ABSTRACT
          
            The bacterium
            Nocardia terpenica
            IFM 0406 is known as the producer of the immunosuppressant brasilicardin A. Here, we report the completely sequenced genome of strain IFM 0406, which facilitates the heterologous expression of the brasilicardin biosynthetic gene cluster but also unveils the intriguing biosynthetic capacity of the strain to produce secondary metabolites.
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Botas Muñoz, Alma María

Buchmann, Anina

Eitel, Michael

Fernández Cortés, Jesús

Gross, Harald S.

Koch, Pierre

Krawiec, Michal

Morís Varas, Francisco

Méndez Fernández, María del Carmen

Núñez González, Luz Elena

Schwarz, Paul N.

Stegmann, Evi

Wohlleben, Wolfgang

Wolanski, Marcin

Zakrzewska Czerwinska, Jolanta

PubMed

This work, including the efforts of Carmen Méndez, was funded by Ministerio de Economía y Competitividad (This work, including the efforts of Carmen Mendez, was funded by Ministerio de Economía y Competitividad (MINECO) (PCIN-2014-066). This work, including the efforts of Francisco Morris, was funded by Ministerio de Economía y Competitividad (MINECO) (PCIN-2014-097). This work, including the efforts of Harald Gross and Pierre Koch, was funded by Bundesministerium für Bildung und Forschung (BMBF) (FKZ 031A568A). This work, including the efforts of Wolfgang Wohlleben, was funded by Bundesministerium für Bildung und Forschung (BMBF) (FKZ 031A568B). This work, including the efforts of Jolanta Zakrzewska-Czerwinska, was funded by MNiSW | Narodowe Centrum Badani Rozwoju (NCBR) (ERA-NET-IB/NeBrasCa/10/2015).). This work, including the efforts of Francisco Morris, was funded by Ministerio de Economía y Competitividad (MINECO) (PCIN-2014-097). This work, including the efforts of Harald Gross and Pierre Koch, was funded by Bundesministerium für Bildung und Forschung (BMBF) (FKZ 031A568A). This work, including the efforts of Wolfgang Wohlleben, was funded by Bundesministerium für Bildung und Forschung (BMBF) (FKZ 031A568B). This work, including the efforts of Jolanta Zakrzewska-Czerwinska, was funded by MNiSW | Narodowe Centrum Badan´ i Rozwoju (NCBR) (ERA-NET-IB/NeBrasCa/10/2015)

