrates for biocatalyst provision and product synthesis, respectively. Downstream processing of PCA was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. This resulted in a maximum final purity of 95.4%. The established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

Enhancing l-glutamine production in Corynebacterium glutamicum by rational metabolic engineering combined with a two-stage pH control strategy

l-glutamine is a semi-essential amino acid widely used in the food and pharmaceutical industries. The microbial synthesis of l-glutamine is limited by lack of effective strains with high titer and safety. First, ARTP mutagenesis combined with high-throughput screening generated an l-glutamine-producing strain of Corynebacterium glutamicum with titer of 25.7 ± 2.7 g/L. Subsequently, a series of rational metabolic approaches were used to further improve l-glutamine production, which included increasing the carbon flow to l-glutamine (proB and NCgl1221 knockout), enhancing the catalytic efficiency of the key enzyme (glnE knockout and glnA screening and overexpression) and reinforcement of ATP regeneration (ppk overexpression and RBS optimization). Finally, we proposed a two-stage pH control strategy to address the inconsistent effect of pH on cell growth and l-glutamine production. These combined strategies led to a 186.0% increase of l-glutamine titer compared to that of the initial strain, reaching 73.5 ± 3.1 g/L with a yield of 0.368 ± 0.034 g/g glucose.

History-Driven Genetic Modification Design Technique Using a Domain-Specific Lexical Model for the Acceleration of DBTL Cycles for Microbial Cell Factories.

The development of microbes for conducting bioprocessing via synthetic biology involves design-build-test-learn (DBTL) cycles. To aid the designing step, we developed a computational technique that suggests next genetic modifications on the basis of relatedness to the user's design history of genetic modifications accumulated through former DBTL cycles conducted by the user. This technique, which comprehensively retrieves well-known designs related to the history, involves searching text for previous literature and then mining genes that frequently co-occur in the literature with those modified genes. We further developed a domain-specific lexical model that weights literature that is more related to the domain of metabolic engineering to emphasize genes modified for bioprocessing. Our technique made a suggestion by using a history of creating a Corynebacterium glutamicum strain producing shikimic acid that had 18 genetic modifications. Inspired by the suggestion, eight genes were considered by biologists for further modification, and modifying four of these genes proved experimentally efficient in increasing the production of shikimic acid. These results indicated that our proposed technique successfully utilized the former cycles to suggest relevant designs that biologists considered worth testing. Comprehensive retrieval of well-tested designs will help less-experienced researchers overcome the entry barrier as well as inspire experienced researchers to formulate design concepts that have been overlooked or suspended. This technique will aid DBTL cycles by feeding histories back to the next genetic design, thereby complementing the designing step.

(Optochemical) Control of Synthetic Microbial Coculture Interactions on a Microcolony Level.

Synthetic microbial cocultures carry enormous potential for applied biotechnology and are increasingly the subject of fundamental research. So far, most cocultures have been designed and characterized based on bulk cultivations without considering the potentially highly heterogeneous and diverse single-cell behavior. However, an in-depth understanding of cocultures including their interacting single cells is indispensable for the development of novel cultivation approaches and control of cocultures. We present the development, validation, and experimental characterization of an optochemically controllable bacterial coculture on a microcolony level consisting of two Corynebacterium glutamicum strains. Our coculture combines an l-lysine auxotrophic strain together with a l-lysine-producing variant carrying the genetically IPTG-mediated induction of l-lysine production. We implemented two control approaches utilizing IPTG as inducer molecule. First, unmodified IPTG was supplemented to the culture enabling a medium-based control of the production of l-lysine, which serves as the main interacting component. Second, optochemical control was successfully performed by utilizing photocaged IPTG activated by appropriate illumination. Both control strategies were validated studying cellular growth on a microcolony level. The novel microfluidic single-cell cultivation strategies applied in this work can serve as a blueprint to validate cellular control strategies of synthetic mono- and cocultures with single-cell resolution at defined environmental conditions.

