Aromatic amino acid auxotrophs constructed by recombinant marker exchange in Methylophilus methylotrophus AS1 cells expressing the aroP-encoded transporter of Escherichia coli.

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 c

SUBMITTER: Yomantas YA 

PROVIDER: S-EPMC2798654 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature