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Autophagy-like processes are involved in lipid droplet degradation in Auxenochlorella protothecoides during the heterotrophy-autotrophy transition.

ABSTRACT: Autophagy is a cellular degradation process that recycles cytoplasmic components in eukaryotes. Although intensively studied in yeast, plants, and mammals, autophagy in microalgae is not well understood. Auxenochlorella protothecoides is a green microalga that has the ability to grow either autotrophically when under light or heterotrophically when in media containing glucose. The two growth modes are inter-convertible and transition between them is accompanied by drastic changes in morphology and cellular composition; however, the mechanisms underlying these changes are unknown. In this study, we identified autophagy-related genes and characterized their roles in the degradation of lipid droplets during the heterotrophy-to-autotrophy (HA) transition in A. protothecoides. Most of the proteins constituting the eukaryotic "core machinery" were conserved in A. protothecoides. Two proteins, Atg4 and Atg8, were further investigated. A. protothecoides ATG4 was cloned from a cDNA library and expressed within yeast, and was able to functionally restore the autophagy pathway in atg4? yeast during nitrogen starvation. Furthermore, Atg8, which displayed high sequence identity with its yeast homolog, was able to conjugate to phosphatidylethanolamine (PE) in vitro and was recruited to the phagophore assembly site in yeast. We also identified a C-terminal glycine residue, G118, that was the cleavage site for Atg4. Finally, we used confocal and transmission electron microscopy to reveal that autophagic-like vacuoles were detectable in algal cells during the HA transition. Our data suggested that the lipid droplets in heterotrophic cells were engulfed directly by the autophagic-like vacuole instead of via autophagosomes.

SUBMITTER: Zhao L

PROVIDER: S-EPMC4132264 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Project description:Background:The yield of microalgae biomass is the key to affect the accumulation of fatty acids. A few microalgae can assimilate organic carbon to improve biomass yield. In mixotrophic cultivation, microalgae can use organic carbon source and light energy simultaneously. The preference of the main energy source by microalgae determines the biomass yield. Auxenochlorella protothecoides is an oleaginous mixotrophic microalga that can efficiently assimilate glucose and accumulate a large amount of biomass and fatty acids. The current study focused on the effect of light on the growth and glucose assimilation of A. protothecoides. Results:In this study, we found that the uptake and metabolism of glucose in A. protothecoides could be inhibited by light, resulting in a reduction of biomass growth and lipid accumulation. We employed comparative proteomics to study the influence of light on the regulation of glucose assimilation in A. protothecoides. Proteomics revealed that proteins involving in gene translation and photosynthesis system were up-regulated in the light, such as ribulose-phosphate 3-epimerase and phosphoribulokinase. Calvin cycle-related proteins were also up-regulated, suggesting that light may inhibit glucose metabolism by enhancing the production of glyceraldehyde-3-phosphate (G3P) in the Calvin cycle. In addition, the redox homeostasis-related proteins such as thioredoxin reductase were up-regulated in the light, indicating that light may regulate glucose uptake by changing the redox balance. Moreover, the increase of NADH levels and redox potential of the medium under illumination might inhibit the activity of the glucose transport system and subsequently reduce glucose uptake. Conclusions:A theoretical model of how glucose assimilation in A. protothecoides is negatively influenced by light was proposed, which will facilitate further studies on the complex mechanisms underlying the transition from autotrophy to heterotrophy for improving biomass accumulation.

Project description:Despite the capacity to accumulate ~70% w/w of lipids, commercially produced unicellular green alga A. protothecoides may become compromised due to the high cost of phosphate fertilizers. To address this limitation A. protothecoides was selected for adaptation to conditions of 100× and 5× lower phosphate and peptone, respectively, compared to 'wild-type media'. The A. protothecoides showed initial signs of adaptation by 45-50 days, and steady state growth at ~100 days. The low phosphate (P)-adapted strain produced up to ~30% greater biomass, while total lipids (~10% w/w) remained about the same, compared to the wild-type strain. Metabolomic analyses indicated that the low P-adapted produced 3.3-fold more saturated palmitic acid (16:0) and 2.2-fold less linolenic acid (18:3), compared to the wild-type strain, resulting in an ~11% increase in caloric value, from 19.5kJ/g for the wild-type strain to 21.6kJ/g for the low P-adapted strain, due to the amounts and composition of certain saturated fatty acids, compared to the wild type strain. Biochemical changes in A. protothecoides adapted to lower phosphate conditions were assessed by comparative RNA-Seq analysis, which yielded 27,279 transcripts. Among them, 2,667 and 15 genes were significantly down- and up-regulated, at >999-fold and >3-fold (adjusted p-value <0.1), respectively. The expression of genes encoding proteins involved in cellular processes such as division, growth, and membrane biosynthesis, showed a trend toward down-regulation. At the genomic level, synonymous SNPs and Indels were observed primarily in coding regions, with the 40S ribosomal subunit gene harboring substantial SNPs. Overall, the adapted strain out-performed the wild-type strain by prioritizing the use of its limited phosphate supply for essential biological processes. The low P-adapted A. protothecoides is expected to be more economical to grow over the wild-type strain, based on overall greater productivity and caloric content, while importantly, also requiring 100-fold less phosphate.

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Autophagy-like processes are involved in lipid droplet degradation in Auxenochlorella protothecoides during the heterotrophy-autotrophy transition.

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