Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype

Abstract

Background: Improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids.

Results: We sequenced the nuclear genome and methylome of this oleaginous diatom to identify the genetic traits that enable substantial accumulation of triacylglycerol. The genome is comprised of highly methylated repetitive sequence, which does not significantly change under silicon starved lipid induction, and data further suggests the primary role of DNA methylation is to suppress DNA transposition. Annotation of pivotal glycolytic, lipid metabolism, and carbohydrate degradation processes reveal an expanded enzyme repertoire in C. cryptica that would allow for an increased metabolic capacity toward triacylglycerol production. Identification of previously unidentified genes, including those involved in carbon transport and chitin metabolism, provide potential targets for genetic manipulation of carbon flux to further increase its lipid phenotype. New genetic tools were developed, bringing this organism on a par with other microalgae in terms of genetic manipulation and characterization approaches.

Conclusions: Functional annotation and detailed cross-species comparison of key carbon rich processes in C. cryptica highlights the importance of enzymatic subcellular compartmentation for regulation of carbon flux, which is often overlooked in photosynthetic microeukaryotes. The availability of the genome sequence, as well as advanced genetic manipulation tools enable further development of this organism for deployment in large-scale production systems.

Keywords: Algae biofuel; Carbon metabolism; Cyclotella cryptica; DNA methylation; Diatom; Genome sequence.

Fig. 1

Lipid accumulation in Cyclotella cryptica under silicon deprivation. Grayscale image of C. cryptica in (a) silicon replete medium, 0 h lipid uninduced, or (b) 48 h silicon deplete, lipid induced. c, d Respective composite images of chlorophyll autofluorescence (red) and fluorescent lipophilic dye BODIPY (green). e, f Differential interference contrast image of silicon replete (e) and 96 h silicon deplete, lipid induced, with red arrows identifying lipid droplets (f). Scale bars 5 μm

Fig. 2

Cyclotella cryptica genetic components. Percentages reflect group relative to the total number of gene models in the species. a Comparison of genes between C. cryptica and T. pseudonana (JGI version 3). Over half the genes in C. cryptica (Cc) are not found in T. pseudonana (Tp). b Classification of C. cryptica specific genes (n = 11,244) relative to other diatom genomes. c Relative copy numbers of genes comparing C. cryptica and T. pseudonana (n = 9877). Percentages shown are based on the Cc total gene models. d Classification of T. pseudonana specific genes relative to other diatom genomes (n = 1899)

Fig. 3

DNA methylation in Cyclotella cryptica. a Percent genomic methylation under both conditions according to cytosine motif (denoted at left, H = A, T, or C nucleotides). b Fraction methylation across genomic contig g111188_00083. Blue silicon replete, red silicon deplete 48 h. Augustus V3 models depicted in black, Repeat Modeler data depicted in gray. c Fraction methylation across intergenic, repeat, and d gene body, exon, intron regions. Outliers in c and d have been removed

Fig. 4

Methylation based on evolutionary origin of genes from DarkHorse analysis. a Enrichment analysis of methylated genes given their taxonomic group. Enrichment of methylation within a taxonomic group was determined by subtracting the proportion of genes methylated within a taxonomic group from the proportion of genes methylated across the C. cryptica genome. Groups with numbers below 0 have a lower proportion of methylated genes in the taxonomic group compared to the proportion of this group in the overall genome. b Percent of methylated and unmethylated genes relative to taxonomic group. Top numbers are the total number of genes from that group in the genome

Fig. 5

In silico targeting predictions of all nuclear gene models in C. cryptica. Percentages are listed in the chart. See also Additional file 2, subcellular targeting. targeting predictions used for this specific analysis were SignalP 3.0, TargetP, ChloroP, HECTAR, Predotar, and ASAFind [, , –58]

