Unraveling the cellulolytic and hemicellulolytic potential of two novel Streptomyces strains

Abstract

The Streptomyces spp. are notorious plant biomass decomposers in soil environments, but only few strains were biochemically and genetically characterized. Here, we employed functional screening along with genomic sequencing for identification of novel lignocellulolytic Streptomyces strains. Streptomyces strains isolated from soil were functional screened based on their cellulolytic and hemicellulolytic capacities by enzymatic plate assays containing carboxymethylcellulose (CMC) and beechwood xylan as sole carbon source. Subsequently, genomes of Streptomyces strains were sequenced, annotated, and interpreted to correlate their genetic contents with biochemical properties. Among the 80 bacterial isolates that were screened for enzymatic activity, two Streptomyces strains (named as F1 and F7) exhiting higher endoglucanase and endoxylanase activities were selected for biochemical and genomic characterization. After cultivation on steam-pretreated sugarcane bagasse-based medium, the supernatant of the strains F1 and F7 exhibited enzymatic activity against different substrates, such as arabinan, rye arabinoxylan, β-glucan, starch, CMC, xylan, and chitin. Furthermore, strain F7 was able to degrade pectin, mannan, and lichenan. The genomic analysis of both strains revealed a diversity of carbohydrate-active enzymes. The F1 and F7 genomes encode 33 and 44 different types of glycosyl hydrolases families, respectively. Moreover, the genomic analysis also identified genes related to degradation of lignin-derived aromatic compounds. Collectively, the study revealed two novel Streptomyces strains and further insights on the degradation capability of lignocellulolytic bacteria, from which a number of technologies can arise, such as saccharification processes.

Introduction

The depletion of fossil fuels and environmental concerns has driven global politics towards the development of renewable energy resources. Lignocellulosic biomass represents the largest reservoir for renewable energy and value-added chemical production (Gomez et al. 2008; Himmel et al. 2010; Van Dyk and Pletschke 2012). The term lignocellulosic biomass refers to nonfood materials available from plants, including rice straw, cotton straw, corn stover, sugarcane bagasse, wood, grass, and others (Gomez et al. 2008; Sanderson 2011; Van Dyk and Pletschke 2012).

The transformation of the lignocellulosic biomass into fermentable sugars and biofuel production is a challenging process. Generally, lignocellulosic biomasses are composed of variable contents of cellulose, hemicellulose, and lignin. These polymers are highly organized and interlinked among themselves into recalcitrant structure (Gomez et al. 2008; Himmel et al. 2010). However, in nature ecosystems, lignocellulosic biomass is constantly converted by microorganisms, which efficiently degrade lignocellulosic material (Himmel et al. 2010; McGuire and Treseder 2010; Koeck et al. 2014). The biomass-degrading microorganisms are found in various environments, including hot spring pool (Hamilton-Brehm et al. 2010; Brumm et al. 2015), cow rumen (Hess et al. 2011), biogas reactor (Hanreich et al. 2013; Tomazetto et al. 2015), and soil (Zhou et al. 2014; Jiménez et al. 2016; López-Mondéjar et al. 2016). These environments represent resource for isolation of novel microorganisms and enzymes involved on plant biomass conversion. For instance, Streptomyces have been reported to play an important role in the carbon cycle and plant biomass deconstruction in soils (Chater et al. 2010; Bontemps et al. 2013; Větrovský et al. 2014; Book et al. 2016). Indeed, Streptomyces species genomes have revealed several carbohydrate-active enzymes (CAZymes), including cellulases, hemicellulases, and lytic polysaccharide monooxygenases (Book et al. 2014, 2016; Pinheiro et al. 2016). Moreover, a recent study described three Streptomyces strains (pl6, pl88, and pr55) isolated from soil capable to metabolize polysaccharides and poplar lignin (Větrovský et al. 2014).

Although several Streptomyces strains have been described as plant biomass decomposer, the molecular and biochemical understanding on how these microorganisms degrade lignocellulosic biomass remain rather fragmented and restricted to a few strains, e.g., I1.2 (Pinheiro et al. 2016), SirexAA-E, SDPB6 (Book et al. 2014), ATexAB-D23, and LaPpAH-95 (Book et al. 2016).

