Genome and Methylome of a Candidate Biofuel Organism Provides Insights into Carbon Flux and Regulation Jesse Traller PhD Candidate Scripps Institution of Oceanography, UC San Diego Algal Biomass Summit, September 30th, 2014 Co Authors: David Lopez, Shawn Cokus, Marco Morcelli, Michael Thompson, Sean Gallaher, Kalpana Manandhar Shrestha, Olia Gaidarenko, Orna Cook, Sophie Alvarez, Sabeeha Merchant, Richard Sayre, Matteo Pellegrini, and Mark Hildebrand Cyclotella cryptica 1978-1996 ncma.bigelow.org Previous work in C. cryptica sets the stage for further studies C. cryptica accumulates lipid under Si deplete conditions (Roessler, 1988) Fundamental characterization of key enzymes involved in carbon flux (Roessler, 1987) First stable nuclear transformation of a diatom (Dunahay et al., 1995) Outdoor growth in large open raceway ponds (Laws et. al,1987) Image: Sarah Lerch 3 main goals Further describe diatom and algal cell biology Facilitate C. cryptica’s use for the production of biofuel. Advance our understanding of how carbon metabolism and more specifically neutral lipid accumulation are controlled in microalgae. Chlorophyll Autofluorescence Lipid (BODIPY) Fluorescence Silicon Limitation: Hours 0, 20, 44, 68, 76, 93, 100 Traller and Hildebrand, Algal Research, 2013 A massive accumulation of triacylglycerol (TAG) under both conditions Silicon Limitation T=0 T=24 Nitrogen Limitation T=48 T=72 T=96 BODIPY Chlorophyll Scale Bars, 5μm Traller and Hildebrand, Algal Research, 2013 Apply high throughput ‘omic’ technologies to investigate the cellular response involved in lipid accumulation in Cyclotella cryptica Methylome DNA methylation: CH3 CH3 CH3 Exponential T=0 and Silicon starved T=48 CH3 Time course: early and late stages i) silicon starvation ii) nitrogen starvation Figure adapted from R. Goodacre, 2005 Cyclotella cryptica 8x10um 183.9 Mbp ~23,500 genes 9,000 genes, high confidence Gene Density: ~16.5% 53% Repetitive 53% of DNA is methylated Chlamydomonas reinhardtii 10um diameter 112 Mbp 17,741 genes Gene Density: 58% 16.7% Repetitive 10% of DNA is methylated Thalassiosira pseudonana 4x5um 31Mbp 11,390 genes Gene Density: 64.1% 2% Repetitive No methylation data Nannochloropsis gaditana 2-3um diameter 29Mbp 9,052 genes Gene Density: ~50% 10% Repetitive (N. oceanica) No methylation data Phaeodactylum tricornutum 2X10um 27Mbp 10,025 genes Gene Density: 57.3% 15% Repetitive 5% of DNA is methylated C. cryptica genome is 53% methylated …still ~2000 methylated genes g095264_00087 All gene models High Confidence Fraction Methylation Repeat Base Pairs x1000 Gene rich, low methylation Repeat rich, high methylation David Lopez, Marco Morcelli What genes are methylated? No enrichment of genes involved in major metabolic processes Methylated genes are overwhelmingly found in repeat rich regions David Lopez et. al., 2011 (>10000 RPKM) Relative gene expression Exceptions to the rule…methylated genes with high gene expression Fraction Methylation across gene (>.50 = methylated) Comparing methylation in induced and non-induced cultures T=0 Exponential phase T=48 Silicon starved, lipid induced Absolute Value of Per-Site Methylation Difference |Me48 – Me0| C. cryptica Cell Metabolism Subcellular localization of genes Plastid, 73% (1687 genes) importance of the 27% (620 genes) subcellular localization in the cell We can begin to understand the Periplastid, Are there key enzymes or pathways localized to the periplastid space? er pathways partitioned across different How are key enzymes and chlcell? 