- Email: [email protected]
Complete genome sequence of Bacillus sp. 275, producing extracellular cellulolytic, xylanolytic and ligninolytic enzymes
Journal of Biotechnology 254 (2017) 59–62
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short genome communications
Complete genome sequence of Bacillus sp. 275, producing extracellular cellulolytic, xylanolytic and ligninolytic enzymes
MARK
⁎
Gyeongtaek Gonga,b, Seil Kimc, Sun-Mi Leea,d,e, Han Min Woof, Tai Hyun Parkb,g, , ⁎⁎ Youngsoon Uma,d, a
Clean Energy Research Center, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea Interdisciplinary Program of Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea c Division of Metrology for Quality of Life, Center for Bioanalysis, Korea Research Institute of Standards and Science, 267 Gajeong-Ro, Yuseong-Gu, Daejeon 34113, Republic of Korea d Clean Energy and Chemical Engineering, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea e Green School (Graduate School of Energy and Environment), Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea f Department of Food Science and Biotechnology, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea g School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Keywords: Complete genome sequencing Lignocellulose degradation Bacillus sp. 275 Cellulose Xylan Lignin
Technologies for degradation of three major components of lignocellulose (e.g. cellulose, hemicellulose and lignin) are needed to efficiently utilize lignocellulose. Here, we report Bacillus sp. 275 isolated from a mudflat exhibiting various lignocellulolytic activities including cellulase, xylanase, laccase and peroxidase in the cell culture supernatant. The complete genome of Bacillus sp. 275 strain contains 3832 protein cording sequences and an average G + C content of 46.32% on one chromosome (4045,581bp) and one plasmid (6389bp). The genes encoding enzymes related to the degradation of cellulose, xylan and lignin were detected in the Bacillus sp. 275 genome. In addition, the genes encoding glucosidases that hydrolyze starch, mannan, galactoside and arabinan were also found in the genome, implying that Bacillus sp. 275 has potentially a wide range of uses in the degradation of polysaccharide in lignocellulosic biomasses.
Lignocellulosic biomass is one of the most attractive carbon resources due to its abundance and renewability. The degradation of recalcitrant lignocellulosic biomass is the first obstacle encountered toward the production of alternative renewable fuels and chemicals. Furthermore, the utilization of all the major components of lignocellulose, cellulose, hemicellulose and lignin, is essential for the efficient and complete use of lignocellulose (Beckham et al., 2016; Brown and Chang, 2014; Moreira and Filho, 2016). Among the lignocellulose degradation technologies, microbial degradation is one of the promising methods for the use of cellulose, xylan and lignin (Mathews et al., 2015; Oh et al., 2015; Perez et al., 2002; Salvachúa et al., 2015). Generally, Bacillus is a genus of rod-shaped, Gram-positive, aerobic or facultative anaerobic bacteria. Also, the genus Bacillus consists of a very diverse group of over 300 species (Euzéby, 2017; Feng et al., 2016). Several Bacillus strains have been reported to degrade lignin or lignin model compounds (Bandounas et al., 2011; Huang et al., 2013; Zhu et al., 2014). Moreover, Bacillus sp. 55S5 isolated from peat exhibited not only the capability of lignin modification but also cellulase
⁎
and xylanase activities (Maki et al., 2012). Similarly, Bacillus sp. R2 isolated from the Red Sea also had cellulase, xylanase, pectinase and peroxidase activities (Khelil et al., 2016). Those results imply that Bacillus species have potential in degrading lignocellulose components including not only lignin but also cellulose and hemicellulose. In this study, we report a newly isolated Bacillus sp. 275 as a potential lignocellulose-degrading bacterium producing extracellular cellulolytic, xylanolytic and ligninolytic enzymes. The complete genome of Bacillus sp. 275 was also sequenced and investigated to find the genes related to lignocellulose degradation. Bacillus sp. 275 isolated from a mudflat showed relatively high lignocellulolytic activities such as cellulase, xylanase and ligninase on solid agar plates compared to 88 other bacterial isolates belonging to Bacillus, Streptomyces, Burkholderia and Pseudomonas (Gong et al., 2017). To analyze the amount of extracellular enzymes and lignocellulolytic activities, Bacillus sp. 275 was grown in LB medium containing 10 g/L of tryptone, 5 g/L of yeast extract and 5 g/L of NaCl for 24 h at 30 °C and a cell-free supernatant was collected. As shown in Table 1, the lignocellulolytic activities of Bacillus sp. 275
Corresponding author at: Interdisciplinary Program of Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea. Corresponding author at: Clean Energy Research Center, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea. E-mail addresses: [email protected] (T.H. Park), [email protected], [email protected] (Y. Um).
⁎⁎
http://dx.doi.org/10.1016/j.jbiotec.2017.05.021 Received 3 April 2017; Received in revised form 25 May 2017; Accepted 26 May 2017 Available online 31 May 2017 0168-1656/ © 2017 Elsevier B.V. All rights reserved.
