Abstract
Vanillin and syringaldehyde obtained from lignin are essential intermediates for the production of basic chemicals using microbial cell factories. However, in contrast to vanillin, the microbial conversion of syringaldehyde is poorly understood. Here, we identified an aromatic aldehyde dehydrogenase (ALDH) gene responsible for syringaldehyde catabolism from 20 putative ALDH genes of Sphingobium sp. strain SYK-6. All these genes were expressed in Escherichia coli, and nine gene products, including previously characterized BzaA, BzaB, and vanillin dehydrogenase (LigV), exhibited oxidation activities for syringaldehyde to produce syringate. Among these genes, SLG_28320 (desV) and ligV were most highly and constitutively transcribed in the SYK-6 cells. Disruption of desV in SYK-6 resulted in a significant reduction in growth on syringaldehyde and in syringaldehyde oxidation activity. Furthermore, a desV ligV double mutant almost completely lost its ability to grow on syringaldehyde. Purified DesV showed similar kcat/Km values for syringaldehyde (2100 s−1·mM−1) and vanillin (1700 s−1·mM−1), whereas LigV substantially preferred vanillin (8800 s−1·mM−1) over syringaldehyde (1.4 s−1·mM−1). These results clearly demonstrate that desV plays a major role in syringaldehyde catabolism. Phylogenetic analyses showed that DesV-like ALDHs formed a distinct phylogenetic cluster separated from the vanillin dehydrogenase cluster.
Highlights
Pathway and lower ring-cleavage pathways have been identified and characterized[5]
These results suggest that an alternative aromatic ALDH gene is involved in the catabolism of SN in SYK-6
When an extract of SYK-6 cells grown in Lysogeny broth (LB) (300 μg of protein/ml) was incubated with 300 μM SN in the presence of 500 μM NAD+, the extract converted SN into syringate with a specific activity of 34 ± 0.3 nmol·min−1·mg−1
Summary
Pathway and lower ring-cleavage pathways have been identified and characterized[5] Among these genes, the VN dehydrogenase gene (ligV), which is essential for the catabolism of VN, was isolated by shotgun cloning[25]. Disruption of ligV minimally affected the growth of SYK-6 on SN25 These results suggest that an alternative aromatic ALDH gene is involved in the catabolism of SN in SYK-6. BzaA and bzaB, whose products were capable of converting a wide range of benzaldehyde derivatives including SN, were isolated[28]. Disruption of these genes in SYK-6 had only a slight impact on its growth on SN28. Gene disruption experiments identified the actual SN dehydrogenase gene, and enzymatic properties of SN dehydrogenase and LigV were characterized
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