Identification and Overexpression of Endogenous Transcription Factors to Enhance Lipid Accumulation in the Commercially Relevant Species Chlamydomonas pacifica

Abstract

Sustainable low-carbon energy solutions are critical to mitigating global carbon emissions. Algae-based platforms offer potential by converting carbon dioxide into valuable products while aiding carbon sequestration. However, scaling algae cultivation faces challenges like contamination in outdoor systems. Previously, our lab evolved Chlamydomonas pacifica, an extremophile green alga, which tolerates high temperature, pH, salinity, and light, making it ideal for large-scale bioproduct production, including biodiesel. Here, we enhanced lipid accumulation in evolved C. pacifica by identifying and overexpressing key endogenous transcription factors through genome-wide in-silico analysis and in-vivo testing. These factors include Lipid Remodeling Regulator 1 (CpaLRL1), Nitrogen Response Regulator 1 (CpaNRR1), Compromised Hydrolysis of Triacylglycerols 7 (CpaCHT7), and Phosphorus Starvation Response 1 (CpaPSR1). Under nitrogen deprivation, CpaLRL1, CpaNRR1, and CpaCHT7 overexpression enhanced lipid accumulation compared to wildtype. However, CpaPSR1 increased lipid accumulation compared to wildtype in normal media despite causing no effect under nitrogen depravation, highlighting the difference in function based on media conditions. Notably, lipid analysis of CpaPSR1 under normal media conditions revealed a 2.4-fold increase in triglycerides (TAGs) compared to the wild type, highlighting its potential for biodiesel production. This approach provides a framework for transcription factor-focused metabolic engineering in algae, advancing bioenergy and biomaterial production.

Keywords: biofuels; biotechnology; computational modeling; microalgae; nitrogen-deprivation; sustainability; transcription factors.

Figure 1:. Overview and comparative analysis of transcription factors in Chlamydomonas pacifica with other algae and higher plant species.

(A) Distribution of transcription factors across different Clusters of Orthologous Genes (COG) categories, indicating their potential functions in Chlamydomonas pacifica. (B) Bar chart showing the distribution of transcription factors across various families in Chlamydomonas pacifica, highlighting the abundance of different types within the species. (C) PCA plot illustrating the transcription factor profiles of Chlamydomonas pacifica compared with those of various other algae and higher plant species. (D) A zoomed-in PCA plot focusing on the Chlorophyta group, highlighting the close relationship of Chlamydomonas pacifica with other green algae species. (E) Network plot showing the interactions between transcription factors of Chlamydomonas pacifica, Chlamydomonas reinhardtii, and other Chlorophyta species. The labels for species in panel (C,D) are as follows: Chlamydomonas reinhardtii (Cre), Chlamydomonas pacifica (Cpa), Volvox carteri (Vca), Chlorella sp. NC64A (Cnc), Coccomyxa sp. C-169 (Cvu), Micromonas pusilla CCMP1545 (Mpu), Micromonas sp. RCC299 (Mrc), Ostreococcus lucimarinus CCE9901 (Olu), Ostreococcus sp. RCC809 (Orc), and Ostreococcus tauri (Ota).

Figure 2:. Pipeline for predicting transcription factors involved in starch and lipid biosynthesis in C. pacifica.

The schematic illustrates the prediction of transcription factors involved in starch and lipid biosynthesis in C. pacifica. In Step 1, transcription factors were predicted using three different tools, and a comprehensive database of validated transcription factors was created from 10 different genera based on literature. In Step 2, a phylogenetic tree analysis was conducted on the transcription factors from C. pacifica and the curated validated database within the same transcription factor family. This analysis resulted in the identification of 10 predicted transcription factors involved in the biosynthetic pathways. The color coding of various transcription factors signifies whether they have been experimentally tested in this study.

Figure 3:. Conservation of transcription factor domain and genes in starch metabolism.

(A) Alignment showing MYB and coiled-coil domains from Chlamydomonas reinhardtii’s PSR1 (CrePSR1), Arabidopsis thaliana’s PHR1 (AtPHR1), and an orthologous predicted CpaPSR1 transcription factor from Chlamydomonas pacifica (anno1.g8834.t1), highlighting amino acid conservation. (B) An illustration of orthologous genes related to starch synthesis and breakdown is marked in green text. It features the enriched motifs within the promoter sequences of two of these genes that are similar to the consensus motif sequence of CrePSR1. The schematic is adapted from Bajhaiya et al., 2016. The enzyme label SBE represents Starch Branching Enzyme, and the orthologous gene definitions are described in Supplementary Table S5.

Figure 4:. Cell growth measured as chlorophyll fluorescence and cell density in transgenic lines.

(A), (B) Growth curves depicting chlorophyll levels and culture density of transcription factor overexpressed lines compared to the wildtype strain, respectively. Each line represents three replicates. A.U. stands for arbitrary units. O.D. stands for optical density.

Figure 5:. Flow cytometry data showing enhanced lipid production.

(A), (B) Flow cytometry analysis of Nile red-stained cells was conducted to quantify lipid content in transcription factor overexpressed lines compared to the wildtype strain under normal minimal or nitrogen-deprived media conditions. The statistics displayed in the top right corner of each plot correspond to populations above the threshold of 7.7×10⁴. PE-A refers to the phycoerythrin-area channel. “Normal” and “-N” represent normal and nitrogen-deprived media conditions, respectively. Each plot shows curves corresponding to three biological replicates. ‘*’ represents significant upward differences of the entire distribution from wildtype (Wilcoxon ranked sum test, p < 0.001) and each media is compared separately.

Figure 6:. Confocal microscopy of cells in different media conditions.

Cells in normal minimal and nitrogen-deprived media, respectively. The scale bar represents 5 μm. Blue and magenta correspond to Chlorophyll and triacylglycerol, respectively.

Figure 7:. GC-MS analysis of algae-derived FAME biodiesel.

Comparison of biodiesel composition of overexpressed lines with evolved wildtype C. pacifica in normal minimal media through relative peak area integration percentages in gas chromatography-mass spectrometry analysis (n = 2).

Figure 8:. Proposed model showing how transcription factors shape triglyceride and starch accumulation in Chlamydomonas pacifica through increased production and decreased degradation.

The metabolic pathways for (A) TAG and (B) starch biosynthesis and initial degradation in C. pacifica. Arrows from TFs represents putative upregulation, and perpendicular tipped lines represent putative downregulation. Abbreviations: ACP, acyl carrier protein; DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglyceride.