Nishimura et al. BMC Microbiology (2018) 18:154 https://doi.org/10.1186/s12866-018-1309-2 RESEARCHARTICLE Open Access Molecular cloning and characterization of vanillin dehydrogenase from Streptomyces sp. NL15-2K Motohiro Nishimura* , Susumu Kawakami and Hideaki Otsuka Abstract Background: Streptomyces 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. Results: The 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. Conclusions: Our 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. Keywords: Vanillin dehydrogenase, Streptomyces, Biotransformation, Salicylaldehyde dehydrogenase Background medicines, etc. The global vanillin demand exceeds 16,000 Lignin is a crosslinked phenylpropanoid polymer that is tons a year [1], and is expected to grow further in the fu- the most abundant component of plant biomass after cel- ture. Natural vanillin is extracted from the cured seedpods lulose. Partial decay of lignin yields numerous aromatic of Vanilla planifolia. However, the amount that can be monomers that can be used in the cosmetics, food, supplied to the global market from this source is negligible pharmaceutical, and chemical industries. For example, because of the extremely high production cost. Therefore, vanillin is an essential aromatic compound extensively most of the vanillin supplied to the market is chemically used as a flavoring agent in foods and beverages, and as synthesized. However, together with the current increasing an aromatic additive in perfumes, air fresheners, awareness of consumer health and safety, the demand for natural vanillin is growing. * Correspondence: [email protected] As an alternative to chemical synthesis of vanillin, bio- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, transformation of plant constituents such as ferulic acid Japan and eugenol to vanillin by various microbial species was © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nishimura et al. BMC Microbiology (2018) 18:154 Page 2 of 12 proposed. Such microbial species include fungi, e.g., ornamentation (spiny) of strain NL15-2K are clearly dif- Pycnoporus cinnabarinus [2, 3], and bacteria, e.g., Bacil- ferent from those (Retinaculiaperti-type; smooth) of the lus [4, 5], Pseudomonas [6, 7], Rhodococcus [8], and S. capoamus type strain. Analysis of the degradation Streptomyces [9], or recombinant Escherichia coli strains products of lignin-derived aromatic compounds added carrying genes encoding enzymes that mediate the bio- to the medium as the sole carbon source revealed that transformation [10–14]. However, biotransformation to this strain possesses an extensive catabolic network for vanillin by using a whole-cell biocatalyst frequently diverse compounds, including the pathway transforming entails a reduction in the vanillin yield either because of ferulic acid to vanillin [18]. Thus, we have been charac- the toxicity of the highly reactive aromatic aldehyde terizing the enzymes, and their encoding genes, involved group of vanillin or because of the degradation of vanil- in the catabolism of lignin-derived aromatic compounds lin to vanillic acid (Fig. 1). Methods employing resins to in Streptomyces sp. NL15-2K. absorb the produced vanillin have been devised to in- VDH, one of the key enzymes involved in the catabol- crease the yield [2, 9, 14]. Further, the inactivation of ism of lignin-derived aromatic compounds, degrades vdh, which encodes vanillin dehydrogenase (VDH), has vanillin to vanillic acid. Although extensive studies of been demonstrated in Amycolatopsis sp. ATCC 39116 vdh genes have been performed [6, 15, 19–26], only a [15] and Pseudomonas fluorescens BF13 [6]. Although few studies reported both, the gene analysis and vanillin accumulation was markedly enhanced in both characterization of VDH enzyme [15, 25, 26]. No infor- cases, a detectable amount of vanillic acid was generated mation for VDH from Streptomyces species is available. and coupled to a slow degradation of vanillin; in these As noted above, Streptomyces sp. NL15-2K could poten- studies, it was proposed that vanillic acid was formed by tially be used as a biocatalyst for vanillin production. a nonspecific aldehyde dehydrogenase (ALDH) or oxi- However, to achieve this goal, it is essential to first dase. Besides the biotransformation to vanillin, other in- characterize the VDH that degrades the produced vanil- triguing approaches reported for vanillin production lin and the encoding gene. Here, we isolated the vdh include a method involving the use of immobilized en- gene from Streptomyces sp. NL15-2K and comprehen- zymes [16] and de novo biosynthesis from glucose [17]. sively characterized the gene product. Streptomycetes are gram-positive bacteria with a high G + C content of DNA. These bacteria are responsible for the production of the majority of antibiotics used Methods worldwide. Most Streptomyces species are found in the Materials and chemicals soil, where they play an environmentally critical role in Vanillin (4-hydroxy-3-methoxybenzaldehyde), vanillic the recycling of organic materials because they have acid (4-hydroxy-3-methoxybenzoic acid), ferulic acid evolved complex and efficient enzyme systems that (4-hydroxy-3-methoxycinnamic acid), isovanillin (3-hy- break down and catabolize diverse organic substances, droxy-4-methoxybenzaldehyde), protocatechualdehyde including lignin and lignin-derived aromatic compounds. (3,4-dihydroxybenzaldehyde), benzaldehyde, p-hydroxy- Therefore, Streptomyces species can potentially serve as benzaldehyde, m-anisaldehyde (3-methoxybenzaldehyde), biocatalysts for the production of valuable compounds, veratraldehyde (3,4-dimethoxybenzaldehyde), syringalde- such as vanillin, from inexpensive plant constituents. hyde (4-hydroxy-3,5-dimethoxybenzaldehyde), and salicy- Previously, we isolated Streptomyces sp. NL15-2K from a laldehyde (2-hydroxybenzaldehyde) were purchased from forest soil by screening for bacteria capable of cataboliz- Wako Pure Chemicals (Osaka, Japan). NAD(P)+ and lactate ing lignin-derived aromatic compounds [18]. Based on dehydrogenase from the rabbit muscle were purchased 16S rRNA gene phylogeny, this strain is most closely re- from Oriental Yeast Co. Ltd. (Osaka, Japan). Oligonucleo- lated to Streptomyces capoamus NBRC 13411. However, tides were purchased from Life Technologies Japan Ltd. spore chain morphology (Spira-type) and spore surface (Tokyo, Japan). Vanillin Vanillate-O- Protocatechuate dehydrogenase demethylase 3,4-dioxygenase Ring fission Vanillin Vanillic acid Protocatechuic acid Fig. 1 Degradation pathway of vanillin. The major pathway common to bacteria that degrade vanillin is shown Nishimura et al. BMC Microbiology (2018) 18:154 Page 3 of 12 Bacterial strains, vectors, and cultivation media at 80 °C for 5 min, and 10-μL aliquot of the mixture was Streptomyces sp. NL15-2K used in this study was iso- analyzed by HPLC by using a Kinetex 5 u C18 100A col- lated from a forest soil in Canada and reported as a bac- umn (4.6 × 250 mm; Phenomenex Inc., Torrance, CA). terium capable of degrading lignin-related