Safety and efficacy of a feed additive consisting of l-valine produced by Corynebacterium glutamicumCGMCC 7.366 for all animal species (Ningxia Eppen Biotech Co., Ltd.).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of the feed additive consisting of l‐valine produced by fermentation using a non‐genetically modified strain of Corynebacterium glutamicum (CGMCC 7.366). The additive is intended to be used in feed and water for drinking for all animal species and categories. The production strain is considered suitable for the qualified presumption of safety (QPS) approach to safety assessment. The FEEDAP Panel concludes that l‐valine produced using C. glutamicum CGMCC 7.366 is safe for the target species when supplemented in appropriate amounts to the diet according to the nutritional needs of the target species. The FEEDAP Panel has concerns on the use of amino acids in water for drinking for hygienic reasons, and due to the risk of imbalances when administered simultaneously via feed. The use of l‐valine produced using C. glutamicum CGMCC 7.366 in animal nutrition is considered safe for the consumer and for the environment. No conclusion could be drawn on the potential of l‐valine produced using C. glutamicum CGMCC 7.366 to be toxic by inhalation, irritant to the skin or eyes, or a dermal sensitiser due to the lack of data. The additive l‐valine produced by fermentation using C. glutamicum CGMCC 7.366 is regarded as an efficacious source of the essential amino acid l‐valine for non‐ruminant nutrition. For the supplemental l‐valine to be as efficacious in ruminants as in non‐ruminant species, it requires protection against degradation in the rumen.

Need for speed: evaluation of dilute and shoot-mass spectrometry for accelerated metabolic phenotyping in bioprocess development

With the utilization of small-scale and highly parallelized cultivation platforms embedded in laboratory robotics, microbial phenotyping and bioprocess development have been substantially accelerated, thus generating a bottleneck in bioanalytical bioprocess sample analytics. While microscale cultivation platforms allow the monitoring of typical process parameters, only limited information about product and by-product formation is provided without comprehensive analytics. The use of liquid chromatography mass spectrometry can provide such a comprehensive and quantitative insight, but is often limited by analysis runtime and throughput. In this study, we developed and evaluated six methods for amino acid quantification based on two strong cation exchanger columns and a dilute and shoot approach in hyphenation with either a triple-quadrupole or a quadrupole time-of-flight mass spectrometer. Isotope dilution mass spectrometry with 13C15N labeled amino acids was used to correct for matrix effects. The versatility of the methods for metabolite profiling studies of microbial cultivation supernatants is confirmed by a detailed method validation study. The methods using chromatography columns showed a linear range of approx. 4 orders of magnitude, sufficient response factors, and low quantification limits (7–443 nM) for single analytes. Overall, relative standard deviation was comparable for all analytes, with < 8% and < 11% for unbuffered and buffered media, respectively. The dilute and shoot methods with an analysis time of 1 min provided similar performance but showed a factor of up to 35 times higher throughput. The performance and applicability of the dilute and shoot method are demonstrated using a library of Corynebacterium glutamicum strains producing l-histidine, obtained from random mutagenesis, which were cultivated in a microscale cultivation platform.Graphical abstract

Genome Sequence of the Bacteriophage CL31 and Interaction with the Host Strain Corynebacterium glutamicum ATCC 13032.

In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4%). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage Φ16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the ∆resmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

Dynamic Co-Cultivation Process of Corynebacterium glutamicum Strains for the Fermentative Production of Riboflavin