Journal of Biotechnology 254 (2017) 59–62
G. Gong et al.
siamensis KCTC 13613T. Meanwhile, the average nucleotide identity (ANI) values of the strain 275 compared to B. velezensis FZB42 were found to be over 98%. B. velezensis FZB42 had formerly classified as B. amyloliquefaciens subsp. plantarum FZB42T and it had been the closest type strain to Bacillus sp. 275 based on the 16S rRNA sequences until B. amyloliquefaciens subsp. plantarum FZB42T was re-classified to B. velezensis FZB42 (Dunlap et al., 2016). Based on these results that the closest relative to the strain 275 could not be clearly defined, the strain 275 was named as Bacillus sp. 275. A phylogenetic tree of Bacillus sp. 275 based on 16S rRNA sequences is shown in Fig. 2. Bacillus sp. 275 was closely related to B. siamensis KCTC 13613T and B. velezensis CR-502T as described earlier. Furthermore, the tree showed that B. amyloliquefaciens DSM 7T formed a distinct branch together with B. siamensis KCTC 13613T, B. velezensis CR502T and Bacillus sp. 275. The complete genomes of B. siamensis KCTC 13613T and B. velezensis CR-502T have not been reported. Therefore, instead of B. siamensis KCTC 13613T and B. velezensis CR-502T, B. siamensis SDLI1 (the only B. siamensis strain with the complete genome sequences available) and B. velezensis FZB42 whose complete genome sequences have been reported were selected for the comparative genome analysis with Bacillus sp. 275. Additionally, the complete genome of B. amyloliquefaciens DSM 7T was also compared with that of Bacillus sp. 275. The COG functional categories of the four complete genome sequences are shown in Table 3. Among the 3832 CDS of Bacillus sp. 275, 3431 CDS were classified into COG categories (Tatusov et al., 2000). The major categories of Bacillus sp. 275 were transcription (K), amino acid transport and metabolism (E), carbohydrate transport and metabolism (G), energy production and conversion (C), cell wall/membrane/envelope biogenesis (M) and inorganic ion transport and metabolism (P). Especially, the number of genes in carbohydrate transport and metabolism (G) related to lignocellulose degradation were 241. This value is similar to that of B. velezensis FZB42, but higher than those of B. siamensis SDLI1 and B. amyloliquefaciens DSM 7T by 9% and 3%, respectively. The genes related to the degradation of lignocellulose were detected in the genome of Bacillus sp. 275 (Table 4). Endoglucanase, β-glucanase, glucohydrolase and glucosidase were found in the complete genome of Bacillus sp. 275. Regarding xylan degradation, glycoside hydrolase 43 family protein (1,4-β-xylosidase), arabinoxylan arabinofuranohydrolase, glucuronoxylanase and α-N-arabinofuranoside were found. The genes encoding deferrochelatase (dye decolorizing peroxidase) and laccase involved in lignin degradation were also observed in the genome of Bacillus sp. 275. In addition, Bacillus sp. 275 has other glycosidases including α-amylase, β-mannosidase, endo-1,4-β-galactanase, 6-phospho-β-galactosidase, arabinan endo-1,5-α-L-arabinosidase and endo-α-(1- > 5)-L-arabinanase. The existence of these genes implies that strain 275 has the potential for utilizing or degrading starch, mannan, galactoside and arabinan. Most of the genes related to the degradation of lignocellulose were highly observed in all strains (Table 4). However, in the genome of B. amyloliquefaciens DSM 7T, endoglucanase, α-amylase and 1,4-β-xylosidase were not found. The distribution of lignocellulose degradationrelated genes between Bacillus sp. 275 and B. siamensis SDLI1 were also somewhat different. In our study, all lignocellulose-degrading genes in Bacillus sp. 275 were found in B. velezensis FZB42. B. velezensis has been mainly studied for its relationship with plant and fungi, because B. velezensis produces antifungal secondary metabolites (Kim et al., 2017). In conclusion, Bacillus sp. 275 has the cellulolytic, xylanolytic and ligninolytic enzyme activities and also has genes encoding lignocellulolytic enzymes. The complete genome information of Bacillus sp. 275 would enhance a better understanding on the degradation of lignocellulose in the genus Bacillus. These results would provide insight
Table 1 Lignocellulolytic enzyme activities in the cell-free supernatant of Bacillus sp. 275 strain. Enzyme feature
Activity (U/g protein)
Cellulasea Xylanaseb Laccasec Peroxidasec
1245.8 ± 7.2 138.4 ± 0.8 1.3 ± 0.3 0.5 ± 0.1
Protein concentration (74.48 ± 0.96 mg/L) in the supernatants was measured using the Bradford protein assay reagent (Bio-Rad, CA, USA). a Cellulase (endo-cellulase) activity was quantified with the Cellulase assay kit (CELLG5 method) (Megazyme, Ireland). b Xylanase activity was measured by the 3,5-dinitrosalicylic acid (DNS) colorimetric method (Teather and Wood, 1982) after adding xylan (10 g/L) as a substrate to the supernatant and reacting at 50 °C for 10 min. c Peroxidase and laccase activities were assayed by the detecting oxidation of (2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) [ABTS] (ε420nm = 3.6 m−1 cm−1) in 100 mM acetate buffer (pH 3.0) at room temperature with or without H2O2. Peroxidase activity was calculated by subtracting the oxidation activity without H2O2 from that with H2O2. One unit of enzyme activity was defined as the amount of enzyme required to release one micromole of product per minute. All experiments were performed in triplicate.
