Kumar et al. Biotechnol Biofuels (2018) 11:154 https://doi.org/10.1186/s13068-018-1148-2 Biotechnology for Biofuels RESEARCH Open Access Genomic and proteomic analysis of lignin degrading and polyhydroxyalkanoate accumulating β‑proteobacterium Pandoraea sp. ISTKB Madan Kumar1, Sandhya Verma2, Rajesh Kumar Gazara2, Manish Kumar1, Ashok Pandey3, Praveen Kumar Verma2* and Indu Shekhar Thakur1* Abstract Background: Lignin is a major component of plant biomass and is recalcitrant to degradation due to its complex and heterogeneous aromatic structure. The biomass-based research mainly focuses on polysaccharides component of biomass and lignin is discarded as waste with very limited usage. The sustainability and success of plant polysac- charide-based biorefnery can be possible if lignin is utilized in improved ways and with minimal waste generation. Discovering new microbial strains and understanding their enzyme system for lignin degradation are necessary for its conversion into fuel and chemicals. The Pandoraea sp. ISTKB was previously characterized for lignin degradation and successfully applied for pretreatment of sugarcane bagasse and polyhydroxyalkanoate (PHA) production. In this study, genomic analysis and proteomics on aromatic polymer kraft lignin and vanillic acid are performed to fnd the impor- tant enzymes for polymer utilization. Results: Genomic analysis of Pandoraea sp. ISTKB revealed the presence of strong lignin degradation machinery and identifed various candidate genes responsible for lignin degradation and PHA production. We also applied label-free quantitative proteomic approach to identify the expression profle on monoaromatic compound vanillic acid (VA) and polyaromatic kraft lignin (KL). Genomic and proteomic analysis simultaneously discovered Dyp-type peroxidase, peroxidases, glycolate oxidase, aldehyde oxidase, GMC oxidoreductase, laccases, quinone oxidoreductase, dioxyge- nases, monooxygenases, glutathione-dependent etherases, dehydrogenases, reductases, and methyltransferases and various other recently reported enzyme systems such as superoxide dismutases or catalase–peroxidase for lignin degradation. A strong stress response and detoxifcation mechanism was discovered. The two important gene clus- ters for lignin degradation and three PHA polymerase spanning gene clusters were identifed and all the clusters were functionally active on KL–VA. Conclusions: The unusual aerobic ‘-CoA’-mediated degradation pathway of phenylacetate and benzoate (reported only in 16 and 4–5% of total sequenced bacterial genomes), peroxidase-accessory enzyme system, and fenton chem- istry based are the major pathways observed for lignin degradation. Both ortho and meta ring cleavage pathways for aromatic compound degradation were observed in expression profle. Genomic and proteomic approaches provided *Correspondence: [email protected]; [email protected]; [email protected] 1 School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, 110067, India 2 National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/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://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kumar et al. Biotechnol Biofuels (2018) 11:154 Page 2 of 23 validation to this strain’s robust machinery for the metabolism of recalcitrant compounds and PHA production and provide an opportunity to target important enzymes for lignin valorization in future. Keywords: Genomics, Lignin, Polyhydroxyalkanoate, Gene cluster, Label-free quantifcation, Vanillic acid Background industry [6, 9–11]. Te application of advanced ‘omics’ Te genus Pandoraea is a very recently classifed genus approach such as genomics, transcriptomics, and pro- proposed in the year 2000. Bacteria belonging to genus teomics to individual microbial strains or microbial Pandoraea are Gram-negative, non-sporulating, and community will help in identifcation and functional motile bacteria with single polar fagellum [1]. Te characterization of novel ligninolytic enzymes in the genus belongs to Burkholderiaceae family and class near future [12–14]. With the increase in genomic data β-proteobacteria. Te Pandoraea genus was earlier misi- of bacteria and fungi, the biomass degrading potential dentifed and grouped together with Burkholderia or across diferent taxa can be identifed that will further Ralstonia [1] Tis genus contains fve species (Pandoraea enhance our understanding related to lignin degra- pnomenusa, Pandoraea sputorum, Pandoraea