Journal of Biotechnology 204 (2015) 3–4
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of the lipase producing strain Burkholderia glumae PG1 Sonja Voget a,1 , Andreas Knapp b,1 , Anja Poehlein a , Christel Vollstedt c , Wolfgang Streit c , Rolf Daniel a , Karl-Erich Jaeger b,∗ a Department of Genomic and Applied Microbiology and Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, Georg-August University Göttingen, Germany b Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf and Forschungszentrum Jülich GmbH, Germany c Department of Microbiology and Biotechnology, Biocenter Klein Flottbek, University of Hamburg, Germany
a r t i c l e
i n f o
Article history: Received 25 March 2015 Accepted 27 March 2015 Available online 4 April 2015 Keywords: Lipase Rhamnolipid Burkholderia glumae Genome
a b s t r a c t The Gram-negative proteobacterium Burkholderia glumae PG1 produces a lipase of biotechnological interest, which is used for the production of enantiopure pharmaceuticals. In order to better understand the underlying mechanisms and provide a basis for further studies, we present here the complete genome sequence of B. glumae PG1. © 2015 Elsevier B.V. All rights reserved.
Members of the genus Burkholderia are widely distributed, covering diverse ecological niches (Coenye and Vandamme, 2003; Compant et al., 2008). Two major phylogenetic clusters can be distinguished (Suárez-Moreno et al., 2012; Ussery et al., 2009): the non-pathogenic PBE (Plant-Beneficial-Environmental) cluster and the pathogenic cluster, which comprises the Burkholderia cepacia complex (BCC), the Burkholderia pseudomallei/thailandensis/mallei (Bptm) group and the phytopathogenic subgroup. Burkholderia glumae PG1 (formerly Pseudomonas glumae PG1) is a member of the phytopathogenic subgroup and all B. glumae strains identified so far cause panicle blight of rice, which is an increasing worldwide problem for rice production (Ham et al., 2011). Bacterial lipases are an important group of biotechnological relevant enzymes in the food, pharmaceutical, dairy and agrochemical industries (Anobom et al., 2014; Jaeger and Eggert, 2002). B. glumae PG1 produces the extracellular lipase LipA which is used by the company BASF SE for the production of enantiopure pharmaceuticals (Boekema et al., 2007; Frenken et al., 1992). As an alternative to the opportunistic human pathogen Pseudomonas aeruginosa whose lipase production has been extensively studied (Rosenau and Jaeger, 2000), B. glumae PG1 is regarded as suitable
∗ Corresponding author. Tel.: +49 2461 613716. E-mail address: [email protected] (K.-E. Jaeger). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.jbiotec.2015.03.022 0168-1656/© 2015 Elsevier B.V. All rights reserved.
for bulk enzyme production. Here, we report the complete genome sequence of B. glumae PG1. Genomic DNA of B. glumae PG1 was isolated with the MasterPure DNA purification kit (Epicentre, Madison, USA). The extracted DNA was used to construct whole genome shotgun fosmid and plasmid libraries. All inserts were sequenced using ABI3730xl Sequencers and BigDye Terminator v3.1 chemistry (Life Technologies, Darmstadt, Germany). Approximately 90,000 generated sequences were assembled into contigs with the Phrap assembly tool (http://www.phrap.org). Primer walking on plasmids, fosmid clones and PCR based techniques were used to close remaining gaps and to solve misassembled regions caused by the high number of repetitive sequences. All manual editing steps were performed using the GAP4 software package v4.6 (Staden, 1996). The finished complete genome of B. glumae PG1 consists of 7,896,538 bp, and is organized in two replicons of 4,163,767 bp (chromosome 1) and 3,732,771 bp (chromosome 2) (Table 1). Coding sequences (CDS) and open reading frames (ORFs) were predicted with YACOP (Tech and Merkl, 2003) and manually curated. The IMG/ER system (Markowitz et al., 2012) was used for automatic annotation followed by manual inspection. In total 6502 protein coding genes were predicted (Table 1) including the lipAB operon coding for the lipase LipA and the lipase-specific foldase LipB. Secretion of enzymes by Gram-negative bacteria requires complex transport systems. The genome of B. glumae PG1 contains the genes encoding a Sec-translocase complex as well as a type
4
S. Voget et al. / Journal of Biotechnology 204 (2015) 3–4
Table 1 Genome features of B. glumae PG1.
supported by the Bundesministerium für Bildung und Forschung (BMBF).
