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Draft Genome Sequence of Cutaneotrichosporon curvatus DSM 101032 (Formerly Cryptococcus curvatus), an Oleaginous Yeast Producing Polyunsaturated Fatty Acids Thomas Hofmeyer,a Silke Hackenschmidt,a Florian Nadler,a Andrea Thürmer,b Rolf Daniel,b Johannes Kabischa Junior Research Group Drop-In Biofuels, Ernst-Moritz-Arndt-Universität, Greifswald, Germany;a Göttingen Genomics Laboratory, Göttingen, Germanyb
Cutaneotrichosporon curvatus DSM 101032 is an oleaginous yeast that can be isolated from various habitats and is capable of producing substantial amounts of polyunsaturated fatty acids. Here, we present the first draft genome sequence of any C. curvatus species. Received 28 March 2016 Accepted 29 March 2016 Published 12 May 2016 Citation Hofmeyer T, Hackenschmidt S, Nadler F, Thürmer A, Daniel R, Kabisch J. 2016. Draft genome sequence of Cutaneotrichosporon curvatus DSM 101032 (formerly Cryptococcus curvatus), an oleaginous yeast producing polyunsaturated fatty acids. Genome Announc 4(3):e00362-16. doi:10.1128/genomeA.00362-16. Copyright © 2016 Hofmeyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Johannes Kabisch, [email protected].
C
utaneotrichosporon curvatus, until recently known as Cryptococcus curvatus, is an oleaginous yeast of great interest for biotechnological applications due to the increasing demand of microbial lipids as a feedstock for the production of sustainable and renewable fuels and fine chemicals (1, 2). The fungus is widespread and can be isolated, for example, from foodstuff, like raw milk and lettuce, or from marine sediments (3–5). C. curvatus has the capability to utilize a broad spectrum of different renewable carbon sources, like glucose, sucrose, lactose, xylose, glycerol, arabinose, and cellobiose (6). Furthermore, it was shown to be capable of utilizing these and other substrates, like pretreatment-derived hemicellulose, organic waste from the food industry, or active sludge to produce lipids in numerous batch and fed-batch cultivations (7–10). In this context, the C. curvatus genome sequencing revealed several hits of coding sequences for enzymes required for the degradation of renewable polymers, such as cellulose (e.g., putative cellulases, Ccurv_10480 and Ccurv_12500) and chitin (putative chitinase, Ccurv_32040), as well as enzymes for the metabolism of the resulting monomers. Under nitrogen-limiting conditions, C. curvatus can accumulate lipids up to 60% of the dry cell weight (11). The fatty acid profile of this study’s strain contains about 50% unsaturated fatty acids, among them ␣-linolenic acid (C18:3, n-3); hence, C. curvatus can be denoted as a natural omega-3 fatty acid producer (data not shown). However, no genome sequence has been publicly available. Here, we report the draft genome sequence of C. curvatus DSM 101032, alongside a JBrowse (12)-based genome browser of the functionally annotated genome, which is available at http://genomes.chemie.uni-greifswald.de/. The analyzed strain, a progeny of C. curvatus PYCC 2565, is deposited at DSMZ as DSM 101032. The genomic DNA of C. curvatus was prepared according to the rapid preparation of yeast DNA protocol (13, 14) and treated with RNase (NEB), according to the manufacturer’s instructions. Strain taxonomy was confirmed by amplification and sequencing of internal transcribed spacer (ITS) DNA with the ITS1 and ITS4 primers (15). Shotgun libraries were generated using the Nextera XT DNA
May/June 2016 Volume 4 Issue 3 e00362-16
sample preparation kit, according to the manufacturer’s instructions. The whole genome of C. curvatus was sequenced with the Genome Analyzer IIx (Illumina, San Diego, CA) by the Göttingen Genomics Laboratory (G2L [http://appmibio.uni-goettingen.de /index.php?sec⫽g2l]). The libraries were sequenced in a 112-bp paired-end single-indexed run. The resulting 19.9 ⫻ 106 pairedend reads were assembled using SPAdes 3.5.0, yielding 366 large contigs (ⱖ10,000 bp), an N50 contig size of 59,188 bp, and a total length of approximately 18.28 Mbp, with a G⫹C content of 59.41% (16). Open reading frames were determined using AUGUSTUS 2.7, with the species set to Cryptococcus neoformans (17). The draft genome sequence presented notable progress in the further understanding of C. curvatus and will lead to the development of useful biomolecular tools and protocols (18). These data will indubitably enable a better exploitation of C. curvatus metabolism and open up new avenues on the road to a renewable production of single-cell oil. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. LDEP00000000. The version described in this paper is version LDEP01000000. ACKNOWLEDGMENTS We thank Frieder Schauer for providing C. curvatus PYCC 2565 from his strain collection (Institute of Microbiology, Ernst-Moritz-ArndtUniversität, Greifswald, Germany).