Table 2 Genome features of Bacillus sp. 275 strain. Features
Chromosome
Plasmid
Genome size (bp) G + C content (%) Total number of genes Protein-coding genes (CDS) rRNA genes tRNA genes
4,045,581 46.33 4017 3827 27 86
6389 43.25 6 5 0 0
were observed in the supernatant of the liquid culture broth. The activity of cellulase and xylanase from 74.48 mg/L of extracellular proteins were 1245.8 U/g and 138.4 U/g, respectively. Regarding the lignin degradation, the activities of laccase and peroxidase, which were known to be related to the degradation of lignin or lignin model compounds (Rahmanpour et al., 2016; Roth and Spiess, 2015; Yoshida and Sugano, 2015), were detected. The genome of Bacillus sp. 275 was sequenced using a combination of the PacBio RSII system (Pacific Biosciences, CA, USA), Illumina MiSeq (100-bp paired-end) and the Roche 454 sequencing TITAN technology. All the reads were assembled using the Roche gsAssembler 2.6, CLC Genomics Workbench 6.5.1 and PacBio SMRT Analysis 2.1 with a genome coverage of 276 folds. The complete genome of Bacillus sp. 275 consisted of one 4,045,581 bp chromosome and one 6389 bp plasmid with 3832 protein cording sequences (CDS), 86 tRNA genes, 27 rRNA genes and an average G + C content of 46.32% (Table 2 and Fig. 1). The NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) was used to annotate the genes of Bacillus sp. 275. Strain 275 was initially classified as B. siamensis based on the most hit taxon strain with the 16S rRNA sequence data in the Eztaxon server (http://www.ezbiocloud.net) (Kim et al., 2012) compared to B. siamensis KCTC 13613T with a pairwise similarity of 99.93% (e.g., 1471 bp matching out of 1472 bp). However, the number of mismatching nucleotide between the strain 275 and B. velezensis CR-502T was also only one bp (e. g., 1402 bp matching out of 1403 bp) as same as the B.
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Journal of Biotechnology 254 (2017) 59–62
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Fig. 1. Circular genome map of Bacillus sp. 275. The circular genome map consists of 5 circles. From the outer circle inward, each circle displays information about the genome of rRNA/tRNA, Reverse CDS, Forward CDS, G + C Ratio and GC Skew.
into bacterial degradation of all components in lignocellulose and possibly a new use for Bacillus sp. 275 in the conversion of lignocellulose into useful chemicals and fuels.
CP019627 (plasmid). The strain is available from the Korean Collection for Type Culture (KCTC) under the accession number KCTC 33887.
Nucleotide sequence and strain accession numbers
Conflict of interest The authors declare no conflict of interest.
The genome sequence of Bacillus sp. 275 was deposited into the GenBank under the accession number CP019626 (chromosome) and
Fig. 2. Phylogenetic tree derived from 16S rRNA gene sequence data of Bacillus sp. 275 and its relatives. The tree was generated by the NeighborJoining method. Evolutionary analyses were conducted in MEGA7 and the distances were computed using the Maximum Composite Likelihood method. Bacillus cereus ATCC 14579 was used as the out group. Only bootstrap values above 60% were shown from 1000 replications. Bar, 0.005 nucleotide substitutions per site.
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National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT (Information & Communication Technology) & Future Planning (2016M3D3A1A01913249). The authors also appreciate the support from the Korea Institute of Energy Technology Evaluation and Planning (Project No. 20143010091880).
Table 3 COG functional categories of the complete genome sequences of Bacillus sp. 275, B. siamensis SDLI1, B. amyloliquefaciens DSM 7T and B. velezensis FZB42. Code
J K L D O M N P T C G E F H I Q S Total
Functional annotation
Strains (number of genes)
Translation, ribosomal structure and biogenesis Transcription Replication, recombination and repair Cell cycle control, cell division, chromosome partitioning Posttranslational modification, protein turnover, chaperones Cell wall/membrane/envelope biogenesis Cell motility Inorganic ion transport and metabolism Signal transduction mechanisms Energy production and conversion Carbohydrate transport and metabolism Amino acid transport and metabolism Nucleotide transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Secondary metabolites biosynthesis, transport and catabolism Function unknown
275
SDLI1
DSM 7T
FZB42
159
163
161
159
261 159 35
255 157 36
271 155 36
261 140 36
100
103
108
102
183
210
206
195
47 181
42 179
45 180
42 188
140 184 241
138 179 222
136 176 235
143 180 244
284 77 102 110 91
284 90 108 110 83
299 84 108 104 67
293 80 111 108 87
1077 3431
1174 3533
1200 3571
1074 3443
