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Corynebacterium glutamicum with the ability to simultaneously utilize glucose/pentose mixed sugars was metabolically engineered to overproduce shikimate, a valuable hydroaromatic compound used as a starting material for the synthesis of the anti-influenza drug oseltamivir. To achieve this, the shikimate kinase and other potential metabolic activities for the consumption of shikimate and its precursor dehydroshikimate were inactivated. Carbon flux toward shikimate synthesis was enhanced by overexpression of genes for the shikimate pathway and the non-oxidative pentose phosphate pathway. Subsequently, to improve the availability of the key aromatics precursor phosphoenolpyruvate (PEP) toward shikimate synthesis, the PEP: sugar phosphotransferase system (PTS) was inactivated and an endogenous myo-inositol transporter IolT1 and glucokinases were overexpressed. Unexpectedly, the resultant non-PTS strain accumulated 1,3-dihydroxyacetone (DHA) and glycerol as major byproducts. This observation and metabolome analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed reaction as a limiting step in glycolysis. Consistently, overexpression of GAPDH significantly stimulated both glucose consumption and shikimate production. Blockage of the DHA synthesis further improved shikimate yield. We applied an aerobic, growth-arrested and high-density cell reaction to the shikimate production by the resulting strain and notably achieved the highest shikimate titer (141 g/l) and a yield (51% (mol/mol)) from glucose reported to date after 48 h in minimal medium lacking nutrients required for cell growth. Moreover, comparable shikimate productivity could be attained through simultaneous utilization of glucose, xylose, and arabinose, enabling efficient shikimate production from lignocellulosic feedstocks. These findings demonstrate that C. glutamicum has significant potential for the production of shikimate and derived aromatic compounds. 相似文献
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The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and l-lysine production drastically improved. Moreover, increasing the flux through l-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and l-methionine biosynthesis, further improved l-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the l-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45 % by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., l-threonine, l-methionine and l-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce l-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The l-lysine productivity was 2.73 g l?1 h?1 and the α was 47.06 % after 48 h. However, the attenuation of MurE was not beneficial to increase the l-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through l-lysine biosynthetic pathway and DCW are beneficial to improve l-lysine production in C. glutamicum. 相似文献
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Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is a natural bioactive phenolic acid potentially valuable as a pharmaceutical raw material owing to its diverse pharmacological activities. Corynebacterium glutamicum forms PCA as a key intermediate in a native pathway to assimilate shikimate/quinate through direct conversion of the shikimate pathway intermediate 3-dehydroshikimate (DHS), which is catalyzed by qsuB-encoded DHS dehydratase (the DHS pathway). PCA can also be formed via an alternate pathway extending from chorismate by introducing heterologous chorismate pyruvate lyase that converts chorismate into 4-hydroxybenzoate (4-HBA), which is then converted into PCA catalyzed by endogenous 4-HBA 3-hydroxylase (the 4-HBA pathway). In this study, we generated three plasmid-free C. glutamicum strains overproducing PCA based on the markerless chromosomal recombination by engineering each or both of the above mentioned two PCA-biosynthetic pathways combined with engineering of the host metabolism to enhance the shikimate pathway flux and to block PCA consumption. Aerobic growth-arrested cell reactions were performed using the resulting engineered strains, which revealed that strains dependent on either the DHS or 4-HBA pathway as the sole PCA-biosynthetic route produced 43.8 and 26.2 g/L of PCA from glucose with a yield of 35.3% and 10.0% (mol/mol), respectively, indicating that PCA production through the DHS pathway is significantly efficient compared to that produced through the 4-HBA pathway. Remarkably, a strain simultaneously using both DHS and 4-HBA pathways achieved the highest reported PCA productivity of 82.7 g/L with a yield of 32.8% (mol/mol) from glucose in growth-arrested cell reaction. These results indicated that simultaneous engineering of both DHS and 4-HBA pathways is an efficient method for PCA production. The generated PCA-overproducing strain is plasmid-free and does not require supplementation of aromatic amino acids and vitamins due to the intact shikimate pathway, thereby representing a promising platform for the industrial bioproduction of PCA and derived chemicals from renewable sugars. 相似文献
