Characterization of the Aspartate Family Amino Acids Biosynthetic Enzymes in<scp>L</scp>-Threonine- and<scp>L</scp>-Lysine-producing Mutants of<i>Methylobacillus glycogens</i>

Motoyama, Hiroaki; Anazawa, Hideharu; Teshiba, Sadao

doi:10.1271/bbb.57.461

Cited by 5 publications

(15 citation statements)

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“…40) The activity of M. glycogenes AK was also reduced by L-lysine although inhibition was only moderate. 13) But the M. methylotrophus AK studied here was completely insensitive to L-lysine on its own and moderately sensitive to L-threonine. This mode of regulation appears to be novel, although the major form of inhibition by the combination of L-lysine and Lthreonine is the same as that of C. glutamicum.…”

Section: Discussionmentioning

confidence: 64%

“…Some of the enzymes and corresponding genes of L-threonine biosynthesis in M. glycogenes have also been characterized. 13,14) Among methylotrophic bacteria, M. methylotrophus possesses several advantages for industrial applications. 3,[15][16][17] These are: (1) high growth yield and growth rate, (2) applicability of recombinant DNA technology, (3) absence of spore formation and easy handling in culture and storage, and (4) moderately high optimal growth temperature.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 64%

mentioning

confidence: 99%

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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“…Nucleotide 733 is C in ATCC 21276 and AL119, whereas it is T in DHL122, which altered the amino acid residue L88 in ATCC 21276 and AL119 to F88 in DHL122. The mutation in this amino acid residue was considered to cause the partial desensitization of DDPS of DHL122 to feedback inhibition by L-lysine (14).…”

Section: Resultsmentioning

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%

“…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. Enzymatic analysis revealed that aspartokinase, the common regulatory enzyme in the biosynthesis of L-lysine and L-threonine, was insensitive to the feedback inhibition by L-lysine in AL119 and DHL122 and that dihydrodipicolinate synthase (DDPS), the key regulatory enzyme located at the branch point of L-lysine and L-threonine biosynthesis, was partially insensitive to the feedback inhibition by L-lysine in DHL122 (14). We concluded that desensitization of these regulatory enzymes led to the production of L-threonine and L-lysine.…”

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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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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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l-Lysine biosynthetic pathway of Methylophilus methylotrophus and construction of an l-lysine producer

Tsujimoto

Gunji

Ogawa-Miyata

et al. 2006

Journal of Biotechnology

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

Cited by 5 publications

References 5 publications

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

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

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

l-Lysine biosynthetic pathway of Methylophilus methylotrophus and construction of an l-lysine producer

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