Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP -Encoded Transporter of Escherichia coli
Autor: | Elena G. Abalakina, Evgueni R. Gak, Yurgis A. V. Yomantas, Sergey V. Mashko, Svetlana M. Kazakova, Natalya V. Gorshkova, Irina L. Tokmakova |
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Rok vydání: | 2010 |
Předmět: |
DNA
Bacterial Amino Acid Transport Systems Auxotrophy Molecular Sequence Data Mutagenesis (molecular biology technique) Biology medicine.disease_cause Applied Microbiology and Biotechnology Bacteriophage mu Amino Acids Aromatic chemistry.chemical_compound Methods medicine Aromatic amino acids Amino acid transporter Escherichia coli Gene Recombination Genetic Genetics chemistry.chemical_classification Base Sequence Ecology Escherichia coli Proteins Sequence Analysis DNA Amino acid Mutagenesis Insertional Biochemistry chemistry Methylophilus methylotrophus Food Science Biotechnology |
Zdroj: | Applied and Environmental Microbiology. 76:75-83 |
ISSN: | 1098-5336 0099-2240 |
Popis: | 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. |
Databáze: | OpenAIRE |
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