FUNDING INFORMATION This work, including the efforts of Johannes Kabisch, was funded by Fachagentur Nachwachsende Rohstoffe e. V. (22007413).
REFERENCES 1. Lennen RM, Pfleger BF. 2013. Microbial production of fatty acid-derived fuels and chemicals. Curr Opin Biotechnol 24:1044 –1053. http:// dx.doi.org/10.1016/j.copbio.2013.02.028. 2. Liu X-Z, Wang Q-M, Göker M, Groenewald M, Kachalkin AV, Lumbsch HT, Millanes AM, Wedin M, Yurkov AM, Boekhout T, Bai F-Y. 2015. Towards an integrated phylogenetic classification of the Tre-
Genome Announcements
genomea.asm.org 1
Hofmeyer et al.
3.
4. 5.
6. 7.
8.
9.
10.
mellomycetes. Stud Mycol 81:85–147. http://dx.doi.org/10.1016/ j.simyco.2015.12.001. Cocolin L, Aggio D, Manzano M, Cantoni C, Comi G. 2002. An application of PCR-DGGE analysis to profile the yeast populations in raw milk. Int Dairy J 12:407– 411. http://dx.doi.org/10.1016/S0958 -6946(02)00023-7. Magnuson JA, King AD, Jr, Török T. 1990. Microflora of partially processed lettuce. Appl Environ Microbiol 56:3851–3854. Takishita K, Tsuchiya M, Reimer JD, Maruyama T. 2006. Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima knoll methane seep. Extremophiles 10:165–169. http://dx.doi.org/ 10.1007/s00792-005-0495-7. Fonseca Á, Boekhout T, Fell JW. 2011. Cryptococcus Vuillemin (1901), p 1661–1737. In Kurtzman CP, Fell JW, Boekhout T (ed), The yeasts: a taxonomic study, 5th ed. Elsevier, London, United Kingdom. Gonzalez-Garcia Y, Hernandez R, Zhang G, Escalante FME, Holmes W, French WT. 2013. Lipids accumulation in Rhodotorula glutinis and Cryptococcus curvatus growing on distillery wastewater as culture medium. Environ Prog Sustain Energy 32:69 –74. http://dx.doi.org/10.1002/ep.10604. Ryu B-G, Kim J, Kim K, Choi Y-E, Han J-I, Yang J-W. 2013. High-celldensity cultivation of oleaginous yeast Cryptococcus curvatus for biodiesel production using organic waste from the brewery industry. Bioresour Technol 135:357–364. http://dx.doi.org/10.1016/j.biortech.2012.09.054. Seo Yh, Lee Ig, Han Ji. 2013. Cultivation and lipid production of yeast Cryptococcus curvatus using pretreated waste active sludge supernatant. Bioresour Technol 135:304 –308. http://dx.doi.org/10.1016/ j.biortech.2012.10.024. Yu X, Zheng Y, Dorgan KM, Chen S. 2011. Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid. Bioresour Technol 102:6134 – 6140. http://dx.doi.org/ 10.1016/j.biortech.2011.02.081.