References Bandounas, L., Wierckx, N.J., de Winde, J.H., Ruijssenaars, H.J., 2011. Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential. BMC Biotechnol. 11, 94. Beckham, G.T., Johnson, C.W., Karp, E.M., Salvachua, D., Vardon, D.R., 2016. Opportunities and challenges in biological lignin valorization. Curr. Opin. Biotechnol. 42, 40–53. Brown, M.E., Chang, M.C., 2014. Exploring bacterial lignin degradation. Curr. Opin. Chem. Biol. 19, 1–7. Dunlap, C.A., Kim, S.J., Kwon, S.W., Rooney, A.P., 2016. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp plantarum and ‘Bacillus oryzicola' are later heterotypic synonyms of Bacillus velezensis based on phylogenomics. Int. J. Syst. Evol. Microbiol. 66, 1212–1217. Euzéby, J.P., 2017. List of Prokaryotic Names with Standing in Nomenclature. http:// www.bacterio.net/bacillus.html (Accessed on 23 May 2017). Feng, L., Liu, D., Sun, X., Wang, G., Li, M., 2016. Bacillus cavernae sp. nov. isolated from cave soil. Int. J. Syst. Evol. Microbiol. 66, 801–806. Gong, G., Lee, S.M., Woo, H.M., Park, T.H., Um, Y., 2017. Influences of media compositions on characteristics of isolated bacteria exhibiting lignocellulolytic activities from various environmental sites. Appl. Biochem. Biotechnol. http://dx.doi.org/10. 1007/s12010-017-2474-8. Huang, X.F., Santhanam, N., Badri, D.V., Hunter, W.J., Manter, D.K., Decker, S.R., Vivanco, J.M., Reardon, K.F., 2013. Isolation and characterization of lignin-degrading bacteria from rainforest soils. Biotechnol. Bioeng. 110, 1616–1626. Khelil, O., Choubane, S., Cheba, B.A., 2016. Polyphenols content of spent coffee grounds subjected to physico-chemical pretreatments influences lignocellulolytic enzymes production by Bacillus sp. R2. Bioresour. Technol. 211, 769–773. Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H., Yi, H., Won, S., Chun, J., 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62, 716–721. Kim, S.Y., Lee, S.Y., Weon, H.Y., Sang, M.K., Song, J., 2017. Complete genome sequence of Bacillus velezensis M75, a biocontrol agent against fungal plant pathogens, isolated from cotton waste. J. Biotechnol. 241, 112–115. Maki, M.L., Idrees, A., Leung, K.T., Qin, W., 2012. Newly isolated and characterized bacteria with great application potential for decomposition of lignocellulosic biomass. J. Mol. Microbiol. Biotechnol. 22, 156–166. Mathews, S.L., Pawlak, J., Grunden, A.M., 2015. Bacterial biodegradation and bioconversion of industrial lignocellulosic streams. Appl. Microbiol. Biotechnol. 99, 2939–2954. Moreira, L.R., Filho, E.X., 2016. Insights into the mechanism of enzymatic hydrolysis of xylan. Appl. Microbiol. Biotechnol. 100, 5205–5214. Oh, Y.H., Eom, I.Y., Joo, J.C., Yu, J.H., Song, B.K., Lee, S.H., Hong, S.H., Park, S.J., 2015. Recent advances in development of biomass pretreatment technologies used in biorefinery for the production of bio-based fuels, chemicals and polymers. Korean J. Chem. Eng. 32, 1945–1959. Perez, J., Munoz-Dorado, J., de la Rubia, T., Martinez, J., 2002. Biodegradation and biological treatments of cellulose: hemicellulose and lignin: an overview. Int. Microbiol. 5, 53–63. Rahmanpour, R., Rea, D., Jamshidi, S., Fulop, V., Bugg, T.D., 2016. Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds. Arch. Biochem. Biophys. 594, 54–60. Roth, S., Spiess, A.C., 2015. Laccases for biorefinery applications: a critical review on challenges and perspectives. Bioprocess. Biosyst. Eng. 38, 2285–2313. Salvachúa, D., Karp, E.M., Nimlos, C.T., Vardon, D.R., Beckham, G.T., 2015. Towards lignin consolidated bioprocessing: simultaneous lignin depolymerization and product generation by bacteria. Green Chem. 17, 4951–4967. Tatusov, R.L., Galperin, M.Y., Natale, D.A., Koonin, E.V., 2000. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28, 33–36. Teather, R.M., Wood, P.J., 1982. Use of Congo red polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43, 777–780. Yoshida, T., Sugano, Y., 2015. A structural and functional perspective of DyP-type peroxidase family. Arch. Biochem. Biophys. 574, 49–55. Zhu, D., Tanabe, S.H., Xie, C., Honda, D., Sun, J., Ai, L., 2014. Bacillus ligniniphilus sp. nov.: an alkaliphilic and halotolerant bacterium isolated from sediments of the South China Sea. Int. J. Syst. Evol. Microbiol. 64, 1712–1717.
Table 4 Comparison of genes encoding lignocellulose-degrading enzymes in Bacillus sp. 275 and other Bacillus strains. SDLI1
DSM 7T
FZB42
AQP94520 AQP96424 AQP97176 WP_077392024 AQP95497 AQP96452 WP_077392079 AQP98009 AQP97260 AQP95939 AQP95972
O O O O O X O O X X O
X O O O O O O O O O X
O O O O O O O O O O O
AQP94597
O
X
O
AQP94515
O
O
O
WP_077391892 AQP97388 WP_077392066 AQP97416 AQP97396 AQP96449 AQP95141 WP_077391940
O X O O O X O O
O O O O O O O O
O O O O O O O O
AQP96486 AQP94816
O O
O O
O O
Bacillus sp. 275 Predicted function Cellulose-related endoglucanase β-glucanase glucohydrolase glucohydrolase 6-phospho-α-glucosidase 6-phospho-β-glucosidase 6-phospho-β-glucosidase 6-phospho-β-glucosidase α-glucosidase aryl-phospho-β-D-glucosidase α-amylase Hemicellulose-related glycoside hydrolase 43 family protein arabinoxylan arabinofuranohydrolase glucuronoxylanase arabinan endo-1,5-α-L-arabinosidase endo-α-(1- > 5)-L-arabinanase α-N-arabinofuranoside α-N-arabinofuranoside β-mannosidase endo-1,4-β-galactanase 6-phospho-β-galactosidase Lignin-related deferrochelatase laccase
Accession No.
