Metabolomic analysis reveals stress tolerance mechanisms in common bean (Phaseolus vulgaris L.) related to treatment with a biostimulant obtained from Corynebacterium glutamicum.

Microbial biostimulants have emerged as a sustainable alternative to increase the productivity and quality of important crops. Despite this, the effects of the treatment on plant metabolism are poorly understood. Thus, this study investigated the metabolic response of common bean (Phaseolus vulgaris) related to the treatment with a biostimulant obtained from the extract of Corynebacterium glutamicum that showed positive effects on the development, growth, and yield of crops previously. By untargeted metabolomic analysis using UHPLC-MS/MS, plants and seeds were subjected to treatment with the biostimulant. Under ideal growth conditions, the plants treated exhibited higher concentration levels of glutamic acid, nicotiflorin and glycosylated lipids derived from linolenic acid. The foliar application of the biostimulant under water stress conditions increased the chlorophyll content by 17% and induced the accumulation of flavonols, mainly quercetin derivatives. Also, germination seed assays exhibited longer radicle lengths for seeds treated compared to the untreated control even in the absence of light (13-18% increase, p-value <0.05). Metabolomic analysis of the seeds indicated changes in concentration levels of amino acids (tryptophan, phenylalanine, tyrosine, glutamine, and arginine) and their derivatives. The results point out the enhancement of abiotic stress tolerance and the metabolic processes triggered in this crop associated with the treatment with the biostimulant, giving the first insights into stress tolerance mechanisms in P. vulgaris.

Revealing Corynebacterium glutamicum proteoforms through top-down proteomics.

Corynebacterium glutamicum is a bacterium widely employed in the industrial production of amino acids as well as a broad range of other biotechnological products. The present study describes the characterization of C. glutamicum proteoforms, and their post-translational modifications (PTMs) employing top-down proteomics. Despite previous evidence of PTMs having roles in the regulation of C. glutamicum metabolism, this is the first top-down proteome analysis of this organism. We identified 1125 proteoforms from 273 proteins, with 60% of proteins presenting at least one mass shift, suggesting the presence of PTMs, including several acetylated, oxidized and formylated proteoforms. Furthermore, proteins relevant to amino acid production, protein secretion, and oxidative stress were identified with mass shifts suggesting the presence of uncharacterized PTMs and proteoforms that may affect biotechnologically relevant processes in this industrial workhorse. For instance, the membrane proteins mepB and SecG were identified as a cleaved and a formylated proteoform, respectively. While in the central metabolism, OdhI was identified as two proteoforms with potential biological relevance: a cleaved proteoform and a proteoform with PTMs corresponding to a 70 Da mass shift.

Review transglutaminases: part II-industrial applications in food, biotechnology, textiles and leather products.

Because of their protein cross-linking properties, transglutaminases are widely used in several industrial processes, including the food and pharmaceutical industries. Transglutaminases obtained from animal tissues and organs, the first sources of this enzyme, are being replaced by microbial sources, which are cheaper and easier to produce and purify. Since the discovery of microbial transglutaminase (mTGase), the enzyme has been produced for industrial applications by traditional fermentation process using the bacterium Streptomyces mobaraensis. Several studies have been carried out in this field to increase the enzyme industrial productivity. Researches on gene expression encoding transglutaminase biosynthesis were performed in Streptomyces lividans, Escherichia coli, Corynebacterium glutamicum, Yarrowia lipolytica, and Pichia pastoris. In the first part of this review, we presented an overview of the literature on the origins, types, mediated reactions, and general characterizations of these important enzymes, as well as the studies on recombinant microbial transglutaminases. In this second part, we focus on the application versatility of mTGase in three broad areas: food, pharmacological, and biotechnological industries. The use of mTGase is presented for several food groups, showing possibilities of applications and challenges to further improve the quality of the end-products. Some applications in the textile and leather industries are also reviewed, as well as special applications in the PEGylation reaction, in the production of antibody drug conjugates, and in regenerative medicine.

L-lysine production improvement: a review of the state of the art and patent landscape focusing on strain development and fermentation technologies.

