Molecular cloning and characterization of vanillin dehydrogenase from Streptomyces sp. NL15-2K

Abstract

BackgroundStreptomyces sp. NL15-2K, previously isolated from the forest soil, features an extensive catabolic network for lignin-derived aromatic compounds, including pathways transforming ferulic acid to vanillin, vanillic acid, and protocatechuic acid. To successfully use Streptomyces sp. NL15-2K as a biocatalyst for vanillin production, it is necessary to characterize the vanillin dehydrogenase (VDH) that degrades the produced vanillin to vanillic acid, as well as the gene encoding this enzyme. Here, we cloned the VDH-encoding gene (vdh) from strain NL15-2K and comprehensively characterized its gene product.ResultsThe vdh open reading frame contains 1488 bp and encodes a 496-amino-acid protein with a calculated molecular mass of 51,705 Da. Whereas the apparent native molecular mass of recombinant VDH was estimated to be 214 kDa by gel filtration analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a subunit molecular mass of ca. 56 kDa, indicating that VDH is a homotetramer. The recombinant enzyme showed optimal activity at 45 °C and pH 9.5. The VDH substrate specificity followed this order: vanillin (100%) > protocatechualdehyde (91%) > benzaldehyde (79%) > p-hydroxybenzaldehyde (56%) > isovanillin (49%) ≈ salicylaldehyde (48%) > anisaldehyde (15%) ≈ veratraldehyde (12%). Although peptide mass fingerprinting and BLAST searches indicated that this enzyme is a salicylaldehyde dehydrogenase (SALDH), the determined kinetic parameters clearly demonstrated that the enzyme is a vanillin dehydrogenase. Lastly, phylogenetic analysis revealed that VDH from Streptomyces sp. NL15-2K forms an independent branch in the phylogenetic tree and, hence, is evolutionarily distinct from other VDHs and SALDHs whose activities have been confirmed experimentally.ConclusionsOur findings not only enhance the understanding of the enzymatic properties of VDH and the characteristics of its amino acid sequence, but also contribute to the development of Streptomyces sp. NL15-2K into a biocatalyst for the biotransformation of ferulic acid to vanillin.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1309-2) contains supplementary material, which is available to authorized users.

Regulation of vanillate and syringate catabolism by a MarR-type transcriptional regulator DesR in Sphingobium sp. SYK-6

“…These aromatic compounds derived from G- and S-lignins are degraded through vanillate (VA) and syringate (SA), respectively 12,16 . In addition to SYK-6 catabolism, it has been reported that lignin-derived aromatic compounds, including ferulate, sinapate, vanillin, and syringaldehyde, are degraded via VA and SA in many microorganisms 9,17–20 . Therefore, VA and SA are considered to be the key intermediate metabolites in the microbial degradation of lignin.…”

Isolation and characterization of Lignin-derived monomer degraders under acidic conditions from tropical peatland

“…The lower conversion rate on strain S38 reveals the correlation of the strain isolate source of undisturbed peatlands with lower lignin monomers such as vanillin compared with the strain S46 isolate source on disturbed peatlands. Different theories exist in the literature regarding vanillin regarding some of the degradation products after conversion from vanillic acid to protocatechuic acid (Nishimura et al, 2018), while another found is guaiacol (Fig. S6), as reported by previous research (Álvarez-Rodríguez et al, 2003;Ito et al, 2016;Witthuhn et al, 2012).…”

Genomic analysis of dibenzofuran-degradingPseudomonas veroniistrain Pvy reveals its biodegradative versatility

“…These enzymes transform the secondary metabolite vanillin to vanillic acid, which is further converted to protocatechuate. Vanillin dehydrogenases from various bacteria were reported to exhibit broad substrate specificity toward various aromatic aldehydes, such as isovanillin, veratraldehyde, anisaldehyde, protocatechualdehyde, p -hydroxybenzaldehyde, salicylaldehyde, and benzaldehyde ( Masai et al 2007 ; Mitsui et al 2010 ; Graf et al 2016 ; Nishimura et al 2018 ). The genes fadD and fadB encode for a putative feruloyl-CoA synthase and vanillin synthase, respectively, enabling the catabolism of ferulic acid through its conversion to vanillin, which is further transformed by vanillin dehydrogenase and vanillate O -demethylase ( Overhage et al 1999 ).…”