O = detected; X = not detected.
Acknowledgements This research was supported by C1 Gas Refinery Program through the
62
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short genome communications
Complete genome sequence of Bacillus sp. 275, producing extracellular cellulolytic, xylanolytic and ligninolytic enzymes
MARK
⁎
Gyeongtaek Gonga,b, Seil Kimc, Sun-Mi Leea,d,e, Han Min Woof, Tai Hyun Parkb,g, , ⁎⁎ Youngsoon Uma,d, a
Clean Energy Research Center, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea Interdisciplinary Program of Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea c Division of Metrology for Quality of Life, Center for Bioanalysis, Korea Research Institute of Standards and Science, 267 Gajeong-Ro, Yuseong-Gu, Daejeon 34113, Republic of Korea d Clean Energy and Chemical Engineering, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea e Green School (Graduate School of Energy and Environment), Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea f Department of Food Science and Biotechnology, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea g School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Keywords: Complete genome sequencing Lignocellulose degradation Bacillus sp. 275 Cellulose Xylan Lignin
Technologies for degradation of three major components of lignocellulose (e.g. cellulose, hemicellulose and lignin) are needed to efficiently utilize lignocellulose. Here, we report Bacillus sp. 275 isolated from a mudflat exhibiting various lignocellulolytic activities including cellulase, xylanase, laccase and peroxidase in the cell culture supernatant. The complete genome of Bacillus sp. 275 strain contains 3832 protein cording sequences and an average G + C content of 46.32% on one chromosome (4045,581bp) and one plasmid (6389bp). The genes encoding enzymes related to the degradation of cellulose, xylan and lignin were detected in the Bacillus sp. 275 genome. In addition, the genes encoding glucosidases that hydrolyze starch, mannan, galactoside and arabinan were also found in the genome, implying that Bacillus sp. 275 has potentially a wide range of uses in the degradation of polysaccharide in lignocellulosic biomasses.
Lignocellulosic biomass is one of the most attractive carbon resources due to its abundance and renewability. The degradation of recalcitrant lignocellulosic biomass is the first obstacle encountered toward the production of alternative renewable fuels and chemicals. Furthermore, the utilization of all the major components of lignocellulose, cellulose, hemicellulose and lignin, is essential for the efficient and complete use of lignocellulose (Beckham et al., 2016; Brown and Chang, 2014; Moreira and Filho, 2016). Among the lignocellulose degradation technologies, microbial degradation is one of the promising methods for the use of cellulose, xylan and lignin (Mathews et al., 2015; Oh et al., 2015; Perez et al., 2002; Salvachúa et al., 2015). Generally, Bacillus is a genus of rod-shaped, Gram-positive, aerobic or facultative anaerobic bacteria. Also, the genus Bacillus consists of a very diverse group of over 300 species (Euzéby, 2017; Feng et al., 2016). Several Bacillus strains have been reported to degrade lignin or lignin model compounds (Bandounas et al., 2011; Huang et al., 2013; Zhu et al., 2014). Moreover, Bacillus sp. 55S5 isolated from peat exhibited not only the capability of lignin modification but also cellulase
⁎
and xylanase activities (Maki et al., 2012). Similarly, Bacillus sp. R2 isolated from the Red Sea also had cellulase, xylanase, pectinase and peroxidase activities (Khelil et al., 2016). Those results imply that Bacillus species have potential in degrading lignocellulose components including not only lignin but also cellulose and hemicellulose. In this study, we report a newly isolated Bacillus sp. 275 as a potential lignocellulose-degrading bacterium producing extracellular cellulolytic, xylanolytic and ligninolytic enzymes. The complete genome of Bacillus sp. 275 was also sequenced and investigated to find the genes related to lignocellulose degradation. Bacillus sp. 275 isolated from a mudflat showed relatively high lignocellulolytic activities such as cellulase, xylanase and ligninase on solid agar plates compared to 88 other bacterial isolates belonging to Bacillus, Streptomyces, Burkholderia and Pseudomonas (Gong et al., 2017). To analyze the amount of extracellular enzymes and lignocellulolytic activities, Bacillus sp. 275 was grown in LB medium containing 10 g/L of tryptone, 5 g/L of yeast extract and 5 g/L of NaCl for 24 h at 30 °C and a cell-free supernatant was collected. As shown in Table 1, the lignocellulolytic activities of Bacillus sp. 275
Corresponding author at: Interdisciplinary Program of Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea. Corresponding author at: Clean Energy Research Center, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea. E-mail addresses: [email protected] (T.H. Park), [email protected], [email protected] (Y. Um).
⁎⁎
http://dx.doi.org/10.1016/j.jbiotec.2017.05.021 Received 3 April 2017; Received in revised form 25 May 2017; Accepted 26 May 2017 Available online 31 May 2017 0168-1656/ © 2017 Elsevier B.V. All rights reserved.
