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

Abstract

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...

Characterization of mutations induced by N-methyl-N′-nitro-N-nitrosoguanidine in an industrial Corynebacterium glutamicum strain

“…Some of these mutants have been shown to be genetically deregulated with respect to relevant 2 biosynthetic pathways. However, recent more detailed analysis has revealed that mutations responsible for deregulation, such as the mutations in thrA [4], dapA [5], lysC [6], and gnd [7], resulted in only partial desensitization of the enzymes, despite continual efforts of strain improvement. This made us realize again that NTG mutagenesis is not necessarily the best to achieve high desensitization of regulatory enzymes.…”

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

“…Several reports on manipulation of the aspartate pathway for L-lysine overproduction exist for both M. glycogenes and M. methylotrophus. The M. glycogenes hom gene, encoding HD, and thrC gene, encoding threonine synthase, have been cloned and characterized (27), and overexpression of a mutant dapA gene in this bacterium resulted in L-lysine production at 8 g/liter in fermentors (28). Besides these examples, genetic and biochemical information on L-lysine biosynthesis in this bacterium is lacking.…”

“…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 metabolism have been found to be important for achieving the efficient precursor supply needed for L-lysine overproduction (22,40).…”

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

“…The mutation in G49 AK was in the region involved in the allosteric regulation of AK. 37) With respect to DDPS, the predicted amino acid sequence of the M. methylotrophus product had identities of 79% with that of M. glycogenes, 38) also an obligate methylotroph, and 55% with that of E. coli (292 aa), and relatively low identities of 45%, 39%, and 30% with those of B. subtilis (290 aa), C. glutamicum (301 aa), and P. aeruginosa (293 aa) respectively. A comparison of the amino acid sequences of the five dapA genes is shown in Fig.…”

“…This mutation occurred in a position outside the region (residues no. 79-88 of DDPS in E. coli) reported to cause desensitization of DDPSs from E. coli and plants, 38,[50][51][52] and the position was novel. Recently, the crystal structure of complex of E. coli DDPS with Llysine has been determined.…”