Characterization of a Unique MutantlysEGene, Originating fromCorynebacterium glutamicum, Encoding a Product That Induces<scp>L</scp>-Lysine Production inMethylophilus methylotrophus

Gunji, Yoshiya; Ito, Hisao; Hara, Masaki; Yasueda, Hisashi

doi:10.1271/bbb.60339

Cited by 6 publications

(1 citation statement)

References 14 publications

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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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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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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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Amino Acid Production:L‐Lysine

Nagai

Ito

Yasueda

2009

Encyclopedia of Industrial Biotechnology

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L ‐Lysine is an essential amino acid for humans and animals and is exclusively used as a feed additive for swine and poultry throughout the world, since most grains used for the feed are deficient in L ‐lysine in nutritional amino acid balance. This amino acid has been extensively produced by fermentation using gram‐positive coryneform bacteria including Corynebacterium glutamicum . To date, many studies on strain improvement have been carried out using various methods including conventional mutagenesis and screening, genetic engineering, and metabolic engineering. Recently, the complete genome sequence of C. glutamicum has been determined, and the genetic information is very valuable for strain development for higher performance, in coordination with various “omics” analyses. Besides C. glutamicum , other bacteria including Escherichia coli have also been considered as promising producers of amino acids with high interest. Together with strain development, the improvements of fermentation and purification technologies also contribute to the success of the industrial production of L ‐lysine. It is estimated that the current annual production of L ‐lysine is more than 850,000 metric tons throughout the world.

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Microbial Metabolite Export

Eggeling¹

2010

Encyclopedia of Industrial Biotechnology

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Microbial metabolite export is an essential issue in industrial biotechnology. The reason is simple: otherwise, the development of advanced engineered strains might fail. Export belongs to the entire reaction sequence of substrate uptake and conversion to the valuable product finally accumulating in the medium. The relevance of export is probably best studied in the case of amino acid production with Escherichia coli or Corynebacterium glutamicum where specific exporters, when deleted, prevent amino acid production, or when overexpressed, increase amino acid production. However, antibiotic production also relies on exporters as the final step in cellular synthesis of these important metabolites. Another area where the importance of exporters is assessed is the use of whole cells for bioconversions occurring in organic solvents. This review covers these areas, gives a tabular overview on exporters recognized in biotechnology and describes also the varied methodology of exporter identification.

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Characterization of a Unique MutantlysEGene, Originating fromCorynebacterium glutamicum, Encoding a Product That InducesL-Lysine Production inMethylophilus methylotrophus

Cited by 6 publications

References 14 publications

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

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

Amino Acid Production:L‐Lysine

Microbial Metabolite Export

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