Isolation of a mutant auxotrophic for L-Alanine and identification of three major aminotransferases that synthesize L-Alanine In Escherichia Coli

Hiroshi Yoneyama, Hatsuhiro Hori, Sang Jo Lim, Tatsuya Murata, Tasuke Ando, Emiko Isogai, Ryoichi Katsumata

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

For Escherichia coli, it has been assumed that Lalanine is synthesized by alanine-valine transaminase (AvtA) in conjunction with an unknown alanine aminotransferase( s). We isolated alanine auxotrophs from a prototrophic double mutant deficient in AvtA and YfbQ, a novel alanine aminotransferase, by chemical mutagenesis. A shotgun cloning experiment identified two genes, uncharacterized yfdZ and serC, that complemented the alanine auxotrophy. When the yfdZ- or serC-mutation was introduced into the double mutant, one triple mutant (avtA yfbQ yfdZ) showed alanine auxotrophy, and another (avtA yfbQ serC), prototrophy. In addition, we found that four independent alanine auxotrophs possessed a point mutation in yfdZ but not in serC. We also found that yfdZ expression was induced in minimal medium. Furthermore, yfbQ-bearing plasmid conferred the ability to excrete alanine on the mutant lacking D-amino acid dehydrogenase-encoding gene, dadA. From these results, we concluded that E. coli synthesizes L-alanine by means of three aminotransferases, YfbQ, YfdZ, and AvtA.

Original languageEnglish
Pages (from-to)930-938
Number of pages9
JournalBioscience, Biotechnology and Biochemistry
Volume75
Issue number5
DOIs
Publication statusPublished - 2011

Keywords

  • Alanine auxotroph
  • Aminotransferase
  • Escherichia coli
  • Mutagenesis
  • Shotgun cloning

ASJC Scopus subject areas

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

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