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Role of the Bacillus methanolicus citrate synthase II gene, citY, in regulating the secretion of glutamate in L-lysine-secreting mutants.

ABSTRACT: The thermotolerant, restrictive methylotroph Bacillus methanolicus MGA3 (ATCC 53907) can secrete 55 g of glutamate per liter (maximum yield, 0.36 g/g) at 50 degrees C with methanol as a carbon source and a source of ammonia in fed-batch bioreactors. A homoserine dehydrogenase mutant, 13A52-8A66, secreting up to 35 g of L-lysine per liter in fed-batch fermentations had minimal 2-oxoglutarate dehydrogenase activity [7.3 nmol min(-1) (mg of protein)(-1)], threefold-increased pyruvate carboxylase activity [535 nmol min(-1) (mg of protein)(-1)], and elevated citrate synthase (CS) activity [292 nmol min(-1) (mg of protein)(-1)] and simultaneously secreted glutamate (20 to 30 g per liter) and L-lysine. The flow of carbon from oxaloacetate is split between transamination to aspartate and formation of citrate. To investigate the regulation of this branch point, the B. methanolicus gene citY encoding a CSII protein with activity at 50 degrees C was cloned from 13A52-8A66 into a CS-deficient Escherichia coli K2-1-4 strain. A citY-deficient B. methanolicus mutant, NCS-L-7, was also isolated from the parent strain of 13A52-8A66 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, followed by selection with monofluoroacetate disks on glutamate plates. Characterization of these strains confirmed that citY in strain 13A52-8A66 was not altered and that B. methanolicus possessed several forms of CS. Analysis of citY cloned from NCS-L-7 showed that the reduced CS activity resulted from a frameshift mutation. The level of glutamate secreted by NCS-L-7 was reduced sevenfold and the ratio of L-lysine to glutamate secreted was increased 4.5-fold compared to the wild type in fed-batch cultures with glutamate feeding. This indicates that glutamate secretion in L-lysine-overproducing mutants can be altered in favor of increased L-lysine secretion by regulating in vivo CS activity.

SUBMITTER: Brautaset T

PROVIDER: S-EPMC165195 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Role of the Bacillus methanolicus citrate synthase II gene, citY, in regulating the secretion of glutamate in L-lysine-secreting mutants.

Brautaset Trygve T Williams Mark D MD Dillingham Richard D RD Kaufmann Christine C Bennaars Assumpta A Crabbe Edward E Flickinger Michael C MC

Applied and environmental microbiology 20030701 7

The thermotolerant, restrictive methylotroph Bacillus methanolicus MGA3 (ATCC 53907) can secrete 55 g of glutamate per liter (maximum yield, 0.36 g/g) at 50 degrees C with methanol as a carbon source and a source of ammonia in fed-batch bioreactors. A homoserine dehydrogenase mutant, 13A52-8A66, secreting up to 35 g of L-lysine per liter in fed-batch fermentations had minimal 2-oxoglutarate dehydrogenase activity [7.3 nmol min(-1) (mg of protein)(-1)], threefold-increased pyruvate carboxylase ac ...[more]

PMID: 12839772

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Project description:Bacillus subtilis forms robust biofilms in the presence of large amounts of carbon sources, such as glycerol. However, little is known about the importance of the metabolic systems, or the relationship between metabolic systems and regulatory systems, involved in biofilm formation. Glutamate synthase, encoded by gltAB, is an enzyme that converts 2-ketoglutarate (a tricarboxylic acid [TCA] cycle intermediate) and glutamine into glutamate, which is a general amino group donor in metabolism. Here, we show that a ?gltA mutant exhibited early arrest of biofilm formation in complex medium containing glycerol. This phenotype was not due to glutamate auxotrophy. Consistent with its biofilm formation phenotype, the ?gltA mutant exhibited an early decrease in expression of the epsA and tapA operons, which are responsible for production of biofilm matrix polymers. This resulted from decreased activity of their regulator, Spo0A, as evidenced by reduced expression of other Spo0A-regulated genes in the ?gltA mutant. The ?gltA mutation prevented biofilm formation only in the presence of large amounts of glycerol. Moreover, limited expression of citrate synthase (but not other TCA enzymes) restored biofilm-forming ability to the ?gltA mutant. These results indicate that the ?gltA mutant accumulates an inhibitory intermediate (citrate) in the TCA cycle in the presence of large amounts of glycerol. The ?gltA mutant formed biofilms when excess iron was added to the medium. Taken together, the data suggest that accumulation of citrate ions by the ?gltA mutant causes iron shortage due to chelation, which prevents activation of Spo0A and causes defective biofilm formation.IMPORTANCE Bacillus subtilis, a model organism for bacterial biofilm formation, forms robust biofilms in a medium-dependent manner. Although the regulatory network that controls biofilm formation has been well studied, the importance of the underlying metabolic systems remains to be elucidated. The present study demonstrates that a metabolic disorder in a well-conserved metabolic system causes accumulation of an inhibitory metabolic intermediate that prevents activation of the system that regulates biofilm formation. These findings increase our understanding of the coordination between cellular metabolic status and the regulatory networks governing biofilm formation.

Project description:The thermotolerant methylotroph Bacillus methanolicus MGA3 was originally isolated from freshwater marsh soil. Due to its ability to use methanol as sole carbon and energy source, B. methanolicus is increasingly explored as a cell factory for the production of amino acids, fine chemicals, and proteins of biotechnological interest. During high cell density fermentation in industrial settings with the membrane-permeable methanol as the feed, the excretion of low molecular weight products synthesized from it will increase the osmotic pressure of the medium. This in turn will impair cell growth and productivity of the overall biotechnological production process. With this in mind, we have analyzed the core of the physiological adjustment process of B. methanolicus MGA3 to sustained high osmolarity surroundings. Through growth assays, we found that B. methanolicus MGA3 possesses only a restricted ability to cope with sustained osmotic stress. This finding is consistent with the ecophysiological conditions in the habitat from which it was originally isolated. None of the externally provided compatible solutes and proline-containing peptides affording osmostress protection for Bacillus subtilis were able to stimulate growth of B. methanolicus MGA3 at high salinity. B. methanolicus MGA3 synthesized the moderately effective compatible solute L-glutamate in a pattern such that the cellular pool increased concomitantly with increases in the external osmolarity. Counterintuitively, a large portion of the newly synthesized L-glutamate was excreted. The expression of the genes (gltAB and gltA2) for two L-glutamate synthases were upregulated in response to high salinity along with that of the gltC regulatory gene. Such a regulatory pattern of the system(s) for L-glutamate synthesis in Bacilli is new. Our findings might thus be generally relevant to understand the production of the osmostress protectant L-glutamate by those Bacilli that exclusively rely on this compatible solute for their physiological adjustment to high osmolarity surroundings.

Dataset Information

Role of the Bacillus methanolicus citrate synthase II gene, citY, in regulating the secretion of glutamate in L-lysine-secreting mutants.

Publications

Role of the Bacillus methanolicus citrate synthase II gene, citY, in regulating the secretion of glutamate in L-lysine-secreting mutants.

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