Construction and application of a synthetic promoter library for Corynebacterium glutamicum / 生物工程学报

Promoter is an important genetic tool for fine-tuning of gene expression and has been widely used for metabolic engineering. Corynebacterium glutamicum is an important chassis for industrial biotechnology. However, promoter libraries that are applicable to C. glutamicum have been rarely reported, except for a few developed based on synthetic sequences containing random mutations. In this study, we constructed a promoter library based on the native promoter of odhA gene by mutating the -10 region and the bystanders. Using a red fluorescent protein (RFP) as the reporter, 57 promoter mutants were screened by fluorescence imaging technology in a high-throughput manner. These mutants spanned a strength range between 2.4-fold and 19.6-fold improvements of the wild-type promoter. The strongest mutant exhibited a 2.3-fold higher strength than the widely used strong inducible promoter Ptrc. Sequencing of all 57 mutants revealed that 55 mutants share a 1-4 bases shift (4 bases shift for 68% mutants) of the conserved -10 motif "TANNNT" to the 3' end of the promoter, compared to the wild-type promoter. Conserved T or G bases at different positions were observed for strong, moderate, and weak promoter mutants. Finally, five promoter mutants with different strength were employed to fine-tune the expression of γ-glutamyl kinase (ProB) for L-proline biosynthesis. Increased promoter strength led to enhanced L-proline production and the highest L-proline titer of 6.4 g/L was obtained when a promoter mutant with a 9.8-fold higher strength compared to the wild-type promoter was used for ProB expression. The use of stronger promoter variants did not further improve L-proline production. In conclusion, a promoter library was constructed based on a native C. glutamicum promoter PodhA. The new promoter library should be useful for systems metabolic engineering of C. glutamicum. The strategy of mutating native promoter may also guide the construction of promoter libraries for other microorganisms.

Optimization of CRISPR/Cas9-based multiplex base editing in Corynebacterium glutamicum / 生物工程学报

As a new CRISPR/Cas-derived genome engineering technology, base editing combines the target specificity of CRISPR/Cas and the catalytic activity of nucleobase deaminase to install point mutations at target loci without generating DSBs, requiring exogenous template, or depending on homologous recombination. Recently, researchers have developed a variety of base editing tools in the important industrial strain Corynebacterium glutamicum, and achieved simultaneous editing of two and three genes. However, the multiplex base editing based on CRISPR/Cas9 is still limited by the complexity of multiple sgRNAs, interference of repeated sequence and difficulty of target loci replacement. In this study, multiplex base editing in C. glutamicum was optimized by the following strategies. Firstly, the multiple sgRNA expression cassettes based on individual promoters/terminators was optimized. The target loci can be introduced and replaced rapidly by using a template plasmid and Golden Gate method, which also avoids the interference of repeated sequence. Although the multiple sgRNAs structure is still complicated, the editing efficiency of this strategy is the highest. Then, the multiple gRNA expression cassettes based on Type Ⅱ CRISPR crRNA arrays and tRNA processing were developed. The two strategies only require one single promoter and terminator, and greatly simplify the structure of the expression cassette. Although the editing efficiency has decreased, both methods are still applicable. Taken together, this study provides a powerful addition to the genome editing toolbox of C. glutamicum and facilitates genetic modification of this strain.

Engineering the C4 pathway of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid / 生物工程学报

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.

Advances and prospects in metabolic engineering for the production of amino acids / 生物工程学报

Fermentative production of amino acids is one of the pillars of the fermentation industry in China. Recently, with the fast development of metabolic engineering and synthetic biology technologies, the metabolic engineering for production of amino acids has been flourishing. Conventional forward metabolic engineering, reversed metabolic engineering based on omics data and in silico simulation, and evolutionary metabolic engineering mimicking the natural evolution, have shown increasingly promising applications. A series of highly efficient and robust amino acids-producing strains have been developed and applied in the industrial production of amino acids. The increasingly fierce market competition has put forward new requirements for strain breeding and selection, such as developing high value-added amino acids, dynamic regulation of cellular metabolism, and adapting to the requirements of new process. This review summarizes the advances and prospects in metabolic engineering for the production of amino acids.