L-lysine is an essential amino acid used in various industrial sectors but mainly in food and animal feed. Intense research has been directed toward increasing its productivity. This literature review presents the state of the art and patent landscape of the industrial production of L-lysine, with a focus on the strain development and fermentation technologies, through geographic, social, and chronological analysis, using the text mining technique. The geographic analysis showed a greater tendency for countries with industrial plants with large production capacity to submit patents or publish articles, while the social analysis reflected the close relationship between educational units and companies. The technologies of each document were divided into optimization of fermentation parameters, conventional mutation, and genetic engineering. Corynebacterium glutamicum and Escherichia coli present the most attractive industrial phenotypes, and their cultivation occurs mainly in fed-batch processes with control parameters carefully selected to enhance metabolism. These strains are generally modified by conventional approaches (e.g., mutagenesis and selection of auxotrophic and/or regulatory mutants) or by genetic engineering technologies. The combination of both these approaches enables genomic breeding and the construction of strains with industrial potential, capable of accumulating more than 120 g/L of L-lysine. From the analysis of these approaches, we developed a descriptive flow of substrate uptake, amino acid metabolism, and mechanisms of excretion of a lysine-producing model cell. It is expected that the various mechanisms of L-lysine production, here shown and described, will become a guide that aids in increasing amino acid productivity without interfering with the strain stability.

Comprehensive assessment of the L-lysine production process from fermentation of sugarcane molasses.

L-Lysine is an essential amino acid that can be produced by chemical processes from fossil raw materials, as well as by microbial fermentation, the latter being a more efficient and environmentally friendly procedure. In this work, the production process of L-lysine-HCl is studied using a systematic approach based on modeling and simulation, which supports decision making in the early stage of process design. The study considers two analysis stages: first, the dynamic analysis of the fermentation reactor, where the conversion of sugars from sugarcane molasses to L-lysine with a strain of Corynebacterium glutamicum is carried out. In this stage, the operation mode (either batch or fed batch) and operating conditions of the fermentation reactor are defined to reach the maximum technical criteria. Afterwards, the second analysis stage relates to the industrial production process of L-lysine-HCl, where the fermentation reactor, upstream processing, and downstream processing are included. In this stage, the influence of key parameters on the overall process performance is scrutinized through the evaluation of several technical, economic, and environmental criteria, to determine a profitable and sustainable design of the L-lysine production process. The main results show how the operating conditions, process design, and selection of evaluation criteria can influence in the conceptual design. The best plant design shows maximum product yield (0.31 g L-lysine/g glucose) and productivity (1.99 g/L/h), achieving 26.5% return on investment (ROI) with a payback period (PBP) of 3.8 years, decreasing water and energy consumption, and with a low potential environmental impact (PEI) index.

High-level production of Bacillus cereus phospholipase C in Corynebacterium glutamicum.

Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme.

CMRegNet-An interspecies reference database for corynebacterial and mycobacterial regulatory networks.

BACKGROUND: Organisms utilize a multitude of mechanisms for responding to changing environmental conditions, maintaining their functional homeostasis and to overcome stress situations. One of the most important mechanisms is transcriptional gene regulation. In-depth study of the transcriptional gene regulatory network can lead to various practical applications, creating a greater understanding of how organisms control their cellular behavior. DESCRIPTION: In this work, we present a new database, CMRegNet for the gene regulatory networks of Corynebacterium glutamicum ATCC 13032 and Mycobacterium tuberculosis H37Rv. We furthermore transferred the known networks of these model organisms to 18 other non-model but phylogenetically close species (target organisms) of the CMNR group. In comparison to other network transfers, for the first time we utilized two model organisms resulting into a more diverse and complete network of the target organisms. CONCLUSION: CMRegNet provides easy access to a total of 3,103 known regulations in C. glutamicum ATCC 13032 and M. tuberculosis H37Rv and to 38,940 evolutionary conserved interactions for 18 non-model species of the CMNR group. This makes CMRegNet to date the most comprehensive database of regulatory interactions of CMNR bacteria. The content of CMRegNet is publicly available online via a web interface found at http://lgcm.icb.ufmg.br/cmregnet .

Regulation of expression of sodA and msrA genes of Corynebacterium glutamicum in response to oxidative and radiative stress.

Promoters of genes encoding superoxide dismutase (sodA) and peptide methionine sulfoxide reductase (msrA) from Cory-nebacterium glutamicum were cloned and sequenced. Promoter region analysis of sodA-msrA was unable to identify putative sites of fixed eventual regulators except for possible sites of fixed OxyR and integra-tion host factor. A study of the regulation of these genes was performed using the lacZ gene of Escherichia coli as a reporter placed under the control of sequences downstream of sodA and msrA. In silico analysis was used to identify regulators in the genome of C. glutamicum, which revealed the absence of homologs of soxRS and arcA and the presence of inactive oxyR and putative candidates of the homologs of ahpC, ohrR, integration host factor, furA, IdeR, diphtheria toxin repressor, and mntR.