Journal of Biotechnology 254 (2017) 59–62
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siamensis KCTC 13613T. Meanwhile, the average nucleotide identity (ANI) values of the strain 275 compared to B. velezensis FZB42 were found to be over 98%. B. velezensis FZB42 had formerly classified as B. amyloliquefaciens subsp. plantarum FZB42T and it had been the closest type strain to Bacillus sp. 275 based on the 16S rRNA sequences until B. amyloliquefaciens subsp. plantarum FZB42T was re-classified to B. velezensis FZB42 (Dunlap et al., 2016). Based on these results that the closest relative to the strain 275 could not be clearly defined, the strain 275 was named as Bacillus sp. 275. A phylogenetic tree of Bacillus sp. 275 based on 16S rRNA sequences is shown in Fig. 2. Bacillus sp. 275 was closely related to B. siamensis KCTC 13613T and B. velezensis CR-502T as described earlier. Furthermore, the tree showed that B. amyloliquefaciens DSM 7T formed a distinct branch together with B. siamensis KCTC 13613T, B. velezensis CR502T and Bacillus sp. 275. The complete genomes of B. siamensis KCTC 13613T and B. velezensis CR-502T have not been reported. Therefore, instead of B. siamensis KCTC 13613T and B. velezensis CR-502T, B. siamensis SDLI1 (the only B. siamensis strain with the complete genome sequences available) and B. velezensis FZB42 whose complete genome sequences have been reported were selected for the comparative genome analysis with Bacillus sp. 275. Additionally, the complete genome of B. amyloliquefaciens DSM 7T was also compared with that of Bacillus sp. 275. The COG functional categories of the four complete genome sequences are shown in Table 3. Among the 3832 CDS of Bacillus sp. 275, 3431 CDS were classified into COG categories (Tatusov et al., 2000). The major categories of Bacillus sp. 275 were transcription (K), amino acid transport and metabolism (E), carbohydrate transport and metabolism (G), energy production and conversion (C), cell wall/membrane/envelope biogenesis (M) and inorganic ion transport and metabolism (P). Especially, the number of genes in carbohydrate transport and metabolism (G) related to lignocellulose degradation were 241. This value is similar to that of B. velezensis FZB42, but higher than those of B. siamensis SDLI1 and B. amyloliquefaciens DSM 7T by 9% and 3%, respectively. The genes related to the degradation of lignocellulose were detected in the genome of Bacillus sp. 275 (Table 4). Endoglucanase, β-glucanase, glucohydrolase and glucosidase were found in the complete genome of Bacillus sp. 275. Regarding xylan degradation, glycoside hydrolase 43 family protein (1,4-β-xylosidase), arabinoxylan arabinofuranohydrolase, glucuronoxylanase and α-N-arabinofuranoside were found. The genes encoding deferrochelatase (dye decolorizing peroxidase) and laccase involved in lignin degradation were also observed in the genome of Bacillus sp. 275. In addition, Bacillus sp. 275 has other glycosidases including α-amylase, β-mannosidase, endo-1,4-β-galactanase, 6-phospho-β-galactosidase, arabinan endo-1,5-α-L-arabinosidase and endo-α-(1- > 5)-L-arabinanase. The existence of these genes implies that strain 275 has the potential for utilizing or degrading starch, mannan, galactoside and arabinan. Most of the genes related to the degradation of lignocellulose were highly observed in all strains (Table 4). However, in the genome of B. amyloliquefaciens DSM 7T, endoglucanase, α-amylase and 1,4-β-xylosidase were not found. The distribution of lignocellulose degradationrelated genes between Bacillus sp. 275 and B. siamensis SDLI1 were also somewhat different. In our study, all lignocellulose-degrading genes in Bacillus sp. 275 were found in B. velezensis FZB42. B. velezensis has been mainly studied for its relationship with plant and fungi, because B. velezensis produces antifungal secondary metabolites (Kim et al., 2017). In conclusion, Bacillus sp. 275 has the cellulolytic, xylanolytic and ligninolytic enzyme activities and also has genes encoding lignocellulolytic enzymes. The complete genome information of Bacillus sp. 275 would enhance a better understanding on the degradation of lignocellulose in the genus Bacillus. These results would provide insight
Table 1 Lignocellulolytic enzyme activities in the cell-free supernatant of Bacillus sp. 275 strain. Enzyme feature
Activity (U/g protein)
Cellulasea Xylanaseb Laccasec Peroxidasec
1245.8 ± 7.2 138.4 ± 0.8 1.3 ± 0.3 0.5 ± 0.1
Protein concentration (74.48 ± 0.96 mg/L) in the supernatants was measured using the Bradford protein assay reagent (Bio-Rad, CA, USA). a Cellulase (endo-cellulase) activity was quantified with the Cellulase assay kit (CELLG5 method) (Megazyme, Ireland). b Xylanase activity was measured by the 3,5-dinitrosalicylic acid (DNS) colorimetric method (Teather and Wood, 1982) after adding xylan (10 g/L) as a substrate to the supernatant and reacting at 50 °C for 10 min. c Peroxidase and laccase activities were assayed by the detecting oxidation of (2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) [ABTS] (ε420nm = 3.6 m−1 cm−1) in 100 mM acetate buffer (pH 3.0) at room temperature with or without H2O2. Peroxidase activity was calculated by subtracting the oxidation activity without H2O2 from that with H2O2. One unit of enzyme activity was defined as the amount of enzyme required to release one micromole of product per minute. All experiments were performed in triplicate.
