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

Abstract

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.

Disruption ofmetFIncreased L-Lysine Production byMethylophilus methylotrophusfrom Methanol

“…8) We attempted to obtain the threonine auxotrophs for the purpose of avoiding feedback inhibition of aspartokinase by threonine. Petek et al have reported that decreasing biomass might allow increased precursor supply from central metabolism into the L-lysine biosynthetic pathway by minimizing the loss of carbon required for cell growth.…”

“…M. methylotrophus strains were grown on a modified minreral salts medium (SEIIa) at 37 C in test tubes and flasks. 8) Streptomycin (50 mg/l) was used to maintain plasmid pSEA10 in M. methylotrophus.…”

“…Each transformant was selected on SEIIa media containing 1 g/l of methionine and 20 mg/ ml of Streptomycine. 8) Enzyme assay. Homoserine kinase acitivity was assayed by monitoring ADP production by a pyruvate kinase-lactate dehydrogenase coupled assay.…”

“…[8][9][10] The introduction of the L-lysine exporter lysE24, a mutated lysE gene originating in Corynebacterium glutamicum 2256, greatly improved L-lysine production in M. methylotrophus. M. methylotrophus, carrying both lysE24 and dapA24, produced more L-lysine than the parent producer.…”

Phage Mu-driven two-plasmid system for integration of recombinant DNA in the Methylophilus methylotrophus genome

“…Cells of M. methylotrophus AS1 were grown at 37°C on a mineral medium SEIIa (Gunji et al 2004) O, 9.7 mg/l, methanol 2%, pH 7.0. Bactoagar (1.2%, Difco, USA) and methanol 1% were applied to the solid media.…”