Recent advances in developing enabling technologies for Corynebacterium glutamicum metabolic engineering / 生物工程学报

Corynebacterium glutamicum is an important workhorse of industrial biotechnology, especially for amino acid bioindustry. This bacterium is being used to produce various amino acids at a level of over 6 million tons per year. In recent years, enabling technologies for C. glutamicum metabolic engineering have been developed and improved, which accelerated construction and optimization of microbial cell factoriers, expanding spectra of substrates and products, and facilitated basic researches on C. glutamicum. With these technologies, C. glutamicum has become one of the ideal microbial chasses. This review summarizes recent key technological developments of enabling technologies for C. glutamicum metabolic engineering and focuses on establishment and applications of CRISPR-based genome editing, gene expression regulation, adaptive laboratory evolution, and biosensor technologies.

Advances in stress tolerance mechanisms and synthetic biology for the industrial robustness of Corynebacterium glutamicum / 生物工程学报

As a model industrial host and microorganism with the generally regarded as safe (GRAS) status, Corynebacterium glutamicum not only produces amino acids on a large scale in the fermentation industry, but also has the potential to produce various new products. C. glutamicum usually encounters various stresses in the process of producing compounds, which severely affect cell viability and production performance. The development of synthetic biology provides new technical means for improving the robustness of C. glutamicum. In this review, we discuss the tolerance mechanisms of C. glutamicum to various stresses in the fermentation process. At the same time, we highlight new synthetic biology strategies for boosting C. glutamicum robustness, including discovering new stress-resistant elements, modifying transcription factors, and using adaptive evolution strategies to mine stress-resistant functional modules. Finally, prospects of improving the robustness of engineered C. glutamicum strains ware provided, with an emphasis on biosensor, screening and design of transcription factors, and utilizing the multiple regulatory elements.

Advances in gene editing of Corynebacterium glutamate / 生物工程学报

Corynebacterium glutamicum, an important microorganism to produce amino acids and organic acids, has been widely applied in food and medicine fields. Therefore, using editing tools to study the function of unknown genes in C. glutamicum has great significance for systematic development of industrial strain with efficient and novel production capability. Recently, gene editing has been greatly developed. Traditional gene editing based on homologous recombination and gene editing mediated by nuclease are successfully applied in C. glutamicum. Among these, the CRISPR system has been developed to be a main tool used for gene knockout of C. glutamicum due to its advantages of efficiency, simplicity and good target specificity. However, more efficient and reliable knockout system is still urgently demanded, to help develop high-performing strains in industrial application.

Expression, purification and characterization of catalase from Corynebacterium glutamicum / 生物工程学报

Catalase catalyzes the decomposition of H₂O₂ to H₂O and O₂, and has a wide range of industrial applications. However, most catalases used in the textile and paper industries are often subjected to high-alkaline challenges which makes it necessary to develop alkaline catalase. In this study, a catalase from Corynebacterium glutamicum was expressed in Escherichia coli, and the expression conditions were optimized. The recombinant catalase was purified by Ni-chelating affinity chromatography, and the recombinant enzyme was characterized. The optimal conditions of producing the recombinant catalase were: an IPTG concentration of 0.2 mmol/L, a culturing temperature of 25 °C and a culturing time of 11 h. The purified catalase had a specific activity of 55 266 U/mg, and it had a high activity in the pH range of 4.0 to11.5, with the highest activity at pH 11.0. When treated in pH 11.0 for 3 h, the enzyme retained 93% of its activity, indicating that the enzyme was qualified with a favorable stability under high-alkaline condition. The recombinant catalase had maximal activity at 30 °C, and showed a satisfactory thermal stability at a range of 25 °C to 50 °C. The apparent Km and Vmax values of purified catalase were 25.89 mmol/L and 185.18 mmol/(minmg), respectively. Besides, different inhibitors, such as sodium dodecyl sulfate (SDS), urea, NaN₂, β-mercaptoethanol, and EDTA had different degrees of inhibition on enzyme activity. The catalase from C. glutamicum shows high catalytic efficiency and high alkaline stability, suggesting its potential utilization in industrial production.

Amino acid exporters and metabolic modification of Corynebacterium glutamicum - a review / 生物工程学报

Amino acids are important compounds with a wide range of applications in the food, medicine and chemical industries. Corynebacterium glutamicum is a powerful workhorse commonly used in industrial amino acid production, with the scale of more than one million tons. In addition to its efficient anabolism, the effective exporters also ensure the high amino acid production by C. glutamicum. In this review, the research progress of amino acid exporter of C. glutamicum is summarized, to provide the foundation for further improving amino acid production by C. glutamicum via metabolic engineering.

Effect of key notes of TCA cycle on L-glutamate production / 生物工程学报

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.

Optimization of base editing in Corynebacterium glutamicum / 生物工程学报

In recent years, CRISPR/Cas9-mediated base editing has been developed to a powerful genome editing tool, providing advantages such as without introducing double-stranded DNA break, a donor template and relying on host homologous recombination repair pathway, and has been widely applied in animals, plants, yeast and bacteria. In previous study, our group developed a multiplex automated base editing method (MACBETH) in the important industrial model strain Corynebacterium glutamicum. In this study, to further optimize the method and improve the base editing efficiency in C. glutamicum, we first constructed a green fluorescent protein (GFP) reporter-based detection system. The point mutation in the inactivated GFP protein can be edited to restore the GFP fluorescence. By combining with flow cytometry analysis, the base-editing efficiency can be quickly calculated. Then, the base editor with the target gRNA was constructed, and the editing efficiency with the initial editing condition was (13.11±0.21)%. Based on this result, the editing conditions were optimized and the result indicated that the best medium is CGXII, the best initial OD₆₀₀ of induction is 0.05, the best induction time is 20 h, and the best IPTG concentration is 0.01 mmol/L. After optimization, the editing efficiency was improved to (30.35±0.75)%, which was 1.3-fold of that in initial condition. Finally, endogenous genomic loci of C. glutamicum were selected to assess if the optimized condition can improve genome editing in other loci. Editing efficiency of different loci in optimized condition were improved to 1.7-2.5 fold of that in original condition, indicating the effectiveness and versatility of the optimized condition. Our research will promote the better application of base editing technology in C. glutamicum.

Secretory production of xylanase in Corynebacterium glutamicum using its endogenous elements / 生物工程学报

We constructed bicistronic expression system containing AH6 promoter, 5' UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno (SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat (CgR0949) and Sec (CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 ℃, respectively. Its activity was 80% of initial activity after pretreatment at 4 ℃ for 24 h at pH 4-11, 95% after incubation below 50 ℃ for 15 min, and 20% when the temperature above 60 ℃. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C. glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.

Anti-Helicobacter pylori, Anti-apoptotic, and Cytoprotective Effects of Threonine Synthesized from Corynebacterium glutamicum in Gastric Epithelial Cells

BACKGROUND/AIMS: Among irritants causing gastric ulcer, Helicobacter pylori (H. pylori) might be pivotal, after which eradication became essential way in either inhibiting ulcerogenesis or preventing ulcer recurrence. Since threonine is essential in either mucus synthesis or cytoprotection, we hypothesized that the dietary threonine from Corynebacterium glutamicum (C. glutamicum) can mitigate the cytotoxicity of H. pylori infection.MATERIALS AND METHODS: RGM-1 cells were challenged with 100 multiplicity of infection H. pylori for 6 hours, during which threonine alone or combination with Corynebacterium sp. was administered and compared for anti-Helicobacter, anti-inflammation, anti-oxidative, and cytoprotective actions.RESULTS: Threonine alone or combination of threonine and C. glutamicum yielded significant bacteriostatic outcomes. The increased expressions of interleukin (IL)-1β, IL-8, Cox-2, and iNOS mRNA after H. pylori infection were significantly decreased with either threonine alone or the combination of threonine and C. glutamicum. The elevated expressions of NF-kB, HIF-1a, and c-jun after H. pylori infection were all significantly decreased with the combination of threonine and broth from C. glutamicum (P < 0.05), leading to significant decreases in 2′,7′-dichlorofluorescein-diacetate (P < 0.01). Tracing further host antioxidative response, the attenuated expression of heme oxygenase-1, Nrf2, and dehydrogenase quinone-1 after H. pylori infection was significantly preserved with combination of threonine and C. glutamicum. H. pylori infection led to significant increases in apoptosis accompanied with Bcl-2 decreases and Bax increases, while the combination of threonine and C. glutamicum significantly attenuated apoptosis, in which attenuated EGF, TGF-β, and VEGF were significantly regulated, while β-catenin did not change.CONCLUSIONS: Threonine synthesized from C. glutamicum significantly alleviated the cytotoxicity of H. pylori in gastric epithelial cells.