In this sense, soil samples from distinct locations were used as inoculum on Streptomyces selective-agar plates for isolation of their species. Isolates obtained were screened based on their cellulolytic and hemicellulolytic capacities by enzymatic plate assays using carboxymethylcellulose (CMC) and beechwood xylan as sole carbon source. Among the 80 bacterial isolates, two Streptomyces strains demonstrated ability to secrete a wide range of enzymes against plant-based polysaccharides. To further investigate the mechanisms of lignocellulosic biomass degradation, the genomes of both strains were sequenced and analyzed regarding their CAZymes profiles. Their genomes encode several CAZymes confirming the cellulolytic and hemicellulolytic phenotypes. In addition, genomic analysis indicated that both strains presented gene cluster related to lignin degradation.

Material and methods

Streptomyces strains isolation

Eighty bacterial isolates were isolated from soil samples collected from different places at Brazil (São Paulo, Brazil: 22°49′8.861″ S, 47°3′39.085″ W; Minas Gerais, Brazil: 19°57′08.1″ S, 44°12′55.7″ W; Minas Gerais, Brazil: 20°36′29.624″ S, 46°2′30.739″ W, Table S1). Briefly, soil samples corresponding to the upper 10 cm were collected and transported to the laboratory into sterile bags. These samples were mixed with calcium carbonate (1:1), ground with a pestle and air-dried (El-Nakeeb and Lechevalier 1963). For each sample collected, 4 g of air-dried soil was mixed with 40-mL sterilized water and stirred vigorously. Several dilutions (10⁻¹ to 10⁻⁴) were spread on isolation medium ISP 4 agar plates (Shirling and Gottlieb 1966) and incubated at 30 °C for 3 days. Single colonies were transferred to medium ISP 2 agar plates (Shirling and Gottlieb 1966) and grown at 30 °C for 6–8 days. The procedure was repeated until pure cultures were obtained. The isolated strains were stored at − 80 °C as mixtures of mycelial fragments in 20% (v/v) glycerol or in medium ISP 2.

Qualitative and quantitative screening

To screen cellulolytic and hemicellulolytic microorganisms among the isolate ones, they were cultivated on Bushnell Haas Broth (BHB, Sigma Aldrich®) mineral salts medium agar plates (g/L: K₂HPO₄ 1, KH₂PO₄ 1, CaCl₂·2H₂O 0.02, NH₄NO₃ 1, FeCl₃ 0.05, MgSO₄·7H₂O 0.2, and agar 15, pH 7.0) supplemented with 0.5% (w/v) of beechwood xylan or carboxymethylcellulose (CMC). After incubation at 30 °C for 48–72 h, agar plates were stained with Congo red and destained with 1-M NaCl (Teather and Wood 1982). Colonies showing clear halos were selected as indicative for the CMC (endoglucanase activity) and xylan (endoxylanase) degradation. Of the 80 strains screened, 48 showed a visual enzymatic activity (Table S1). The 14 strains exhibiting the highest enzymatic activities index (E.A.I) were selected for further analysis described as follows.

These strains were selected and initially grown on medium ISP 2 agar plates at 30 °C for 7 days. Each culture was inoculated 10⁶ spores mL⁻¹ (1%, v/v) into 25 mL of BHB medium and supplemented with beechwood xylan or CMC (0.5%, w/v) and incubated at 30 °C for 6 days in a shaker (New Brunswick Scientific, New Jersey, USA) at 180 rpm. Supernatants were taken daily to monitor the corresponding enzymatic activity (Table S2) using the DNS method as described in the section below (“Enzymatic assays” section). All experiments were done in biological triplicates.

