Botryococcus Braunii
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Fang et al. Biotechnol Biofuels (2015) 8:130 DOI 10.1186/s13068-015-0307-y RESEARCH ARTICLE Open Access Transcriptomic analysis of a moderately growing subisolate Botryococcus braunii 779 (Chlorophyta) in response to nitrogen deprivation Lei Fang1,6†, Deying Sun2,7†, Zhenyu Xu1, Jing He3, Shuyuan Qi1, Xin Chen4, Wee Chew5 and Jianhua Liu1,3* Abstract Background: The colonial microalga Botryococcus braunii has been brought to people’s attention for its conspicuous ability to accumulate a variety of lipids including hydrocarbons. B. braunii strains are classified into 3 races based on the types of hydrocarbons. A and B races are known to accumulate high level of lipids. However, their extreme slow growth rate has impeded its application for renewable biofuel production. Results: In this study, we report the transcriptomic response of a moderately growing subisolate from the culture of Botryococcus sp. CCALA-779 upon nitrogen deprivation (ND). We show that the subisolate has an average growth 1 1 rate of 0.52 g l− day− under photoautotrophic growth conditions and lipid content is enhanced to 75 % of CDW upon ND. Both rDNA sequence and hydrocarbon composition analyses indicate that the subisolate belongs to A race B. braunii. Hence, it is designated as B. braunii 779. We show that B. braunii 779 transcriptome shares homology to majority of the A race but not B race B. braunii ESTs, suggesting that transcriptomes of A race differ from that of B race. We found that many homologous ESTs between A races 779 and Bot-88 are unknown sequences, implying that A race contains many unknown genes. Pathway-based transcriptomic analysis indicates that energy metabo- lisms are among the top expressed functions in log-phase cells, indicating that the slow growth rate is a result that energy flow is directed to lipid biosynthesis but not population growth. Upon ND, reconfiguration of metabolisms for reducing power is apparent, suggesting that B. braunii 779 is rapidly adapting under ND condition by transcriptomic reprogramming. Conclusions: Taken together, our result shows that the subisolate B. braunii 779, similar to the Gottingen strain, is useful for biofuel production. Difference between transcriptomes of A and B races implies that different races of B. braunii strains belong to different sub-species. Furthermore, there are many novel genes that are unique to A race, suggesting that sequences of many enzymes involved in hydrocarbon biosynthesis are not currently known. We propose that B. braunii transcriptomes provide a rich source for discovery of novel genes involved in hydrocarbon biosynthesis. Keywords: Botryococcus braunii, Green microalgae, Hydrocarbon, Transcriptome, Response to nitrogen deprivation *Correspondence: [email protected] †Lei Fang and Deying Sun contributed equally to this work 3 Ocean Research Centre of Zhoushan, Zhejiang University, 10 Tiyu Road, Room 502, Zhoushan 316021, Zhejiang, China Full list of author information is available at the end of the article © 2015 Fang et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fang et al. Biotechnol Biofuels (2015) 8:130 Page 2 of 21 Background 19 out of 55 sequences encoding enzymes potentially Microalgae have been getting renewed interests for its involved in biosynthesis of VLCFA have been discov- CO2 mitigation and lipid production. Photosynthetic ered in A race B. braunii Bot-88 [24] and 100 curated and microalgae, like green plants, utilize photon energy to machine-assembled sequences potentially involved in convert CO2 (in presence of water) into sugar and oxy- biosynthesis of botryococcenes and squalene have been gen. Lipid yield from oleaginous algae is believed to be revealed in B race B. braunii Showa [21]. However, it is at least an order of magnitude higher than that of many unclear why not all the enzymes that are involved in bio- energy plants [1, 2], besides that cultivation of microalgae synthesis of VLCFA are found in the transcriptomes of A can avoid competing arable lands and irrigation water race B. braunii. for crops [3, 4]. Many oleaginous microalgae accumulate ND is one of the most widely used methods for triacylglycerols (or TAG) that need to be transesterified enhancement of storage lipids in a number of TAG-con- with methanol to methyl esters of fatty acids and glycerol, taining green microalgae [2, 5, 22]. In A race B. braunii, for example, before being refined to biodiesel [5]. On the nitrogen limitation enhances accumulation of oleic acid other hand, oleaginous green colonial microalga Botryo- [26], but not hydrocarbons [27]. Global transcriptional coccus braunii has been brought to people’s attention for profiling of microalgal cells in response