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Metabolic Engineering of Corynebacterium glutamicum for Trehalose Overproduction: Role of the TreYZ Trehalose Biosynthetic Pathway
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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. 相似文献
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Mimitsuka T Sawai H Hatsu M Yamada K 《Bioscience, biotechnology, and biochemistry》2007,71(9):2130-2135
Cadaverine, the expected raw material of polyamides, is produced by decarboxylation of L-lysine. If we could produce cadaverine from the cheapest sugar, and as a renewable resource, it would be an effective solution against global warming, but there has been no attempt to produce cadaverine from glucose by fermentation. We focused on Corynebacterium glutamicum, whose L-lysine fermentation ability is superior, and constructed a metabolically engineered C. glutamicum in which the L-homoserine dehydrogenase gene (hom) was replaced by the L-lysine decarboxylase gene (cadA) of Escherichia coli. In this recombinant strain, cadaverine was produced at a concentration of 2.6 g/l, equivalent to up to 9.1% (molecular yield) of the glucose transformed into cadaverine in neutralizing cultivation. This is the first report of cadaverine fermentation by C. glutamicum. 相似文献
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Peters-Wendisch P Stolz M Etterich H Kennerknecht N Sahm H Eggeling L 《Applied and environmental microbiology》2005,71(11):7139-7144
Although L-serine proceeds in just three steps from the glycolytic intermediate 3-phosphoglycerate, and as much as 8% of the carbon assimilated from glucose is directed via L-serine formation, previous attempts to obtain a strain producing L-serine from glucose have not been successful. We functionally identified the genes serC and serB from Corynebacterium glutamicum, coding for phosphoserine aminotransferase and phosphoserine phosphatase, respectively. The overexpression of these genes, together with the third biosynthetic serA gene, serA(delta197), encoding an L-serine-insensitive 3-phosphoglycerate dehydrogenase, yielded only traces of L-serine, as did the overexpression of these genes in a strain with the L-serine dehydratase gene sdaA deleted. However, reduced expression of the serine hydroxymethyltransferase gene glyA, in combination with the overexpression of serA(delta197), serC, and serB, resulted in a transient accumulation of up to 16 mM L-serine in the culture medium. When sdaA was also deleted, the resulting strain, C. glutamicum delta sdaA::pK18mobglyA'(pEC-T18mob2serA(delta197)CB), accumulated up to 86 mM L-serine with a maximal specific productivity of 1.2 mmol h(-1) g (dry weight)(-1). This illustrates a high rate of L-serine formation and also utilization in the C. glutamicum wild type. Therefore, metabolic engineering of L-serine production from glucose can be achieved only by addressing the apparent key position of this amino acid in the central metabolism. 相似文献
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The capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate was explored, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolize it. Expression of the Aspergillus terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in strain ATCC13032 led to the production of 1.4 mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440 mU mg−1 and 30 mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60 mM (7.8 g l−1), a molar yield of 0.4 mol mol−1, and a volumetric productivity of 2.1 mmol l−1 h−1. 相似文献
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2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter(-1)) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter(-1)) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate. 相似文献
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Yota Tsuge Hideo Kawaguchi Shogo Yamamoto Yoshiko Nishigami Masahiro Sota Chiaki Ogino 《Bioscience, biotechnology, and biochemistry》2018,82(7):1252-1259