Repositorio Institucional de la Universidad de Oviedo

High-Quality Draft Genome Sequence of the ActinobacteriumNocardia terpenica IFM 0406, Producer of the ImmunosuppressantBrasilicardins, Using Illumina and PacBio TechnologiesAnina Buchmann,a,b Michael Eitel,c Pierre Koch,c Paul N. Schwarz,d Evi Stegmann,b,d Wolfgang Wohlleben,b,d Marcin Wolan´ski,eMichał Krawiec,e Jolanta Zakrzewska-Czerwin´ska,e,f Carmen Méndez,g Alma Botas,g Luz Elena Núñez,h Francisco Morís,hJesus Cortés,h Harald Grossa,bDepartment of Pharmaceutical Biology, Institute of Pharmaceutical Sciences, University of Tübingen, Tübingen, Germanya; German Centre for Infection Research (DZIF),Partner Site Tübingen, Tübingen, Germanyb; Department of Medicinal and Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, University of Tübingen,Tübingen, Germanyc; Department of Microbiology and Biotechnology, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen,Germanyd; Faculty of Biotechnology, University of Wrocław, Wrocław, Polande; Ludwik Hirszfeld Insitute of Immunology and Experimental Therapy, Polish Academy ofSciences, Wrocław, Polandf; Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Oviedo, Spaing;EntreChem S.L., Oviedo, SpainhThe bacteriumNocardia terpenica IFM 0406 is known as the producer of the immunosuppressant brasilicardin A. Here, we re-port the completely sequenced genome of strain IFM 0406, which facilitates the heterologous expression of the brasilicardin bio-synthetic gene cluster but also unveils the intriguing biosynthetic capacity of the strain to produce secondary metabolites.Received 17 October 2016 Accepted 21 October 2016 Published 15 December 2016Citation Buchmann A, Eitel M, Koch P, Schwarz PN, Stegmann E, Wohlleben W, Wolan´ski M, Krawiec M, Zakrzewska-Czerwin´ska J, Méndez C, Botas A, Núñez LE, Morís F, CortésJ, Gross H. 2016. High-quality draft genome sequence of the actinobacterium Nocardia terpenica IFM 0406, producer of the immunosuppressant brasilicardins, using Illuminaand PacBio technologies. Genome Announc 4(6):e01391-16 doi:10.1128/genomeA.01391-16.Copyright © 2016 Buchmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.Address correspondence to Harald Gross, harald.gross@uni-tuebingen.de.Actinobacteria of the genus Nocardia are often found to behuman and animal pathogens that cause pulmonary, cutane-ous and subcutaneous diseases (1–3). However, species in thisgenus also produce a plethora of potent bioactive natural com-pounds with therapeutic potential. For example, Nocardia ter-penica IFM 0406 (formerly referred to as Nocardia brasiliensis IFM0406) is known to produce the antifungal and immunosuppres-sant brasilinolides A–C (4, 5) and brasilicardins AD (6–9). Theimmunosuppressant brasilicardin A is of particular interest be-cause its potency is superior to today’s standard immunosuppres-sive drugs for organ transplantation, but it is less toxic, since itmediates its activity by a different mode of action (10). The devel-opment of this promising lead structure is currently hampereddue to supply issues. The producer strain only shows a low pro-duction titer and is furthermore categorized as a biosafety-level 2organism. Both of these facts make the fermentative production ofbrasilicardin A expensive and elaborate. Due to its complex stere-ochemistry, a total synthesis has not been achieved to date (11–13). Therefore, we want to overcome this hurdle by heterologousexpression of the corresponding biosynthetic gene cluster (14) in anonpathogenic producer strain. In order to obtain the full biosyn-thetic pathway, including the related precursor metabolic path-ways, we sequenced the genome of N. terpenica IFM 0406.Genome sequencing was carried out using the Illumina HiSeq2500 and Pacific Biosciences RS II sequencing platforms.Genomic DNA was isolated (ZR fungal/bacterial DNA mini prepkit, Zymo Research) and a paired-end library with bar coding wasconstructed (Nextera DNA sample preparation kit). The HiSeqpaired-end data were processed and filtered, and a quality analysiswas performed. This resulted in 6,564,821 reads with an averagelength of 509 bp (approx. 360 coverage). The quality of theIllumina FASTQ sequences was enhanced by trimming off low-quality bases, which was followed by de novo assembly using CLCGenomics Workbench version 7.0.4 to yield contigs. The optimalk-mer size was determined using KmerGenie (15). In addition, a10-kb genomic sublibrary of strain IFM 0406 was produced andsequenced with PacBioSMRT technology. The resultant continu-ous long-read (CLR) data were filtered using the SMRT Analysissoftware suite. In summary, one run produced 113,652 filteredreads with average read lengths of 3,262 bp (approx. 40 cover-age). The contigs were linked and placed into super-scaffoldsbased on the alignment of the PacBio CLR reads using BLASR (16)and SSPACE-LongRead scaffolder version 7.0.4 (17). The gappedregions within the super-scaffolds were closed using GapFillerversion 1.10 (18).The final gap-closed genome comprises three scaffolds with atotal size of 9,276,856 bp and a GC content of 68.5%. The ge-nome contains 8,755 open reading frames, including 76 tRNAgenes and nine rRNA operons. Bioinformatic analyses using anti-SMASH version 3.0 (19) revealed that IFM 0406 possessed, besidesthe putative brasilicardin (14) and brasilinolide (20) gene cluster, 47further orphan natural product biosynthesis gene clusters. Insightinto the genome of strain IFM 0406 not only reveals a high potentialto produce secondary metabolites, but will also enable and facilitatethe heterologous expression and biotechnological production of bra-silicardins in a nonpathogenic host system.Accession number(s). This genome sequence has been de-posited in EMBL/GenBank under the accession numbercrossmarkGenome AnnouncementsNovember/December 2016 Volume 4 Issue 6 e01391-16 genomea.asm.org 1 on March 30, 2017 by UOV BIBLIOTECA UNIVERSITARIAhttp://genomea.asm.org/Downloaded from LWGR00000000. The version described in this paper is the firstversion.ACKNOWLEDGMENTWe thank BaseClear BV (Leiden, Netherlands) for sequencing the Nocar-dia terpenica genome, especially Walter Pirovano and his team for helpfuladvice.FUNDING INFORMATIONThis work, including the efforts of Carmen Mendez, was funded byMinisterio de Economía y Competitividad (MINECO) (PCIN-2014-066). This work, including the efforts of Francisco Morris, wasfunded by Ministerio de Economía y Competitividad (MINECO)(PCIN-2014-097). This work, including the efforts of Harald Grossand Pierre Koch, was funded by Bundesministerium für Bildung undForschung (BMBF) (FKZ 031A568A). This work, including the effortsof Wolfgang Wohlleben, was funded by Bundesministerium fürBildung und Forschung (BMBF) (FKZ 031A568B). This work,including the efforts of Jolanta Zakrzewska-Czerwin´ska, was fundedby MNiSW | Narodowe Centrum Badan´ i Rozwoju (NCBR)(ERA-NET-IB/NeBrasCa/10/2015).REFERENCES1. Wilson JW. 2012. Nocardiosis: updates and clinical overview. Mayo ClinProc 87:403– 407. http://dx.doi.org/10.1016/j.mayocp.2011.11.016.2. Xia L, Cai J, Wang B, Huang Y, Jian J, Lu Y. 2015. Draft genomesequence of Nocardia seriolae ZJ0503, a fish pathogen isolated fromTrachinotus ovatus in China. Genome Announc 3(1):e01223-14. http://dx.doi.org/10.1128/genomeA.01223-14.3. Tan CK, Lai CC, Lin SH, Liao CH, Chou CH, Hsu HL, Huang YT,Hsueh PR. 2010. Clinical and microbiological characteristics of nocardi-osis including those caused by emerging Nocardia species in Taiwan,1998 –2008. Clin Microbiol Infect 16:966 –972. http://dx.doi.org/10.1111/j.1469-0691.2009.02950.x.4. 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Genome Announcements

High-quality draft genome sequence of the actinobacterium Nocardia terpenica IFM 0406, producer of the immunosuppressant brasilicardins, using illumina and PacBio technologies

Anina Buchmann

Michael Eitel

Pierre Koch

Paul N. Schwarz

Evi Stegmann

Wolfgang Wohlleben

Marcin Wolański

Michał Krawiec

Jolanta Zakrzewska-Czerwińska

Carmen Méndez

Alma Botas

Luz Elena Núñez

Francisco Morís

Jesus Cortés

Harald Gross

Crossref

High-Quality Draft Genome Sequence of the Actinobacterium Nocardia terpenica IFM 0406, Producer of the Immunosuppressant Brasilicardins, Using Illumina and PacBio Technologies

http://dx.doi.org/10.1128/genomeA.01391-16

High-quality draft genome sequence of the actinobacterium Nocardia terpenica IFM 0406, producer of the immunosuppressant brasilicardins, using illumina and PacBio technologies

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