2 genomea.asm.org
11. Meng X, Yang J, Xu X, Zhang L, Nie Q, Xian M. 2009. Biodiesel production from oleaginous microorganisms. Renew Energ 34:1–5. http://dx.doi.org/10.1016/j.energy.2008.09.006. 12. Skinner ME, Uzilov AV, Stein LD, Mungall CJ, Holmes IH. 2009. JBrowse: a next-generation genome browser. Genome Res 19:1630 –1638. http://dx.doi.org/10.1101/gr.094607.109. 13. Green MR, Sambrook J. 2012. Isolation and quantification of DNA, p 64 – 65. In Green MR, Sambrook J (ed), Molecular cloning: a laboratory manual, 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 14. Hoffman CS, Winston F. 1987. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272. http://dx.doi.org/10.1016/0378 -1119(87)90131-4. 15. Innis MA, Gelfand DH, Sninsky JJ, White TJ. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p 315–322. In Innis MA, Gelfand DH, Sninsky JJ, White TJ (ed), PCR protocols: a guide to methods and applications. Academic Press, Inc., San Diego, CA. 16. Nurk S, Bankevich A, Antipov D, Gurevich A, Korobeynikov A, Lapidus A, Prjibelsky A, Pyshkin A, Sirotkin A, Sirotkin Y, Stepanauskas R, McLean J, Lasken R, Clingenpeel S, Woyke T, Tesler G, Alekseyev M, Pevzner P. 2013. Research in computational molecular biology, p 158 –170. In Deng M, Jiang R, Sun F, Zhang X (ed), Assembling genomes and mini-metagenomes from highly chimeric reads. Springer, Heidelberg, Germany. 17. Stanke M, Waack S. 2003. Gene prediction with a hidden Markov model and a new intron submodel. Bioinformatics 19(Suppl 2):ii215. http:// dx.doi.org/10.1093/bioinformatics/btg1080. 18. Wagner JM, Alper HS. 2016. Synthetic biology and molecular genetics in non-conventional yeasts: current tools and future advances. Fungal Genet Biol 89:126 –136. http://dx.doi.org/10.1016/j.fgb.2015.12.001.
Genome Announcements
May/June 2016 Volume 4 Issue 3 e00362-16
Draft Genome Sequence of Cutaneotrichosporon curvatus DSM 101032 (Formerly Cryptococcus curvatus), an Oleaginous Yeast Producing Polyunsaturated Fatty Acids Thomas Hofmeyer,a Silke Hackenschmidt,a Florian Nadler,a Andrea Thürmer,b Rolf Daniel,b Johannes Kabischa Junior Research Group Drop-In Biofuels, Ernst-Moritz-Arndt-Universität, Greifswald, Germany;a Göttingen Genomics Laboratory, Göttingen, Germanyb
Cutaneotrichosporon curvatus DSM 101032 is an oleaginous yeast that can be isolated from various habitats and is capable of producing substantial amounts of polyunsaturated fatty acids. Here, we present the first draft genome sequence of any C. curvatus species. Received 28 March 2016 Accepted 29 March 2016 Published 12 May 2016 Citation Hofmeyer T, Hackenschmidt S, Nadler F, Thürmer A, Daniel R, Kabisch J. 2016. Draft genome sequence of Cutaneotrichosporon curvatus DSM 101032 (formerly Cryptococcus curvatus), an oleaginous yeast producing polyunsaturated fatty acids. Genome Announc 4(3):e00362-16. doi:10.1128/genomeA.00362-16. Copyright © 2016 Hofmeyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Johannes Kabisch, [email protected].
C
utaneotrichosporon curvatus, until recently known as Cryptococcus curvatus, is an oleaginous yeast of great interest for biotechnological applications due to the increasing demand of microbial lipids as a feedstock for the production of sustainable and renewable fuels and fine chemicals (1, 2). The fungus is widespread and can be isolated, for example, from foodstuff, like raw milk and lettuce, or from marine sediments (3–5). C. curvatus has the capability to utilize a broad spectrum of different renewable carbon sources, like glucose, sucrose, lactose, xylose, glycerol, arabinose, and cellobiose (6). Furthermore, it was shown to be capable of utilizing these and other substrates, like pretreatment-derived hemicellulose, organic waste from the food industry, or active sludge to produce lipids in numerous batch and fed-batch cultivations (7–10). In this context, the C. curvatus genome sequencing revealed several hits of coding sequences for enzymes required for the degradation of renewable polymers, such as cellulose (e.g., putative cellulases, Ccurv_10480 and Ccurv_12500) and chitin (putative chitinase, Ccurv_32040), as well as enzymes for the metabolism of the resulting monomers. Under nitrogen-limiting conditions, C. curvatus can accumulate lipids up to 60% of the dry cell weight (11). The fatty acid profile of this study’s strain contains about 50% unsaturated fatty acids, among them ␣-linolenic acid (C18:3, n-3); hence, C. curvatus can be denoted as a natural omega-3 fatty acid producer (data not shown). However, no genome sequence has been publicly available. Here, we report the draft genome sequence of C. curvatus DSM 101032, alongside a JBrowse (12)-based genome browser of the functionally annotated genome, which is available at http://genomes.chemie.uni-greifswald.de/. The analyzed strain, a progeny of C. curvatus PYCC 2565, is deposited at DSMZ as DSM 101032. The genomic DNA of C. curvatus was prepared according to the rapid preparation of yeast DNA protocol (13, 14) and treated with RNase (NEB), according to the manufacturer’s instructions. Strain taxonomy was confirmed by amplification and sequencing of internal transcribed spacer (ITS) DNA with the ITS1 and ITS4 primers (15). Shotgun libraries were generated using the Nextera XT DNA
May/June 2016 Volume 4 Issue 3 e00362-16
sample preparation kit, according to the manufacturer’s instructions. The whole genome of C. curvatus was sequenced with the Genome Analyzer IIx (Illumina, San Diego, CA) by the Göttingen Genomics Laboratory (G2L [http://appmibio.uni-goettingen.de /index.php?sec⫽g2l]). The libraries were sequenced in a 112-bp paired-end single-indexed run. The resulting 19.9 ⫻ 106 pairedend reads were assembled using SPAdes 3.5.0, yielding 366 large contigs (ⱖ10,000 bp), an N50 contig size of 59,188 bp, and a total length of approximately 18.28 Mbp, with a G⫹C content of 59.41% (16). Open reading frames were determined using AUGUSTUS 2.7, with the species set to Cryptococcus neoformans (17). The draft genome sequence presented notable progress in the further understanding of C. curvatus and will lead to the development of useful biomolecular tools and protocols (18). These data will indubitably enable a better exploitation of C. curvatus metabolism and open up new avenues on the road to a renewable production of single-cell oil. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. LDEP00000000. The version described in this paper is version LDEP01000000. ACKNOWLEDGMENTS We thank Frieder Schauer for providing C. curvatus PYCC 2565 from his strain collection (Institute of Microbiology, Ernst-Moritz-ArndtUniversität, Greifswald, Germany).
FUNDING INFORMATION This work, including the efforts of Johannes Kabisch, was funded by Fachagentur Nachwachsende Rohstoffe e. V. (22007413).
REFERENCES 1. Lennen RM, Pfleger BF. 2013. Microbial production of fatty acid-derived fuels and chemicals. Curr Opin Biotechnol 24:1044 –1053. http:// dx.doi.org/10.1016/j.copbio.2013.02.028. 2. Liu X-Z, Wang Q-M, Göker M, Groenewald M, Kachalkin AV, Lumbsch HT, Millanes AM, Wedin M, Yurkov AM, Boekhout T, Bai F-Y. 2015. Towards an integrated phylogenetic classification of the Tre-
Genome Announcements
genomea.asm.org 1
Hofmeyer et al.
3.
4. 5.
6. 7.
8.
9.
