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Complete genome sequence of Pseudomonas sp. strain VLB120 a solvent tolerant, styrene degrading bacterium, isolated from forest soil
Journal of Biotechnology 168 (2013) 729–730
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of Pseudomonas sp. strain VLB120 a solvent tolerant, styrene degrading bacterium, isolated from forest soil夽 Kirsten A.K. Köhler e , Christian Rückert a , Sarah Schatschneider d , Frank-Jörg Vorhölter a , Rafael Szczepanowski a , Lars M. Blank e,1 , Karsten Niehaus d , Alexander Goesmann c , Alfred Pühler b , Jörn Kalinowski a , Andreas Schmid e,∗ a
Technology Platform Genomics, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstr. 27, 33615 Bielefeld, Germany Senior Research Group Genome Research of Industrial Microorganisms, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstr. 27, 33615 Bielefeld, Germany c Bioinformatics Resource Facility, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstr. 27, 33615 Bielefeld, Germany d Proteomics and Metabolomics, Department of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany e Laboratory of Chemical Biotechnology, TU Dortmund University, Emil-Figge-Str. 66, D-44227 Dortmund, Germany b
a r t i c l e
i n f o
Article history: Received 9 September 2013 Accepted 4 October 2013 Available online 24 October 2013
a b s t r a c t Pseudomonas sp. VLB120 was isolated in Stuttgart, Germany, as a styrene degrading organism. The complete genome sequence includes genomic information of solvent tolerance mechanisms, metabolic pathways for various organic compounds, and the megaplasmid pSTY. © 2013 Elsevier B.V. All rights reserved.
Keywords: Pseudomonas VLB120 Solvent tolerance Styrene Biofilm Genome sequence
Pseudomonas sp. VLB120 is a strictly aerobic, Gram negative, rod shaped, motile bacterium that has been assigned to the genus Pseudomonas of the class Gammaproteobacteria, family Pseudomonadaceae. It was first isolated at the University of Stuttgart (Inst. of Microbiology, H.-J. Knackmuss) from forest soil, with styrene as sole carbon and energy source (Panke et al., 1998). Pseudomonas sp. VLB120 can utilize a wide range of carbon sources including short and mid chain length alcohols and can grow in the presence of a second phase of toxic organics solvents such as octanol, toluene, and styrene (Park et al., 2007). The genetic information available hints to an even broader tolerance spectrum as homologues to two well characterized organic solvent efflux pumps are present (TtgABC and TtgGHI, (Rojas et al., 2001)), other
夽 GenBank/EMBL/DDBJ Accession Nr: CP003961 (chromosome), CP003962 (plasmid pSTY). The strain is available from the corresponding author upon request. ∗ Corresponding author at: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Str. 66, D-44227 Dortmund, Germany. Tel.: +49 231 755 7380; fax: +49 231 755 7382. E-mail address: [email protected] (A. Schmid). 1 Present address: iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, Worringerweg 1, D-52074 Aachen, Germany. 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.10.016
short- and long-term tolerance mechanisms, such as alterations of the membrane glycerophospholipid composition are also described (Ruhl et al., 2012). These traits were mainly used for the highly enantiomeric selective epoxidation of styrene to (S)-styrene oxide (ee > 99.8%). This efficient biotransformation using Pseudomonas sp. VLB120C as biocatalyst was described using stirred tank reactors (Park et al., 2007), as well as biofilm reactors (Gross et al., 2010; Halan et al., 2011). Catalytic biofilms have the advantage of natural and strong self-immobilization (Gross et al., 2007). This, together with very low maintenance requirements (Ebert et al., 2011) provides a significant technical advantage over previous whole cell biocatalytic concepts. Pseudomonas sp. VLB120 was also used as donor for genetic material to construct recombinant styrene epoxidation biocatalysts (Panke et al., 1998, 2000, 2002). The Pseudomonas sp. VLB120 genome has been sequenced by a combination of next-generation and Sanger sequencing techniques. Two different sequencing libraries have been prepared for 454 sequencing (3K and 8K paired-ends) and sequenced with two quarters and one quarter of a sequencing plate on a Roche Genome Analyser FLX, respectively. The 454 sequencing yielded a 39.5 fold genome coverage. For validation and homopolymer error
730
K.A.K. Köhler et al. / Journal of Biotechnology 168 (2013) 729–730
Table 1 Genome features of Pseudomonas sp. VLB120. Features
Chromosome
Megaplasmid pSTY
Length (bp) G + C content CDS rRNA genes tRNA genes
5,644,569 61.83% 5117 22 79
321,653 56.77% 365 – –
correction, an additional paired-end library was prepared and sequenced on an Illumina MiSeq, yielding a 40.9 fold coverage. Sequencing data were assembled using Newbler v2.6 to 2 scaffolds and 160 contigs. Genome finishing was done using the software Consed (Gordon, 2003) and 155 Sanger reads were used to generate a complete genome sequence. Annotation was done using the PGAAP pipeline (NCBI, 2010). The Pseudomonas sp. VLB120 genome consists of two replicons, a circular chromosome of 5,644,569 base pairs and the megaplasmid pSTY of 321,653 base pairs in length. The chromosome has a G + C content of 61.83%, whereas the G + C content for the megaplasmid is 56.77%. In total, 5482 protein-coding sequences were predicted, with 5117 located on the chromosome and 365 on the megaplamid. The chromosome contains 22 genes for rRNAs and 79 for tRNAs (Table 1). The rRNAs are organized in 6 gene clusters: 4 operons in the order of 16S-(2 tRNAs)-23S-5S, a fifth operon lacking the tRNA genes, and a rRNA gene cluster organized in the order 16S-(2 tRNAs)-23S-5S-16S-(2 tRNAs)-23S-5S-5S. Acknowledgements The authors thank Yvonne Kutter and Anika Winkler for technical assistance. We acknowledge financial support by the
Ministry of Innovation, Science, Research and Technology of North Rhine-Westphalia (Bio.NRW, Technology Platforms BiocatalysisRedoxCell and PolyOmics). References Ebert, B.E., Kurth, F., Grund, M., Blank, L.M., Schmid, A., 2011. Response of Pseudomonas putida KT2440 to increased NADH and ATP demand. Appl. Environ. Microb. 77, 6597–6605. Gordon, D., 2003. Viewing and editing assembled sequences using Consed. Curr. Protoc. Bioinformatics, Chapter 11: Unit 11.2. Gross, R., Hauer, B., Otto, K., Schmid, A., 2007. Microbial biofilms: new catalysts for maximizing productivity of long-term biotransformations. Biotechnol. Bioeng. 98, 1123–1134. Gross, R., Lang, K., Buhler, K., Schmid, A., 2010. Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis. Biotechnol. Bioeng. 105, 705–717. Halan, B., Schmid, A., Buehler, K., 2011. Real-time solvent tolerance analysis of Pseudomonas sp. strain VLB120C catalytic biofilms. Appl. Environ. Microb. 77, 1563–1571. NCBI, 2010. NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP). http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html Panke, S., Witholt, B., Schmid, A., Wubbolts, M.G., 1998. Towards a biocatalyst for (S)styrene oxide production: characterization of the styrene degradation pathway of Pseudomonas sp. strain VLB120. Appl. Environ. Microb. 64, 2032–2043. Panke, S., Wubbolts, M.G., Schmid, A., Witholt, B., 2000. Production of enantiopure styrene oxide by recombinant Escherichia coli synthesizing a two-component styrene monooxygenase. Biotechnol. Bioeng. 69, 91–100. Panke, S., Held, M., Wubbolts, M.G., Witholt, B., Schmid, A., 2002. Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase. Biotechnol. Bioeng. 80, 33–41. Park, J.B., Buhler, B., Panke, S., Witholt, B., Schmid, A., 2007. Carbon metabolism and product inhibition determine the epoxidation efficiency of solvent-tolerant Pseudomonas sp. strain VLB120C. Biotechnol. Bioeng. 98, 1219–1229. Rojas, A., Duque, E., Mosqueda, G., Golden, G., Hurtado, A., Ramos, J.L., Segura, A., 2001. Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E. J. Bacteriol. 183, 3967–3973. Ruhl, J., Hein, E.M., Hayen, H., Schmid, A., Blank, L.M., 2012. The glycerophospholipid inventory of Pseudomonas putida is conserved between strains and enables growth condition-related alterations. Microb. Biotechnol. 5, 45–58.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of Pseudomonas sp. strain VLB120 a solvent tolerant, styrene degrading bacterium, isolated from forest soil夽 Kirsten A.K. Köhler e , Christian Rückert a , Sarah Schatschneider d , Frank-Jörg Vorhölter a , Rafael Szczepanowski a , Lars M. Blank e,1 , Karsten Niehaus d , Alexander Goesmann c , Alfred Pühler b , Jörn Kalinowski a , Andreas Schmid e,∗ a