Fig. 6

Comparative analysis of key carbon metabolic pathways between C. cryptica and T. pseudonana. Different colored boxes denote subcellular location of a given enzyme based on bioinformatic targeting predictions. Number within the box indicates how many enzyme copies are targeted to that location. Numbers to the right of boxes are total gene copy number found in the genome. Question marks indicate weak targeting and/or inconsistencies between targeting prediction programs. Superscript numbers on enzymes indicate the number of genes found with partial sequence in C. cryptica at the N-terminus, thereby not containing targeting prediction. Red numbers are methylated genes. In the case of MGAT or DGTT, one of the four ER predicted genes is methylated. AAK14816-like putative glycerol-3-phosphate acyltransferase, ACC acetyl-CoA carboxylase, AGPAT 1-acyl-glycerol-3-phosphate acyltransferase, BGS 1,3 β-glucan synthase, DGAT diacylglycerol acyltransferase, DGTT diacylglycerol acyltransferase type 2, ENO enolase, ENR enoyl-ACP reductase, FBA fructose bisphosphate aldolase, FBP fructose 1,6 bisphosphatase, GAPDH glyceraldehyde 3-phosphate dehydrogenase, GLK glucokinase, GPI glucose-6-phosphate isomerase, GPAT glycerol-3-phosphate acyltransferase, HD 3-hydroxyacyl-ACP dehydratase, KAR 3-ketoacyl-ACP reductase, KAS 3-ketoacyl-ACP synthase, LCLAT1 lysocardiolipin acyltransferase 1, LPLAT lysophospholipid acyltransferase, MAT malonyl-CoA-ACP transacylase, MDH malate dehydrogenase, ME malic enzyme, MGAT monoacylglycerol acyltransferase, PAP phosphatidic acid phosphatase, PYC pyruvate carboxylase, PDRP pyruvate phosphate dikinase regulatory protein, PEPC phosphoenolpyruvate carboxylase, PEPCK phosphoenolpyruvate carboxykinase, PEPS phosphoenolpyruvate synthase, PFK phosphofructokinase, PGAM phosphoglycerate mutase, PGK phosphoglycerate kinase, PGM phosphoglucomutase, PK pyruvate kinase, PPDK pyruvate phosphate dikinase, TPI triose phosphate isomerase, UAP UDP-N-acetylglucosamine pyrophosphorylase, UGP UTP-glucose-1-phosphate uridylyltransferase

Fig. 7

Metabolic overview highlighting key findings from Cyclotella cryptica genome. Red numbers are referred to in the text. Enzymatic abbreviations are listed in Fig. 5. ED Entner–Doudoroff, IM inner membrane, OM outer membrane, PEP phosphoenolpyruvate, Fru6P fructose-6-phosphate, 3PG glycerate 3-phosphate, OAA oxaloacetate, PPP pentose phosphate pathway

Fig. 8

Chitin metabolism in C. cryptica. a Genes and their predicted subcellular localization. Organizational schematic as described in Fig. 6. b Hypothetical chitin biosynthesis and degradation pathway in C. cryptica. Dashed line indicates that the enzyme is not found in C. cryptica

Fig. 9

Phylogenetic analysis of hexose transporters in C. cryptica (Cyccr) and T. pseudonana (Thaps). All genes contain PF00083 Sugar (and other) transporter domain. Highlighted gene is hypothetical heterotrophic glucose transporter in C. cryptica and with nearest matches to that transporter found in the diatoms T. oceanica, F. cylindrus, the haptophyte E. huxleyi, and stramenopile N. gaditana. Phylogeny was generated using default parameters in RAxML_GUI v1.3 and is mid-point rooted. Abbreviations and accession numbers are found in Additional file 4

Fig. 10

Genetic manipulation in C. cryptica. Conditional expression using the nitrate reductase promoter. a GFP expression in ASW media, with nitrate as the nitrogen source. b No GFP expression in repressive modified ASW media, with ammonia as the nitrogen source. Chlorophyll and GFP are falsely colored red and green, respectively. Composite images are 3D reconstructions. Scale bars 5 μm