Cultivation and enzymatic production

Based on endoglucanase and endoxylanase activity results, the two strains exhibiting the highest enzymatic activities, namely F1 and F7, were cultivated on steam-pretreated sugarcane bagasse (SCB) as a sole carbon source for enzyme production. SCB was pretreated by steam explosion at 200 °C for 15 min, as described by Rocha et al. (2012). The strains were grown on medium ISP 2 agar plates at 30 °C for 7 days, and then 10⁶ spores mL⁻¹ (1%, v/v) was inoculated into 80 mL of BHB medium supplemented with 1.0% (w/v) steam-pretreated SCB. The flasks were incubated at 30 °C for 10 days in a rotary shaker at 180 rpm. Samples of the culture supernatants were taken daily to evaluate their enzymatic activity against plant-based polysaccharides as described below.

Enzymatic assays

The enzymatic activity (International Units, U) was performed using distinct substrates. All the polysaccharides were purchased from Sigma-Aldrich or Megazyme [beechwood xylan, rye arabinoxylan, β-glucan (barley), sugar beet arabinan, debranched arabinan, carboxymethylcellulose, tamarind xyloglucan, starch, icelandic moss lichenan, chitin from shrimp shells, arabinogalactan, mannan (ivory nut), and citrus pectin]. The enzymatic reactions were carried out in a miniaturized fashion by mixing 20 μL of culture media supernatant, 50 μL of the distinct substrates (0.5%, w/v), and 30 μL of sodium phosphate buffer (0.1 M) at pH 6. Reactions were incubated at 50 °C in a Thermostat® (Eppendorf, Hamburg, Germany) for 30 min or 18 h and stopped with the addition of 100 μL of 3,5 dinitrosalicylic acid (DNS) following immediately by boiling for 5 min at 99 °C (Miller 1959). The solutions were analyzed at 540 nm using the Infinite M200®spectrophotometer (Tecan, Switzerland) to measure the release of reducing sugars. One unit (1 U) of enzymatic activity corresponded to the formation of 1 μmol of reducing sugar equivalent per minute under the assay conditions. Total protein was measured using microtiter plates with Bio-Rad protein assay reagent (Bio-Rad Laboratories, USA), employing a procedure based on Bradford’s method (Bradford 1976). Bovine serum albumin was used as standard. All experiments were done in biological triplicates.

Identification of Streptomyces strains

The strains F1 and F7 were grown in ISP 2 broth at 30 °C in flasks agitated at 180 rpm for 3 days, and their cells harvested by centrifugation at 10,000 rpm for 10 min. The cell pellets were washed twice with sterile water. Genomic DNA was isolated using FastDNA SPIN Kit for soil (MP Biomedicals, Irvine, CA) according to the manufacturer’s instructions. The quality of the total DNA was assessed by electrophoresis in a 0.8% agarose gel stained with ethidium bromide and visualized using UV transilluminator. The DNA concentration was measured by fluorimetry (Qubit® 2.0 Fluorometer-Life Technologies. Carlsbad, California, EUA) using the BR Qubit® dsDNA Assay (Life Technologies, Carlsbad, California, EUA).

To determine the relatedness of strains with their closest described relative Streptomyces, 16S rRNA gene sequences were amplified using polymerase chain reaction (PCR) with primers 27f (5′ AGAGTTTGATCMTGGCTCAG 3′) and 1492r (5′ TACCTTGTTACGACTT 3′). All PCR reactions were performed in 50 μL containing 50 ng of individual genomic DNA, 1-mM dNTP, 10 pmoL each primer, 2-mM MgCl₂, and 1 U of Taq DNA polymerase (Thermo Fischer Scientific, Waltham, USA). The PCR conditions consisted as follows: an initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 60 s, 55 °C for 60 s, and 72 °C for 2 min, and a final extension at 72 °C for 4 min. PCR products were purified using Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare Bio-Sciences, Pittsburgh, USA) and sequenced using a DNA ABI PRISM 377 Genetic Analyzer system (Applied Biosystems, USA). BLASTn search analysis revealed that strains F1 and F7 shared more than 97% 16S rRNA gene sequences identity with members of the genus Streptomyces.

Genome sequencing, assembly, and annotation

Genomic DNA of both strains was used for the construction of libraries using Nextera® DNA Library Preparation Kit (Illumina, San Diego, CA), according to the manufacturer’s protocol. The genomic libraries were sequenced on Illumina HiSeq sequencing platform at NGS sequencing facility at CTBE, generating approximately 8 and 6 million 2 × 100-bp reads, respectively.