Technology Platform Genomics, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstr. 27, 33615 Bielefeld, Germany Senior Research Group Genome Research of Industrial Microorganisms, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstr. 27, 33615 Bielefeld, Germany c Bioinformatics Resource Facility, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstr. 27, 33615 Bielefeld, Germany d Proteomics and Metabolomics, Department of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany e Laboratory of Chemical Biotechnology, TU Dortmund University, Emil-Figge-Str. 66, D-44227 Dortmund, Germany b
a r t i c l e
i n f o
Article history: Received 9 September 2013 Accepted 4 October 2013 Available online 24 October 2013
a b s t r a c t Pseudomonas sp. VLB120 was isolated in Stuttgart, Germany, as a styrene degrading organism. The complete genome sequence includes genomic information of solvent tolerance mechanisms, metabolic pathways for various organic compounds, and the megaplasmid pSTY. © 2013 Elsevier B.V. All rights reserved.
Keywords: Pseudomonas VLB120 Solvent tolerance Styrene Biofilm Genome sequence
Pseudomonas sp. VLB120 is a strictly aerobic, Gram negative, rod shaped, motile bacterium that has been assigned to the genus Pseudomonas of the class Gammaproteobacteria, family Pseudomonadaceae. It was first isolated at the University of Stuttgart (Inst. of Microbiology, H.-J. Knackmuss) from forest soil, with styrene as sole carbon and energy source (Panke et al., 1998). Pseudomonas sp. VLB120 can utilize a wide range of carbon sources including short and mid chain length alcohols and can grow in the presence of a second phase of toxic organics solvents such as octanol, toluene, and styrene (Park et al., 2007). The genetic information available hints to an even broader tolerance spectrum as homologues to two well characterized organic solvent efflux pumps are present (TtgABC and TtgGHI, (Rojas et al., 2001)), other
夽 GenBank/EMBL/DDBJ Accession Nr: CP003961 (chromosome), CP003962 (plasmid pSTY). The strain is available from the corresponding author upon request. ∗ Corresponding author at: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Str. 66, D-44227 Dortmund, Germany. Tel.: +49 231 755 7380; fax: +49 231 755 7382. E-mail address: [email protected] (A. Schmid). 1 Present address: iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, Worringerweg 1, D-52074 Aachen, Germany. 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.10.016
short- and long-term tolerance mechanisms, such as alterations of the membrane glycerophospholipid composition are also described (Ruhl et al., 2012). These traits were mainly used for the highly enantiomeric selective epoxidation of styrene to (S)-styrene oxide (ee > 99.8%). This efficient biotransformation using Pseudomonas sp. VLB120C as biocatalyst was described using stirred tank reactors (Park et al., 2007), as well as biofilm reactors (Gross et al., 2010; Halan et al., 2011). Catalytic biofilms have the advantage of natural and strong self-immobilization (Gross et al., 2007). This, together with very low maintenance requirements (Ebert et al., 2011) provides a significant technical advantage over previous whole cell biocatalytic concepts. Pseudomonas sp. VLB120 was also used as donor for genetic material to construct recombinant styrene epoxidation biocatalysts (Panke et al., 1998, 2000, 2002). The Pseudomonas sp. VLB120 genome has been sequenced by a combination of next-generation and Sanger sequencing techniques. Two different sequencing libraries have been prepared for 454 sequencing (3K and 8K paired-ends) and sequenced with two quarters and one quarter of a sequencing plate on a Roche Genome Analyser FLX, respectively. The 454 sequencing yielded a 39.5 fold genome coverage. For validation and homopolymer error
