AEM Accepts, published online ahead of print on 30 October 2009

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Appl. Environ. Microbiol. doi:10.1128/AEM.02217-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Yurgis A.V. Yomantas, Irina L. Tokmakova, Natalya V. Gorshkova, Elena G. Abalakina, Svetlana M. Kazakova, Evgueni R. Gak, and Sergey V. Mashko^*

Ajinomoto-Genetika Research Institute, 117545, Moscow, Russian Federation

^* To whom correspondence should be addressed. Email: sergey_mashko{at}agri.ru.

The isolation of auxotrophic mutants which is 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 aroP gene, 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 trpE, tyrA, pheA and aroG genes 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, introducing of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.