Metabolic engineering of L-valine synthesis and secretory pathways in Corynebacterium glutamicum for higher production / 生物工程学报

Corynebacterium glutamicum is the main industrial strain to produce L-valine by microbial fermentation. In this study, a low L-alanine producing C. glutamicum strain VWB-2 was constructed by knocking out the alanine aminotransferase encoding gene alaT in a high L-valine producing strain VWB-1. Meanwhile, a site-directed mutagenesis (ilvBN₁ (M13)) was done on the regulatory subunit of acetohydroxyacid synthase (ilvBN), a key enzyme in the L-valine synthesis pathway. Furthermore, the overexpression of the genes involved in the biosynthesis of L-valine, the mutated ilvBN₁ (M13), the acetohydroxy acid isomerase coding genes ilvC, the dihydroxy-acid dehydratase coding gene ilvD and branched-chain amino acid aminotransferase coding gene ilvE, could all promote the L-valine production of VWB-1 by strengthening the carbon flow towards L-valine. With the overexpression of the branched chain amino acid transporter coding gene brnFE and its regulator lrp₁, the L-valine producing capability of VWB-1 was further enhanced. The finally obtained engineered strain VWB-2/pEC-XK99E-ilvBN₁ (M13)CE-lrp₁-brnFE could produce 461.4 mmol/L L-valine in a 5 L fermentor with a sugar acid conversion rate of 0.312 g/g glucose.

Screening efficient constitutive promoters in Corynebacterium glutamicum based on time-series transcriptome analysis / 生物工程学报

Promoter, an essential regulatory element, is widely used for metabolic engineering of industrial strains. Corynebacterium glutamicum is an important industrial workhorse to produce various amino acids. However, strong constitutive promoters that are applicable to C. glutamicum are rarely reported. In this study, we first performed a time-series transcriptome analysis of a glutamate hyper-producing strain C. glutamicum SL4 by using RNA-Seq. Overall, we picked 10 samples at different time during the fermentation process. By analyzing the time-series transcriptome data, we selected 10 candidate genes with the highest transcriptional level. These genes were all transcribed stably during the fermentation process. We subsequently cloned the promoter sequences and evaluated the promoters' strength in strain SL4 using a red fluorescent protein reporter system. To evaluate the universality of the promoters in different C. glutamicum strains, we further tested the performance of some promoters in wild type C. glutamicum strains, including ATCC 13869 and ATCC 13032. The strongest promoter was further characterized using LacZ as a reporter in all the three C. glutamicum strains. Finally, we successfully obtained three constitutive promoters with universality, PcysK, PgapA and PfumC. PcysK is the most efficient promoter among the three C. glutamicum strains. In strains SL4 and ATCC 13869, the strength of PcysK is 2-fold of the strong inducible promoter Ptac using the red fluorescent protein as a reporter and 4-fold of Ptac using LacZ as a reporter. Moreover, the strength of PcysK reaches 30%-40% of Ptac in strain ATCC 13032. The promoter PcysK is identified as a strong promoter for the first time, which can be used as an efficient biobrick for metabolic engineering of synthesis pathways in C. glutamicum.

