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Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp - PubMed

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp

Jianhua Fan et al. Plant Physiol. 2015 Dec.

Abstract

The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels.

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Figures

Figure 1.
Figure 1.

C. pyrenoidosa FACHB-9 as a research model for the starch-to-lipid switch and scalable production of lipids. A, Macromolecule profile under different culture models with heterotrophic cells as seed. B, Production of biomass and lipids by photoautotrophic growth in a 2-L column photobioreactor with 2% CO2. Comparisons of lipid production for heterotrophic and photoautotrophic seed for subsequent photoautotrophic culture are also presented. P-P indicates photoautotrophic culture with photoautotrophic cells as seed, and H-P indicates photoautotrophic culture with heterotrophic cells as seed. C, Dynamics of algal growth and cellular components during SHDP. Arrows represent the six sampling time points for mRNA-seq (0, 24, 72, 74, 80, and 96 h). Dcw, Dry cell weight.

Figure 2.
Figure 2.

Phylogenetic analysis of the C. pyrenoidosa genome. A, Venn diagram representation of shared/unique genes of C. pyrenoidosa compared with red algae, diatoms, Eustigmatophyceae, and green algae. B, Venn diagram representation of shared/unique genes of C. pyrenoidosa compared with with other sequenced Chlorophyceae algae and land plants. C, Functional profiles of the six Chlpy-pairwise-cores and the six Chlpy-pairwise-accessories. D, Whole-genome-based phylogeny of Chlorophyta alga. A maximum-likelihood (consensus) tree generated based on the 1,685 single-copy orthologous gene sets identified from the seven selected green algal phylogenomes. At each branching point on the tree, the number of orthologous genes found within the corresponding branch is shown. The number of single-copy orthologous genes pairwise to C. pyrenoidosa is indicated along with strain names. Red alga, Cyanidioschyzon merolae; diatom, P. tricornutum; green alga, C. reinhardtii; Eustigmatophyceae, N. oceanica; Prasinophyceae, Micromonas RCC299; Streptophyta, Arabidopsis; Trebouxiophyceae, C. variabilis; and Chlorophyceae, C. reinhardtii.

Figure 3.
Figure 3.

Selection pressure on protein-coding sequences in green algae. For each of the 1,685 seven-way single-copy orthologous gene sets identified among seven selected Chlorophyta species, Phylogenetic Analysis by Maximum Likelihood (PAML) model M0 was used to estimate a single ω (Ka/Ks, ratio of nonsynonymous to synonymous nucleotide divergence) that is fixed across the reconstructed whole-genome phylogeny. The associated GO slim terms that have at least three genes are shown for biological process (A), molecular function (B), and cellular component (C). rRNA, Ribosomal RNA.

Figure 4.
Figure 4.

Clustering results of gene expression patterns in C. pyrenoidosa when shifting heterotrophic cells to photoautotrophy. The differentially expressed genes were clustered into nine groups using the k-means clustering method and visualized with TM4 software. The horizontal axis indicates the time points of the culture process, and the vertical axis is the log2 expression ratio. Fold expression changes between different time points (heterotrophy 24 h/heterotrophy 0 h, heterotrophy 72 h/heterotrophy 0 h, phototrophy 2 h/heterotrophy 72 h, phototrophy 8 h/heterotrophy 72 h, and phototrophy 24 h/heterotrophy 72 h) were calculated using the log2 ratios.

Figure 5.
Figure 5.

Overview of metabolic pathways and regulation during the heterotrophy to photoautotrophy transition in C. pyrenoidosa. KEGG pathways in blue, red, or green are present in the C. pyrenoidosa genome. Light-gray background lines indicate KEGG pathways not encoded by the C. pyrenoidosa genome. Genes that are up- or down-regulated (compared with heterotrophy 72 h) are labeled in red and green, respectively. Deeper red and green indicates greater fold changes of differential expression.

Figure 6.
Figure 6.

