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Low-phosphate-selected Auxenochlorella protothecoides redirects phosphate to essential pathways while producing more biomass.

ABSTRACT: 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.

SUBMITTER: Park SH

PROVIDER: S-EPMC6007911 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Low-phosphate-selected Auxenochlorella protothecoides redirects phosphate to essential pathways while producing more biomass.

Park Sang-Hyuck SH Kyndt John J Chougule Kapeel K Park Jeong-Jin JJ Brown Judith K JK

PloS one 20180619 6

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)-adap ...[more]

PMID: 29920531

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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:Renewable biodiesel produced by microalgae has great potential as a portable source of high-density energy that can replace certain traditional hydrocarbon sources. One limitation of scaling up algal cultivation to industrial levels is the availability of key macronutrients, particularly inorganic phosphorus (Pi), which is a finite and dwindling resource. Here, Auxenochlorella protothecoides was adapted to low Pi conditions through continuous cultivation in 100 times less Pi media for 285 days, or ~41 generations. The adapted populations demonstrated significantly higher growth rates than wild type (WT) cells in low Pi, with average maximum growth rates of 0.72 d-1 and 0.54 d-1, respectively in batch-culture experiments. Based on UPLC/MS analyses, the total lipid content of the adapting A. protothecoides populations showed a shift from their typical profile in nutrient replete media to the accumulation of non-phosphorus glycerolipids, with 306% MGDG and 189% SQDG, relative to WT at initial exposure, followed by a decline, and a steady increase until the final time point was reached. Transcriptome profiling by Illumina RNA-seq collected at 5 time points throughout the experiment, and results of the lipid analyses revealed a trend of increased transcript levels during the first ~11 generations of adaptation, followed by an overall decrease in gene expression after ~34 generations. The short-term changes in gene expression were associated with shifts in major metabolic pathways including carbon metabolism, oxidative phosphorylation, glycolysis, and gluconeogenesis. By comparison, certain transcripts showing decreased expression, reflected increased fatty acid turnover, and a stable decrease in photosynthesis-related gene expression. These results illustrate the utility of laboratory-directed evolution for the selection of microalgae populations with altered cultivation traits, revealing distinct phases of adaptation, based on expression profiles. The results further demonstrate the use of metabolic engineering to select a microalga variant after only ~40 generations of growth in low-phosphate conditions that utilizes Pi more efficiently for growth than its wild type parent population and produces 1.22 times more biomass in batch growth experiments.

Dataset Information

Low-phosphate-selected Auxenochlorella protothecoides redirects phosphate to essential pathways while producing more biomass.

Publications

Low-phosphate-selected Auxenochlorella protothecoides redirects phosphate to essential pathways while producing more biomass.

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