Cloning and characterization of the ferulic acid catabolic genes of Sphingomonas paucimobilis SYK-6.
Sphingomonas paucimobilis SYK-6 degrades ferulic acid to vanillin, and it is further metabolized through the protocatechuate 4,5-cleavage pathway. We obtained a Tn5 mutant of SYK-6, FA2, which was able to grow on vanillic acid but not on ferulic acid. A cosmid which complemented the growth deficiency of FA2 on ferulic acid was isolated. The 5.2-kb BamHI-EcoRI fragment in this cosmid conferred the transformation activity of ferulic acid to vanillin on Escherichia coli host cells. A sequencing analysis revealed the genes ferB and ferA in this fragment; these genes consist of 852- and 2,127-bp open reading frames, respectively. The deduced amino acid sequence of ferB showed 40 to 48% identity with that of the feruloyl-coenzyme A (CoA) hydratase/lyase genes of Pseudomonas and Amycolatopsis ferulic acid degraders. On the other hand, the deduced amino acid sequence of ferA showed no significant similarity to the feruloyl-CoA synthetase genes of other ferulic acid degraders. However, the deduced amino acid sequence of ferA did show 31% identity with pimeloyl-CoA synthetase of Pseudomonas mendocina 35, which has been classified as a new superfamily of acyl-CoA synthetase (ADP forming) with succinyl-CoA synthetase (L. B. Sánchez, M. Y. Galperin, and M. Müller, J. Biol. Chem. 275:5794-5803, 2000). On the basis of the enzyme activity of E. coli carrying each of these genes, ferA and ferB were shown to encode a feruloyl-CoA synthetase and feruloyl-CoA hydratase/lyase, respectively. p-coumaric acid, caffeic acid, and sinapinic acid were converted to their corresponding benzaldehyde derivatives by the cell extract containing FerA and FerB, thereby indicating their broad substrate specificities. We found a ferB homolog, ferB2, upstream of a 5-carboxyvanillic acid decarboxylase gene (ligW) involved in the degradation of 5,5'-dehydrodivanillic acid. The deduced amino acid sequence of ferB2 showed 49% identity with ferB, and its gene product showed feruloyl-CoA hydratase/lyase activity with a substrate specificity similar to that of FerB. Insertional inactivation of each fer gene in S. paucimobilis SYK-6 suggested that the ferA gene is essential and that ferB and ferB2 genes are involved in ferulic acid degradation.
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan. emasai@vos.nagaokaut.ac.jp
Eiji
E
Masai
Kyo
K
Harada
Xue
X
Peng
Hirotaka
H
Kitayama
Yoshihiro
Y
Katayama
Masao
M
Fukuda
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism*; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial / Q000502:physiology*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D003373:Coumaric Acids; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C004999:ferulic acid; D003066:Coenzyme A Ligases; C400923:feruloyl-coenzyme A synthetase