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Volker F. Wendisch - One of the best experts on this subject based on the ideXlab platform.
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transport and metabolic engineering of the cell factory corynebacterium Glutamicum
Fems Microbiology Letters, 2018Co-Authors: Fernando Perezgarcia, Volker F. WendischAbstract:Corynebacterium Glutamicum has a long and successful history in the biotechnological production of the amino acids l-glutamate and l-lysine. In the recent years, C. Glutamicum has been engineered for the production of a broad catalog of value-added compounds including organic acids, vitamins, terpenoids and proteins. Moreover, this bacterium has been engineered to realize a flexible carbon source concept enabling product formation from various second generation feedstocks without competing uses in human and animal nutrition. In this review, we highlight transport engineering to improve product export and substrate uptake or to avoid loss of intermediates by excretion as well as the application of new metabolic engineering concepts for C. Glutamicum strain development including the use of designed synthetic Escherichiacoli-C. Glutamicum consortia. As examples, pathway extension of l-lysine and l-glutamate biosynthesis to produce derived value-added chemicals is described. The described examples of C. Glutamicum strain engineering reflect strategies to cope with the increasing complexity of biotechnological processes that are required for successful applications in the bioeconomy.
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engineering of corynebacterium Glutamicum for minimized carbon loss during utilization of d xylose containing substrates
Journal of Biotechnology, 2014Co-Authors: Andreas Radek, Volker F. Wendisch, Michael Bott, Karin Krumbach, Jochem Gatgens, Wolfgang Wiechert, Stephan Noack, Jan MarienhagenAbstract:Abstract Biomass-derived d -xylose represents an economically interesting substrate for the sustainable microbial production of value-added compounds. The industrially important platform organism Corynebacterium Glutamicum has already been engineered to grow on this pentose as sole carbon and energy source. However, all currently described C. Glutamicum strains utilize d -xylose via the commonly known isomerase pathway that leads to a significant carbon loss in the form of CO 2 , in particular, when aiming for the synthesis of α-ketoglutarate and its derivatives (e.g. l -glutamate). Driven by the motivation to engineer a more carbon-efficient C. Glutamicum strain, we functionally integrated the Weimberg pathway from Caulobacter crescentus in C. Glutamicum . This five-step pathway, encoded by the xylXABCD -operon, enabled a recombinant C. Glutamicum strain to utilize d -xylose in d -xylose/ d -glucose mixtures. Interestingly, this strain exhibited a tri-phasic growth behavior and transiently accumulated d -xylonate during d -xylose utilization in the second growth phase. However, this intermediate of the implemented oxidative pathway was re-consumed in the third growth phase leading to more biomass formation. Furthermore, C. Glutamicum pEKEx3- xylXABCD Cc was also able to grow on d -xylose as sole carbon and energy source with a maximum growth rate of μ max = 0.07 ± 0.01 h −1 . These results render C. Glutamicum pEKEx3- xylXABCD Cc a promising starting point for the engineering of efficient production strains, exhibiting only minimal carbon loss on d -xylose containing substrates.
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production and glucosylation of c50 and c40 carotenoids by metabolically engineered corynebacterium Glutamicum
Applied Microbiology and Biotechnology, 2014Co-Authors: Sabine A E Heider, Petra Peterswendisch, Roman Netzer, Marit Stafnes, Trygve Brautaset, Volker F. WendischAbstract:The yellow-pigmented soil bacterium Corynebacterium Glutamicum ATCC13032 is accumulating the cyclic C50 carotenoid decaprenoxanthin and its glucosides. Carotenoid pathway engineering was previously shown to allow for efficient lycopene production. Here, engineering of C. Glutamicum for production of endogenous decaprenoxanthin as well as of the heterologous C50 carotenoids C.p.450 and sarcinaxanthin is described. Plasmid-borne overexpression of genes for lycopene cyclization and hydroxylation from C. Glutamicum, Dietzia sp., and Micrococcus luteus, in a lycopene-producing platform strain constructed here, resulted in accumulation of these three C50 carotenoids to concentrations of about 3–4 mg/g CDW. Chromosomal deletion of a putative carotenoid glycosyltransferase gene cg0730/crtX in these strains entailed production of non-glucosylated derivatives of decaprenoxanthin, C.p.450, and sarcinaxanthin, respectively. Upon introduction of glucosyltransferase genes from M. luteus, C. Glutamicum, and Pantoea ananatis, these hydroxylated C50 carotenoids were glucosylated. We here also demonstrate production of the C40 carotenoids β-carotene and zeaxanthin in recombinant C. Glutamicum strains and co-expression of the P. ananatis crtX gene was used to obtain glucosylated zeaxanthin. Together, our results show that C. Glutamicum is a potentially valuable host for production of a wide range of glucosylated C40 and C50 carotenoids.
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carotenoid biosynthesis and overproduction in corynebacterium Glutamicum
BMC Microbiology, 2012Co-Authors: Sabine A E Heider, Petra Peterswendisch, Volker F. WendischAbstract:Background: Corynebacterium Glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. Results: Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 e-cyclase, respectively. However, the genome of C. Glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. Glutamicum ΔcrtB, thus, C. Glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. Glutamicum ΔcrtI. The potential of C. Glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03±0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. Glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4±0.3 mg/g CDW were obtained. Conclusion: C. Glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. Glutamicum may serve as a potential host for carotenoid production.
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lysine and glutamate production by corynebacterium Glutamicum on glucose fructose and sucrose roles of malic enzyme and fructose 1 6 bisphosphatase
Metabolic Engineering, 2005Co-Authors: Tobias Georgi, Doris Rittmann, Volker F. WendischAbstract:Abstract In the biotechnological production of l -lysine and l -glutamate by Corynebacterium Glutamicum media based on glucose, fructose or sucrose are typically used. Glutamate production by C. Glutamicum ATCC13032 was very similar on glucose, fructose, glucose plus fructose and sucrose. In contrast, lysine production of genetically defined C. Glutamicum strains was significantly higher on glucose than on the other carbon sources. To test whether malic enzyme or fructose-1,6-bisphosphatase might limit growth and lysine on fructose, glucose plus fructose or sucrose, strains overexpressing either malE which encodes the NADPH-dependent malic enzyme or the fructose-1,6-bisphosphatase gene fbp were generated. Overexpression of malE did not improve lysine production on any of the tested carbon sources. Upon overexpression of fbp lysine yields on glucose and/or fructose were unchanged, but the lysine yield on sucrose increased twofold. Thus, fructose-1,6-bisphosphatase was identified as a limiting factor for lysine production by C. Glutamicum with sucrose as the carbon source.
Luis M Mateos - One of the best experts on this subject based on the ideXlab platform.
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06 Letek-Gil RG20.01.qxp
2020Co-Authors: Michal Letek, Efren Ordonez, Jose Vaquera, J A Gil, María Fiuza, Pilar Honrubia-marcos, Luis M MateosAbstract:Summary. Of the five promoters detected for the ftsZ gene in Corynebacterium Glutamicum, three were located within the coding region of the upstream ftsQ gene and two within the intergenic ftsQ-ftsZ region. The most distant ftsZ promoter showed activity in Escherichia coli and controlled high-level transcriptional expression of ftsZ in C. Glutamicum. Quantitative Western blotting showed that all five promoters were active during the exponential growth phase and down-regulated during stationary phase. This tightly controlled expression of ftsZ in C. Glutamicum indicated that small changes in the amount of FtsZ protein strongly affect bacterial cell viability. The controlled overexpression of ftsZ in C. Glutamicum, using the promoter of the gntK gene (PgntK), resulted in approximately 5-fold overproduction of FtsZ, an increase in cell diameter, and a highly variable localization of the protein as spirals or tangles throughout the cell. These results suggest that the intracellular concentration of FtsZ is critical for productive septum formation in C. Glutamicum. Our observations provide insight into the mechanisms used by the coryneform group, which lacks actin homologs and many regulators of cell division, to control cell morphology. [Int Microbiol 2007; 10(4): 271-282
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untitled
2020Co-Authors: Michal Letek, Efren Ordonez, Jose Vaquera, J A Gil, María Fiuza, Pilar Honrubia-marcos, Luis M MateosAbstract:Summary. Of the five promoters detected for the ftsZ gene in Corynebacterium Glutamicum, three were located within the coding region of the upstream ftsQ gene and two within the intergenic ftsQ-ftsZ region. The most distant ftsZ promoter showed activity in Escherichia coli and controlled high-level transcriptional expression of ftsZ in C. Glutamicum. Quantitative Western blotting showed that all five promoters were active during the exponential growth phase and down-regulated during stationary phase. This tightly controlled expression of ftsZ in C. Glutamicum indicated that small changes in the amount of FtsZ protein strongly affect bacterial cell viability. The controlled overexpression of ftsZ in C. Glutamicum, using the promoter of the gntK gene (PgntK), resulted in approximately 5-fold overproduction of FtsZ, an increase in cell diameter, and a highly variable localization of the protein as spirals or tangles throughout the cell. These results suggest that the intracellular concentration of FtsZ is critical for productive septum formation in C. Glutamicum. Our observations provide insight into the mechanisms used by the coryneform group, which lacks actin homologs and many regulators of cell division, to control cell morphology. [Int Microbiol 2007; 10(4): 271-282
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diviva is required for polar growth in the mreb lacking rod shaped actinomycete corynebacterium Glutamicum
Journal of Bacteriology, 2008Co-Authors: Michal Letek, Efren Ordonez, Jose Vaquera, Klas Flardh, William Margolin, Luis M MateosAbstract:The actinomycete Corynebacterium Glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVACg) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVACg-depleted C. Glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. Glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVACg localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. Glutamicum DivIVA is not involved in cell division or chromosome segregation.
