The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics.
Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the beta-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.
Koetsier
Martijn J
MJ
Biochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9747 AG Groningen, The Netherlands.
Gombert
Andreas K
AK
Fekken
Susan
S
Bovenberg
Roel A L
RA
van den Berg
Marco A
MA
Kiel
Jan A K W
JA
Jekel
Peter A
PA
Janssen
Dick B
DB
Pronk
Jack T
JT
van der Klei
Ida J
IJ
Daran
Jean-Marc
JM
D000272:Adipates / Q000378:metabolism*; D002511:Cephalosporins / Q000096:biosynthesis*; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D010408:Penicillium chrysogenum / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D000272:Adipates; D002511:Cephalosporins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D003066:Coenzyme A Ligases