Disruption of<i>metF</i>Increased L-Lysine Production by<i>Methylophilus methylotrophus</i>from Methanol

Ishikawa, Kohei; Asahara, Takayuki; Gunji, Yoshiya; Yasueda, Hisashi; Asano, Kozo

doi:10.1271/bbb.70818

Cited by 14 publications

(11 citation statements)

References 22 publications

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“…In anther example, a Methylophilus methylotrophus carrying a mutant lysE (lysE24) and a mutant dapA (dapA24) was able to produce 11.3 g/L L-lysine from methanol as a carbon source, which is tenfold higher than that produced by the parent producer carrying only dapA24 or lysE24 [85]. This mutant strain was shown to be carrying a beneficial mutation in the metF gene encoding 5,10-methylenetetra-hydrofolate reductase [86]. The metF-deleted M. methylotrophus overexpressing both dapA24 and lysE24 was able to produce 1.2 g/L L-lysine, which is 50% higher than that produced by the wild-type M. methylotrophus overexpressing the same mutant genes, suggesting that metF deletion is beneficial for enhanced production of L-lysine in M. methylotrophus [86].…”

Section: L-lysine Production Strainsmentioning

confidence: 99%

Metabolic pathways and fermentative production of L‐aspartate family amino acids

Park

Lee

2010

Biotechnology Journal

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The L-aspartate family amino acids (AFAAs), L-threonine, L-lysine, L-methionine and L-isoleucine have recently been of much interest due to their wide spectrum of applications including food additives, components of cosmetics and therapeutic agents, and animal feed additives. Among them, L-threonine, L-lysine and L-methionine are three major amino acids produced currently throughout the world. Recent advances in systems metabolic engineering, which combine various high-throughput omics technologies and computational analysis, are now facilitating development of microbial strains efficiently producing AFAAs. Thus, a thorough understanding of the metabolic and regulatory mechanisms of the biosynthesis of these amino acids is urgently needed for designing system-wide metabolic engineering strategies. Here we review the details of AFAA biosynthetic pathways, regulations involved, and export and transport systems, and provide general strategies for successful metabolic engineering along with relevant examples. Finally, perspectives of systems metabolic engineering for developing AFAA overproducers are suggested with selected exemplary studies.

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Section: L-lysine Production Strainsmentioning

confidence: 99%

Metabolic pathways and fermentative production of L‐aspartate family amino acids

Park

Lee

2010

Biotechnology Journal

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“…Also, expression of a mutant L-lysine exporter gene, lysE24, resulted in increased L-lysine production by this bacterium (17). Disruption of the metF gene (encoding 5,10-methylene-tetrahydrofolate reductase), involved in methionine biosynthesis, had a slightly positive effect on lysine synthesis (22). It should be noted that the production levels obtained with recombinant M. methylotrophus strains were generally low (up to 1 g/liter in fermentors).…”

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confidence: 93%

“…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).…”

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confidence: 99%

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

Nærdal

Netzer

Ellingsen

et al. 2011

Appl Environ Microbiol

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We investigated the regulation and roles of six aspartate pathway genes in L-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by L-lysine and induced 2-fold by DL-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by DL-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the L-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has-in addition to a hom-1 mutation-chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by mesodiaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for L-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased L-lysine production levels.The essential amino acid L-lysine is widely used as a feed additive in animal farming, and currently Ͼ1,000,000 tons of L-lysine-HCl per year are produced worldwide by fermentation processes, using classical mutant strains of Corynebacterium glutamicum and its relatives (1,7,34). L-Lysine is a product of the aspartate pathway (Fig. 1), and several of the genes and enzymes involved are feedback regulated. The regulatory mechanisms are allosteric inhibition of the enzymes and transcriptional repression of the genes, including RNA riboswitch mechanisms at the mRNA level (15, 32). The aspartate pathway has been investigated particularly well in C. glutamicum, with the overall aim of identifying enzymes representing ratelimiting steps for L-lysine overproduction. 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 metabol...

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“…As for the L-lysine (Lys) synthesis, systematic research was carried out by specialists at Ajinomoto Co., Inc., Japan, beginning with the investigation of the Lys biosynthetic pathway in M. methylotrophus AS1 (23,61) and continuing with the con-struction and improvement of a Lys producer (22,24,33,34). This was followed by optimization of fed-batch fermentation for overproduction of Lys from methanol (35).…”

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confidence: 99%

Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP -Encoded Transporter of Escherichia coli

Yomantas

Tokmakova

Gorshkova

et al. 2010

Appl Environ Microbiol

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The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium Methylophilus methylotrophus AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the Escherichia coli gene aroP, which encodes the common aromatic amino acid transporter, into the genome of M. methylotrophus. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp. M. methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro using a Km r marker, and electroporated into an aroP carrier recipient strain. This approach led to the construction of a set of marker-less M. methylotrophus AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.The nonpathogenic Gram-negative bacterium Methylophilus methylotrophus is able to grow efficiently using C 1 substrates (methanol, methylamine, or trimethylamine) as the sole source of carbon and energy, and it uses the ribulose monophosphate pathway for fixation of formaldehyde produced by the oxidation of methanol (36). Methanol has received considerable attention by the fermentation industry as an alternative substrate to the more generally used sugars from agricultural crops. It can be synthesized either from petrochemicals or renewable resources, such as biogas (48), and therefore the production of methanol does not compete directly with human food supplies. Methylotrophs can therefore be considered potentially useful strains for industrial biotechnology. M. methylotrophus AS1 is an obligate methylotroph originally isolated from activated sludge, and it has been deposited in the National Collections of Industrial, Marine and Food Bacteria (NCIMB; no. 10515). This organism was extensively studied in the 1970s and has been industrialized on a large scale for the manufacturing of single-cell proteins (SCP) from methanol (56, 63). During that period, a significant amount of research was conducted on the direct production of amino acids by fermentation from methanol (3, 58). Although initially promising, these efforts ultimately proved relatively unsatisfactory and impractical, due primarily to the rather poor set of genetic tools that had been developed for methylotrophs.Over the last 5 years, several genomes of methylotrophs have been sequenced (8,20,29,37,65,67), and significant progress in elucidating their metabolism has been achieved (14). The number of tools available for the genetic and met...

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Disruption ofmetFIncreased L-Lysine Production byMethylophilus methylotrophusfrom Methanol

Cited by 14 publications

References 22 publications

Metabolic pathways and fermentative production of L‐aspartate family amino acids

Metabolic pathways and fermentative production of L‐aspartate family amino acids

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

Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP -Encoded Transporter of Escherichia coli

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