Reducing lactate secretion by ldhA Deletion in L-glutamate- producing strain Corynebacterium glutamicum GDK-9

L-lactate is one of main byproducts excreted in to the fermentation medium. To improve L-glutamate production and reduce L-lactate accumulation, L-lactate dehydrogenase-encoding gene ldhA was knocked out from L-glutamate producing strain Corynebacterium glutamicum GDK-9, designated GDK-9ΔldhA. GDK-9ΔldhA produced approximately 10.1% more L-glutamate than the GDK-9, and yielded lower levels of such by-products as α-ketoglutarate, L-lactate and L-alanine. Since dissolved oxygen (DO) is one of main factors affecting L-lactate formation during L-glutamate fermentation, we investigated the effect of ldhA deletion from GDK-9 under different DO conditions. Under both oxygen-deficient and high oxygen conditions, L-glutamate production by GDK-9ΔldhA was not higher than that of the GDK-9. However, under micro-aerobic conditions, GDK-9ΔldhA exhibited 11.61% higher L-glutamate and 58.50% lower L-alanine production than GDK-9. Taken together, it is demonstrated that deletion of ldhA can enhance L-glutamate production and lower the unwanted by-products concentration, especially under micro-aerobic conditions.

Reducing lactate secretion by ldhA Deletion in L-glutamate- producing strain Corynebacterium glutamicum GDK-9

L-lactate is one of main byproducts excreted in to the fermentation medium. To improve L-glutamate production and reduce L-lactate accumulation, L-lactate dehydrogenase-encoding gene ldhA was knocked out from L-glutamate producing strain Corynebacterium glutamicum GDK-9, designated GDK-9ΔldhA. GDK-9ΔldhA produced approximately 10.1% more L-glutamate than the GDK-9, and yielded lower levels of such by-products as α-ketoglutarate, L-lactate and L-alanine. Since dissolved oxygen (DO) is one of main factors affecting L-lactate formation during L-glutamate fermentation, we investigated the effect of ldhA deletion from GDK-9 under different DO conditions. Under both oxygen-deficient and high oxygen conditions, L-glutamate production by GDK-9ΔldhA was not higher than that of the GDK-9. However, under micro-aerobic conditions, GDK-9ΔldhA exhibited 11.61% higher L-glutamate and 58.50% lower L-alanine production than GDK-9. Taken together, it is demonstrated that deletion of ldhA can enhance L-glutamate production and lower the unwanted by-products concentration, especially under micro-aerobic conditions.

Reducing lactate secretion by ldhA Deletion in L-glutamate- producing strain Corynebacterium glutamicum GDK-9.

L-lactate is one of main byproducts excreted in to the fermentation medium. To improve L-glutamate production and reduce L-lactate accumulation, L-lactate dehydrogenase-encoding gene ldhA was knocked out from L-glutamate producing strain Corynebacterium glutamicum GDK-9, designated GDK-9ΔldhA. GDK-9ΔldhA produced approximately 10.1% more L-glutamate than the GDK-9, and yielded lower levels of such by-products as α-ketoglutarate, L-lactate and L-alanine. Since dissolved oxygen (DO) is one of main factors affecting L-lactate formation during L-glutamate fermentation, we investigated the effect of ldhA deletion from GDK-9 under different DO conditions. Under both oxygen-deficient and high oxygen conditions, L-glutamate production by GDK-9ΔldhA was not higher than that of the GDK-9. However, under micro-aerobic conditions, GDK-9ΔldhA exhibited 11.61% higher L-glutamate and 58.50% lower L-alanine production than GDK-9. Taken together, it is demonstrated that deletion of ldhA can enhance L-glutamate production and lower the unwanted by-products concentration, especially under micro-aerobic conditions.

Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032).

Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD(+) release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 µM NAD(+) to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.

Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: cloning and expression of gntP and gntK in Escherichia coli.

Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral medium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constitutively expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli M1-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: Cloning and expression of gntP and gntK in Escherichia coli

Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral médium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constituvely expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli Ml-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

Transcriptional regulation of catabolic pathways for aromatic compounds in Corynebacterium glutamicum.

