Identification of novel benzoylformate decarboxylases by growth selection.
A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the beta-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.
Henning
Helge
H
Institute of Molecular Enzyme Technology, Heinrich Heine University Duesseldorf, Research Centre Juelich, D-52426 Jülich, Germany.
Leggewie
Christian
C
Pohl
Martina
M
Müller
Michael
M
Eggert
Thorsten
T
Jaeger
Karl-Erich
KE
D000595:Amino Acid Sequence; D001547:Benzaldehydes / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism*; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D015723:Gene Library; D015698:Genomic Library; D006038:Glyoxylates / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D008333:Mandelic Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D017422:Sequence Analysis, DNA; D012987:Soil / Q000032:analysis; D012988:Soil Microbiology
D001547:Benzaldehydes; D003470:Culture Media; D004269:DNA, Bacterial; D006038:Glyoxylates; D008333:Mandelic Acids; D012987:Soil; C012482:phenylglyoxylic acid; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase; C032175:benzaldehyde