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cell growth and cell division in the rod shaped actinomycete corynebacterium Glutamicum
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2008Co-Authors: Michal Letek, Efren Ordonez, Luis M Mateos, María Fiuza, Almudena F Villadangos, Astrid Ramos, Jose A GilAbstract:Bacterial cell growth and cell division are highly complicated and diversified biological processes. In most rod-shaped bacteria, actin-like MreB homologues produce helicoidal structures along the cell that support elongation of the lateral cell wall. An exception to this rule is peptidoglycan synthesis in the rod-shaped actinomycete Corynebacterium Glutamicum, which is MreB-independent. Instead, during cell elongation this bacterium synthesizes new cell-wall material at the cell poles whereas the lateral wall remains inert. Thus, the strategy employed by C. Glutamicum to acquire a rod-shaped morphology is completely different from that of Escherichia coli or Bacillus subtilis. Cell division in C. Glutamicum also differs profoundly by the apparent absence in its genome of homologues of spatial or temporal regulators of cell division, and its cell division apparatus seems to be simpler than those of other bacteria. Here we review recent advances in our knowledge of the C. Glutamicum cell cycle in order to further understand this very different model of rod-shape acquisition.
Michal Letek - One of the best experts on this subject based on the ideXlab platform.
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06 Letek-Gil RG20.01.qxp
2020Co-Authors: Michal Letek, Efren Ordonez, Jose Vaquera, J A Gil, María Fiuza, Pilar Honrubia-marcos, Luis M MateosAbstract:Summary. Of the five promoters detected for the ftsZ gene in Corynebacterium Glutamicum, three were located within the coding region of the upstream ftsQ gene and two within the intergenic ftsQ-ftsZ region. The most distant ftsZ promoter showed activity in Escherichia coli and controlled high-level transcriptional expression of ftsZ in C. Glutamicum. Quantitative Western blotting showed that all five promoters were active during the exponential growth phase and down-regulated during stationary phase. This tightly controlled expression of ftsZ in C. Glutamicum indicated that small changes in the amount of FtsZ protein strongly affect bacterial cell viability. The controlled overexpression of ftsZ in C. Glutamicum, using the promoter of the gntK gene (PgntK), resulted in approximately 5-fold overproduction of FtsZ, an increase in cell diameter, and a highly variable localization of the protein as spirals or tangles throughout the cell. These results suggest that the intracellular concentration of FtsZ is critical for productive septum formation in C. Glutamicum. Our observations provide insight into the mechanisms used by the coryneform group, which lacks actin homologs and many regulators of cell division, to control cell morphology. [Int Microbiol 2007; 10(4): 271-282
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untitled
2020Co-Authors: Michal Letek, Efren Ordonez, Jose Vaquera, J A Gil, María Fiuza, Pilar Honrubia-marcos, Luis M MateosAbstract:Summary. Of the five promoters detected for the ftsZ gene in Corynebacterium Glutamicum, three were located within the coding region of the upstream ftsQ gene and two within the intergenic ftsQ-ftsZ region. The most distant ftsZ promoter showed activity in Escherichia coli and controlled high-level transcriptional expression of ftsZ in C. Glutamicum. Quantitative Western blotting showed that all five promoters were active during the exponential growth phase and down-regulated during stationary phase. This tightly controlled expression of ftsZ in C. Glutamicum indicated that small changes in the amount of FtsZ protein strongly affect bacterial cell viability. The controlled overexpression of ftsZ in C. Glutamicum, using the promoter of the gntK gene (PgntK), resulted in approximately 5-fold overproduction of FtsZ, an increase in cell diameter, and a highly variable localization of the protein as spirals or tangles throughout the cell. These results suggest that the intracellular concentration of FtsZ is critical for productive septum formation in C. Glutamicum. Our observations provide insight into the mechanisms used by the coryneform group, which lacks actin homologs and many regulators of cell division, to control cell morphology. [Int Microbiol 2007; 10(4): 271-282
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diviva is required for polar growth in the mreb lacking rod shaped actinomycete corynebacterium Glutamicum
Journal of Bacteriology, 2008Co-Authors: Michal Letek, Efren Ordonez, Jose Vaquera, Klas Flardh, William Margolin, Luis M MateosAbstract:The actinomycete Corynebacterium Glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVACg) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVACg-depleted C. Glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. Glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVACg localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. Glutamicum DivIVA is not involved in cell division or chromosome segregation.
