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The Path to Triacylglyceride Obesity in the sta6 Strain of Chlamydomonas reinhardtii

ABSTRACT: When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen-starved in acetate and then "boosted" after two days with additional acetate, the cells become "obese" after eight days, with triacylglyceride-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, sta6 and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the two days prior and two days post boost, and the data were compared with published reports using other strains and growth conditions. During the two hours post boost, ~425 genes are up-regulated ≥2-fold and ~875 genes are down-regulated ≥2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes in the glyoxylate and Calvin Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boost. Four genes--encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)--are selectively up-regulated in sta6. Although the bulk rate of acetate depletion from the medium is not boost-enhanced, three candidate acetate permease-encoding genes in the GPR1_FUN34_YaaH superfamily are boost-up-regulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is down-regulated by boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in sta6.

ORGANISM(S): Chlamydomonas reinhardtii

PROVIDER: GSE55253 | GEO | 2014/03/09

SECONDARY ACCESSION(S): PRJNA239005

REPOSITORIES: GEO

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Project description:When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen-starved in acetate and then "boosted" after two days with additional acetate, the cells become "obese" after eight days, with triacylglyceride-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, sta6 and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the two days prior and two days post boost, and the data were compared with published reports using other strains and growth conditions. During the two hours post boost, ~425 genes are up-regulated M-bM-^IM-%2-fold and ~875 genes are down-regulated M-bM-^IM-%2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes in the glyoxylate and Calvin Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boost. Four genes--encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)--are selectively up-regulated in sta6. Although the bulk rate of acetate depletion from the medium is not boost-enhanced, three candidate acetate permease-encoding genes in the GPR1_FUN34_YaaH superfamily are boost-up-regulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is down-regulated by boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in sta6. Here we report studies on gene-expression patterns during the path to obesity. The Merchant/Pellegrini and Los Alamos laboratories recently generated and analyzed RNA-Seq transcriptomes of cw15, sta6, and several complemented sta6 strains during two days of N-starvation (0M-bM-^FM-^R48 h). In collaboration with these groups, the Goodenough lab generated a second pair of transcriptomes using cw15 and sta6, tracing 0M-bM-^FM-^R48 h gene expression patterns under a different set of culture conditions and taking the time course out to 96 h, with an intervening acetate boost. Analysis of these data was deeply informed by cross-comparisons with the Blaby et al. data. Three additional RNA-Seq studies of wild-type strains were also considered.

Project description:Background Lytic polysaccharide monooxygenases (LPMOs) are often studied in simple models involving activity measurements of a single LPMO or a blend thereof with hydrolytic enzymes towards an insoluble substrate. However, the contribution of LPMOs to polysaccharide breakdown in complex cocktails of hydrolytic and oxidative enzymes, similar to fungal secretomes, remains elusive. Typically, two starch-specific AA13 LPMOs are encoded by mainly Ascomycota genomes. Here, we investigate the impact of LPMO loss on the growth and degradation of starches of varying resistance to amylolytic hydrolases by Aspergillus nidulans. Results Deletion of the genes encoding AnAA13A that possesses a CBM20 starch-binding module, AnAA13B (lacking a CBM20) or both AA13s genes resulted in reduction in growth on solid media with resistant, but not soluble processed potato starch. Larger size and amount of residual starch granules were observed for the AA13 KO strains as compared to the reference and the impairment of granular starch degradation was more severe for the AnAA13A KO based on a microscopic analysis. After five days of growth on raw potato starch in liquid media, the mount of residual starch was about 5-fold higher for the AA13 KO strains compared to the reference, which underscores the importance of LPMOs for degradation of especially resistant starches. Proteomic analyses revealed substantial changes in the secretomes of the AA13 double KO, followed by the AnAA13A deficient strains, whereas no significant changes in the proteome were observed for the AnAA13B deficient strain. Conclusions This study shows that the loss of AA13, especially the starch-binding AnAA13A, impairs degradation of resistant potato starch, but has limited impact less-resistant wheat starch and has no impact at all on processed solubilised starch. The effects of LPMO loss are more pronounced at the later stages of fungal growth, when the less-accessible regions of the substrate accumulate. The striking impact of the loss of a single LPMO against a whole secretome offers insight into the crucial role played by AA13 in the degradation of resistant starch and presents a methodological framework to analyse the contribution of distinct LPMOs towards complex substrates under in vivo conditions.

Project description:Zymomonas mobilis is an excellent ethanologenic bacterium. Biomass pretreatment and saccharification provides access to simple sugars, but also produces inhibitors such as acetate and furfural. Our previous work has identified and confirmed the genetic change of a 1.5-kb deletion in the sodium acetate tolerant Z. mobilis mutant (AcR) leading to constitutively elevated expression of a sodium proton antiporter encoding gene nhaA, which contributes to the sodium acetate tolerance of AcR mutant. In this study, we further investigated the responses of AcR and wild-type ZM4 to sodium acetate stress in minimum media using both transcriptomics and a metabolic labeling approach for quantitative proteomics the first time. Proteomic measurements at two time points identified about eight hundreds proteins, or about half of the predicted proteome. Extracellular metabolite analysis indicated AcR overcame the acetate stress quicker than ZM4 with a concomitant earlier ethanol production in AcR mutant, although the final ethanol yields and cell densities were similar between two strains. Transcriptomic samples were analyzed for four time points and revealed that the response of Z. mobilis to sodium acetate stress is dynamic, complex and involved about one-fifth of the total predicted genes from all different functional categories. The modest correlations between proteomic and transcriptomic data may suggest the involvement of posttranscriptional control. In addition, the transcriptomic data of forty-four microarrays from four experiments for ZM4 and AcR under different conditions were combined to identify strain-specific, media-responsive, growth phase-dependent, and treatment-responsive gene expression profiles. Together this study indicates that minimal medium has the most dramatic effect on gene expression compared to rich medium followed by growth phase, inhibitor, and strain background. Genes involved in protein biosynthesis, glycolysis and fermentation as well as ATP synthesis and stress response play key roles in Z. mobilis metabolism with consistently strong expression levels under different conditions.

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The Path to Triacylglyceride Obesity in the sta6 Strain of Chlamydomonas reinhardtii

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