norimber- dation [12, 13]. Te lignin degrading bacterial isolate gensis, Pandoraea apista, and Pandoraea pulmonicola) belongs to actinobacteria, alpha proteobacteria, beta and four genomospecies of thiosulfate-oxidizing (Pan- proteobacteria, gamma proteobacteria, delta proteobac- doraea thiooxydans) and oxalate-oxidizing species as teria, bacteroides, and archaea [7]. Te novel bacterial Pandoraea vervacti, Pandoraea faecigallinarum, and enzymes responsible for lignin degradation and their Pandoraea oxalativorans. Pandoraea is a taxonomically mechanism of action have also been described [15]. In distinct genus having close similarity with Burkholderia recent years, LC–MS-based proteomics studies have and Ralstonia. Pandoraea has been isolated from various been widely performed. Quantitative LC–MS-based environments such as soil, landfll site, sediments, clinical proteomics such as label free and ITRAQ labeling-based samples (only P. apista, P. pnomenusa, and P. sputorum quantifcation methods are generally used to identify isolated until date), and water [1–4]. Te Burkholderia the novel enzymes and their level of expression in a par- and Ralstonia are very much explored and established ticular process [16–18]. genera with their promising potential environmental We have earlier sequenced the genome of Pando- and industrial applications. Pandoraea is a relatively new raea sp. ISTKB and the sequence has been submitted to genus, so there are very few fndings available about their NCBI with accession number MAOS00000000.1 which biotechnological potential. Te species from this genus is openly available [19]. In the present study, we describe have been documented for utilization of polychlorinated the comprehensive analysis of the Pandoraea sp. ISTKB biphenyl, dichloromethane, dyes, lignin, oxalate, thiosul- genome. Te bioinformatics analysis was performed fate, and quorum sensing [3–6]. At present, the genomic to identify a large set of genes and pathways putatively insights for Pandoraea are limiting and such studies responsible for lignin degradation and PHA production. would eventually help to widen the biotechnological pro- Te important gene clusters responsible for lignin degra- spective of this genus. dation and PHA production were also highlighted. Tis Lignin is a complex aromatic heteropolymer and it strain has already been shown to utilize monoaromatic is the most abundant aromatic polymer available on lignin derivatives with great ease compared to polymeric earth. In nature, lignin is degraded mainly by bacte- kraft lignin for PHA production [20]. Terefore, the pro- ria and fungi. Fungi have been studied extensively for teomic study of Pandoraea sp. ISTKB was performed lignin degradation and only a few bacterial species have for identifcation of set of a proteins expressed during its been reported for lignin degradation [7, 8]. Compared growth on monoaromatic vanillic acid (VA) and aromatic to fungi, bacteria ofer advantage as its genome size is polymer lignin, i.e., kraft lignin (KL) that can be overex- small, genetic manipulations, and large-scale recom- pressed for enhanced KL utilization. VA was selected, binant expression of important enzymes can be per- because most of the lignin linkages proceed through gen- formed with a greater ease. Terefore, the focus again eration of vanillin or VA as nodal point during the course shifted to bacteria for the identifcation of novel strains of degradation [21]. Proteomic studies provide insight and enzymes for lignin degradation. Te discovery of into the protein profle and also complement the genom- novel ligninolytic microbes, enzymes, and their bio- ics analysis. Genomic and proteomic analyses would chemical characterization will help in deconstruction of enable us to understand the novel enzymes and pathways biomass for their application in biofuel and bioproduct responsible for lignin degradation and biovalorization. Kumar et al. Biotechnol Biofuels (2018) 11:154 Page 3 of 23 Results oxidoreductase activity. Abundance of ion binding and Salient features of Pandoraea sp. ISTKB genome small molecule-binding proteins indicates their role in Te Pandoraea sp. ISTKB was previously character- transcriptional regulation and transportation of mol- ized for lignin degradation and successfully applied for ecules across cell membrane. Representation of trans- pretreatment of sugarcane bagasse and polyhydroxyal- ferase and hydrolase in good proportion indicates their kanoate