Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779

Abstract

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.

Figure 1. Rooted neighbor joining tree of 18s rRNA sequences of different Nannochloropsis species using Eustigmatos vischeri as an outgroup.

Labels refer to strain identification numbers from the respective culture collections, if applicable the synonym is given as 2^nd name. CCMP, Provasoli Guillard Culture Collection for Marine Phytoplankton, USA; CCAP, Culture Collection of Algae and Protozoa, UK; MBIC, Marine Biotechnology Institute Culture Collection, Japan, AS3-9 from .

Figure 2. Hybrid assembly strategy using Illumina and 454 reads.

N50: the length N for which 50% of all bases in the sequences are in a sequence of length L

Figure 3. Gene Ontology.

(A) Blast2Go functional annotation results overview. No Blast, Number of sequences without blast search performed; No Blast Hit, Number of sequences without blastp hits at the given threshold (e-value<10⁻⁴); No Mapping, Number of blast hits that did not map to the Blast2GO database; No Annot., Number of mapped hits that did not retrieve GO annotations from the Blast2GO database; Annot., Number of sequences that did retrieve one or more GO annotations from the Blast2GO database; Total, Total number of analyzed sequences. (B) The distribution of GO annotations by GO level shows the respective number of added GO annotations in relation to their GO level for each category (P biological process, F molecular function, C cellular component). (C) Results distribution after implementation of InterProScan results. Before, Total number of added GO terms after Blast2GO annotation; after, Total number of GO annotations after implementation of InterProScan results; confirmed, Number of initial GO annotations confirmed by InterProScan result; too general, Number of GO annotations removed after InterProScan because of a lack of specificity.

Figure 4. Maximum likelihood analysis of a MUSCLE alignment of 19 of the identified putative VCP protein sequences from Nannochloropsis oceanica CCMP1779 (Nanno) and the annotated LHC and LHCSR-like sequences from Phaeodactylum tricornutum (Phatr), Cyclotella cryptica (Cycry), Thalassiosira pseudonana (Thaps), Aureococcus anophagefferens (Auran), and the LHCSR-like sequences from Chlamydomonas reinhardtii (Cre), Ostreococcus tauri (Ossta), Physcomitrella patens (Ppa), Scenedesmus obliquus (Sceob), and Volvox carteri (Volca).

Nannochloropsis VCP model 19 (Nanno_19) was not included in the analysis because the sequence is incomplete, causing it to be erroneously assigned as an out-group.

Figure 5. Hydrogen production.

H₂ accumulation was measured at 3, 24, and 48 hours after adding aerobically- or anaerobicially-incubated cells to air-tight sample vials. The vials contained growth media supplemented with 10 mM methyl viologen and 100 mM Na₂S₂O₄ (+MV) or unsupplemented growth media (−MV). A sample without cells was used as a negative control. (n≥3).

Figure 6. Accumulation of oil.

(A) TAG accumulation over time shown as fatty acids esterified to TAG (TAG FA) over total fatty acids (FAtotal) following nitrogen deprivation, and (B) characteristic changes in the fatty acid profile. Fatty acids are designated based on number of carbon atoms: number of double bonds. The accumulation of TAG and the formation of lipid droplets can be observed in ultra-structural changes following nitrogen starvation (C, N-replete; D, N-depleted).

Figure 7. Lipid assembly and modification.

(A) Proposed pathway of desaturation and elongation of fatty acyl chains in the ER of Nannochloropsis. EPA, eicosapentaenoic acid (B) Proposed plastid (green) and ER (lilac) pathway and genes putatively involved in the synthesis of TAG in Nannochloropsis. Numbers indicate count of putative genes identified. Number of ER acyltransferases cannot be assigned unambiguously, multiple candidate genes are listed in Table S13. G3P, glycerol-3-phosphate, LPA, lysophosphatidic acid, PA, phosphatidic acid, MAG, monoacylglycerol, DAG, diacylglycerol, TAG, triacylglycerol, PL, polar glycerolipid. GPAT, glycerol-3-phosphate acyltransferase, LPAT, lysophosphatidic acid acyltransferase, PAP, phosphatidic acid phosphatase, LIPIN, Lipin, MGAT/DGAT mono-/diacylglycerolacyltransferase, PDAT, phospholipid-diacylglycerolacyltransferase.