Organization of threonine biosynthesis genes from the obligate methylotroph Methylobacillus flagellatus The GenBank accession numbers for the aspC-hom-thrC-thyA gene cluster and thrB gene sequences from M. flagellatus reported in this paper are L78665 and L78666, respectively.

Марченко, Г. Н.; Marchenko, Natalia D.; Tsygankov, Y. D.; Chistoserdov, Andrei Y.

doi:10.1099/00221287-145-11-3273

Cited by 11 publications

(6 citation statements)

References 41 publications

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“…Wild-type B. methanolicus MGA3 (ATCC 53907) and homoserine dehydrogenase mutants have been shown to secrete substantial quantities of -glutamic acid and -lysine when grown at 50 mC in fed-batch cultures on methanol as the sole source of carbon and energy (Hanson et al, 1996 ;Lee et al, 1996). In contrast to Gram-negative methylotrophs such as Methylobacillus flagellatus (Marchenko et al, 1999) that also have been reported to secrete amino acids and vitamins, the single cell membrane of B. methano-S. B. Pluschkell and M. C. Flickinger licus should be an advantage for rapid secretion of high levels of amino acids. To maximize the yield of amino acids, the fraction of methanol carbon dissimilated as CO # must be minimized.…”

Section: Introductionmentioning

confidence: 99%

Dissimilation of [13C]methanol by continuous cultures of Bacillus methanolicus MGA3 at 50 °C studied by 13C NMR and isotope-ratio mass spectrometry

Pluschkell

Flickinger

2002

Microbiology

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Using a continuous culture of Bacillus methanolicus MGA3 limited by 100 mM methanol in the feed and growing at a dilution rate Dl025 h N1 , transients in dissolved methanol were studied to determine the effects of methanol toxicity and the pathway of methanol dissimilation to CO 2 . Steady-state cultures were disturbed by pulses of methanol resulting in a rapid change in concentration of 64-128 mM. B. methanolicus MGA3 responded to a sudden increase in available methanol by a transient decline in the biomass concentration in the reactor. In most cases the culture returned to steady state between 4 and 12 h after pulse addition. However, at a methanol pulse of 128 mM, complete biomass washout occurred and the culture did not return to steady state. Integrating the response curves of the dry biomass concentration over a 12 h time period showed that a methanol pulse can cause an average transient decline in the biomass yield of up to 22 %. 13 C NMR experiments using labelled methanol indicated that the transient partial or complete biomass washout was probably caused by toxic accumulation of formaldehyde in the culture. These experiments also showed accumulation of formate, indicating that B. methanolicus possesses formaldehyde dehydrogenase and formate dehydrogenase activity resulting in a methanol dissimilation pathway via formate to CO 2 . Studies using isotope-ratio mass spectrometry provided further evidence of a methanol dissimilation pathway via formate. B. methanolicus MGA3, growing continuously under methanol limitation, consumed added formate at a rate of approximately 085 mmol l N1 h N1 . Furthermore, significant accumulation of 13 CO 2 in the reactor exhaust gas was measured in response to a pulse addition of [ 13 C]formic acid to the bioreactor. This indicates that B. methanolicus dissimilates methanol carbon to CO 2 in order to detoxify formaldehyde by both a linear pathway to formate and a cyclic mechanism as part of the RuMP pathway.

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Section: Introductionmentioning

confidence: 99%

Dissimilation of [13C]methanol by continuous cultures of Bacillus methanolicus MGA3 at 50 °C studied by 13C NMR and isotope-ratio mass spectrometry

Pluschkell

Flickinger

2002

Microbiology

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“…B. methanolicus HSD Ϫ strain 13A52-8A66 can secrete over 60 g of L-lysine plus glutamate (glutamate) per liter, with a mass yield of 0.50 to 0.63 g/g. However, no mutants that secrete only L-lysine have been found with this approach; all simultaneously secrete significant quantities of glutamate and smaller amounts of alanine.Limited knowledge exists for most aerobic methylotrophs of the genetic basis of the regulation of pyruvate metabolism, the activity of tricarboxylic acid (TCA) cycle enzymes during rapid growth on methanol (7,13,54), and particularly regulation of the flow of carbon to amino acid biosynthesis (20,(31)(32)(33)46). To accomplish high-level L-lysine production, the percentage of carbon dissimilated to CO 2 must be minimized to generate an abundant supply of aspartate (37).…”

