Methylophilus Methylotrophus AS1 Research Articles

Disruption ofmetFIncreased L-Lysine Production byMethylophilus methylotrophusfrom Methanol

Methionine auxotrophic mutants of Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine, and mutated lysE (lysE24) encoding the L-lysine exporter from Corynebacterium glutamicum 2256, produced higher amounts of L-lysine from methanol as sole carbon source than did other amino acid auxotrophic mutants. Especially, the M. methylotrophus 102 strain, carrying both dapA24 and lysE24, produced L-lysine in more than 1.5 times amounts higher than the parent. A single-base substitution was identified in this auxotroph in codon-329 of the open reading frame of metF, encoding 5,10-methylene-tetra-hydrofolate reductase. We constructed a metF disruptant mutant carrying both dapA24 and lysE24, and confirmed increases in L-lysine production. This is the first report to the effect that metF deficient increased L-lysine production in methylotroph.

Enhancement of l-lysine production in methylotroph Methylophilus methylotrophus by introducing a mutant LysE exporter

The obligate methylotroph Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine could secrete L-lysine into the medium, but also maintained a high concentration of intracellular L-lysine. To improve the yield from excretion, we attempted to introduce an L-lysine/L-arginine exporter (LysE) from Corynebacterium glutamicum 2256 into M. methylotrophus. We were unable to stably transform M. methylotrophus with a plasmid expressing the wild type lysE gene, but happened to obtain a transformant carrying a spontaneously mutated lysE gene (designated lysE24) which could induce L-lysine production even in the wild type strain. The transformant also possessed increased tolerance to S-(2-aminoethyl)-L-cysteine (an L-lysine analog). lysE24 has a single-base insertion mutation in the middle of the lysE gene, and its product is presumably quite different in structure from wild-type LysE. When lysE24 was introduced into an L-lysine producer of M. methylotrophus carrying dapA24, the level of intracellular L-lysine fell. During fermentation, M. methylotrophus carrying both lysE24 and dapA24 produced 10-fold more L-lysine (11.3 gl(-1) in jar-fermentation) than the parent producer carrying only dapA24 or lysE24. These results show the importance of the factor (lysE24) involved in the excretion of L-lysine on its overproduction in M. methylotrophus.

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

Previously, we showed that the enzymes aspartokinase (AK) and dihydrodipicolinate synthase (DDPS), which are involved in L-lysine biosynthesis in the Gram-negative obligate methylotroph Methylophilus methylotrophus AS1, were inhibited by allosteric effectors, including L-lysine. To elucidate further the regulation of L-lysine biosynthesis in M. methylotrophus, we cloned the genes encoding three other enzymes involved in this pathway, L-aspartate-beta-semialdehyde dehydrogenase, dihydrodipicolinate reductase (DDPR) and diaminopimelate decarboxylase, and examined their properties. DDPR was markedly inhibited by L-lysine. Based on this and our previous results, we constructed an L-lysine-producing strain of M. methylotrophus by introducing well-characterized genes encoding desensitized forms of AK and DDPS, as well as dapB (encoding DDPR) from Escherichia coli, using a broad host range plasmid. L-Lysine production was significantly increased by employing an S-(2-aminoethyl)-L-cysteine (L-lysine analog)-resistant mutant as the host. This derivative accumulated L-lysine at a concentration of 1 g l(-1) of medium using methanol as a carbon source.

Characterization of the L-lysine biosynthetic pathway in the obligate methylotroph Methylophilus methylotrophus.

The L-lysine biosynthetic pathway of the gram-negative 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)-L-cysteine (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.

Electroporation of Pink-Pigmented methylotrophic bacteria

Electroporation conditions were determined for electroporating the broad-host-range plasmid pHX200V-47-ml (22.4 kb) into four pink-pigmented methylotrophic strains (Methylophilus methylotrophus AS1,Methylobacterium extorquens AM1,Methylobacterium organophilum XX, andPseudomonas sp. M27). For these methylotrophs, a high electric-field strength (15 kV/cm) and high DNA concentration (18.75 ng/μI competent cells) were necessary to produce plasmid-containing cells. The addition of 30% polyethylene glycol (PEG) to the electrocompetent cells resulted in a five-to six-fold increase in electroporation efficiency in strainsM. methylotrophus AS1, M.extorquens AM1, andM. organophilum XX, and also allowed successful electroporation ofPseudomonas sp. M27 (no electroporants were obtained without PEG). The maximum electroporation efficiencies for M.methylotrophus AS1, M.extorquens AM1, M.organophilum XX, andPseudomonas sp. M27 were 1.31 X 103, 2.57 X 103, 5.25 x 103, and 1.2 x 104 electroporants/(μg DNA, respectively). This, along with our previous article (1), are the first reports of a plasmid being successfully electroporated into these strains.

Creating auxotrophic mutants in Methylophilus methylotrophus AS1 by combining electroporation and chemical mutagenesis.

Stable auxotrophic mutants of the methylotroph Methylophilus methylotrophus AS1 were obtained by a novel mutagenesis technique in which electroporation is used to transport the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) across the cell membrane. By combining chemical mutagenesis with electroporation and screening single colonies for auxotrophy in 36 different amino acids and growth factors, 3 auxotrophs per 156 colonies screened were obtained, whereas no auxotrophs were found with chemical mutagenesis alone. MNNG mutagen toxicity was also increased in the methylotroph with this novel mutagenesis technique (death rate 96% compared to 79%). This technique did not increase the mutation rate for strain Escherichia coli BK6 which responds well to simple exposure to the mutagen.

Identification of methanol-regulated promoter sequences from the facultative methylotrophic bacterium Methylobacterium organophilum XX

A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless xylE gene of Pseudomonas as the reporter gene. Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene moxF (methanol oxidation) in front of the xylE gene in pHX200V-47 resulted in high-level expression of the xylE gene product--catechol 2,3-dioxygenase--in Methylobacterium organophilum XX. The specific activity of the enzyme was four times higher in methanol-grown M. organophilum XX culture than in succinate-grown culture. Interestingly, the insertion of the same fragment in the opposite orientation in front of the xylE gene (pHX200V-74) also led to elevated catechol 2,3-dioxygenase activity. This promoter activity was also methanol regulated. A total of 21 methanol-regulated promoter clones were identified that originate from three gene clusters (groups V, VI and VII) on the M. organophilum XX chromosome involved in methanol oxidation. Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria: Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1 and Methylobacterium sp. DM4. The reporter gene (xylE) was functionally expressed in all three bacteria with the aid of a proper promoter. Transcriptional fusions of methanol-regulated promoters with the xylE gene were mobilized into Mox- mutants of M. organophilum XX and M. extorquens AM1 to study the roles of methanol oxidation genes, especially regulatory genes. It appeared that vector pHX200 is an efficient promoter probe with wide host-range and an excellent tool for studies of structure and function of promoters/regulators.

Comparative Complementation Mapping of Methylophilus spp. Using Cosmid Gene Libraries and Prime Plasmids

Cosmid gene libraries of the obligate methylotrophs Methylophilus viscogenes and Methylophilus methylotrophus AS1 were generated by ligating Sau3A partial digests of chromosomal DNA into the broad-host-range cosmid vector pLA2917. Genetic linkage groups were identified for each organism by complementation following mobilization of the recombinant cosmids to a wide range of Pseudomonas aeruginosa and Escherichia coli auxotrophs. The linkage data thus obtained confirmed and extended those previously established by prime plasmid complementation, and the two methylotrophs showed very similar arrangements for four of the gene clusters studied. However, comparison of DNA sequences which complemented the same auxotrophic markers failed to demonstrate restriction fragment identity or substantial DNA-DNA homology between the two methylotrophs.

Expression inEscherichia coliandPseudomonas aeruginosaofMethylophilus methylotrophusAS1 genes carried on prime plasmids

Prime plasmids carrying chromosomal fragments of Methylophilus methylotrophus AS1 were transferred to Pseudomonas aeruginosa and Escherichia coli auxotrophs. It was shown that with argF (ornithine transcarbamylase), pyrB (aspartate transcarbamylase) and trpB (tryptophan synthetase, β-subunit) mutants of E. coli and P. aeruginosa complementation was accompanied by the synthesis of the corresponding M. methylotrophus gene product. These results support the basis of complementation mapping by prime plasmids previously used for M. methylotrophus. The level of expression of the M. methylotrophus genes varied in the two hosts but the regulation of the M. methylotrophus genes under these conditions was less responsive to cultural conditions than their expression in M. methylotrophus.

Expression in Escherichia coli and Pseudomonas aeruginosa of Methylophilus methylotrophus AS1 genes carried on prime plasmids

Prime plasmids carrying chromosomal fragments of Methylophilus methylotrophus AS1 were transferred to Pseudomonas aeruginosa and Escherichia coli auxotrophs. It was shown that with argF (ornithine transcarbamylase), pyrB (aspartate transcarbamylase) and trpB (tryptophan synthetase, β-subunit) mutants of E. coli and P. aeruginosa complementation was accompanied by the synthesis of the corresponding M. methylotrophus gene product. These results support the basis of complementation mapping by prime plasmids previously used for M. methylotrophus. The level of expression of the M. methylotrophus genes varied in the two hosts but the regulation of the M. methylotrophus genes under these conditions was less responsive to cultural conditions than their expression in M. methylotrophus.

Regulation and routes of biosynthesis of serine and arginine in Methylophilus methylotrophus AS1

Whole cell extracts and partially purified enzyme preparations of the obligate methylotroph Methylophilus methylotrophus AS1 have been studied for the presence of key enzymes of serine and arginine synthesis. Arginine biosynthesis proceeds via the N-acetylornithine glutamate transacetylase pathway, as found in the fluorescent pseudomonads. Serine synthesis proceeds by the phosphorylated pathway, as in Escherichia coli. There is no evidence for the repression of enzyme synthesis in either of these pathways, however, strict feedback inhibition occurs for the first step in each pathway.

Regulation and routes of biosynthesis of serine and arginine inMethylophilus methylotrophusAS1

Whole cell extracts and partially purified enzyme preparations of the obligate methylotroph Methylophilus methylotrophus AS1 have been studied for the presence of key enzymes of serine and arginine synthesis. Arginine biosynthesis proceeds via the N-acetylornithine glutamate transacetylase pathway, as found in the fluorescent pseudomonads. Serine synthesis proceeds by the phosphorylated pathway, as in Escherichia coli. There is no evidence for the repression of enzyme synthesis in either of these pathways, however, strict feedback inhibition occurs for the first step in each pathway.

Genetic mapping in Methylophilus methylotrophus AS1.

Prime plasmids have been isolated from Methylophilus methylotrophus AS1 using the IncP-1 plasmid pMO172. Such plasmids can complement mutant function when transferred to appropriate strains of Pseudomonas aeruginosa PAO. From the range of particular functions complemented by each prime plasmid, a preliminary map of the M. methylotrophus AS1 genome with four groups of linked markers was obtained. Physical examination of two of these prime plasmids showed that each had acquired an additional DNA segment. Southern hybridization experiments showed there was sequence homology between the additional DNA segments and M. methylotrophus AS1 DNA, confirming that these prime plasmids carried a segment of the M. methylotrophus AS1 genome. Mapping by complementation may be applicable to other bacteria in which mutants suitable for selecting recombinants are not readily available.