Amino Acid Production from Methanol by<i>Methylobacillus glycogenes</i>Mutants: Isolation of<scp>L</scp>-Glutamic Acid Hyper-producing Mutants from<i>M. glycogenes</i>Strains, and Derivation of<scp>L</scp>-Threonine and<scp>L</scp>-Lysine-producing Mutants from Them

Motoyama, Hiroaki; Anazawa, Hideharu; Katsumata, Ryoichi; Araki, Kazumi; Teshiba, Sadao

doi:10.1271/bbb.57.82

Cited by 30 publications

(23 citation statements)

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“…M. glycogenes ATCC 21276 and the derived mutants were cultivated as described by us previously (13). Methanol was used as a sole carbon source.…”

Section: Methodsmentioning

confidence: 99%

“…Bacterial growth was measured by the increase in absorbance at 660 nm, and amino acids in culture supernatants were measured by high-pressure liquid chromatography as described previously (13). Cell extracts of the M. glycogenes transconjugants were prepared and DDPS activities were measured as described previously (14).…”

Section: Methodsmentioning

confidence: 99%

“…We isolated L-glutamic acid-hyperproducing mutants from the obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and 21371 and subsequently derived L-lysine-and Lthreonine-producing mutants from L-glutamic acid-producing mutants (13). An L-threonine-producing mutant, AL119, was isolated among mutants resistant to both DL-␣-amino-␤-hydroxy-valeric acid and L-lysine, and an L-lysine-and L-threonine-coproducing mutant, DHL119, was isolated among 2,6-diamino-4-hexenoic acid hydrochloride-resistant mutants derived from AL119.…”

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Overproduction of l -Lysine from Methanol by Methylobacillus glycogenes Derivatives Carrying a Plasmid with a Mutated dapA Gene

Motoyama

Yano

Terasaki

et al. 2001

Appl Environ Microbiol

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The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by Llysine, was cloned from an L-threonine-and L-lysine-coproducing mutant of the obligate methylotroph Methylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapA mutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an L-threonine producing mutant, elevated the specific activity of DDPS 20-fold and L-lysine production 2-to 3-fold with concomitant reduction of L-threonine in test tube cultures. AL119 containing the dapA gene produced 8 g of L-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M r of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition by L-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector, L-lysine.Methanol, a compound easily synthesized from natural gas, is an attractive raw material for microbial industries. Using methanol as a carbon source, production costs could be greatly reduced and purification and waste treatment processes could be simplified. A number of production processes for useful compounds with methylotrophs (methanol-utilizing microorganisms) have been studied. Production of single-cell protein by a gram-negative methylotroph, Methylophilus methylotrophus, was extensively studied in the 1970s and finally industrialized (1). Efficient production systems for recombinant proteins were also constructed with the methanol-utilizing yeast Pichia pastoris (4).Many attempts have also been made to use methanol in amino acid production; however, successful studies were limited. Lee et al. (9) reported the production of 47 g of L-lysine per liter by a gram-positive methylotroph, Bacillus methanolicus. Izumi et al. (7) reported efficient conversion of glycine to L-serine by a gram-negative methylotroph, Hyphomicrobium methylovorum. Breeding of amino acid-producing mutants requires isolation of mutants desensitized in the feedback regulation of the biosynthetic enzymes for a desired amino acid and blocked in metabolic pathways of by-products. Isolation of mutants from methylotrophs is usually difficult, due to unknown reasons, preventing the use of methanol in amino acid production.We isolated L-glutamic acid-hyperproducing mutants from the obligate methylotro...

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“…M. glycogenes ATCC 21276 and the derived mutants were cultivated as described by us previously (13). Methanol was used as a sole carbon source.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Overproduction of l -Lysine from Methanol by Methylobacillus glycogenes Derivatives Carrying a Plasmid with a Mutated dapA Gene

Motoyama

Yano

Terasaki

et al. 2001

Appl Environ Microbiol

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“…10) Motoyama et al succeeded in isolating L-glutamate producing mutants of the gram-negative Methylobacillus glycogenes, 11) which is phylogenetically close to M. methylotrophus. 12) These researchers also derived L-threonine and L-lysine producing variants.…”

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Characterization of theL-Lysine Biosynthetic Pathway in the Obligate MethylotrophMethylophilus methylotrophus

Gunji

Tsujimoto

Shimaoka

et al. 2004

Bioscience, Biotechnology, and Biochemistry

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The L-lysine biosynthetic pathway of the gramnegative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase. The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine. In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-Lcysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition. The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative. Comparison of the sequences revealed a point mutation in each of these genes in G49. The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.

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“…The L-glutamic acid producers were mutagenized, and L-threonine-producing and L-Iysine-producing mutants were isolated among the L-threonine analog-or L-Iysine analog-resistant strains. 1) In the L-threonine-producing and L-Iysine-producing mutants from Corynebacterium glutamicum, 2,3) or in the L-threonine-producing mutants from Escherichia coli,4) the regulation of key enzymes in L-threonine and L-Iysine biosynthetic pathway were known to be altered, leading to the overproduction ofL-threonine and L-Iysine, respectively ... We assumed that the L-threonine or L-Iysine biosynthetic enzymes of L-threonine-producing and L-Iysine-producing mutants of M. glycogenes might be altered in the feedback regulation by L-threonine or L-Iysine.…”

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confidence: 99%

Characterization of the Aspartate Family Amino Acids Biosynthetic Enzymes inL-Threonine- andL-Lysine-producing Mutants ofMethylobacillus glycogens

Motoyama¹,

Anazawa²,

Teshiba³

1993

Bioscience, Biotechnology, and Biochemistry

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Amino Acid Production from Methanol byMethylobacillus glycogenesMutants: Isolation ofL-Glutamic Acid Hyper-producing Mutants fromM. glycogenesStrains, and Derivation ofL-Threonine andL-Lysine-producing Mutants from Them

Cited by 30 publications

References 1 publication

Overproduction of l -Lysine from Methanol by Methylobacillus glycogenes Derivatives Carrying a Plasmid with a Mutated dapA Gene

Overproduction of l -Lysine from Methanol by Methylobacillus glycogenes Derivatives Carrying a Plasmid with a Mutated dapA Gene

Characterization of theL-Lysine Biosynthetic Pathway in the Obligate MethylotrophMethylophilus methylotrophus

Characterization of the Aspartate Family Amino Acids Biosynthetic Enzymes inL-Threonine- andL-Lysine-producing Mutants ofMethylobacillus glycogens

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