Corynebacterium glutamicum is a gram-positive soil microorganism able to utilize a large variety of aromatic compounds as the sole carbon source. The corresponding catabolic routes are associated with multiple ring-fission dioxygenases and among other channeling reactions, include the gentisate pathway, the protocatechuate and catechol branches of the beta-ketoadipate pathway and two potential hydroxyquinol pathways. Genes encoding the enzymatic machinery for the bioconversion of aromatic compounds are organized in several clusters in the C. glutamicum genome. Expression of the gene clusters is under specific transcriptional control, apparently including eight DNA-binding proteins belonging to the AraC, IclR, LuxR, PadR, and TetR families of transcriptional regulators. Expression of the gentisate pathway involved in the utilization of 3-hydroxybenzoate and gentisate is positively regulated by an IclR-type activator. The metabolic channeling of ferulate, vanillin and vanillate into the protocatechuate branch of the beta-ketoadipate pathway is controlled by a PadR-like repressor. Regulatory proteins of the IclR and LuxR families participate in transcriptional regulation of the branches of the beta-ketoadipate pathway that are involved in the utilization of benzoate, 4-hydroxybenzoate and protocatechuate. The channeling of phenol into this pathway may be under positive transcriptional control by an AraC-type activator. One of the potential hydroxyquinol pathways of C. glutamicum is apparently repressed by a TetR-type regulator. This global analysis revealed that transcriptional regulation of aromatic compound utilization is mainly controlled by single regulatory proteins sensing the presence of aromatic compounds, thus representing single input motifs within the transcriptional regulatory network of C. glutamicum.

Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction: role of the TreYZ trehalose biosynthetic pathway.

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.

Transcriptional regulation of catabolic pathways for aromatic compounds in Corynebacterium glutamicum

Corynebacterium glutamicum is a gram-positive soil microorganism able to utilize a large variety of aromatic compounds as the sole carbon source. The corresponding catabolic routes are associated with multiple ring-fission dioxygenases and among other channeling reactions, include the gentisate pathway, the protocatechuate and catechol branches of the beta-ketoadipate pathway and two potential hydroxyquinol pathways. Genes encoding the enzymatic machinery for the bioconversion of aromatic compounds are organized in several clusters in the C. glutamicum genome. Expression of the gene clusters is under specific transcriptional control, apparently including eight DNA-binding proteins belonging to the AraC, IclR, LuxR, PadR, and TetR families of transcriptional regulators. Expression of the gentisate pathway involved in the utilization of 3-hydroxybenzoate and gentisate is positively regulated by an IclR-type activator. The metabolic channeling of ferulate, vanillin and vanillate into the protocatechuate branch of the beta-ketoadipate pathway is controlled by a PadR-like repressor. Regulatory proteins of the IclR and LuxR families participate in transcriptional regulation of the branches of the beta-ketoadipate pathway that are involved in the utilization of benzoate, 4-hydroxybenzoate and protocatechuate. The channeling of phenol into this pathway may be under positive transcriptional control by an AraC-type activator. One of the potential hydroxyquinol pathways of C. glutamicum is apparently repressed by a TetR-type regulator. This global analysis revealed that transcriptional regulation of aromatic compound utilization is mainly controlled by single regulatory proteins sensing the presence of aromatic compounds, thus representing single input motifs within the transcriptional regulatory network of C. glutamicum.

Heterologous expression of Escherichia coli ppsA (phosphoenolpyruvate synthetase) and galU (UDP-glucose pyrophosphorylase) genes in Corynebacterium glutamicum, and its impact on trehalose synthesis.

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on a Corynebacterium glutamicum strain expressing the Escherichia coli enzyme UDP-glucose pyrophosphorylase (GalU). Biochemical network analysis suggest a further bottleneck for trehalose synthesis resulting from the coupling of phosphotransferase (PTS) mediated glucose uptake, and glucose catabolism in C. glutamicum. To overcome this coupling, we propose the expression of E. coli phosphoenolpyruvate synthetase (PpsA), in addition to GalU expression, in C. glutamicum. Although GalU expression improved trehalose synthesis in C. glutamicum, the simultaneous expression of GalU and PpsA did not result in a further increase in trehalose yield, but resulted in an increased catabolic rate of glucose, which could be ascribed to the operation of a futile cycle between phosphoenolpyruvate and pyruvate. The impact of GalU and PpsA expression on polysaccharide content, side product excretion and metabolic fluxes is discussed, as well as alternative ways to decouple glucose uptake and catabolism, in order to increase trehalose yield.