Raw fastq files were quality checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and processed with Trimmomatic version 0.32 (Bolger et al. 2014) to quality trimming, adaptor removal, and minimum length filtering. High-quality reads were further analyzed using ProDeGe (Tennessen et al. 2015) to remove possible contaminants and then assembled using Spades version 3.6.2 (Bankevich et al. 2012), applying several kmers (k = 21, 27, 33, 55, 77) and careful mode option as parameters.

The draft genome sequences of strains F1 and F7 were uploaded into the IMG/ER annotation pipeline (Markowitz et al. 2009) for automatic prediction of genes using Prodigal (Hyatt et al. 2010). Functional annotation of predicted proteins was obtained using Prokka automatic pipeline (Seemann 2014). Briefly, it uses hierarchical searches against different databases such as bacterial proteins from RefSeq genomes, UniProt, Pfam, and TIGRFAM models. The Aragorn tool (Laslett and Canback 2004) was used to predict tRNA genes whereas rRNA genes were identified using RNAmmer 1.2. Signal peptides, and transmembrane helices were predicted by SignalP and TMHMM, respectively. To determine the carbohydrate-active enzymes (CAZYmes) profiles for strains F1 and F7, putative protein sequences were annotated using dbCAN database version 4 (Yin et al. 2012) using HMMER3.1b package. Results obtained were manually assessed based on their functional annotation prediction and conserved protein domains to identify the corresponding best matching CAZymes.

Phylogenetic analysis

The taxonomic assignment of strains F1 and F7 within Bacteria domain was determined based on 16S rRNA sequence analysis and multilocus phylogenetic analysis. Phylogenetic tree was reconstructed based on 16S rRNA gene sequences of strains isolated and closest related species with validly published name. All sequences were aligned using the ClustalX tool (Larkin et al. 2007) and manually refined. From those aligned, pairwise distances were calculated using the maximum composite likelihood approach, and phylogenetic tree was constructed applying the neighbor-joining method, as implemented in MEGA version 6 (Tamura et al. 2013). Bootstrap tests with 1000 replications/iterations were calculated to assess the node confidence level.

Multilocus phylogenetic analysis was done as previous described Book et al. (2014). Briefly, TIGRFAM models were used to search for single-copy genes conserved among 172 Streptomyces genome sequences available at RefSeq database, and the genome of Kitasatospora setae was considered as an out group. The sequences recovered from each genome were aligned using Mafft v7.299 (Katoh and Standley 2013) and the resulting alignment was concatenated using FASconCAT-G v1.02 (Kück and Longo 2014). The phylogenetic tree was generated using FastTree version 2.1.8 (Price et al. 2010) under WAG as a substitution model and the final tree was visualized using iTOL version 3 (Letunic and Bork 2007).

In addition, the relatedness among strains F1 and F7 and their closest relative Streptomyces strains was determined based on average nucleotide identity (gANI) and the fraction of orthologous genes (Aligment Fraction, AF) calculated with the MiSI (Microbial Species Identifier) method (Varghese et al. 2015).

Statistical analysis

Statistical analysis of enzymatic activities was performed using parametric analysis of variance (ANOVA) with Tukey’s multiple comparisons test (α = 0.05). Calculation was made by Minitab v18 (Minitab Inc., PA, USA).

Nucleotide sequence accession number

The draft genome sequences of strains F1 and F7 are available at the NCBI with the accession numbers FKJI03000000 and FKJH01000000, respectively.

Results and discussion

Strain screening and enzymatic activity evaluation

Of the 80 bacterial isolates from soil samples grown on CMC—or beechwood xylan-agar plates, 48 exhibited degradation halos around them indicating endoglucanase and endoxylanase activities (Supplementary file 1: Table S1). Among them, 14 strains exhibiting the largest degradation halos were selected for further analysis using submerged fermentation.