to ND using next- its conspicuous ability to accumulate a variety of lipids generation sequencing (or NGS) technologies allows including hydrocarbons that are found in petroleum identification of gene regulatory networks involved in deposits and can be refined directly without the need of adaptation and survival [22, 28, 29]. esterification because they are not fatty acids [4, 6]. In this study, we report (1) the characterization of a However, B. braunii has suffered from an extreme slow moderately growing subisolate derived from the Bot- growth (i.e., population doubling time is approximately ryococcus sp. CCALA779 culture, (2) biochemical 5–7 days) that has hampered from its application for bio- analysis of the lipids and hydrocarbons accumulated in fuel production [6]. The slow growth has also prevented the subisolate, and (3) transcriptomic profiling of the it from genetic studies. Hence, many researches have subisolate in response to ND. We show that the subiso- focused on the analysis of hydrocarbon compositions late can reach a maximal growth rate of 1.23 g l−1 d−1 −1 −1 and the discovery of DNA sequences encoding enzymes and an average growth rate of 0.52 g l d in 2× BB involved in biosynthesis of hydrocarbons [7–12]. medium [30] under photoautotrophic growth condi- Depending on the types of hydrocarbons accumulated in tions. Lipid contents are enhanced by nitrogen depriva- cells and cell wall matrix, B. braunii is classified into three tion (ND). The 18S rRNA sequence-based phylogenetic principal races, A, B, and L. A race produces very long analysis places the subisolate to the group of A race. chain fatty acid (or VLCFA)-derived odd-carbon-num- Consistent with this, GC–MS analysis of hydrocarbon bered n-alkadiene and alkatriene, B race produces mainly composition in the subisolate reveals the presence of botryococcenes (or triterpenoids), and L race produces heptacosdiene and heptacostriene. Four giga-nucleo- lycopadiene (or tetraterpenoid) [13–16]. Several genes tide short-read sequences of RNA (or cDNA) generated encoding enzymes such as triterpene methyltransferases using Illumina sequencing platform were assembled by TMTs [8], 1-deoxy-D-xylulose 5-phosphate synthase Trinity software. Approximately 20 % (i.e., 12,292) of DXS [10], squalene synthase-like SSL botryococcenes the non-redundant transcriptome was found to have synthase [9, 17], Botryococcus squalene synthase BSS best-hit in the 6 algal genomes that are comprehensively [18], and squalene epoxidases SQE [11] have been cloned annotated (http://genome.jgi-psf.org). Comparison to and their enzymatic activities have been tested. other previously reported transcriptomes of other B. Genomic sequences of a number of photosynthetic braunii strains implies that the similarity between tran- microalgae have been completed (http://genome.jgi-psf. scriptomes of A and B races is very low (i.e., at a level org). These comprehensively annotated genomes have of 15 %). On the other hand, majority of the homolo- provided rich sources for annotation of novel genomes gous ESTs between A races are unknown sequences. and transcriptomes. Using the comprehensively anno- Taken together, our results indicate that the moder- tated genomes, de novo transcriptome analysis without ately growing subisolate belong to the A race B. braunii. reference genomic sequences has permitted the discov- Transcriptomes between A and B races B. braunii dif- ery of genes and reconstitution of metabolic pathways fer dramatically, suggesting that A and B races belong in some non-model microalgae [19–22]. De novo tran- to different sub-species. Additionally, many ESTs in scriptomic analyses in B. braunii have extended our A race transcriptomes are unknown sequences, pro- knowledge on potential enzymes involved in biosynthesis viding a useful resource for discovery of novel genes. of hydrocarbons in B. braunii [21, 23–25]. For example, We propose that the B. braunii 779 is similar to other Fang et al. Biotechnol Biofuels (2015) 8:130 Page 3 of 21 well-characterized strains such as Gottingen strain [27], a attractive for biofuel production. 4 2xBB y Results 3 g/L) Accumulation of lipid and hydrocarbon in a moderate densit 2 1xBB growing Botryococcus subisolate is enhanced by ND (CDW Cell 1 A moderately growing subisolate from a culture of Bot- ryococcus sp. CCALA 779 on solid BB medium was 0 obtained. Photoautotrophic growth rate of the subisolate 04812 Time (day) in liquid BB medium was examined under the condition μ −2 −1 of 250 mol photon m s . The growth curve indicated b that this subisolate exhibited a maximum growth rate of +N -N 0.61 g l−1 d−1 (i.e., gram of cell dry weight or CDW per −1 liter per day) and a maximum cell density of 1.98 g l in DIC terms of CDW per liter (Fig.