730
K.A.K. Köhler et al. / Journal of Biotechnology 168 (2013) 729–730
Table 1 Genome features of Pseudomonas sp. VLB120. Features
Chromosome
Megaplasmid pSTY
Length (bp) G + C content CDS rRNA genes tRNA genes
5,644,569 61.83% 5117 22 79
321,653 56.77% 365 – –
correction, an additional paired-end library was prepared and sequenced on an Illumina MiSeq, yielding a 40.9 fold coverage. Sequencing data were assembled using Newbler v2.6 to 2 scaffolds and 160 contigs. Genome finishing was done using the software Consed (Gordon, 2003) and 155 Sanger reads were used to generate a complete genome sequence. Annotation was done using the PGAAP pipeline (NCBI, 2010). The Pseudomonas sp. VLB120 genome consists of two replicons, a circular chromosome of 5,644,569 base pairs and the megaplasmid pSTY of 321,653 base pairs in length. The chromosome has a G + C content of 61.83%, whereas the G + C content for the megaplasmid is 56.77%. In total, 5482 protein-coding sequences were predicted, with 5117 located on the chromosome and 365 on the megaplamid. The chromosome contains 22 genes for rRNAs and 79 for tRNAs (Table 1). The rRNAs are organized in 6 gene clusters: 4 operons in the order of 16S-(2 tRNAs)-23S-5S, a fifth operon lacking the tRNA genes, and a rRNA gene cluster organized in the order 16S-(2 tRNAs)-23S-5S-16S-(2 tRNAs)-23S-5S-5S. Acknowledgements The authors thank Yvonne Kutter and Anika Winkler for technical assistance. We acknowledge financial support by the
Ministry of Innovation, Science, Research and Technology of North Rhine-Westphalia (Bio.NRW, Technology Platforms BiocatalysisRedoxCell and PolyOmics). References Ebert, B.E., Kurth, F., Grund, M., Blank, L.M., Schmid, A., 2011. Response of Pseudomonas putida KT2440 to increased NADH and ATP demand. Appl. Environ. Microb. 77, 6597–6605. Gordon, D., 2003. Viewing and editing assembled sequences using Consed. Curr. Protoc. Bioinformatics, Chapter 11: Unit 11.2. Gross, R., Hauer, B., Otto, K., Schmid, A., 2007. Microbial biofilms: new catalysts for maximizing productivity of long-term biotransformations. Biotechnol. Bioeng. 98, 1123–1134. Gross, R., Lang, K., Buhler, K., Schmid, A., 2010. Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis. Biotechnol. Bioeng. 105, 705–717. Halan, B., Schmid, A., Buehler, K., 2011. Real-time solvent tolerance analysis of Pseudomonas sp. strain VLB120C catalytic biofilms. Appl. Environ. Microb. 77, 1563–1571. NCBI, 2010. NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP). http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html Panke, S., Witholt, B., Schmid, A., Wubbolts, M.G., 1998. Towards a biocatalyst for (S)styrene oxide production: characterization of the styrene degradation pathway of Pseudomonas sp. strain VLB120. Appl. Environ. Microb. 64, 2032–2043. Panke, S., Wubbolts, M.G., Schmid, A., Witholt, B., 2000. Production of enantiopure styrene oxide by recombinant Escherichia coli synthesizing a two-component styrene monooxygenase. Biotechnol. Bioeng. 69, 91–100. Panke, S., Held, M., Wubbolts, M.G., Witholt, B., Schmid, A., 2002. Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase. Biotechnol. Bioeng. 80, 33–41. Park, J.B., Buhler, B., Panke, S., Witholt, B., Schmid, A., 2007. Carbon metabolism and product inhibition determine the epoxidation efficiency of solvent-tolerant Pseudomonas sp. strain VLB120C. Biotechnol. Bioeng. 98, 1219–1229. Rojas, A., Duque, E., Mosqueda, G., Golden, G., Hurtado, A., Ramos, J.L., Segura, A., 2001. Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E. J. Bacteriol. 183, 3967–3973. Ruhl, J., Hein, E.M., Hayen, H., Schmid, A., Blank, L.M., 2012. The glycerophospholipid inventory of Pseudomonas putida is conserved between strains and enables growth condition-related alterations. Microb. Biotechnol. 5, 45–58.