Genome features Length (bp) Chromosome Plasmids G + C content (%) Total genes Protein-coding genes rRNA tRNA Protein coding genes with function prediction
7,896,538 2 0 68.77 6583 6502 15 66 5656
II secretion system (T2SS). Moreover, a second T2SS is encoded on chromosome 2 which, however, lacks the outer membrane component secretin. In addition, the B. glumae PG1 genome harbours the genes needed for the synthesis of rhamnolipids, an important class of biosurfactants used in the food, cosmetic, pharmaceutical and chemical industries (Müller et al., 2012). Thus, it is obvious that the genome sequence of B. glumae PG1 can serve as the basis for further studies to elucidate its regulatory networks and their biotechnological relevance. Nucleotide sequence accession numbers The bacterial strain B. glumae PG1 is deposited as strain no. CBS 322.89 at the Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands. The closed genome sequence has been deposited at the DDBJ/EMBL/GenBank under the accession CP002580 (chromosome 1) and CP002581 (chromosome 2). Acknowledgments Work in the laboratory of Karl-Erich Jaeger was funded by the Deutsche Forschungsgemeinschaft (DFG) through the Excellence Cluster EXC 1028. The work in the laboratory of Rolf Daniel was
References Anobom, C.D., Pinheiro, A.S., De-Andrade, R.A., Aguieiras, E.C.G., Andrade, G.C., Moura, M.V., Almeida, R.V., Freire, D.M., 2014. From structure to catalysis: recent developments in the biotechnological applications of lipases. Biomed. Res. Int. 2014, 684506, http://dx.doi.org/10.1155/2014/684506. Boekema, B.K.H.L., Beselin, A., Breuer, M., Hauer, B., Koster, M., Rosenau, F., Jaeger, K.-E., Tommassen, J., 2007. Hexadecane and Tween 80 stimulate lipase production in Burkholderia glumae by different mechanisms. Appl. Environ. Microbiol. 73, 3838–3844, http://dx.doi.org/10.1128/AEM.00097-07. Coenye, T., Vandamme, P., 2003. Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ. Microbiol. 5, 719–729. Compant, S., Nowak, J., Coenye, T., Clément, C., Ait Barka, E., 2008. Diversity and occurrence of Burkholderia spp. in the natural environment. FEMS Microbiol. Rev. 32, 607–626, http://dx.doi.org/10.1111/j.1574-6976.2008.00113.x. Frenken, L.G., Egmond, M.R., Batenburg, A.M., Bos, J.W., Visser, C., Verrips, C.T., 1992. Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues. Appl. Environ. Microbiol. 58, 3787–3791. Ham, J.H., Melanson, R.A., Rush, M.C., 2011. Burkholderia glumae: next major pathogen of rice? Mol. Plant Pathol. 12, 329–339, http://dx.doi.org/10.1111/ j. 1364-3703.2010.00676.x. Jaeger, K.-E., Eggert, T., 2002. Lipases for biotechnology. Curr. Opin. Biotechnol. 13, 390–397. Markowitz, V.M., Chen, I.-M.A., Palaniappan, K., Chu, K., Szeto, E., Grechkin, Y., Ratner, A., Jacob, B., Huang, J., Williams, P., Huntemann, M., Anderson, I., Mavromatis, K., Ivanova, N.N., Kyrpides, N.C., 2012. IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 40, D115–D122, http://dx.doi.org/10.1093/nar/gkr1044. Müller, M.M., Kügler, J.H., Henkel, M., Gerlitzki, M., Hörmann, B., Pöhnlein, M., Syldatk, C., Hausmann, R., 2012. Rhamnolipids – next generation surfactants? J. Biotechnol. 162, 366–380, http://dx.doi.org/10.1016/j.jbiotec.2012.05.022. Rosenau, F., Jaeger, K., 2000. Bacterial lipases from Pseudomonas: regulation of gene expression and mechanisms of secretion. Biochimie 82, 1023–1032. Staden, R., 1996. The Staden sequence analysis package. Mol. Biotechnol. 5, 233–241. Suárez-Moreno, Z.R., Caballero-Mellado, J., Coutinho, B.G., Mendonc¸a-Previato, L., James, E.K., Venturi, V., 2012. Common features of environmental and potentially beneficial plant-associated Burkholderia. Microb. Ecol. 63, 249–266, http://dx.doi.org/10.1007/s00248-011-9929-1. Tech, M., Merkl, R., 2003. YACOP: enhanced gene prediction obtained by a combination of existing methods. In Silico Biol. 3, 441–451. Ussery, D., Kiil, K., Lagesen, K., 2009. The genus Burkholderia: analysis of 56 genomic sequences. In: de Reuse, H., Bereswill, S. (Eds.), Genome Dynamics, vol. 6: Microbial Pathogenomics. , pp. 140–157, http://dx.doi.org/10.1159/000235768.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of the lipase producing strain Burkholderia glumae PG1 Sonja Voget a,1 , Andreas Knapp b,1 , Anja Poehlein a , Christel Vollstedt c , Wolfgang Streit c , Rolf Daniel a , Karl-Erich Jaeger b,∗ a Department of Genomic and Applied Microbiology and Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, Georg-August University Göttingen, Germany b Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf and Forschungszentrum Jülich GmbH, Germany c Department of Microbiology and Biotechnology, Biocenter Klein Flottbek, University of Hamburg, Germany
a r t i c l e
i n f o
Article history: Received 25 March 2015 Accepted 27 March 2015 Available online 4 April 2015 Keywords: Lipase Rhamnolipid Burkholderia glumae Genome
a b s t r a c t The Gram-negative proteobacterium Burkholderia glumae PG1 produces a lipase of biotechnological interest, which is used for the production of enantiopure pharmaceuticals. In order to better understand the underlying mechanisms and provide a basis for further studies, we present here the complete genome sequence of B. glumae PG1. © 2015 Elsevier B.V. All rights reserved.