Genomic and transcriptomic features of central carbon metabolism in C. pyrenoidosa. Metabolic steps are represented by arrows. Dashed lines represent multiple metabolic steps. Boxes indicate those nodes where carbon skeletons from amino acid degradation feed into the pathway. Genes encoding the enzymes of these pathways are labeled in red. Up- or down-regulation of mRNA expression under heterotrophic growth (compared with heterotrophy 0 h, leftward arrows) and the heterotrophy to photoautotrophy transition (compared with heterotrophy 72 h, rightward arrows) based on mRNA-seq data are indicated with red upward arrows and green downward arrows, respectively. The full names of the corresponding genes are given in

Supplemental Table S9

.

Figure 7.
Figure 7.

Fatty acid and glycerolipid biosynthesis and regulation in C. pyrenoidosa FACHB-9 as reconstructed from genomic and transcriptomic evidence. Metabolic steps are represented by arrows. Dashed lines represent the presence of multiple metabolic steps. End products are shown in boxes. Genes encoding the enzymes in these pathways are labeled in red. Up- or down-regulation of mRNA expression under heterotrophic growth (compared with heterotrophy 0 h, leftward arrows) and the heterotrophy to photoautotrophy transition (compared with heterotrophy 72 h, rightward arrows) based on mRNA-seq data are indicated with red upward arrows and green downward arrows, respectively. The full names of the corresponding genes are given in

Supplemental Table S9

. Because the glycolytic enolase is coded by only one gene (g2571) in C. pyrenoidosa, it is proposed that in Chlorella spp., the glycolytic enolase is only active in the cytosol, resulting in a specific pattern of carbon flow following fixation that involves export of carbon from the plastid and then reimport for pyruvate generation.

Figure 8.
Figure 8.

Transformation of C. pyrenoidosa FACHB-9 and validation of transgenic strains. A, A schematic map of the pGreen0029-Ubi-eGFP-Nos plasmid. The pGreen0029-Ubi-eGFP-Nos vector contained an expression box of the eGFP gene, controlled by the Ubiquitin gene1 promoter (Ubi-pro) and terminated by the Nos terminator (Nos-T) and an expression box of the neomycin phosphotransferase enzyme (NptII), which conferred resistance to aminoglycoside antibiotics. B, PCR detection of the NptII gene using primers F and R. M, DNA molecular weight marker; 1, wild-type C. pyrenoidosa cells; 3, positive control, the pGreen0029-Ubi-eGFP-Nos plasmid; 2 and 4 to 12, strains of transgenic cells. C, Southern-blot analysis of C. pyrenoidosa transformants. The DNA from the wild type and transformants were digested with PstI and EcoRI and then hybridized with a 464-bp fragment of the partial eGFP gene. M, DNA molecular weight marker; 1, positive control; the pGreen0029-Ubi-eGFP-Nos plasmid; 5, wild-type cells; 2 to 4 and 6 to 9, strains of transgenic cells. D, Identification of the transgenic strains using fluorescence microscopy observation. E, Identification of the negative control (which underwent the entire electroporation protocol without any plasmid DNA) using fluorescence microscopy.

Figure 9.
Figure 9.

Characterization of AtNADK3-overexpressing C. pyrenoidosa FACHB-9 strains under different culture conditions. A, Growth curves of three AtNADK3-overexpressing strains under heterotrophic culture conditions. B, Growth curves of the three AtNADK3-overexpressing strains under photoautotrophic culture conditions. C, Relative expression levels of AtNADK3 in the NADK3-2 strain under different culture conditions. D, NADPH content of the three AtNADK3-overexpressing strains under different culture conditions. E, Total lipid contents of the three AtNADK3-overexpressing strains under different culture conditions. F, The fatty acids content and composition in the NADK3-2 strain under different culture conditions. Data represent the means ±

sd

of three biological replicate experiments and were analyzed by Student’s t test (n = 3). Asterisks indicate a significant difference from wild-type strains (**, P < 0.01). For heterotrophy, 72-h cultivation was carried out. For light induction (100 μmol m–2 s–1) and heat shock (42°C), 24-h cultivation was carried out using heterotrophic cells as seed. DCW, Dry cell weight; WT, wild-type C. pyrenoidosa.

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