Forced evolution of Escherichia coli cells with the ability to effectively utilize non-natural amino acids l-tert-leucine, l-norleucine and γ-methyl-l-leucine

Abstract

In an attempt to develop a novel biocatalyst able to efficiently catalyse the synthesis of non-natural amino acids, Escherichia coli TG1 was treated with 10 mM NaNO2 and then cultured in selective medium supplemented with 20 mM l-tert-leucine. Each culture was grown for 2 weeks and then subcultured into fresh medium with successive decreases of l-tert-leucine concentration at each transfer to a final value of 0.5 mM. The adapted cells resulting from this forced evolution procedure were able to grow in minimal medium with 0.1 mM l-tert-leucine as sole nitrogen source. Both HPLC and TLC verified progressive removal of l-tert-leucine from the medium during bacterial growth. Further studies revealed that the adapted cells metabolized l-tert-leucine by transamination, removing the amino group but leaving the carbon skeleton of the corresponding 2-oxoacid intact. Despite the mutagenesis, when the four obvious candidate amino acid aminotransferase genes were cloned and sequenced, there was no change in these structural genes. The activity of the adapted cells with l-tert-leucine is apparently attributable to the wild-type branched-chain amino acid aminotransferase (IlvAT), presumably expressed at higher levels as a result of a regulatory mutation. With the isolate I-4, the resting cells transaminate l-tert-leucine, l-norleucine, l-norvaline, γ-methyl-l-leucine and dl-homophenylalanine as effectively as does the crude extract. These evolved cells may be useful for synthesizing non-natural amino acids for the pharmaceutical industry. In addition, the adapted cells can also catalyse transamination of naturally occurring hydrophobic amino acids.