Members of the genus Burkholderia are widely distributed, covering diverse ecological niches (Coenye and Vandamme, 2003; Compant et al., 2008). Two major phylogenetic clusters can be distinguished (Suárez-Moreno et al., 2012; Ussery et al., 2009): the non-pathogenic PBE (Plant-Beneficial-Environmental) cluster and the pathogenic cluster, which comprises the Burkholderia cepacia complex (BCC), the Burkholderia pseudomallei/thailandensis/mallei (Bptm) group and the phytopathogenic subgroup. Burkholderia glumae PG1 (formerly Pseudomonas glumae PG1) is a member of the phytopathogenic subgroup and all B. glumae strains identified so far cause panicle blight of rice, which is an increasing worldwide problem for rice production (Ham et al., 2011). Bacterial lipases are an important group of biotechnological relevant enzymes in the food, pharmaceutical, dairy and agrochemical industries (Anobom et al., 2014; Jaeger and Eggert, 2002). B. glumae PG1 produces the extracellular lipase LipA which is used by the company BASF SE for the production of enantiopure pharmaceuticals (Boekema et al., 2007; Frenken et al., 1992). As an alternative to the opportunistic human pathogen Pseudomonas aeruginosa whose lipase production has been extensively studied (Rosenau and Jaeger, 2000), B. glumae PG1 is regarded as suitable
∗ Corresponding author. Tel.: +49 2461 613716. E-mail address: [email protected] (K.-E. Jaeger). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.jbiotec.2015.03.022 0168-1656/© 2015 Elsevier B.V. All rights reserved.
for bulk enzyme production. Here, we report the complete genome sequence of B. glumae PG1. Genomic DNA of B. glumae PG1 was isolated with the MasterPure DNA purification kit (Epicentre, Madison, USA). The extracted DNA was used to construct whole genome shotgun fosmid and plasmid libraries. All inserts were sequenced using ABI3730xl Sequencers and BigDye Terminator v3.1 chemistry (Life Technologies, Darmstadt, Germany). Approximately 90,000 generated sequences were assembled into contigs with the Phrap assembly tool (http://www.phrap.org). Primer walking on plasmids, fosmid clones and PCR based techniques were used to close remaining gaps and to solve misassembled regions caused by the high number of repetitive sequences. All manual editing steps were performed using the GAP4 software package v4.6 (Staden, 1996). The finished complete genome of B. glumae PG1 consists of 7,896,538 bp, and is organized in two replicons of 4,163,767 bp (chromosome 1) and 3,732,771 bp (chromosome 2) (Table 1). Coding sequences (CDS) and open reading frames (ORFs) were predicted with YACOP (Tech and Merkl, 2003) and manually curated. The IMG/ER system (Markowitz et al., 2012) was used for automatic annotation followed by manual inspection. In total 6502 protein coding genes were predicted (Table 1) including the lipAB operon coding for the lipase LipA and the lipase-specific foldase LipB. Secretion of enzymes by Gram-negative bacteria requires complex transport systems. The genome of B. glumae PG1 contains the genes encoding a Sec-translocase complex as well as a type
4
S. Voget et al. / Journal of Biotechnology 204 (2015) 3–4
Table 1 Genome features of B. glumae PG1.
supported by the Bundesministerium für Bildung und Forschung (BMBF).
