| Microalgal biofuels, given its benefit to carbon-neutral, renewable and environmental friendly, has become one of the worldwide focuses. Nevertheless, the mechanisms of how microalgae evolved self-protective strategies against physiologic stress at the molecular level, as well as synthesis and accumulate lipids under unfavorable conditions are largely unknown, which hinders the development of microalgal biofuels. Our group has focused on this field for almost eighteen years and initiated a novel cultivation strategy called "Sequential Heterotrophy-Dilution-Photoinduction"(SHDP) as platform technology for efficient Chlorella biomass production. Based on SHDP model, we attempt to integrate the production of both high value bioproducts and microalgal biofuels as well as CO2biofixation, which is expected to speed up the industrialization process of microalgal bioenergy. However, little physiologic and biochemical information is available, especially regarding the major molecular events that occur in microalgal cells under diverse trophic transitions. Thus, the mechanism for the SHDP process still needs to be fully understood. Chlorella pyrenoidosa is one of the most common and best-studied species among Chlorella genus. Besides serving as the industrial stain for nourishment purpose, C. pyrenoidosa accumulates～55%lipids under certain culture conditions, with a favorable fatty acid profile for biodiesel production. Due to its uniqueness of combining fast growth rate with the high suitability for bio-refining, in combination with successful cultivation at commercial scale and high market acceptance, C pyrenoidosa is therefore an ideal strain for economical, industrial-scale algal biofuels production. However, the lack of available genome sequence information, hinders the further improvement of this organism as a fuel and food resource.Here we report the draft genome sequence of this versatile Chlorophyta C. pyrenoidosa and compare it with other alga species to clarify evolutionary origin, functional significance and ubiquity of these features throughout algal lineages. To well grasp its cellular metabolism, particularly the regulation of biosynthesis and degradation pathways of lipid, protein and carbohydrates, as well as the diverse trophic adaptation affecting carbon partitioning during SHDP process, we sequenced the transcriptome (RNA-seq) of SHDP process in six time points to discover how transcriptional changes in C. pyrenoidosa modulate metabolic flux trends leading to intracellular components dynamic reassortment. Furthermore, we reconstructed the first networks of heterotrophy and central carbon metabolism and glycerolipid biosynthesis pathways in Chlorella. (1) Regular pattern of intracellular components of heterotrophy/photoinduction switching process under the biochemical and cellular level. Three Chlorella species including C. pyrenoidosa, C. vulgaris and C. ellipsoidea were adopted. Analysis of the cell components and electron micrograph studies suggest that the shift from heterotrophic to light-induced growth causes a rapid decrease in cell carbohydrate and corresponding increases in chloroplast proteins, pigments, and lipids. Further outdoor studies were also carried out to confirm the feasibility of SHDP in a large scale approach for microalgal mass culture process. The total lipid could be accumulated to high levels by SHDP within several hours. Maximum lipid content as26.11%of biomass and maximum lipid productivity of89.89mg/L/d were both accomplished by C. pyrenoidosa. The length of fatty acids carbon chain of the Chlorella cultured by SHDP were between C14to C18, which is suitbale for biodiesel production.(2) Features of the whole genome. The56.6Mb draft nuclear genome of C. pyrenoidosa was generated at26×coverage by Roche454Titanium shotgun and paired-end sequencing, followed by assembly into1,336scaffolds. Half of the assembled genome sequences are contained in9scaffolds, each longer than1.39Mb. The majority of genome contigs are unusually GC-rich (66.6%), making the G+C content of C. pyrenoidosa more akin to that of C. variabilis NC64A than to that of other sequenced eukaryotic genomes to date. C. pyrenoidosa is predicted to contain10,284genes, and87.4%of the predicted proteins have homologous sequence in NCBI nr database (e-value<le-5).80.9%of gene models had cDNA support. The predicted C. pyrenoidosa protein-coding genes are extremely rich in introns, have nearly nine introns per gene on average and hence have the largest average number of introns reported so far in chlorophytes; only1.1%genes lack introns. An ab initio approach detected repeated sequences comprising retroelements, DNA transposons, small repeats and low complexity, et al, make up3.1%of the whole genome. The third base of the codons show significant preference for guanine (G) and cytosine (C). We also predicted the subcellular localisation of C. pyrenoidosa proteins, and the results showed that1,109and2,437proteins are targeted to the plastid and to mitochondria, respectively.