Rapid cloning of metK encoding methionine adenosyltransferase from Corynebacterium glutamicum by screening a genomic library on a high density colony-array.
The genes SAM1 and SAM2 encoding the two different methionine adenosyltransferases (EC 2.5.1.6) in Saccharomyces cerevisiae were used as templates to generate specific DNA-probes. This heterologous mixture of DNA-probes was hybridized under low stringency hybridization conditions to a Corynebacterium glutamicum colony-array representing the complete genome. Subsequently, one genomic fragment was isolated which contained the C. glutamicum methionine adenosyltransferase gene metK (1.224 kb). When overproduced in Escherichia coli, MetK (44.2 kDa) of C. glutamicum had methionine adenosyltransferase activity. In addition, overexpression of metK in C. glutamicum led to an increased intracellular S-adenosylmethionine concentration. The metK transcript was detected by reverse transcription PCR in C. glutamicum cells in the exponential growth phase but not in the stationary phase.
Grossmann
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BASF-LYNX Bioscience AG, Im Neuenheimer Feld 515, 69120, Heidelberg, Germany.
Herbster
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Mack
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D015139:Blotting, Southern; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D015698:Genomic Library; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008716:Methionine Adenosyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020411:Oligonucleotide Array Sequence Analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D008716:Methionine Adenosyltransferase