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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

Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus... Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP -Encoded Transporter of Escherichia coli ▿ † ‡ Yurgis A. V. Yomantas , Irina L. Tokmakova , Natalya V. Gorshkova , Elena G. Abalakina , Svetlana M. Kazakova , Evgueni R. Gak and Sergey V. Mashko * Ajinomoto-Genetika Research Institute, Moscow 117545, Russian Federation ABSTRACT 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied and Environmental Microbiology American Society For Microbiology

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

Applied and Environmental Microbiology , Volume 76 (1): 75 – Jan 1, 2010

Abstract

Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP -Encoded Transporter of Escherichia coli ▿ † ‡ Yurgis A. V. Yomantas , Irina L. Tokmakova , Natalya V. Gorshkova , Elena G. Abalakina , Svetlana M. Kazakova , Evgueni R. Gak and Sergey V. Mashko * Ajinomoto-Genetika Research Institute, Moscow 117545, Russian Federation ABSTRACT 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.

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References (72)

Publisher
American Society For Microbiology
Copyright
Copyright © 2010 by the American society for Microbiology.
ISSN
0099-2240
eISSN
1098-5336
DOI
10.1128/AEM.02217-09
pmid
19880640
Publisher site
See Article on Publisher Site

Abstract

Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP -Encoded Transporter of Escherichia coli ▿ † ‡ Yurgis A. V. Yomantas , Irina L. Tokmakova , Natalya V. Gorshkova , Elena G. Abalakina , Svetlana M. Kazakova , Evgueni R. Gak and Sergey V. Mashko * Ajinomoto-Genetika Research Institute, Moscow 117545, Russian Federation ABSTRACT 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.

Journal

Applied and Environmental MicrobiologyAmerican Society For Microbiology

Published: Jan 1, 2010

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