(3) Functional and phylogenetic analyses. C. pyrenoidosa was found to encode all of the know meiosis-specific proteins, transcriptomics evidence of meiosis-specific genes suggests that cryptic meiosis and sexual reproduction might be part of the C. pyrenoidosa life cycle. Homologs involved in cellulose and hemicellulose synthesis were also identified in the genome. Besides, we also identified putative genes involved in forming and remodeling chitin cell walls, which appears to be uncommon in land plants and green algae. Most Chlorella proteins (84%) exhibit significant similarity to green lineage than to red alga and stramenopiles. The distinct difference of functional profiles between "Chlorella-paivwise cores and accessories" and the large size of "Chlorella-unique-accessory" support a model of genome evolution featuring oriented gene gain and loss in C. pyrenoidosa. At Chlorophyta level, single-copy orthologs are strongly constrained, with median estimates of co much less than one. The most constrained biological processes include photosynthesis, organelle organization and protein phosphorylation, whereas the least constrained ones are lipid metabolism, phospholipid biosynthesis, glycolysis and carbohydrate metabolism, suggesting the relative evolutional conservation of essentially photosynthesis ability as well as fast evolving in lipid and carbohydrate metabolisms which corresponding to their physiological diverseness (e.g.+/-oil producing) among chlorophytes. Nevertheless, at species level (Chlorella), average Ka/Ks value is significant higher than at phylum level (based on all single copy orthologs, t-test, p-value<2.2e-16), suggesting higher evolutionary rates at lower phylogentic levels.(4) Transcript Profiling of Chlorella under heterptrophy to photoinduction transition. Suppression subtractive hybridization strategy was firstly employed to screen and characterize genes that are differentially expressed in response to the light-induced shift from heterotrophy to photoautotrophy. The majority of the genes enriched in the subtracted libraries were associated with energy metabolism, amino acid metabolism, protein synthesis, carbohydrate metabolism, and stress defense, which preliminary confirmed the transcriptome regulation is crucial to SHDP process. Further to obtain deep coverage, we used Solexa GA-Ilx platform, and yielding a total of187,503.403trimmed paired-end reads at six time points of SHDP process. The detailed analysis of all RNA-seq data led to the identification of1,578and1,491nuclear encoded genes displaying a more than3fold differential transcript change for at least one of the time points during heterotrophic growth (heterotrophy24-h/heterotrophy0-h, heterotrophy72-h/heterotrophy0-h) and trophic conversion (phototrophy2-h/heterotrophy72-h, phototrophy8-h/heterotrophy72-h, phototrophy24-h/heterotrophy72-h), respectively. All the differentially expressed genes with KEGG protein/EC number identifiers associated with each of time point expression profiles were in turn used to populate metabolic pathway maps, which provide further evidence of heterotrophy-specific metabolism and metabolic flux migratory movement during nutritional conversion. In heterotrophic growth process, many of the genes that are most strongly up-regulated are those involved in citrate cycle and amino acid metabolism, whereas many of the most down-regulated genes are involved in fatty acid biosynthesis and metabolism, pentose phosphate metabolism and nucleotide metabolism. In contrast to heterotrophy. shift from heterotrophy into phototrophy highlights the increased expression of genes involved in carbon fixation and photosynthesis that would be expected owing to light illumination. In addition, genes related to fatty acid biosynthesis, pentose phosphate pathway and starch metabolism are also transcriptionally up-regulated to a significant extent. These analyses confirm the regulation of metabolic pathways of lipid. protein and carbohydrates and trophic conversion affecting carbon repartition that may be of importance for the cellular components phenotype of different nutritional model. At last, Chlorella heterotrophic metabolism, central carbon metabolism, glycerolipids and starch biosynthesis pathways were reconstructed based on the predicted gene models, and changes in transcripts abundance of genes encoding enzymes are also summarized. The above findings provide genomic and transcriptomic foundation for improving microalgal growth at a molecular level to finally increase total lipid yield.The C. pyrenoidosa genome and transcriptome data present here go a long way towards developing this versatile organism as a model species, explaining the distinct nutritional diversity, and providing the possibility to combine genomic and genetic approaches to further improve its capacity of both lipid and nourishment production. |
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