Analysis of the <i>Sh ble</i> gene integrated site in the mitochondrial genome of MT-B.

<p>A: The Southern hybridization of <i>C. reinhardtii</i> MT-B with the ble-probe; B: Restriction map of the mitochondrial genome of <i>C. reinhardtii</i> MT-B showing the positions of all mapped genes and restriction sites. Line 1,2, 3: Total DNA of MT-B were digested with <i>Bam</i> HI, <i>Nde</i> I and <i>Sac</i> II, respectively, Line 4: the genome of CC-2654 digested with <i>Nde</i>I was used as control.</p

Sensitivity analysis of transgenic strains MT-Bs to Zeomycin.

<p>a: wild-type strain CC-124; b: respiratory deficient strain CC-2654; c: transgenic strain MT-B; d: empty control.</p

MOESM2 of Artificial miRNA inhibition of phosphoenolpyruvate carboxylase increases fatty acid production in a green microalga Chlamydomonas reinhardtii

Additional file 2: Figure S2. The growth curve of transgenic C. reinhardtii. aRP1.1, aRP1.2 were derived from individual amicroRNA- PEPC1 transformants, and aRP2.1, aRP2.2, aRP2.3 were from amicroRNA- PEPC2 transformants. C. reinhardtii CC-849 is the wild type. Introduction of amicroRNA- PEPC1 and amicroRNA- PEPC2 has no effect on the growth of transgenic algae

Schematic diagram of homologous recombination events between pBsLPNCB and CC-2654 mtDNA.

<p>The dotted line represents the deletion part of mtDNA in CC-2654 relative to wild-type CC-124. The empty boxes are the mitochondrial genes and the empty arrows above the genes indicate their transcription directions. The crossed solid lines denote the homologous recombination region between the CC-2654 mtDNA and expression vector pBsLPNCB.</p

The expression levels of the <i>Sh ble</i> gene and the mitochondrial genes in transgenic MT-B.

<p>A: The gene organization of MT-B mitochondrial genome: B: RT-PCR results of <i>Sh ble</i> gene and the mitochondrial genes with rrnL7 as internal control; C: Relative mRNA levels analysis using BIO-RAD image software.</p

Western blotting analysis of the transformants MT-B using monoclonal anti-BLE antibody.

<p>The soluble protein of CC-2654 (2) were used as control, the soluble protein of transformants MT-B (1) was detected by Western blotting.</p

<i>Sh ble</i> gene transcripts in transformant MT-B.

<p>Panal A is the RT-PCR analysis of MT-B strains and PCR amplification with total RNA. M: DL2000 marker, Lane 1: cDNA of negative control CC-2654 used as template; 2–4: total RNA isolated from different MT-B strains used as template; lane 5–7: cDNA of different MT-B strains used as template. Panal B is the Northern blot analysis of <i>C. reinhardtii</i> MT-B. Lane 1, total RNA of MT-B was detected with <i>Sh ble</i> probe; lane 2. total RNA of CC-2654 was used as negative control.</p

Growth of transformants MT-Bs on the TAP media with 3 µg/mL Zeomycin (A) and PCR analysis with B1/B2 primers (B).

<p>M, DL2000 marker; lanes1-6, different clones of MT-B; lanes 7–8, negative controls CC-2654 and CC-124; lane 9, water used as negative control.</p

Table_1_Flagella-Associated WDR-Containing Protein CrFAP89 Regulates Growth and Lipid Accumulation in Chlamydomonas reinhardtii.docx

<p>WD40-repeat (WDR) domain-containing proteins are subunits of multi-protein E3 ligase complexes regulating various cellular and developmental activities in eukaryotes. Chlamydomonas reinhardtii serves as a model organism to study lipid metabolism in microalgae. Under nutrition deficient conditions, C. reinhardtii accumulates lipids for survival. The proteins in C. reinhardtii flagella have diverse functions, such as controlling the motility and cell cycle, and environment sensing. Here, we characterized the function of CrFAP89, a flagella-associated WDR-containing protein, which was identified from C. reinhardtii nitrogen deficiency transcriptome analysis. Quantitative real time-PCR showed that the transcription levels of CrFAP89 were significantly enhanced upon nutrient deprivation, including nitrogen, sulfur, or iron starvation, which is considered an effective condition to promote triacylglycerol (TAG) accumulation in microalgae. Under sulfur starvation, the expression of CrFAP89 was 32.2-fold higher than the control. Furthermore, two lines of RNAi mutants of CrFAP89 were generated by transformation, with gene silencing of 24.9 and 16.4%, respectively. Inhibiting the expression of the CrFAP89 gene drastically increased cell density by 112–125% and resulted in larger cells, that more tolerant to nutrition starvation. However, the content of neutral lipids declined by 12.8–19.6%. The fatty acid content in the transgenic algae decreased by 12.4 and 13.3%, mostly decreasing the content of C16:0, C16:4, C18, and C20:1 fatty acids, while the C16:1 fatty acid in the CrFAP89 RNAi lines increased by 238.5 to 318.5%. Suppressed expression of TAG biosynthesis-related genes, such as CrDGAT1 and CrDGTTs, were detected in CrFAP89 gene silencing cells, with a reduction of 16–78%. Overall our results suggest that down-regulating of the expression of CrFAP89 in C. reinhardtii, resulting in an increase of cell growth and a decrease of fatty acid synthesis with the most significant decrease occurring in C16:0, C16:4, C18, and C20:1 fatty acid. CrFAP89 might be a regulator for lipid accumulation in C. reinhardtii.</p

Image_1_Comparative transcriptomic and lipidomic analyses indicate that cold stress enhanced the production of the long C18–C22 polyunsaturated fatty acids in Aurantiochytrium sp..png

Aurantiochytrium sp. belonging to Thraustochytrids are known for their capacity to produce long-chain polyunsaturated fatty acids (PUFAs). However, effects of cold stress accompanied with staged-temperature control on the fatty acid metabolism in Aurantiochytrium sp. were rarely studied. In this study, cold stress (15°C, 5°C) was applied for Aurantiochytrium sp., with the physiological responses (morphology, growth, fatty acid profiling) and gene expression related FA synthesis, lipid metabolism, and regulatory processes was observed. Results showed that there is a significant change for the lipid types under 5°C (251 species) and 15°C (97 species) treatment. The 5°C treatment was benefit for the C18–C22 PUFAs with the yield of docosahexaenoic acid (DHA) increased to 1.25 times. After incubation at 15°C, the accumulation of eicosadienoic acid (EA) (20:2) was increased to 2.00-fold. Based on transcriptomic and qPCR analysis, an increase in genes involved in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways was observed under low-temperature treatment. With upregulation of 3-ketoacyl-CoA synthase (2.44-fold), ketoreductase (2.50-fold), and dTDP-glucose 4,6-Dehydratase (rfbB) (2.31-fold) involved in PKS pathway, the accumulation of DHA was enhanced under 5°C. While, FAS and fatty elongase 3 (ELO) involved in the FAS pathway were upregulated (1.55-fold and 2.45-fold, respectively) to accumulate PUFAs at 15°C. Additionally, glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT), phosphatidic acid phosphatase (PAP), phosphatidylserine synthase (PSS), and phosphatidylserine decarboxylase (PSD) involved in glycerophospholipid biosynthesis were upregulated at 5°C increasing the accumulation of phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). However, glycolysis and the TCA cycle were inhibited under 5°C. This study provides a contribution to the application of two-staged temperature control in the Aurantiochytrium sp. fermentation for producing cold stress-enhancing PUFAs, in order to better understand the function of the key genes for future genetic engineering.</p