Aromatic amino acid auxotrophs constructed by recombinant marker exchange in Methylophilus methylotrophus AS1 cells expressing the aroP-encoded transporter of Escherichia coli.
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 cloned homologous arms longer than 1,000 bp. M. methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro using a Kmr 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, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.
Yomantas
Yurgis A V
YA
Ajinomoto-Genetika Research Institute, Moscow 117545, Russian Federation.
Tokmakova
Irina L
IL
Gorshkova
Natalya V
NV
Abalakina
Elena G
EG
Kazakova
Svetlana M
SM
Gak
Evgueni R
ER
Mashko
Sergey V
SV
D026905:Amino Acid Transport Systems / Q000235:genetics*; D024322:Amino Acids, Aromatic / Q000172:deficiency*; D010583:Bacteriophage mu / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D020630:Methylophilus methylotrophus / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
D026905:Amino Acid Transport Systems; D024322:Amino Acids, Aromatic; C063201:AroP protein, E coli; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins