crossmark
Draft Genome Sequence of the Oleaginous Yeast Cryptococcus albidus var. albidus Shashwat Vajpeyi, Kartik Chandran Department of Earth and Environmental Engineering, Columbia University, New York, New York, USA
We report the complete draft genome sequence of Cryptococcus albidus var. albidus, an oleaginous yeast, which can utilize various organic carbon sources for lipid synthesis. Availability of this genome will help elucidate factors driving lipid accumulation in C. albidus and contribute toward bioprocess development and optimization for engineered lipid production. Received 25 March 2016 Accepted 29 March 2016 Published 19 May 2016 Citation Vajpeyi S, Chandran K. 2016. Draft genome sequence of the oleaginous yeast Cryptococcus albidus var. albidus. Genome Announc 4(3):e00390-16. doi:10.1128/ genomeA.00390-16. Copyright © 2016 Vajpeyi and Chandran. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Kartik Chandran, [email protected].
H
ere, we present the draft genome sequence of Cryptococcus albidus var. albidus, an oleaginous yeast that can utilize a wide variety of organic carbon sources such as glucose (1), acetic acid (2), and butyric acid (3) for the synthesis of lipids, which can be used for the commercial production of biodiesel and commodity chemicals. Notwithstanding documented studies on factors promoting lipid production and accumulation in C. albidus, a lack of knowledge relating to the associated metabolic pathways and mechanisms has limited further efforts toward bioprocess engineering and system optimization. We expect that the presented genome sequence will significantly enhance our understanding of lipid production and accumulation in C. albidus and contribute to efforts for engineered production of related biofuels and biobased chemicals. Cryptococcus albidus (ATCC 10672) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated on YM (yeast and mold) medium in a 100-mL shake flask culture at room temperature (T ⫽ 25°C). Cells were harvested from 100 L culture broth (~2 ⫻ 107 cells) by centrifuging at 16,000 ⫻ g for 10 min at 4°C, followed by automated extraction of genomic DNA from the resulting pellet using Qiacube. A 400-bp genome library of C. albidus was prepared and sequenced using Ion-torrentPGM. The raw sequence data contained 3,163,623 reads with median length of 362 bp, resulting in 44.8-fold coverage. The raw sequence reads were assembled using SPADES assembler version 3.1.0 (4) with a k-mer size of 21. This led to a genome assembly containing 834 contigs (sequence length ⬎500 bp). The total consensus genome length was 24,807,186 bp with a median contig length (N50) of 114,383 bp and a G⫹C% content of 52.7%. Open reading frames (ORFs) were assigned using the evidence-based annotation pipeline AUGUSTUS (5), trained using Cryptococcus neoformans gattii, the closest organism for which the fully sequenced genome is available. A total of 8,637 putative ORFs were identified, and 6,110 known proteins were annotated using NCBI BLASTp (6) (protein vs. protein alignment). Additional functional annotation performed using BlastKOALA (7) revealed essential pathways for metabolism of various carbon sources (including glucose, sucrose, citric, acetic, propionic and
May/June 2016 Volume 4 Issue 3 e00390-16
butyric acids) and for accumulation of carbon compounds (including lipids and glycogen). Inferred pathways also included uptake and assimilation of various nitrogen sources (ammonia, nitrate, nitrite, and urea) and the capability of assimilative reduction of sulfate to sulfide. Specifically pertaining to oleaginity in C. albidus, we identified two copies of the acly gene and 19 copies of the me2 and mdh2 genes coding for enzymes ATP citrate lyase and malate dehydrogenase, respectively, which ensure the continuous cytosolic availability of acetyl-CoA and NADPH for lipid biosynthesis (8). We also identified seven copies of genes idh1, idh2, and idh3 coding for NAD⫹- and NADP⫹-dependent isocitrate dehydrogenase, which diverts carbon flow away from the citric acid cycle to lipid biosynthesis under nitrogen limitation (9), and eight copies of acc1, coding for acetyl-coA carboxylase, which catalyzes carboxylation of acetyl-coA to malonyl-coA, the rate-limiting step in lipid biosynthesis. C. albidus contains both fas2 and fas1 genes coding for ␣- and -subunits of cytosolic type I fatty-acid synthase, which is typical in yeasts (10). Nucleotide sequence accession numbers. The draft genome sequence has been deposited to DDBJ/EMBL/GenBank under the accession number LKPZ00000000. The version discussed in this paper is the first version, LKPZ00000000.1. ACKNOWLEDGMENTS This work was supported by the Bill and Melinda Gates Foundation Water Sanitation and Hygiene program.
FUNDING INFORMATION This work, including the efforts of Kartik Chandran, was funded by the Bill and Melinda Gates Foundation.
REFERENCES 1. Hansson L, Dostálek M. 1986. Lipid formation by Cryptococcus albidus in nitrogen-limited and in carbon-limited chemostat cultures. Appl Microbiol Biotechnol 24:187–192. http://dx.doi.org/10.1007/BF00261535. 2. Li X, Xiong L, Chen X, Huang C, Chen X, Ma L. 2015. Effects of acetic acid on growth and lipid production by Cryptococcus albidus. J Am Oil Chem Soc 92:1113–1118. http://dx.doi.org/10.1007/s11746-015-2685-5. 3. Vajpeyi S, Chandran K. 2015. Microbial conversion of synthetic and food
Genome Announcements
genomea.asm.org 1
Vajpeyi and Chandran
waste-derived volatile fatty acids to lipids. Bioresour Technol 188:49 –55. http://dx.doi.org/10.1016/j.biortech.2015.01.099. 4. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to singlecell sequencing. J Comput Biol 19:455– 477. http://dx.doi.org/10.1089/ cmb.2012.0021. 5. Stanke M, Waack S. 2003. Gene prediction with a hidden Markov model and a new intron submodel. Bioinformatics 19(suppl 2):ii215–ii225. http://dx.doi.org/10.1093/bioinformatics/btg1080. 6. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403– 410. http://dx.doi.org/10.1016/ S0022-2836(05)80360-2.
2 genomea.asm.org
7. Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428:726 –731. http://dx.doi.org/ 10.1016/j.jmb.2015.11.006. 8. Ratledge C. 2004. Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie 86:807– 815. http://dx.doi.org/ 10.1016/j.biochi.2004.09.017. 9. Tang W, Zhang S, Wang Q, Tan H, Zhao ZK. 2009. The isocitrate dehydrogenase gene of oleaginous yeast Lipomyces starkeyi is linked to lipid accumulation. Can J Microbiol 55:1062–1069. http://dx.doi.org/ 10.1139/w09-063. 10. Tehlivets O, Scheuringer K, Kohlwein SD. 2007. Fatty acid synthesis and elongation in yeast. Biochim Biophys Acta 1771:255–270. http:// dx.doi.org/10.1016/j.bbalip.2006.07.004.
Genome Announcements
May/June 2016 Volume 4 Issue 3 e00390-16
Draft Genome Sequence of the Oleaginous Yeast Cryptococcus albidus var. albidus Shashwat Vajpeyi, Kartik Chandran Department of Earth and Environmental Engineering, Columbia University, New York, New York, USA
We report the complete draft genome sequence of Cryptococcus albidus var. albidus, an oleaginous yeast, which can utilize various organic carbon sources for lipid synthesis. Availability of this genome will help elucidate factors driving lipid accumulation in C. albidus and contribute toward bioprocess development and optimization for engineered lipid production. Received 25 March 2016 Accepted 29 March 2016 Published 19 May 2016 Citation Vajpeyi S, Chandran K. 2016. Draft genome sequence of the oleaginous yeast Cryptococcus albidus var. albidus. Genome Announc 4(3):e00390-16. doi:10.1128/ genomeA.00390-16. Copyright © 2016 Vajpeyi and Chandran. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Kartik Chandran, [email protected].
H
ere, we present the draft genome sequence of Cryptococcus albidus var. albidus, an oleaginous yeast that can utilize a wide variety of organic carbon sources such as glucose (1), acetic acid (2), and butyric acid (3) for the synthesis of lipids, which can be used for the commercial production of biodiesel and commodity chemicals. Notwithstanding documented studies on factors promoting lipid production and accumulation in C. albidus, a lack of knowledge relating to the associated metabolic pathways and mechanisms has limited further efforts toward bioprocess engineering and system optimization. We expect that the presented genome sequence will significantly enhance our understanding of lipid production and accumulation in C. albidus and contribute to efforts for engineered production of related biofuels and biobased chemicals. Cryptococcus albidus (ATCC 10672) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated on YM (yeast and mold) medium in a 100-mL shake flask culture at room temperature (T ⫽ 25°C). Cells were harvested from 100 L culture broth (~2 ⫻ 107 cells) by centrifuging at 16,000 ⫻ g for 10 min at 4°C, followed by automated extraction of genomic DNA from the resulting pellet using Qiacube. A 400-bp genome library of C. albidus was prepared and sequenced using Ion-torrentPGM. The raw sequence data contained 3,163,623 reads with median length of 362 bp, resulting in 44.8-fold coverage. The raw sequence reads were assembled using SPADES assembler version 3.1.0 (4) with a k-mer size of 21. This led to a genome assembly containing 834 contigs (sequence length ⬎500 bp). The total consensus genome length was 24,807,186 bp with a median contig length (N50) of 114,383 bp and a G⫹C% content of 52.7%. Open reading frames (ORFs) were assigned using the evidence-based annotation pipeline AUGUSTUS (5), trained using Cryptococcus neoformans gattii, the closest organism for which the fully sequenced genome is available. A total of 8,637 putative ORFs were identified, and 6,110 known proteins were annotated using NCBI BLASTp (6) (protein vs. protein alignment). Additional functional annotation performed using BlastKOALA (7) revealed essential pathways for metabolism of various carbon sources (including glucose, sucrose, citric, acetic, propionic and
May/June 2016 Volume 4 Issue 3 e00390-16
butyric acids) and for accumulation of carbon compounds (including lipids and glycogen). Inferred pathways also included uptake and assimilation of various nitrogen sources (ammonia, nitrate, nitrite, and urea) and the capability of assimilative reduction of sulfate to sulfide. Specifically pertaining to oleaginity in C. albidus, we identified two copies of the acly gene and 19 copies of the me2 and mdh2 genes coding for enzymes ATP citrate lyase and malate dehydrogenase, respectively, which ensure the continuous cytosolic availability of acetyl-CoA and NADPH for lipid biosynthesis (8). We also identified seven copies of genes idh1, idh2, and idh3 coding for NAD⫹- and NADP⫹-dependent isocitrate dehydrogenase, which diverts carbon flow away from the citric acid cycle to lipid biosynthesis under nitrogen limitation (9), and eight copies of acc1, coding for acetyl-coA carboxylase, which catalyzes carboxylation of acetyl-coA to malonyl-coA, the rate-limiting step in lipid biosynthesis. C. albidus contains both fas2 and fas1 genes coding for ␣- and -subunits of cytosolic type I fatty-acid synthase, which is typical in yeasts (10). Nucleotide sequence accession numbers. The draft genome sequence has been deposited to DDBJ/EMBL/GenBank under the accession number LKPZ00000000. The version discussed in this paper is the first version, LKPZ00000000.1. ACKNOWLEDGMENTS This work was supported by the Bill and Melinda Gates Foundation Water Sanitation and Hygiene program.
FUNDING INFORMATION This work, including the efforts of Kartik Chandran, was funded by the Bill and Melinda Gates Foundation.
REFERENCES 1. Hansson L, Dostálek M. 1986. Lipid formation by Cryptococcus albidus in nitrogen-limited and in carbon-limited chemostat cultures. Appl Microbiol Biotechnol 24:187–192. http://dx.doi.org/10.1007/BF00261535. 2. Li X, Xiong L, Chen X, Huang C, Chen X, Ma L. 2015. Effects of acetic acid on growth and lipid production by Cryptococcus albidus. J Am Oil Chem Soc 92:1113–1118. http://dx.doi.org/10.1007/s11746-015-2685-5. 3. Vajpeyi S, Chandran K. 2015. Microbial conversion of synthetic and food
Genome Announcements
genomea.asm.org 1
Vajpeyi and Chandran
waste-derived volatile fatty acids to lipids. Bioresour Technol 188:49 –55. http://dx.doi.org/10.1016/j.biortech.2015.01.099. 4. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to singlecell sequencing. J Comput Biol 19:455– 477. http://dx.doi.org/10.1089/ cmb.2012.0021. 5. Stanke M, Waack S. 2003. Gene prediction with a hidden Markov model and a new intron submodel. Bioinformatics 19(suppl 2):ii215–ii225. http://dx.doi.org/10.1093/bioinformatics/btg1080. 6. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403– 410. http://dx.doi.org/10.1016/ S0022-2836(05)80360-2.
2 genomea.asm.org
7. Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428:726 –731. http://dx.doi.org/ 10.1016/j.jmb.2015.11.006. 8. Ratledge C. 2004. Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie 86:807– 815. http://dx.doi.org/ 10.1016/j.biochi.2004.09.017. 9. Tang W, Zhang S, Wang Q, Tan H, Zhao ZK. 2009. The isocitrate dehydrogenase gene of oleaginous yeast Lipomyces starkeyi is linked to lipid accumulation. Can J Microbiol 55:1062–1069. http://dx.doi.org/ 10.1139/w09-063. 10. Tehlivets O, Scheuringer K, Kohlwein SD. 2007. Fatty acid synthesis and elongation in yeast. Biochim Biophys Acta 1771:255–270. http:// dx.doi.org/10.1016/j.bbalip.2006.07.004.
Genome Announcements
May/June 2016 Volume 4 Issue 3 e00390-16