Development of an L-Lysine Enriched Bran for Animal Nutrition via Submerged Fermentation by Corynebacterium glutamicum using Agroindustrial Substrates

L-Lysine is an essential aminoacid added as supplement for animal feed. The aim of this work was to produce an L-Lysine enriched bran using Brazilian agroindustrial byproducts. Both the raw material costs and purification steps were minimized. Firstly, medium composition for the growth of Corynebacterium glutamicum ATCC 21799 was optimized targeting enhanced L-Lysine production - salt, vitamins and nitrogen sources concentrations were tested and selected. Next, UV mutant strains were generated and the best producers were used in formulated media using sugarcane molasses. It was reached a production of 9.3 g/L of L-Lysine with the optimized formulated media. This L-Lyisne rich broth was then impregnated and cyclically reimpregnated in pre-treated solid matrixes (sugarcane bagasse, citrus pulp, brewer spent grain, soybean husk and wheat bran). After processing, it was generated enriched brans with significant amounts of L-Lysine (13.8%, 7.0%, 8.9%, 5.9% and 8.4%, respectively), which has an interesting market potential for animal feed.

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.

PARÁMETROS FISICOQUÍMICOS PARA LA SÍNTESIS DE ÁCIDO LÁCTICO O ETANOL DE LA BACTERIA (Corynebacterium glutamicum) / Physico-Chemical Parameter for Production of Lactic Acid or Ethanol of (Corynebacterium glutamicum) Bacteria

El interés por obtener productos para la industria de biocombustibles a partir de desechos agrícolas, conduce a la búsqueda de nuevos sistemas biotecnológicos resistentes y costo-efectivos. Corynebacterium glutamicum, es un microorganismo usado para producir amino-ácidos que crece en gran variedad de sustratos y es resistente durante la fermentación, a variaciones de pH, temperatura, presión osmótica y acumulación de alcohol, características que lo hacen candidato a ser mejorado para la síntesis de ácido láctico y etanol. Aún se desconocen aspectos de su fisiología que aumenten su eficiencia en convertir azúcares (C5 y C6) en estos dos metabolitos. Por tanto, este trabajo buscó identificar los parámetros fisicoquímicos que tuvieron un mayor efecto sobre crecimiento bacteriano y síntesis de ácido láctico o etanol en un sistema por lotes. Para lograr este objetivo, ocho variables fueron evaluadas en un modelo estadístico producido en erlenmeyer; con los resultados obtenidos, se hallaron las mejores condiciones que fueron puestas a prueba en un cultivo en biorreactor. La temperatura, concentración de biotina y azúcar fueron las variables de mayor impacto (p< 0,05). Usando las mejores condiciones, 36 °C; 6,1 mg/L de biotina y 50 g/L de glucosa, se obtiene una µmax de 0,394 h-1, 16 g/L de ácido láctico a las 15 h del proceso con un rendimiento del 32%; observándose un mayor consumo de sustrato durante el crecimiento y poca disponibilidad para la fermentación, sugiriendo una alimentación del cultivo al final de la fase exponencial que aumente los rendimientos de producción.

The interest to obtain products for the bio-fuel industry from renewable resources has directed research to find resistant and costs-effective biotechnological systems. Corynebacterium glutamicum, is a microorganism used to produce amino acids, that grows in wide variety of substrates and its resistance during fermentation to pH, temperature, osmotic pressure variations and alcohol aggregate, renders this organism a suitable candidate to improve by genetic modifications lactic acid and ethanol synthesis. However, some aspects of its physiology remain unknown, such us increase lactic acid and ethanol production from C5 and C6 sugars. For this reason, the main aim in our work was to identify the most important variables with impact on culture and the best culture conditions to produce lactic acid or ethanol in batch culture. To achieve this objective, eight variables were tested in culture using a statistical model. The best culture conditions were obtained and tested in a bacth biorreactor system. Temperature, biotin and glucose concentration were the variables with most impact (p< 0.05) in culture. Using optimal conditions, 36 °C; 6.1 mg/L of biotin and 50 g/L of glucose; a µmax of 0.394 h-1, 16 g/L of lactic acid was obtained after 15 h of culture with an efficiency of 32%. High glucose consumption was observed during bacterial growth, which leads to low concentration of substrate for the production process; this suggests a culture feeding at the end of exponential growth phase, which can increase the production yield.

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.