Table 2 Genome features of Bacillus sp. 275 strain. Features
Chromosome
Plasmid
Genome size (bp) G + C content (%) Total number of genes Protein-coding genes (CDS) rRNA genes tRNA genes
4,045,581 46.33 4017 3827 27 86
6389 43.25 6 5 0 0
were observed in the supernatant of the liquid culture broth. The activity of cellulase and xylanase from 74.48 mg/L of extracellular proteins were 1245.8 U/g and 138.4 U/g, respectively. Regarding the lignin degradation, the activities of laccase and peroxidase, which were known to be related to the degradation of lignin or lignin model compounds (Rahmanpour et al., 2016; Roth and Spiess, 2015; Yoshida and Sugano, 2015), were detected. The genome of Bacillus sp. 275 was sequenced using a combination of the PacBio RSII system (Pacific Biosciences, CA, USA), Illumina MiSeq (100-bp paired-end) and the Roche 454 sequencing TITAN technology. All the reads were assembled using the Roche gsAssembler 2.6, CLC Genomics Workbench 6.5.1 and PacBio SMRT Analysis 2.1 with a genome coverage of 276 folds. The complete genome of Bacillus sp. 275 consisted of one 4,045,581 bp chromosome and one 6389 bp plasmid with 3832 protein cording sequences (CDS), 86 tRNA genes, 27 rRNA genes and an average G + C content of 46.32% (Table 2 and Fig. 1). The NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) was used to annotate the genes of Bacillus sp. 275. Strain 275 was initially classified as B. siamensis based on the most hit taxon strain with the 16S rRNA sequence data in the Eztaxon server (http://www.ezbiocloud.net) (Kim et al., 2012) compared to B. siamensis KCTC 13613T with a pairwise similarity of 99.93% (e.g., 1471 bp matching out of 1472 bp). However, the number of mismatching nucleotide between the strain 275 and B. velezensis CR-502T was also only one bp (e. g., 1402 bp matching out of 1403 bp) as same as the B.
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Fig. 1. Circular genome map of Bacillus sp. 275. The circular genome map consists of 5 circles. From the outer circle inward, each circle displays information about the genome of rRNA/tRNA, Reverse CDS, Forward CDS, G + C Ratio and GC Skew.
into bacterial degradation of all components in lignocellulose and possibly a new use for Bacillus sp. 275 in the conversion of lignocellulose into useful chemicals and fuels.
CP019627 (plasmid). The strain is available from the Korean Collection for Type Culture (KCTC) under the accession number KCTC 33887.
Nucleotide sequence and strain accession numbers
Conflict of interest The authors declare no conflict of interest.
The genome sequence of Bacillus sp. 275 was deposited into the GenBank under the accession number CP019626 (chromosome) and
Fig. 2. Phylogenetic tree derived from 16S rRNA gene sequence data of Bacillus sp. 275 and its relatives. The tree was generated by the NeighborJoining method. Evolutionary analyses were conducted in MEGA7 and the distances were computed using the Maximum Composite Likelihood method. Bacillus cereus ATCC 14579 was used as the out group. Only bootstrap values above 60% were shown from 1000 replications. Bar, 0.005 nucleotide substitutions per site.
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National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT (Information & Communication Technology) & Future Planning (2016M3D3A1A01913249). The authors also appreciate the support from the Korea Institute of Energy Technology Evaluation and Planning (Project No. 20143010091880).
Table 3 COG functional categories of the complete genome sequences of Bacillus sp. 275, B. siamensis SDLI1, B. amyloliquefaciens DSM 7T and B. velezensis FZB42. Code
J K L D O M N P T C G E F H I Q S Total
Functional annotation
Strains (number of genes)
Translation, ribosomal structure and biogenesis Transcription Replication, recombination and repair Cell cycle control, cell division, chromosome partitioning Posttranslational modification, protein turnover, chaperones Cell wall/membrane/envelope biogenesis Cell motility Inorganic ion transport and metabolism Signal transduction mechanisms Energy production and conversion Carbohydrate transport and metabolism Amino acid transport and metabolism Nucleotide transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Secondary metabolites biosynthesis, transport and catabolism Function unknown
275
SDLI1
DSM 7T
FZB42
159
163
161
159
261 159 35
255 157 36
271 155 36
261 140 36
100
103
108
102
183
210
206
195
47 181
42 179
45 180
42 188
140 184 241
138 179 222
136 176 235
143 180 244
284 77 102 110 91
284 90 108 110 83
299 84 108 104 67
293 80 111 108 87
1077 3431
1174 3533
1200 3571
1074 3443