Overall, the supernatant of the 14 Streptomyces isolates obtained from submerged fermentations containing CMC or beechwood xylan showed endoglucanase and endoxylanase activities (Supplementary file 1: Table S2). Endoglucanase activity varies between 0.18 to 0.40 U mL⁻¹ and endoxylanase 1.97 to 10.78 U mL⁻¹ using xylan as sole carbon source, and endoglucanase activity between 0.10 to 0.17 U mL⁻¹ and endoxylanase 0.12 to 0.28 U mL⁻¹ using CMC as carbon source. Considering the enzymatic activities (Table S2), two strains (F1 and F7) exhibited the highest endoglucanase and endoxylanase activities and they were selected for enzymatic production and genomic analysis.

Enzymatic assays of the strains F1 and F7 supernatants obtained from cultivation on SCB revealed that these microorganisms were capable to secrete a set of enzymes involved in the hydrolysis of distinct polysaccharides, including arabinan, rye arabinoxylan, β-glucan, starch, CMC, xylan, and chitin (Fig. 1a). The strain F7 was also able to secrete enzymes for pectin, mannan, and lichenan degradation. Overall, the supernatant of strain F7 exhibited higher enzymatic activities for degradation of plant-based polysaccharides than strain F1 during 10 days of the submerged fermentation on SCB (Figs. 1b and 2). No enzymatic activity was detected for degradation of arabinan sugar beet, xyloglucan, and arabinogalactan in both strains, even though the majority of genes encoding enzymes involved in these polysaccharides were predicted (see “CAZYme profiles” section), which could be because SCB was not capable to induce the expression of these enzymes.

Fig. 1

Enzymatic activities detected in the supernatants from strains F1 and F7 grown on steam-pretreated sugarcane bagasse (SCB) as the carbon source. a Bar graph showing the maximum enzymatic activity of each substrate tested. b Variation of the endoglucanase and endoxylanase activities in the supernatants from strains F1 and F7 cultivated for 10 days on SCB. The data represent the means ± standard errors from three technical replicates; lowercase letters indicate values statistically different between strains (Tukey’s multiple comparisons test: p < 0.01). Abbreviations: CMC, carboxymethylcellulose

Fig. 2

Variation of the enzymatic activities detected in the supernatants from strains F1 (a) and F7 (b) cultivated for 10 days on steam-pretreated sugarcane bagasse (SCB) as the carbon source. The data represent the means ± standard errors from three technical replicates

Our findings corroborated with previous studies and the cellulolytic and hemicellulolytic potential of Streptomyces spp. (Chater et al. 2010; Bontemps et al. 2013; Book et al. 2014; Pinheiro et al. 2016). In a recently study using comparative genomic, transcriptomic, and biochemical analysis (Book et al. 2016), 29 Streptomyces strains have shown a relatively high rate of cellulose degrading activity. Pinheiro et al. (2016) reported that Streptomyces spp. were able to secrete a set of enzymes involved in degradation of distinct natural carbohydrates, including cellulose, xylan, mannan, starch, chitin, pectin, and β-glucan.

General features and phylogenetic analysis

Based on cellulolytic and hemicellulolytic profiles of strains F1 and F7, we decided to genetically characterize both strains to uncover their metabolic pathways dedicated to biomass degradation. The genomes of both strains were sequenced on the Illumina HiSeq system using the 2 × 100-pb sequencing strategy. The genomes of strains F1 and F7 resulted in 69 and 66 contigs, respectively, ranging from 10,212 to 760,841 bases (Table 1). Strain F1 consists of an 8.1 megabase pair (Mb) chromosome coding 7262 protein-coding sequences (CDS). The strain F7 genome is 7.33 Mb in size and contains 6463 CDSs. The genomes display a similar GC content of 72%.

Table 1 Genome statistics for strains F1 and F7

The 16S rRNA gene sequence analyses placed strains F1 and F7 within the genera Streptomyces (Fig. 3). Pairwise analysis revealed that strain F1 was closely related to Streptomyces misionensis NRRL B-3230^T sharing 99% 16S rRNA gene sequence identity. Whereas, strain F7 showed 100% 16S rRNA gene sequence identity with Streptomyces viridodiastaticus IFO 13106^T. It is important to underline that the percentage of identity obtained from 16S rRNA gene sequences alignment was calculated disregarding a gap (26 and 31 nucleotides) that are present only in strains F1 and F7.

