Role of the
            <i>Bacillus methanolicus</i>
            Citrate Synthase II Gene,
            <i>citY</i>
            , in Regulating the Secretion of Glutamate in
            <scp>l</scp>
            -Lysine-Secreting Mutants

Brautaset, Trygve; Williams, Mark D.; Dillingham, Richard D.; Kaufmann, Christine; Bennaars, Assumpta; Crabbe, Edward; Flickinger, Michael C.

doi:10.1128/aem.69.7.3986-3995.2003

Cited by 58 publications

(75 citation statements)

References 52 publications

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“…B. methanolicus cells growing exponentially (OD 600 ϭ 1.0) in defined media were harvested and washed twice in ice-cold potassium phosphate buffer (50 mM; pH 7.6). The cells were disrupted by sonication as previously described (7). Coupled HPS-PHI activities were assayed by monitoring the fixation of formaldehyde with ribulose-5-phosphate (28).…”

Section: Methodsmentioning

confidence: 99%

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

et al. 2006

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The natural plasmid pBM19 carries the key mdh gene needed for the oxidation of methanol into formaldehyde by Bacillus methanolicus. Five more genes, glpX, fba, tkt, pfk, and rpe, with deduced roles in the cell primary metabolism, are also located on this plasmid. By using real-time PCR, we show that they are transcriptionally upregulated (6-to 40-fold) in cells utilizing methanol; a similar induction was shown for two chromosomal genes, hps and phi. These seven genes are involved in the fructose bisphosphate aldolase/sedoheptulose bisphosphatase variant of the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation. Curing of pBM19 causes higher methanol tolerance and reduced formaldehyde tolerance, and the methanol tolerance is reversed to wild-type levels by reintroducing mdh. Thus, the RuMP pathway is needed to detoxify the formaldehyde produced by the methanol dehydrogenase-mediated conversion of methanol, and the in vivo transcription levels of mdh and the RuMP pathway genes reflect the methanol tolerance level of the cells. The transcriptional inducer of hps and phi genes is formaldehyde, and not methanol, and introduction of multiple copies of these two genes into B. methanolicus made the cells more tolerant of growth on high methanol concentrations. The recombinant strain also had a significantly higher specific growth rate on methanol than the wild type. While pBM19 is critical for growth on methanol and important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for B. methanolicus when growing on mannitol. Our data contribute to a new and fundamental understanding of the regulation of B. methanolicus methylotrophy.Aerobic methylotrophs are bacteria capable of utilizing reduced one-carbon (C 1 ) compounds as the sole carbon source for growth and energy (2), and the majority of research on these bacteria has focused on their biochemical novelty and commercial viability. A number of gram-positive and thermotolerant Bacillus strains have been isolated and designated Bacillus methanolicus (4,26). This methylotrophic bacterium has a novel NAD-dependent methanol dehydrogenase (MDH), which contains bound NAD, to oxidize methanol into formaldehyde (11). The enzyme has a remarkably high affinity for methanol, and an activator protein called ACT modulates its in vivo activity. ACT activates MDH by hydrolysis, and the expression of these two proteins is reported to be under coordinate (and methanol-induced) control in B. methanolicus (16). Formaldehyde is the key intermediate in C 1 metabolism and can be assimilated via the ribulose monophosphate (RuMP) pathway (Fig. 1). Many RuMP pathway variants are described in the literature, and thermotolerant Bacillus strains are reported to use the fructosebisphosphate aldolase/transaldolase variant (3,4,12).Steady-state cultures of B. methanolicus MGA3 limited by methanol in the feed display sensitivity to minor methanol pulses and respond by a transient decline in biomass concentration. By using 13 C nuclear magnetic resonan...

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Section: Methodsmentioning

confidence: 99%

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

et al. 2006

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“…Late-exponential-phase cell cultures were harvested by centrifugation (Sorvall GSA rotor; 4.500 rpm, 10 min, 4°C) and washed twice in 50 mM potassium phosphate buffer (pH 7.5) containing 5 mM MgSO 4 6-bisphosphatase (and sedoheptulose-1,7-bisphosphatase) gene; rpi, ribose-5-phosphate isomerase gene; rpe, RPE gene; H-6-P, hexulose 6-phosphate; F-1,6-dP, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; X-5-P, xylulose 5-phosphate; E-4-P, erythrose 4-phosphate; S-1,7-dP, sedoheptulose 1,7-bisphosphate; S-7-P, sedoheptulose 7-phosphate, Ri-5-P, ribose 5-phosphate. Sonifer 250) as described previously (11). B. methanolicus cells were incubated at 37°C for 60 min in the presence of lysozyme (5 mg/ml) and 25 U of mutanolysin (10 U/ml; Sigma) before sonication.…”