References Bandounas, L., Wierckx, N.J., de Winde, J.H., Ruijssenaars, H.J., 2011. Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential. BMC Biotechnol. 11, 94. Beckham, G.T., Johnson, C.W., Karp, E.M., Salvachua, D., Vardon, D.R., 2016. Opportunities and challenges in biological lignin valorization. Curr. Opin. Biotechnol. 42, 40–53. Brown, M.E., Chang, M.C., 2014. Exploring bacterial lignin degradation. Curr. Opin. Chem. Biol. 19, 1–7. Dunlap, C.A., Kim, S.J., Kwon, S.W., Rooney, A.P., 2016. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp plantarum and ‘Bacillus oryzicola' are later heterotypic synonyms of Bacillus velezensis based on phylogenomics. Int. J. Syst. Evol. Microbiol. 66, 1212–1217. Euzéby, J.P., 2017. List of Prokaryotic Names with Standing in Nomenclature. http:// www.bacterio.net/bacillus.html (Accessed on 23 May 2017). Feng, L., Liu, D., Sun, X., Wang, G., Li, M., 2016. Bacillus cavernae sp. nov. isolated from cave soil. Int. J. Syst. Evol. Microbiol. 66, 801–806. Gong, G., Lee, S.M., Woo, H.M., Park, T.H., Um, Y., 2017. Influences of media compositions on characteristics of isolated bacteria exhibiting lignocellulolytic activities from various environmental sites. Appl. Biochem. Biotechnol. http://dx.doi.org/10. 1007/s12010-017-2474-8. Huang, X.F., Santhanam, N., Badri, D.V., Hunter, W.J., Manter, D.K., Decker, S.R., Vivanco, J.M., Reardon, K.F., 2013. Isolation and characterization of lignin-degrading bacteria from rainforest soils. Biotechnol. Bioeng. 110, 1616–1626. Khelil, O., Choubane, S., Cheba, B.A., 2016. Polyphenols content of spent coffee grounds subjected to physico-chemical pretreatments influences lignocellulolytic enzymes production by Bacillus sp. R2. Bioresour. Technol. 211, 769–773. Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H., Yi, H., Won, S., Chun, J., 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62, 716–721. Kim, S.Y., Lee, S.Y., Weon, H.Y., Sang, M.K., Song, J., 2017. Complete genome sequence of Bacillus velezensis M75, a biocontrol agent against fungal plant pathogens, isolated from cotton waste. J. Biotechnol. 241, 112–115. Maki, M.L., Idrees, A., Leung, K.T., Qin, W., 2012. Newly isolated and characterized bacteria with great application potential for decomposition of lignocellulosic biomass. J. Mol. Microbiol. Biotechnol. 22, 156–166. Mathews, S.L., Pawlak, J., Grunden, A.M., 2015. Bacterial biodegradation and bioconversion of industrial lignocellulosic streams. Appl. Microbiol. Biotechnol. 99, 2939–2954. Moreira, L.R., Filho, E.X., 2016. Insights into the mechanism of enzymatic hydrolysis of xylan. Appl. Microbiol. Biotechnol. 100, 5205–5214. Oh, Y.H., Eom, I.Y., Joo, J.C., Yu, J.H., Song, B.K., Lee, S.H., Hong, S.H., Park, S.J., 2015. Recent advances in development of biomass pretreatment technologies used in biorefinery for the production of bio-based fuels, chemicals and polymers. Korean J. Chem. Eng. 32, 1945–1959. Perez, J., Munoz-Dorado, J., de la Rubia, T., Martinez, J., 2002. Biodegradation and biological treatments of cellulose: hemicellulose and lignin: an overview. Int. Microbiol. 5, 53–63. Rahmanpour, R., Rea, D., Jamshidi, S., Fulop, V., Bugg, T.D., 2016. Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds. Arch. Biochem. Biophys. 594, 54–60. Roth, S., Spiess, A.C., 2015. Laccases for biorefinery applications: a critical review on challenges and perspectives. Bioprocess. Biosyst. Eng. 38, 2285–2313. Salvachúa, D., Karp, E.M., Nimlos, C.T., Vardon, D.R., Beckham, G.T., 2015. Towards lignin consolidated bioprocessing: simultaneous lignin depolymerization and product generation by bacteria. Green Chem. 17, 4951–4967. Tatusov, R.L., Galperin, M.Y., Natale, D.A., Koonin, E.V., 2000. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28, 33–36. Teather, R.M., Wood, P.J., 1982. Use of Congo red polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43, 777–780. Yoshida, T., Sugano, Y., 2015. A structural and functional perspective of DyP-type peroxidase family. Arch. Biochem. Biophys. 574, 49–55. Zhu, D., Tanabe, S.H., Xie, C., Honda, D., Sun, J., Ai, L., 2014. Bacillus ligniniphilus sp. nov.: an alkaliphilic and halotolerant bacterium isolated from sediments of the South China Sea. Int. J. Syst. Evol. Microbiol. 64, 1712–1717.
Table 4 Comparison of genes encoding lignocellulose-degrading enzymes in Bacillus sp. 275 and other Bacillus strains. SDLI1
DSM 7T
FZB42
AQP94520 AQP96424 AQP97176 WP_077392024 AQP95497 AQP96452 WP_077392079 AQP98009 AQP97260 AQP95939 AQP95972
O O O O O X O O X X O
X O O O O O O O O O X
O O O O O O O O O O O
AQP94597
O
X
O
AQP94515
O
O
O
WP_077391892 AQP97388 WP_077392066 AQP97416 AQP97396 AQP96449 AQP95141 WP_077391940
O X O O O X O O
O O O O O O O O
O O O O O O O O
AQP96486 AQP94816
O O
O O
O O
Bacillus sp. 275 Predicted function Cellulose-related endoglucanase β-glucanase glucohydrolase glucohydrolase 6-phospho-α-glucosidase 6-phospho-β-glucosidase 6-phospho-β-glucosidase 6-phospho-β-glucosidase α-glucosidase aryl-phospho-β-D-glucosidase α-amylase Hemicellulose-related glycoside hydrolase 43 family protein arabinoxylan arabinofuranohydrolase glucuronoxylanase arabinan endo-1,5-α-L-arabinosidase endo-α-(1- > 5)-L-arabinanase α-N-arabinofuranoside α-N-arabinofuranoside β-mannosidase endo-1,4-β-galactanase 6-phospho-β-galactosidase Lignin-related deferrochelatase laccase
Accession No.
O = detected; X = not detected.
Acknowledgements This research was supported by C1 Gas Refinery Program through the
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