Genome features Length (bp) Chromosome Plasmids G + C content (%) Total genes Protein-coding genes rRNA tRNA Protein coding genes with function prediction
7,896,538 2 0 68.77 6583 6502 15 66 5656
II secretion system (T2SS). Moreover, a second T2SS is encoded on chromosome 2 which, however, lacks the outer membrane component secretin. In addition, the B. glumae PG1 genome harbours the genes needed for the synthesis of rhamnolipids, an important class of biosurfactants used in the food, cosmetic, pharmaceutical and chemical industries (Müller et al., 2012). Thus, it is obvious that the genome sequence of B. glumae PG1 can serve as the basis for further studies to elucidate its regulatory networks and their biotechnological relevance. Nucleotide sequence accession numbers The bacterial strain B. glumae PG1 is deposited as strain no. CBS 322.89 at the Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands. The closed genome sequence has been deposited at the DDBJ/EMBL/GenBank under the accession CP002580 (chromosome 1) and CP002581 (chromosome 2). Acknowledgments Work in the laboratory of Karl-Erich Jaeger was funded by the Deutsche Forschungsgemeinschaft (DFG) through the Excellence Cluster EXC 1028. The work in the laboratory of Rolf Daniel was
References Anobom, C.D., Pinheiro, A.S., De-Andrade, R.A., Aguieiras, E.C.G., Andrade, G.C., Moura, M.V., Almeida, R.V., Freire, D.M., 2014. From structure to catalysis: recent developments in the biotechnological applications of lipases. Biomed. Res. Int. 2014, 684506, http://dx.doi.org/10.1155/2014/684506. Boekema, B.K.H.L., Beselin, A., Breuer, M., Hauer, B., Koster, M., Rosenau, F., Jaeger, K.-E., Tommassen, J., 2007. Hexadecane and Tween 80 stimulate lipase production in Burkholderia glumae by different mechanisms. Appl. Environ. Microbiol. 73, 3838–3844, http://dx.doi.org/10.1128/AEM.00097-07. Coenye, T., Vandamme, P., 2003. Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ. Microbiol. 5, 719–729. Compant, S., Nowak, J., Coenye, T., Clément, C., Ait Barka, E., 2008. Diversity and occurrence of Burkholderia spp. in the natural environment. FEMS Microbiol. Rev. 32, 607–626, http://dx.doi.org/10.1111/j.1574-6976.2008.00113.x. Frenken, L.G., Egmond, M.R., Batenburg, A.M., Bos, J.W., Visser, C., Verrips, C.T., 1992. Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues. Appl. Environ. Microbiol. 58, 3787–3791. Ham, J.H., Melanson, R.A., Rush, M.C., 2011. Burkholderia glumae: next major pathogen of rice? Mol. Plant Pathol. 12, 329–339, http://dx.doi.org/10.1111/ j. 1364-3703.2010.00676.x. Jaeger, K.-E., Eggert, T., 2002. Lipases for biotechnology. Curr. Opin. Biotechnol. 13, 390–397. Markowitz, V.M., Chen, I.-M.A., Palaniappan, K., Chu, K., Szeto, E., Grechkin, Y., Ratner, A., Jacob, B., Huang, J., Williams, P., Huntemann, M., Anderson, I., Mavromatis, K., Ivanova, N.N., Kyrpides, N.C., 2012. IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 40, D115–D122, http://dx.doi.org/10.1093/nar/gkr1044. Müller, M.M., Kügler, J.H., Henkel, M., Gerlitzki, M., Hörmann, B., Pöhnlein, M., Syldatk, C., Hausmann, R., 2012. Rhamnolipids – next generation surfactants? J. Biotechnol. 162, 366–380, http://dx.doi.org/10.1016/j.jbiotec.2012.05.022. Rosenau, F., Jaeger, K., 2000. Bacterial lipases from Pseudomonas: regulation of gene expression and mechanisms of secretion. Biochimie 82, 1023–1032. Staden, R., 1996. The Staden sequence analysis package. Mol. Biotechnol. 5, 233–241. Suárez-Moreno, Z.R., Caballero-Mellado, J., Coutinho, B.G., Mendonc¸a-Previato, L., James, E.K., Venturi, V., 2012. Common features of environmental and potentially beneficial plant-associated Burkholderia. Microb. Ecol. 63, 249–266, http://dx.doi.org/10.1007/s00248-011-9929-1. Tech, M., Merkl, R., 2003. YACOP: enhanced gene prediction obtained by a combination of existing methods. In Silico Biol. 3, 441–451. Ussery, D., Kiil, K., Lagesen, K., 2009. The genus Burkholderia: analysis of 56 genomic sequences. In: de Reuse, H., Bereswill, S. (Eds.), Genome Dynamics, vol. 6: Microbial Pathogenomics. , pp. 140–157, http://dx.doi.org/10.1159/000235768.