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cell growth and cell division in the rod shaped actinomycete corynebacterium Glutamicum
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2008Co-Authors: Michal Letek, Efren Ordonez, Luis M Mateos, María Fiuza, Almudena F Villadangos, Astrid Ramos, Jose A GilAbstract:Bacterial cell growth and cell division are highly complicated and diversified biological processes. In most rod-shaped bacteria, actin-like MreB homologues produce helicoidal structures along the cell that support elongation of the lateral cell wall. An exception to this rule is peptidoglycan synthesis in the rod-shaped actinomycete Corynebacterium Glutamicum, which is MreB-independent. Instead, during cell elongation this bacterium synthesizes new cell-wall material at the cell poles whereas the lateral wall remains inert. Thus, the strategy employed by C. Glutamicum to acquire a rod-shaped morphology is completely different from that of Escherichia coli or Bacillus subtilis. Cell division in C. Glutamicum also differs profoundly by the apparent absence in its genome of homologues of spatial or temporal regulators of cell division, and its cell division apparatus seems to be simpler than those of other bacteria. Here we review recent advances in our knowledge of the C. Glutamicum cell cycle in order to further understand this very different model of rod-shape acquisition.
Akihiko Kondo - One of the best experts on this subject based on the ideXlab platform.
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biotransformation of ferulic acid to protocatechuic acid by corynebacterium Glutamicum atcc 21420 engineered to express vanillate o demethylase
AMB Express, 2017Co-Authors: Naoko Okai, Takaya Masuda, Yasunobu Takeshima, Kosei Tanaka, Kenichi Yoshida, Masanori Miyamoto, Chiaki Ogino, Akihiko KondoAbstract:Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a lignin-derived phenolic compound abundant in plant biomass. The utilization of FA and its conversion to valuable compounds is desired. Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a precursor of polymers and plastics and a constituent of food. A microbial conversion system to produce PCA from FA was developed in this study using a PCA-producing strain of Corynebacterium Glutamicum F (ATCC 21420). C. Glutamicum strain F grown at 30 °C for 48 h utilized 2 mM each of FA and vanillic acid (4-hydroxy-3-methoxybenzoic acid, VA) to produce PCA, which was secreted into the medium. FA may be catabolized by C. Glutamicum through proposed (I) non-β-oxidative, CoA-dependent or (II) β-oxidative, CoA-dependent phenylpropanoid pathways. The conversion of VA to PCA is the last step in each pathway. Therefore, the vanillate O-demethylase gene (vanAB) from Corynebacterium efficiens NBRC 100395 was expressed in C. Glutamicum F (designated strain FVan) cultured at 30 °C in AF medium containing FA. Strain C. Glutamicum FVan converted 4.57 ± 0.07 mM of FA into 2.87 ± 0.01 mM PCA after 48 h with yields of 62.8% (mol/mol), and 6.91 mM (1064 mg/L) of PCA was produced from 16.0 mM of FA after 12 h of fed-batch biotransformation. Genomic analysis of C. Glutamicum ATCC 21420 revealed that the PCA-utilization genes (pca cluster) were conserved in strain ATCC 21420 and that mutations were present in the PCA importer gene pcaK.