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confidence: 99%

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

Brautaset

Williams

Dillingham

et al. 2003

Appl Environ Microbiol

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The unusual metabolic properties of aerobic, restrictive, thermotolerant methylotrophic bacteria make them useful for the production of recombinant proteins, vitamins, amino acids, coenzymes, and cytochromes (12). Bacillus methanolicus MGA3 (ATCC 53907) is a gram-positive bacterium with a growth optimum of 50°C to 53°C. Cell extracts of this bacterium grown on methanol contain an NAD-dependent methanol dehydrogenase (3, 13), have 3-hexulose-6-phosphate synthase, fructose bisphosphate aldolase, and transaldolase activity indicative of the fructose bisphosphate aldolase/ transaldolase variant of the ribulose monophosphate pathway, RuMP (4, 14, 44). The precursors (acetyl-coenzyme A, pyruvate, oxaloacetate, and 2-oxoglutarate) for lipids, amino acids, nucleic acids, and assimilation of ammonia are all synthesized from the products of the RuMP pathway (2). The dissimilation of carbon from formaldehyde to CO 2 in B. methanolicus, which may function to detoxify accumulating formaldehyde, has recently been confirmed by 13 C nuclear magnetic resonance and isotope ratio mass spectrometry (39). The theoretical yield of secreted amino acids varies depending on the percentage of formaldehyde carbon dissimilated to CO 2 (39), and therefore control of the feeding of methanol to fed-batch cultures is critical (29).Homoserine dehydrogenase mutants (HSD Ϫ ) of B. methanolicus, which are threonine plus methionine auxotrophs, have been shown to secrete substantial quantities of L-lysine when grown at 50°C in fed-batch cultures on methanol as a sole carbon source and supplied with a source of ammonia (18,29,44). B. methanolicus HSD Ϫ strain 13A52-8A66 can secrete over 60 g of L-lysine plus glutamate (glutamate) per liter, with a mass yield of 0.50 to 0.63 g/g. However, no mutants that secrete only L-lysine have been found with this approach; all simultaneously secrete significant quantities of glutamate and smaller amounts of alanine.Limited knowledge exists for most aerobic methylotrophs of the genetic basis of the regulation of pyruvate metabolism, the activity of tricarboxylic acid (TCA) cycle enzymes during rapid growth on methanol (7,13,54), and particularly regulation of the flow of carbon to amino acid biosynthesis (20,(31)(32)(33)46). To accomplish high-level L-lysine production, the percentage of carbon dissimilated to CO 2 must be minimized to generate an abundant supply of aspartate (37). In Bacillus subtilis, aspartate is synthesized from oxaloacetate by aspartate aminotransferase (glutamate:oxaloacetate transaminase) encoded by aspB, which appears to be constitutively expressed (5).The simultaneous production of glutamate and L-lysine in B. methanolicus mutants indicates a substantial flow of carbon both to aspartate and into the first three reactions of the TCA cycle catalyzed by citrate synthase (CS), aconitase, and isocitrate dehydrogenase (Fig. 1). Since many methylotrophs generate reducing power by the oxidation of formaldehyde to

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“…The gene encoding homoserine dehydrogenase (hom) has been cloned and sequenced from diverse bacteria including Corynebacterium glutamicum [23], Lactobacillus lactis [14], Bacillus subtilis [22], Bacillus sp. ULM1 [16], Pseudomonas aeruginosa [5], Methylobacillus glycogens [21], Methylobacillus Xagellatus [18], Esherichia coli and Salmonella typhimurium [6] and Streptomyces sp. NRRL 5331 [7].…”

Section: Introductionmentioning

confidence: 99%

Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus

Yilmaz

Çaydasi

Özcengiz

2007

J Ind Microbiol Biotechnol

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The aspartate pathway of Streptomyces clavuligerus is an important primary metabolic pathway which provides substrates for beta-lactam synthesis. In this study, the hom gene which encodes homoserine dehydrogenase was cloned from the cephamycin C producer S. clavuligerus NRRL 3585 and characterized. The fully sequenced open reading frame encodes 433 amino acids with a deduced M (r) of 44.9 kDa. The gene was heterologously expressed in the auxotroph mutant Escherichia coli CGSC 5075 and the recombinant protein was purified. The cloned gene was used to construct a plasmid containing a hom disruption cassette which was then transformed into S. clavuligerus. A hom mutant of S. clavuligerus was obtained by insertional inactivation via double crossover, and the effect of hom gene disruption on cephamycin C yield was investigated by comparing antibiotic levels in culture broths of this mutant and in the parental strain. Disruption of hom gene resulted in up to 4.3-fold and twofold increases in intracellular free L-lysine concentration and specific cephamycin C production, respectively, during stationary phase in chemically defined medium.

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Organization of threonine biosynthesis genes from the obligate methylotroph Methylobacillus flagellatus The GenBank accession numbers for the aspC-hom-thrC-thyA gene cluster and thrB gene sequences from M. flagellatus reported in this paper are L78665 and L78666, respectively.

Cited by 11 publications

References 41 publications

Dissimilation of [13C]methanol by continuous cultures of Bacillus methanolicus MGA3 at 50 °C studied by 13C NMR and isotope-ratio mass spectrometry

Dissimilation of [13C]methanol by continuous cultures of Bacillus methanolicus MGA3 at 50 °C studied by 13C NMR and isotope-ratio mass spectrometry

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

Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus

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