Section: Methodsmentioning

confidence: 99%

“…For preparation of crude extracts B. methanolicus strains were grown in SOB containing 0.25 M sucrose, and methanol was added at a concentration of 150 mM 1 h prior to harvest to induce MDH expression. For all other purposes B. methanolicus cells were grown at 50°C in methanol minimal vitamin medium (MVcMY medium) containing 1 mM MgSO 4 , high-salt buffer, vitamins, 0.025% yeast extract (Difco), 200 mM methanol, and trace metals essentially as described previously (11). Mannitol medium was MVcMY medium without methanol supplemented with D-mannitol (10 g/liter; Sigma) as the sole carbon source.…”

Section: Methodsmentioning

confidence: 99%

“…B. methanolicus uses an NAD(P)-dependent methanol dehydrogenase (MDH) to oxidize methanol to formaldehyde, and in addition to entering the RuMP pathway, a linear branch for dissimilation of formaldehyde to CO 2 was recently demonstrated (29). It was previously shown that this bacterium can secrete 55 g of glutamate per liter at 50°C by using methanol as a carbon source in fed-batch fermentation (11), and a homoserine dehydrogenase mutant (13A52-8A66) that secreted up to 35 g of L-lysine per liter at 50°C was described (18). In a previous study (13) it was found that the B. methanolicus NOA2 mutant 13A52 possesses a plasmid with an estimated size of 17 kb, and a plasmid of a similar size was later identified (N. Tsujimoto, H. Yasueda, and S. Sugimoto, 24 October 2000, Japanese patent application JP2000295988) in B. methanolicus PB1 (ϭ NCIMB 13113).…”

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Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

et al. 2004

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Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50°C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

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“…We later showed that this organism has two different HD-encoding genes, hom-1 and hom-2, and that the classical hom-1 mutant B. methanolicus strain M168-20 had 60-fold increased L-lysine production (6). Interestingly, intracellular metabolite analysis and manipulations with pyruvate carboxylase and citrate synthase indicated that the oxaloacetate precursor supply is not a major bottleneck for L-lysine overproduction in B. methanolicus (4,6).…”

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Analysis and Manipulation of Aspartate Pathway Genes for l -Lysine Overproduction from Methanol by Bacillus methanolicus

Nærdal

Netzer

Ellingsen

et al. 2011

Appl Environ Microbiol

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We investigated the regulation and roles of six aspartate pathway genes in L-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by L-lysine and induced 2-fold by DL-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by DL-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the L-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has-in addition to a hom-1 mutation-chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by mesodiaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for L-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased L-lysine production levels.The essential amino acid L-lysine is widely used as a feed additive in animal farming, and currently Ͼ1,000,000 tons of L-lysine-HCl per year are produced worldwide by fermentation processes, using classical mutant strains of Corynebacterium glutamicum and its relatives (1,7,34). L-Lysine is a product of the aspartate pathway (Fig. 1), and several of the genes and enzymes involved are feedback regulated. The regulatory mechanisms are allosteric inhibition of the enzymes and transcriptional repression of the genes, including RNA riboswitch mechanisms at the mRNA level (15, 32). The aspartate pathway has been investigated particularly well in C. glutamicum, with the overall aim of identifying enzymes representing ratelimiting steps for L-lysine overproduction. In particular, the importance of aspartokinase (AK) in controlling L-lysine biosynthesis has been well documented for this bacterium (13) as well as for several other bacteria, including Escherichia coli (25, 30), Lactobacillus plantarum (11, 12), Bacillus subtilis (41), and Methylophilus methylotrophus (38). It has been unraveled that homoserine dehydrogenase (HD), dihydrodipicolinate synthase (DapA), dihydrodipicolinate reductase (DapB), mesodiaminopimelate decarboxylase (LysA), and L-lysine exporter (LysE) can also represent targets for achieving L-lysine overproduction (13,17,21,28,29,38). Moreover, several enzymes of cell primary metabol...

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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

Cited by 58 publications

References 52 publications

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

Analysis and Manipulation of Aspartate Pathway Genes for l -Lysine Overproduction from Methanol by Bacillus methanolicus

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