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production of protocatechuic acid by corynebacterium Glutamicum expressing chorismate pyruvate lyase from escherichia coli
Applied Microbiology and Biotechnology, 2016Co-Authors: Naoko Okai, Yasunobu Takeshima, Chiaki Ogino, Takanori Miyoshi, Hiroaki Kuwahara, Akihiko KondoAbstract:Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) serves as a building block for polymers and pharmaceuticals. In this study, the biosynthetic pathway for PCA from glucose was engineered in Corynebacterium Glutamicum. The pathway to PCA-employed elements of the chorismate pathway by using chorismate-pyruvate lyase (CPL) and 4-hydroxybenzoate hydroxylase (4-HBA hydroxylase). As C. Glutamicum has the potential to synthesize the aromatic amino acid intermediate chorismate and possesses 4-HBA hydroxylase, we focused on expressing Escherichia coli CPL in a phenylalanine-producing strain of C. Glutamicum ATCC21420. To secrete PCA, the gene (ubiC) encoding CPL from E. coli was expressed in C. Glutamicum ATCC 21420 (strain F(UbiC)). The formation of 28.8 mg/L of extracellular 4-HBA (36 h) and 213 ± 29 mg/L of extracellular PCA (80 h) was obtained by the C. Glutamicum strain F(UbiC) from glucose. The strain ATCC21420 was also found to produce extracellular PCA. PCA fermentation was performed using C. Glutamicum strain F(UbiC) in a bioreactor at the optimized pH of 7.5. C. Glutamicum F(UbiC) produced 615 ± 2.1 mg/L of PCA from 50 g/L of glucose after 72 h. Further, fed-batch fermentation of PCA by C. Glutamicum F(UbiC) was performed with feedings of glucose every 24 h. The maximum production of PCA (1140.0 ± 11.6 mg/L) was achieved when 117.0 g/L of glucose was added over 96 h of fed-batch fermentation.
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direct l lysine production from cellobiose by corynebacterium Glutamicum displaying beta glucosidase on its cell surface
Applied Microbiology and Biotechnology, 2013Co-Authors: Noriko Adachi, Chihiro Takahashi, Naoko Onomurota, Rie Yamaguchi, Tsutomu Tanaka, Akihiko KondoAbstract:We constructed beta-glucosidase (BGL)-displaying Corynebacterium Glutamicum, and direct l-lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans, was successfully displayed on the C. Glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600 nm of BGL-displaying C. Glutamicum grown on cellobiose as a carbon source reached 23.5 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. Glutamicum produced 1.08 g/l of l-lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced l-lysine using BGL-secretory C. Glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. Glutamicum.
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direct production of cadaverine from soluble starch using corynebacterium Glutamicum coexpressing α amylase and lysine decarboxylase
Applied Microbiology and Biotechnology, 2009Co-Authors: Toshihiro Tateno, Hideki Fukuda, Yusuke Okada, Takeyuki Tsuchidate, Akihiko KondoAbstract:Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium Glutamicum strain coexpressing Streptococcus bovis 148 α-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli–C. Glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. Glutamicum CSS secreting AmyA. The engineered C. Glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5’-phosphate, which is the coenzyme for CadA. C. Glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert l-lysine to cadaverine, as there was no accumulation of l-lysine in the culture medium during fermentation. Thus, we demonstrated that C. Glutamicum has great potential to produce cadaverine from biomass resources.
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direct production of l lysine from raw corn starch by corynebacterium Glutamicum secreting streptococcus bovis α amylase using cspb promoter and signal sequence
Applied Microbiology and Biotechnology, 2007Co-Authors: Toshihiro Tateno, Hideki Fukuda, Akihiko KondoAbstract:Corynebacterium Glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. Glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. Glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. Glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. Glutamicum secreting AmyA using the cspB promoter and signal sequence.
Marta V Mendes - One of the best experts on this subject based on the ideXlab platform.
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engineering corynebacterium Glutamicum with a comprehensive genomic library and phage based vectors
Metabolic Engineering, 2020Co-Authors: Filipe Marques, Andriy Luzhetskyy, Marta V MendesAbstract:The Gram-positive bacterium Corynebacterium Glutamicum sustains the industrial production of chiral molecules such as L-amino acids. Through heterologous gene expression, C. Glutamicum is becoming a sustainable source of small organic molecules and added-value chemicals. The current methods to implement heterologous genes in C. Glutamicum rely on replicative vectors requiring lasting selection or chromosomal integration using homologous recombination. Here, we present a set of dedicated and transversal tools for genome editing and gene delivery into C. Glutamicum. We generated a cosmid-based library suitable for efficient double allelic exchange, covering more than 94% of the chromosome with an average 5.1x coverage. We employed the library and an iterative marker excision system to generate the carotenoid-free C. GlutamicumBT1-C31-Albino (BCA) host, featuring the attachment sites for actinophages ϕC31 and ϕBT1 for one-step chromosomal integration. As a proof-of-principle, we employed a ϕC31-based integration and a Cre system for the markerless expression of the type III polyketide synthase RppA, and a ϕBT1-based integration system for the expression of the phosphopantetheinylation-dependent non-ribosomal peptide synthetase BpsA in the C. Glutamicum BCA host. The developed genomic library and microbial host, and the characterized molecular tools will contribute to the study of the physiology and the rise of C. Glutamicum as a leading host for drug discovery.