10.
mellomycetes. Stud Mycol 81:85–147. http://dx.doi.org/10.1016/ j.simyco.2015.12.001. Cocolin L, Aggio D, Manzano M, Cantoni C, Comi G. 2002. An application of PCR-DGGE analysis to profile the yeast populations in raw milk. Int Dairy J 12:407– 411. http://dx.doi.org/10.1016/S0958 -6946(02)00023-7. Magnuson JA, King AD, Jr, Török T. 1990. Microflora of partially processed lettuce. Appl Environ Microbiol 56:3851–3854. Takishita K, Tsuchiya M, Reimer JD, Maruyama T. 2006. Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima knoll methane seep. Extremophiles 10:165–169. http://dx.doi.org/ 10.1007/s00792-005-0495-7. Fonseca Á, Boekhout T, Fell JW. 2011. Cryptococcus Vuillemin (1901), p 1661–1737. In Kurtzman CP, Fell JW, Boekhout T (ed), The yeasts: a taxonomic study, 5th ed. Elsevier, London, United Kingdom. Gonzalez-Garcia Y, Hernandez R, Zhang G, Escalante FME, Holmes W, French WT. 2013. Lipids accumulation in Rhodotorula glutinis and Cryptococcus curvatus growing on distillery wastewater as culture medium. Environ Prog Sustain Energy 32:69 –74. http://dx.doi.org/10.1002/ep.10604. Ryu B-G, Kim J, Kim K, Choi Y-E, Han J-I, Yang J-W. 2013. High-celldensity cultivation of oleaginous yeast Cryptococcus curvatus for biodiesel production using organic waste from the brewery industry. Bioresour Technol 135:357–364. http://dx.doi.org/10.1016/j.biortech.2012.09.054. Seo Yh, Lee Ig, Han Ji. 2013. Cultivation and lipid production of yeast Cryptococcus curvatus using pretreated waste active sludge supernatant. Bioresour Technol 135:304 –308. http://dx.doi.org/10.1016/ j.biortech.2012.10.024. Yu X, Zheng Y, Dorgan KM, Chen S. 2011. Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid. Bioresour Technol 102:6134 – 6140. http://dx.doi.org/ 10.1016/j.biortech.2011.02.081.
2 genomea.asm.org
11. Meng X, Yang J, Xu X, Zhang L, Nie Q, Xian M. 2009. Biodiesel production from oleaginous microorganisms. Renew Energ 34:1–5. http://dx.doi.org/10.1016/j.energy.2008.09.006. 12. Skinner ME, Uzilov AV, Stein LD, Mungall CJ, Holmes IH. 2009. JBrowse: a next-generation genome browser. Genome Res 19:1630 –1638. http://dx.doi.org/10.1101/gr.094607.109. 13. Green MR, Sambrook J. 2012. Isolation and quantification of DNA, p 64 – 65. In Green MR, Sambrook J (ed), Molecular cloning: a laboratory manual, 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 14. Hoffman CS, Winston F. 1987. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272. http://dx.doi.org/10.1016/0378 -1119(87)90131-4. 15. Innis MA, Gelfand DH, Sninsky JJ, White TJ. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p 315–322. In Innis MA, Gelfand DH, Sninsky JJ, White TJ (ed), PCR protocols: a guide to methods and applications. Academic Press, Inc., San Diego, CA. 16. Nurk S, Bankevich A, Antipov D, Gurevich A, Korobeynikov A, Lapidus A, Prjibelsky A, Pyshkin A, Sirotkin A, Sirotkin Y, Stepanauskas R, McLean J, Lasken R, Clingenpeel S, Woyke T, Tesler G, Alekseyev M, Pevzner P. 2013. Research in computational molecular biology, p 158 –170. In Deng M, Jiang R, Sun F, Zhang X (ed), Assembling genomes and mini-metagenomes from highly chimeric reads. Springer, Heidelberg, Germany. 17. Stanke M, Waack S. 2003. Gene prediction with a hidden Markov model and a new intron submodel. Bioinformatics 19(Suppl 2):ii215. http:// dx.doi.org/10.1093/bioinformatics/btg1080. 18. Wagner JM, Alper HS. 2016. Synthetic biology and molecular genetics in non-conventional yeasts: current tools and future advances. Fungal Genet Biol 89:126 –136. http://dx.doi.org/10.1016/j.fgb.2015.12.001.
Genome Announcements
May/June 2016 Volume 4 Issue 3 e00362-16
