Project description:Background: 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. Results: 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 2-4 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, and Chlorella variabilis NC64A. The assembled contigs (~181 Mb) includes 481,381 transcripts, 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. Conclusion: Herein, we 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 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. Examine two sets of RNA-Seq data from high lipid-producing mutant and wild-type sample Dt_v1*: the FASTA and annotation files used for D9_1, WT_1 samples Dt_v10*: the FASTA and annotation files used for D9_2, WT_2 samples Dt_v10-hit*: the FASTA and annotation files only including the contigs that can be annotated in Dt_v10 Additional file 1.xls: processed data for sample 1,2 Additional file 4.xls: processed data for sample 3-6

Project description:Plants and microalgae have the ability to harness sunlight into energy dense molecules such as triacylglycerols (TAGs). TAGs hold great importance for human and animal nutrition, and are also a source for renewable energy. Acyl-CoA-dependent TAG synthesis by diacylglycerol acyltransferases (DGATs) in oilseeds has been well characterized, but how the ectopic introduction of TAG synthesis in vegetative tissues of plants affects metabolism is largely unknown. Here we report the identification and characterization of several Chlamydomonas reinhardtii Diacylglycerol Acyltransferase Type Two (DGTT) enzymes and their use in engineering TAG biosynthesis in plant vegetative tissues. Focusing on DGTT2 we demonstrate that it can accept a broad range of substrates. Production of DGTT2 in Arabidopsis results in distinct seedling phenotypes and accumulation of TAG with very long chain fatty acids (VLCFA), in both seedlings (23.4-fold increase) and in the leaves of soil-grown plants (13.3-fold increase). Levels of surface lipids, such as waxes and cutins, are decreased in DGTT2-producing lines, consistent with a decreased carbon/acyl flux to the surface lipid pathway. Global transcript analysis showed that very few genes had altered expression. Little to no change in the transcripts of wax/cutin pathway genes was observed in response to altered carbon partitioning into TAGs in the leaves suggesting that the supply of acyl groups for surface lipid biosynthesis is not transcriptionally adjusted in the transgenic lines. As an indication that the DGTT2-producing plants are richer in feed value, the generalist herbivore Spodoptera exigua reared on the transgenic plants gained more weight. Apparently, the substrate specificity of DGTT2 from Chlamydomonas when produced in the Arabidopsis cells leads to diversion of carbon from surface compounds in TAGs and enhances the nutritional value of leaves. 4 biological replicates DGTT2 transgenic plants are compared to 4 biological replicates of wild type

Project description:Background: 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. Results: 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 2-4 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, and Chlorella variabilis NC64A. The assembled contigs (~181 Mb) includes 481,381 transcripts, 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. Conclusion: Herein, we 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 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.

Project description:The absence of oxygen (O2) is a stress condition for aerobic organisms and requires extensive acclimation responses. Previously, Chlamydomonas reinhardtii has been used as a reference organism for understanding these acclimation responses. In this work, we use RNA-Seq for a whole genome view of the acclimation of the organism to dark-anoxic conditions. To distinguish the responses dependent on the COPPER RESPONSE REGULATOR 1 (CRR1), which is also involved in hypoxic gene regulation, we compared the transcriptome of crr1 mutants to that of complemented strains. Nearly 10% of the genome (~ 1,400 genes) are affected by hypoxia based on pairwise comparisons of all strains and two time-points. Comparing transcript profiles from early (hypoxic) with those from late (anoxic) time-points indicated that the cells activated oxidative energy generation pathways before employing fermentative enzymes. Probable substrates included not only carbohydrates but also amino acids and fatty acids (FAs). Lipid profiling of the C. reinhardtii cells revealed that they degraded FAs but also accumulated triacylglycerols (TAGs). In contrast to N-deprived cells, the TAGs accumulating in hypoxic cells are enriched in desaturated FAs, which distinguishes the contribution of individual pathways for Chlamydomonas TAG accumulation. In crr1 mutants, about 140 genes were aberrantly regulated , re-affirming the importance of CRR1 for the hypoxic response, but indicating also the contribution of additional O2-sensors and signaling strategies to account for the remaining differentially regulated transcripts. We conclude that nitric oxide (NO) dependent signaling cascades, employing both known and novel components, are operative in C. reinhardtii. The transcriptome of four different Chlamydomonas strains (wild type CC-124, crr1 mutant, crr1:CRR1 rescued strain and crr1dCys rescued strain) are profiled by RNA-Seq in the dark at different times after the transition from light-oxic to dark-anoxic conditions

			Action	DRS
		Other
	GSE287670_processed_data.xls.gz	Xls

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Chlamydomonas reinhardtii (red) rich in protoporphyrin Ⅸ exerts anti-diabetic effects in liver tissue

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