9% compartments the 6% cyto 78% Subcellular localization: • Predotar* • TargetP* • SignalP* • HECTAR* • CELLO • PredAlgo • PSORT mito 7% Michael Thompson, Gruber, 2007 Investigation of carbon metabolism genes Cyclotella cryptica • Localization (based on targeting programs) • Isozyme number • Enzyme similarity • • • • Glycolysis/gluconeogenesis Fatty acid/TAG biosynthesis Pyruvate metabolism Chysolaminarin biosynthesis/breakdown Thalassiosira pseudonana Smith, Abbriano, and Hildebrand. Algal Research, 2012 There are more putative MGAT/DGATs C. cryptica C. cryptica Additional copies has mitochondrial of PEPS, PK, pyruvate plastid in PEPS carboxylase. gene isAbility than T. pseudonana, which may differences in to initiate silenced by gluconeogenesis methylation in theexplain mitochondrion? levels of TAG in each organism. The importance of localization in understanding ‘omic’ data Start site of glycolysis Storage Carbohydrate (chrysolaminarian) Glucose ATP Glycolytic intermediates Glu6P Pyruvate kinase Gluconeogenesis Carbon Fixation 3PG CO2 PGAM 2PG ENO Enolase PEP AMP + Pi ADP PYK PEPS ATP + H20 ATP AMP + PPi Fold Protein Abundance ADP 1.8 Proteome (iTRAQ) 1.6 1.4 1.2 1 0.8 0.6 0 PPDK ATP + Pi 20 40 Hour in Si- Media Enolase 1 Pyruvate Kinase 1 Enolase 2 Pyruvate Kinase 2 Pyruvate PDC Acetyl-CoA ATP + HCO3- ACCase ADP + Pi TCA Cycle Malonyl-CoA Fatty Acid & TAG Biosynthesis Fig. adapted from Smith et al., 2012 Summary Cyclotella cryptica is an excellent accumulator of lipid. Collaborative effort to understand this process and create tools to enhance our knowledge. The C. cryptica genome is large, repetitive and highly methylated. Methylation of DNA is primarily used to silence repeats, instead of playing a role in temporal gene regulation. Blueprinting carbon metabolic pathways in the cell allows us to more accurately understand: i) the flow of carbon in the cell ii) importance of subcellular regions in the cell Carefully comparing similarly related species (T. pseudonana and C. cryptica) highlights prospective target sites for genetic manipulation that could test the roles of steps in the process. Acknowledgements David Lopez, Shawn Cokus, Marco Morcelli, Michael Thompson, Sean Gallaher, Kalpana Manandhar-Shrestha, Olia Gaidarenko, Orna Cook, Sarah Smith, Raffaela Abbriano, Trina Norden-Krichmar, Andrew Allen, Sophie Alvarez, Sabeeha Merchant, Richard Sayre, Matteo Pellegrini, and Mark Hildebrand UC Los Angeles, Scripps Inst. of Oceanography, UC San Diego, Donald Danforth Plant Science Center, Los Alamos National Labs Funding: AFOSR, DOE, NSF, Zobell Fellowship, SIO Grad Office Additional stories… N. Schoepp, A. Quigley, F. Fields R. Shrestha, SIT1 promoter overexpression/GFP O. Gaidarenko, 1. localization of plastid targeted enolase. 2. EDA, enzyme involved in Entner Duodoroff Pathway Genome Sequencing: DNA isolated using a CsCl gradient Illumina paired end and mate pair sequencing to give reads ranging in 150-2700bp…ideal for de novo assembly Functional annotation and data! Assembly: ABySS Transcriptome: paired end and single end to create gene models Proteome How to sequence the methylome: Bisulfite Sequencing Top Strand Bottom Strand Original Bottom Strand Compliment to Original bottom Strand Original Top Strand Compliment to original top strand BS Seeker- to map the reads on the genome Chen et al, BMC Genomics 2010; Krueger et al, Nature Methods 2012 iTRAQ