Ultraviolet-absorbing chemicals are useful in cosmetics and skin care to prevent UV-induced skin damage. We demonstrate here that heterologous production of shinorine, which shows broad absorption maxima in the UV-A and UV-B region. A shinorine producing Corynebacterium glutamicum strain was constructed by expressing four genes from Actinosynnema mirum DSM 43827, which are responsible for the biosynthesis of shinorine from sedoheptulose-7-phosphate in the pentose phosphate pathway. Deletion of transaldolase encoding gene improved shinorine production by 5.2-fold. Among the other genes in pentose phosphate pathway, overexpression of 6-phosphogluconate dehydrogenase encoding gene further increased shinorine production by 60% (19.1 mg/L). The genetic engineering of the pentose phosphate pathway in C. glutamicum improved shinorine production by 8.3-fold in total, and could be applied to produce the other chemicals derived from sedoheptulose-7-phosphate. 相似文献
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Xu Meijuan Gao Hui Ma Zhenfeng Han Jin Zheng Keyi Shao Minglong Rao Zhiming 《Amino acids》2022,54(11):1437-1450
Amino Acids - 2-Pyrrolidone is widely used in the textile and pharmaceutical industries. Here, we established a 2-pyrrolidone biosynthesis pathway in Corynebacterium glutamicum, by expressing... 相似文献
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As an important biological methyl group donor, S-adenosyl-l-methionine is used as nutritional supplement or drug for various diseases, but bacterial strains that can efficiently produce S-adenosyl-l-methionine are not available. In this study, Corynebacterium glutamicum strain HW104 which can accumulate S-adenosyl-l-methionine was constructed from C. glutamicum ATCC13032 by deleting four genes thrB, metB, mcbR and Ncgl2640, and six genes metK, vgb, lysCm, homm, metX and metY were overexpressed in HW104 in different combinations, forming strains HW104/pJYW-4-metK-vgb, HW104/pJYW-4-SAM2C-vgb, HW104/pJYW-4-metK-vgb-metYX, and HW104/pJYW-4-metK-vgb-metYX-homm-lysCm. Fermentation experiments showed that HW104/pJYW-4-metK-vgb produced more S-adenosyl-l-methionine than other strains, and the yield achieved 196.7 mg/L (12.15 mg/g DCW) after 48 h. The results demonstrate the potential application of C. glutamicum for production of S-adenosyl-l-methionine without addition of l-methionine. 相似文献
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Sun Dehu Chen Jiuzhou Wang Yu Li Mingyue Rao Deming Guo Yanmei Chen Ning Zheng Ping Sun Jibin Ma Yanhe 《Journal of industrial microbiology & biotechnology》2019,46(2):203-208
Journal of Industrial Microbiology & Biotechnology - Corynebacterium glutamicum is an important platform strain that is wildly used in industrial production of amino acids and various other... 相似文献
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ABSTRACT: 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 epsilon-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 DeltacrtB, 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 DeltacrtI. 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 DeltacrtEb 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. 相似文献
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Metabolic engineering of Corynebacterium glutamicum for production of 1,5-diaminopentane from hemicellulose 总被引:1,自引:0,他引:1
In the present work, the bio-based production of 1,5-diaminopentane (cadaverine), an important building block for bio-polyamides, was extended to hemicellulose a non-food raw material. For this purpose, the metabolism of 1,5-diaminopentane-producing Corynebacterium glutamicum was engineered to the use of the C(5) sugar xylose. This was realized by heterologous expression of the xylA and xylB genes from Escherichia coli, mediating the conversion of xylose into xylulose 5-phosphate (an intermediate of the pentose phosphate pathway), in a defined diaminopentane-producing C. glutamicum strain, recently obtained by systems metabolic engineering. The created mutant, C. glutamicum DAP-Xyl1, exhibited efficient production of the diamine from xylose and from mixtures of xylose and glucose. Subsequently, the novel strain was tested on industrially relevant hemicellulose fractions, mainly containing xylose and glucose as carbon source. A two-step process was developed, comprising (i) enzymatic hydrolysis of hemicellulose from dried oat spelts, and (ii) biotechnological 1,5-diaminopentane production from the obtained hydrolysates with the novel C. glutamicum strain. This now opens a future avenue towards bio-based 1,5-diaminopentane and bio-polyamides thereof from non-food raw materials. 相似文献