Residual streams from lignocellulosic processes contain sugar mixtures of glucose, xylose, and mannose. Here, the industrial workhorse Corynebacterium glutamicum was explored as a research platform for the rational utilization of a multiple sugar substrate. The endogenous manA gene was overexpressed to enhance mannose utilization. The overexpression of the xylA gene from Xanthomonas campestris in combination with the endogenous xylB gene enabled xylose consumption by C. glutamicum. Furthermore, riboflavin production was triggered by overexpressing the sigH gene from C. glutamicum. The resulting strains were studied during batch fermentations in flasks and 2 L lab-scale bioreactors separately using glucose, mannose, xylose, and a mixture of these three sugars as a carbon source. The production of riboflavin and consumption of sugars were improved during fed-batch fermentation thanks to a dynamic inoculation strategy of manA overexpressing strain and xylAB overexpressing strain. The final riboflavin titer, yield, and volumetric productivity from the sugar mixture were 27 mg L−1, 0.52 mg g−1, and 0.25 mg L−1 h−1, respectively. It reached a 56% higher volumetric productivity with 45% less by-product formation compared with an equivalent process inoculated with a single strain overexpressing the genes xylAB and manA combined. The results indicate the advantages of dynamic multi strains processes for the conversion of sugar mixtures.

Safety and efficacy of l-lysine monohydrochloride and concentrated liquid l-lysine (base) produced by fermentation with Corynebacterium glutamicum KCCM 80216 as feed additive for all animal species.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of concentrated liquid l‐lysine (base, minimum 50%) and l‐lysine monohydrochloride (HCl, minimum 99%) produced by fermentation with a genetically modified strain of Corynebacterium glutamicum (KCCM 80216) as nutritional additives for all animal species. Neither the production strain nor its recombinant DNA was detected in the final products. The additives do not pose any safety concern associated with the genetic modification of the production strain. Concentrated liquid l‐lysine (base) and l‐lysine HCl produced by C. glutamicum KCCM 80216 do not represent a risk for the target species, the consumer and the environment. From the results of studies on the safety for the user of concentrated liquid l‐lysine (base) and l‐lysine HCl produced by a different production strain, it was possible to conclude on the safety for the user of the products under assessment. The concentrated liquid l‐lysine (base) and the l‐lysine HCl are not irritant to skin or eyes or skin sensitiser. l‐lysine HCl is not hazardous by inhalation. l‐lysine HCl and concentrated liquid l‐lysine (base) are considered as efficacious sources of the essential amino acid l‐lysine for non‐ruminant animal species. For the supplemental l‐lysine to be as efficacious in ruminants as in non‐ruminant species, it would require protection against degradation in the rumen.

Safety and efficacy of l-lysine monohydrochloride and concentrated liquid l-lysine (base) produced by fermentation with Corynebacterium glutamicumKCTC 12307BP as feed additives for all animal species.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of a concentrated liquid l‐lysine (base, minimum 50%) and a l‐lysine monohydrochloride (HCl, minimum 99%) produced by fermentation with a genetically modified strain of Corynebacterium glutamicum (KCTC 12307BP). Both forms of l‐lysine are intended to be used in feed for all animal species and categories. Neither the production strain nor its recombinant DNA were detected in the final products. The additives do not pose any safety concern associated with the genetic modification of the production strain. Concentrated liquid l‐lysine (base) and l‐lysine HCl produced by the strain C. glutamicum KCTC 12307BP do not represent a risk for the target species, for the consumer, for the user and for the environment. l‐Lysine HCl and concentrated liquid l‐lysine (base) are considered to be efficacious sources of the essential amino acid l‐lysine for non‐ruminant animal species. For the supplemental l‐lysine to be as efficacious in ruminants as in non‐ruminant species, it would require protection against degradation in the rumen.

Expression of the NADPH+-Dependent Formate-Dehydrogenase Gene from Pseudomonas Increases Lysine Production in Corynebacterium glutamicum

The psefdh_D221Q gene encodes a mutant formate dehydrogenase from Pseudomonas (P-seFDG_D221Q), which catalyzes formate oxidation with the simultaneous formation of NADPH. It is expressed in cells of lysine-producing Corynebacterium glutamicum strains. The psefdh_D221Q gene was introduced into C. glutamicum strains as part of an autonomous plasmid or was integrated into the chromosome with the simultaneous inactivation of the host formate-dehydrogenase genes. It was shown that the C. glutamicum strains with NADP+-dependent formate dehydrogenase have an increased level of L-lysine synthesis in the presence of formate if their own formate dehydrogenase is inactivated.

