Application of RNA-Seq for discovery of biofuel producing organisms

For many years, increasing demands for fossil fuels have met with limited supply. As a potential substitute and renewable source of biofuel feedstock, microalgae have received significant attention. However, few of the current algal species produce high lipid yields to be commercially viable. To discover more high yielding strains, next-generation sequencing technology is used to elucidate lipid synthetic pathways and energy metabolism involved in lipid yield. When subjected to manipulation by genetic and metabolic engineering, enhancement of such pathways may further enhance lipid yield.

In this study, transcriptome profiling of a random insertional mutant with enhanced lipid production generated from a non-model marine microalga Dunaliella tertiolecta is presented. D9 mutant has a lipid yield that is 2- to 4-fold higher than that of wild type. Using novel Bag2D-workflow scripts developed and reported here, the non-redundant transcripts from de novo assembly were annotated based on the best hits in five model microalgae, namely Chlamydomonas reinhardtii, Coccomyxa subellipsoidea C-169, Ostreococcus lucimarinus, Volvox carteri, Chlorella variabilis NC64A and a high plant species Arabidopsis thaliana. The assembled contigs (~181 Mb) includes 481,381 contigs, covering 10,185 genes. Pathway analysis showed that a pathway from inositol phosphate metabolism to fatty acid biosynthesis is the most significantly correlated with higher lipid yield in this mutant.

Herein, researchers from the National University of Singapore described a pipeline to analyze RNA-Seq data without pre-existing transcriptomic information. The draft transcriptome of D. tertiolecta was constructed and annotated, which offered useful information for characterizing high lipid-producing mutants. D. tertiolecta mutant was generated with an enhanced photosynthetic efficiency and lipid production. RNA-Seq data of the mutant and wild type were compared, providing biological insights into the expression patterns of contigs associated with energy metabolism and carbon flow pathways. Comparison of D. tertiolecta genes with homologs of five other green algae and a model high plant species can facilitate the annotation of D. tertiolecta and lead to a more complete annotation of its sequence database, thus laying the groundwork for optimization of lipid production pathways based on genetic manipulation.


RNA-Seq data analysis flowchart used in this study. The general pipeline includes sample preparation and harvesting, sequencing, data analyses, and biological interpretation. The red highlighted rectangle illustrates the construction of the D. tertiolecta reference library using Bag2D-workflow scripts. Bag2D: Blast1-annotation1-gene model1-Delete redundant genes-Blast2-annotation2-gene model2 (protein name, transcriptID, geneID)

Availability – The novel Bag2D program package and computationally processed Dunaliella tertiolecta draft transcriptome database and its annotation files are hosted at the author’s GitHub page https://​github.​com/​SPURc-Lab/​NGS-D9 with the step-by-step user manual for public access.

Yao L, Tan TW, Ng YK, Ban KH, Shen H, Lin H, Lee YK. (2015) RNA-Seq transcriptomic analysis with Bag2D software identifies key pathways enhancing lipid yield in a high lipid-producing mutant of the non-model green alga Dunaliella tertiolecta. Biotechnol Biofuels 8:191. [article]

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