Gene cloning, sequencing, and inactivation of the branched-chain aminotransferase of Lactococcus lactis LM0230

Myrta W. Atiles, Edward G. Dudley, James L. Steele

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

A branched-chain aminotransferase gene (ilvE) from Lactococcus lactis LM0230 was identified on a 9-kb chromosomal insert by complementation in Escherichia coli DL39. Sequencing of a 2.0-kbp fragment resulted in the identification of a 1,023-bp open reading frame that could encode a 340- amino-acid protein. Sequence analysis of the deduced amino acid sequence revealed 62% identity to IlvE of Haemophilus influenzae and high similarity to IlvEs from a variety of organisms found in GenBank classified as class IV aminotransferases. Under logarithmic growth in complex medium, ilvE is transcribed monocistronically as a 1.1-kb transcript. Hydrophobicity plot analysis of the deduced amino acid sequence and the lack of a signal peptide sequence suggest IlvE is a cytosolic protein. A derivative of LM0230 lacking IlvE activity was constructed by gene replacement. Comparison of the IlvE- deficient strain's ability to grow in defined media lacking an amino acid but containing its α-keto acid biosynthetic precursor to that of the wild-type strain indicated that IlvE is the only enzyme capable of synthesis of Ile and Val from their biosynthetic precursors. Comparison of the aminotransferase activity of the IlvE mutant to LM0230 revealed that the mutant retained <2, 4.5, 43, 40, and 76% of its aminotransferase activity with Ile, Val, Leu, Met, and Phe, respectively. No difference in growth or acidification rate between LM0230 and the IlvE-deficient strain was observed in milk.

Original languageEnglish (US)
Pages (from-to)2325-2329
Number of pages5
JournalApplied and environmental microbiology
Volume66
Issue number6
DOIs
StatePublished - Jun 1 2000

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Lactococcus lactis
transaminases
Transaminases
Organism Cloning
molecular cloning
inactivation
amino acid
Protein Sequence Analysis
gene
Genes
Keto Acids
Amino Acids
amino acid sequences
Nucleic Acid Databases
Haemophilus influenzae
Growth
Protein Sorting Signals
Hydrophobic and Hydrophilic Interactions
keto acids
mutants

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

Cite this

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abstract = "A branched-chain aminotransferase gene (ilvE) from Lactococcus lactis LM0230 was identified on a 9-kb chromosomal insert by complementation in Escherichia coli DL39. Sequencing of a 2.0-kbp fragment resulted in the identification of a 1,023-bp open reading frame that could encode a 340- amino-acid protein. Sequence analysis of the deduced amino acid sequence revealed 62{\%} identity to IlvE of Haemophilus influenzae and high similarity to IlvEs from a variety of organisms found in GenBank classified as class IV aminotransferases. Under logarithmic growth in complex medium, ilvE is transcribed monocistronically as a 1.1-kb transcript. Hydrophobicity plot analysis of the deduced amino acid sequence and the lack of a signal peptide sequence suggest IlvE is a cytosolic protein. A derivative of LM0230 lacking IlvE activity was constructed by gene replacement. Comparison of the IlvE- deficient strain's ability to grow in defined media lacking an amino acid but containing its α-keto acid biosynthetic precursor to that of the wild-type strain indicated that IlvE is the only enzyme capable of synthesis of Ile and Val from their biosynthetic precursors. Comparison of the aminotransferase activity of the IlvE mutant to LM0230 revealed that the mutant retained <2, 4.5, 43, 40, and 76{\%} of its aminotransferase activity with Ile, Val, Leu, Met, and Phe, respectively. No difference in growth or acidification rate between LM0230 and the IlvE-deficient strain was observed in milk.",
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Gene cloning, sequencing, and inactivation of the branched-chain aminotransferase of Lactococcus lactis LM0230. / Atiles, Myrta W.; Dudley, Edward G.; Steele, James L.

In: Applied and environmental microbiology, Vol. 66, No. 6, 01.06.2000, p. 2325-2329.

Research output: Contribution to journalArticle

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