Elevated, non-proliferative temperatures change the profile of fermentation products in Corynebacterium glutamicum

Although temperature is a crucial factor affecting enzymatic activity on biochemical and biofuel production, the reaction temperature for the generation of these products is usually set at the optimal growth temperature of the host strain, even under non-proliferative conditions. Given that the production of fermentation products only requires a fraction of the cell's metabolic pathways, the optimal temperatures for microbial growth and the fermentative production of a target compound may be different. Here, we investigated the effect of temperature on lactic and succinic acids production, and related enzymatic activities, in wild-type and succinic acid-overproducing strains of Corynebacterium glutamicum. Interestingly, fermentative production of lactic acid increased with the temperature in wild-type: production was 69% higher at 42.5°C, a temperature that exceeded the upper limit for growth, than that at the optimal growth temperature (30°C). Conversely, succinic acid production was decreased by 13% under the same conditions in wild-type. The specific activity of phosphoenolpyruvate carboxylase decreased with the increase in temperature. In contrast, the other glycolytic and reductive TCA cycle enzymes demonstrated increased or constant activity as the temperature was increased. When using a succinic acid over-producing strain, succinic acid production was increased by 34% at 42.5°C. Our findings demonstrate that the profile of fermentation products is dependent upon temperature, which could be caused by the modulation of enzymatic activities. Moreover, we report that elevated temperatures, exceeding the upper limit for cell growth, can be used to increase the production of target compounds in C. glutamicum. KEY POINTS: • Lactate productivity was increased by temperature elevation. • Succinate productivity was increased by temperature elevation when lactate pathway was deleted. • Specific activity of phosphoenolpyruvate carboxylase was decreased by temperature elevation.

Safety and efficacy of l-valine produced by fermentation using Corynebacterium glutamicumCGMCC 7.358 as a feed additive for all animal species.

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on l‐valine produced by fermentation using a non‐genetically modified strain of Corynebacterium glutamicum (CGMCC 7.358). The additive is intended to be used in feed and water for drinking for all animal species and categories. The production strain meets the qualifications for the qualified presumption of safety (QPS) approach to safety assessment and is considered safe. l‐Valine produced using C. glutamicum CGMCC 7.358 is safe for the target species when supplemented in appropriate amounts to the diet according to the nutritional needs of the target species, for the consumer and for the environment. No conclusion could be drawn on the potential of l‐valine produced using C. glutamicum CGMCC 7.358 to be toxic by inhalation, irritant to the skin or eyes, or a dermal sensitiser due to the lack of data. The product l‐valine produced by fermentation using C. glutamicum CGMCC 7.358 is regarded as an efficacious source of the essential amino acid l‐valine for non‐ruminant nutrition. For the supplemental l‐valine to be as efficacious in ruminants as in non‐ruminant species, it requires protection against degradation in the rumen.

Preparation, purification, and characterization of low-molecular-weight hyaluronic acid.

The use and commercial value of hyaluronic acid (HA) as an important element in the pharmaceutical, biomedical, and cosmetics industry is because of its purity. Four recombinant strains of Corynebacterium glutamicum containing different genes were used to produce HA. The production parameters were measured and strain 183.2, with the highest amount of HA (2.15 mg/ml), was selected for further experiments. HA was precipitated by different ratios of ethanol-isopropanol at 4 °C and - 20 °C. Active charcoal (1%) was added to the solvent precipitation mixture at pH 5 and 10. Finally, to achieve more purity and separation, gel filtration chromatography was used. The best result was obtained using an ethanol-isopropanol ratio of 1:1 of at - 20 °C, followed by active charcoal treatment at the acidic pH, and three fractions of the chromatography with molecular weights of 27, 27-110, and < 27 KDa were more analyzed with electrophoresis and FTIR. The present study described a simple, economical, and reproducible method resulting in a high yield for low-MW HA from C. glutamicum.