Fig. 3

Phylogenetic analysis of 16S rRNA gene sequences of strains F1 and F7 (printed in bold) relative to the most closely related strains of the genus Streptomyces. The phylogenetic tree was constructed using neighbor-joining (NJ) algorithm. Bootstrap values higher than 60% are shown (1000 replications for bootstrapping were done). Genbank accession numbers are shown in parentheses and type strain with superscript “T”. The sequence of Streptacidiphilus albus was used as an out group

Previous studies reported that 16S rRNA gene sequence analysis is not suitable to distinguish closely related Streptomyces species since several Streptomyces type strains share completely identical 16S rRNA gene sequences (Guo et al. 2008; Antony-Babu et al. 2017). Therefore, to refine the phylogenetic relationship of the strains isolated within Streptomyces genus, a multilocus phylogenetic tree was constructed based on 288 single-copy conserved genes found across strains F1, F7, and 172 Streptomyces genomes available at RefSeq database (Fig. 4). Indeed, multilocus analysis indicated a phylogenetic relationship different from the analysis based on 16S rRNA gene sequences. While, strain F1 forms a monophyletic clade with Streptomyces griseofuscus NRRL B 5429^T, which is a type species of this genus, strain F7 is closely related to species with currently not validly published names.

Fig. 4

Multilocus phylogenetic analysis of Streptomyces genus. The multilocus phylogenetic tree was generated from a concatenated alignment of 288 conserved genes across all genomes of Streptomyces. Bootstrap values (expressed as percentages of 1000 replicates) higher than 0.8 are shown at branch nodes

In addition, gANI and AF among strains F1 and F7 and their closest related species were calculated to support the clades. Strains F1 and F7 shared a maximum pairwise similarity of 90.28% and 94.30% ANI and AF of 0.73 and 0.82 across Streptomyces strains within their respectively clades (Supplementary file 1: Table S3). Considering the threshold at minimum of 95 or 96.5% of ANI, which corresponds to 70% DNA-DNA hybridization, and AF above 0.6 between two whole genome as strong evidence for same species (Goris et al. 2007; Varghese et al. 2015), values of ANI and AF for strains F1 and F7 alongside the multilocus tree confirmed that both strains represent novel species with genus Streptomyces.

CAZYme profiles

The CAZymes (Cantarel et al. 2009) present in Streptomyces strains F1 and F7 were identified using dbCAN web resource (Yin et al. 2012). Both strains have similar profile of CAZymes genes in their genomes (Table 2). Strain F1 genome encodes 85 glycoside hydrolyses (GH), 31 glycosyltransferases (GT), 18 carbohydrate esterases (CE), one polysaccharide lyases (PL), 31 carbohydrate-binding motifs (CBM), and nine classified as auxiliary activities (AA), which are CAZymes with redox activities (Tables 3 and S4). Whereas, strain F7 genome possesses 100 GHs, 22 GTs, 26 CEs, five PLs, 26 CBMs, and nine AAs. A detailed CAZymes gene prediction indicated that strains F1 and F7 genomes encode 33 and 44 different types of GH families, respectively, which are potentially involved in degradation of starch, chitin, hemicellulose, and cellulose (Table 3 and Supplementary file 2: Table S1). However, only strain F7 genome encodes pectin-, arabinogalactan-, lichenan-, and mannan-degradation from families PL1, PL3, GH35, GH64, GH5, and GH2, respectively.

Table 2 Comparison of CAZyme genomic profile between strains F1 and F7

Table 3 Summarizes of CAZyme profile encoded by strains F1 and F7

As expected, Streptomyces strains F1 and F7 do not encode cellulosomes, which are the enzymatic complex composed of cohesins and dockerins modules and several lignocellulolytic enzymes with CBMs domains (Smith and Bayer 2013). However, both strains possess GHs connected to CBMs, which is not surprising since lignocellulolytic enzymes connected to CBMs are broadly found in Bacteria domain (Tomazetto et al. 2015; López-Mondéjar et al. 2016; Pinheiro et al. 2016). The F7 genome encodes different CAZymes associated with CBMs domains, including endoglucanases, chitinase, xylanases, and α-amylase (Supplementary file 2: Table S1). Whereas, strain F1 genome was found to encode CBMs associated with only chitinase and endoglucanase. CBMs domains bind to plant cell wall polysaccharides and facilitating GHs activity (Campos et al. 2016). In addition, both strains contain genes encoding lytic polysaccharide monooxygenases (LPMOs) belonging to family AA10 (Supplementary file 2: Table S1). Chitinolytic and cellulolytic AA10 genes are frequently found in Streptomyces genomes (Book et al. 2014). Nevertheless, strains F1 and F7 genomes have only AA10 genes related to chitin degradation.

Aromatic compound degradation pathways

Several bacteria have been reported to metabolize lignin, releasing aromatics compounds that are imported into the cell for catabolism (Bugg et al. 2011; Brown and Chang 2014). Among the lignin-degrading bacteria, few Streptomyces strains were described to degrade lignin or lignin-derived compounds, such as catechol, protocatechuate, and gentisate (Ishiyama et al. 2004; Gottschalk et al. 2008; Davis et al. 2013; Větrovský et al. 2014). In this context, the corresponding metabolic pathways in strains F1 and F7 were manually reconstructed from their genome sequence data.

The genomic analyses of strains F1 and F7 revealed several genes involved on protocatechuate and gentisate catabolism (Fig. 5 and Supplementary file 2: Table S2). For gentisate catabolism, identical gene clusters consisting of four genes were found in both strains genomes. Sequence analysis predicted four encoding genes, including DNA-binding transcriptional regulator (IclR family), gentisate 1,2 dioxygenase, fumarylacetoacetate hydrolase, and maleylpyruvate isomerase. For protocatechuate catabolism, all genetic determinants for this aromatic degradation were found only in strain F1. These seven genes are organized in a cluster and flaked by transcriptional regulators. It is important to mention the presence of transcriptional regulator in all these clusters. For instance, the protocatechuate cluster in Streptomyces F7 genome consists of four genes involved on aromatic degradation preceded by a transcriptional regulator. Members of IclR family frequently control genes whose products are involved in the degradation of aromatics (Molina-Henares et al. 2006). These findings suggest that the clusters could be expressed and allow the strains to use these aromatic compounds as carbon sources.

Fig. 5

a) Schematic representation of the gene clusters encoding the gentisate and protocatechuate degradation and their corresponding metabolism found in F1 and F7 genomes. A.1 and A.2, organization of the gentisate and protocatechuate clusters, respectively. The putative functions of the gene products are as follows: kdgR, transcriptional regulator KdgR (MarR family transcriptional regulator); sdgD, gentisate 1,2-dioxygenase; nagK, fumarylacetoacetate hydrolase family; nagL, maleylpyruvate isomerase; nodD, nodulation protein D (MarR Family); catD, 3-oxoadipate enol-lactonase; pcaB, β-carboxymuconatecycloisomerase; pcaB,3-carboxy-cis,cis-muconate cycloisomerase; pcaGH, protocatechuate 3,4-dioxygenase, α and β-subunits; paaJ, 3-oxoadipyl-CoA thiolase; scoAB, 3-oxoacid CoA-transferase subunit A and B; hosA, transcriptional regulator (MarR family). b) Metabolic pathways of the protocatechuate and gentisate degradation (B.1 and B.2, respectively). The enzyme names are shown above the arrows. Abbreviation: TCA cycle, tricarboxylic acid cycle

Concluding remarks

This work disclose two novel Streptomyces strains isolated from soil, able to grow on CMC, xylan, and steam-pretreated SCB as sole carbon sources, and secrete a range of hydrolytic enzymes. The enzymatic assays indicated that strain F7 was more efficient in the degradation of natural carbohydrates than strain F1. This observation was confirmed by genomic analysis: the genome of strain F1 encodes 85 glycoside hydrolases (GHs) which comprise 33 different types of GH families, and strain F7 harbors 100 GHs representing 44 GH families. Finally, the identification of gene clusters encoding enzymes dedicate to aromatic compound degradation suggests that the strains could also perform lignin degradation.