Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors

Abstract

Background: Microalgae can accumulate considerable amounts of lipids under different nutrient-deficient conditions, making them as one of the most promising sustainable sources for biofuel production. These inducible processes provide a powerful experimental basis for fully understanding the mechanisms of physiological acclimation, lipid hyperaccumulation and gene expression in algae. In this study, three nutrient-deficiency strategies, viz nitrogen-, phosphorus- and iron-deficiency were applied to trigger the lipid hyperaccumulation in an oleaginous Chlorella pyrenoidosa. Regular patterns of growth characteristics, lipid accumulation, physiological parameters, as well as the expression patterns of lipid biosynthesis-related genes were fully analyzed and compared.

Results: Our results showed that all the nutrient stress conditions could enhance the lipid content considerably compared with the control. The total lipid and neutral lipid contents exhibit the most marked increment under nitrogen deficiency, achieving 50.32% and 34.29% of dry cell weight at the end of cultivation, respectively. Both photosynthesis indicators and reactive oxygen species parameters reveal that physiological stress turned up when exposed to nutrient depletions. Time-course transcript patterns of lipid biosynthesis-related genes showed that diverse expression dynamics probably contributes to the different lipidic phenotypes under stress conditions. By analyzing the correlation between lipid content and gene expression level, we pinpoint several genes viz. rbsL, me g6562, accA, accD, dgat g2354, dgat g3280 and dgat g7063, which encode corresponding enzymes or subunits of malic enzyme, ACCase and diacylglycerol acyltransferase in the de novo TAG biosynthesis pathway, are highly related to lipid accumulation and might be exploited as target genes for genetic modification.

Conclusion: This study provided us not only a comprehensive picture of adaptive mechanisms from physiological perspective, but also a number of targeted genes that can be used for a systematic metabolic engineering. Besides, our results also represented the feasibility of lipid production through trophic transition cultivation modes, throwing light on a two-stage microalgal lipid production strategy with which heterotrophy stage provides sufficient robust seed and nitrogen-starvation photoautotrophy stage enhances the overall lipid productivity.

Figure 1

Growth and photosynthetic activity of Chlorella pyrenoidosa under different stress conditions. (a) Growth, (b) chlorophyll content, (c) variable-to-maximum fluorescence ratio (Fv/Fm) and (d) NPQ value. N, nitrogen; P, phosphorus; Fe, iron.

Figure 2

The lipid content of Chlorella pyrenoidosa. Lipid content under (a) total nutrient cultivation, (b) nitrogen-deficient cultivation, (c) phosphorus-deficient cultivation and (d) iron-deficient cultivation. TAG, triacylglycerol.

Figure 3

Expression level of l,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCO), malic enzyme (ME) and phosphoenolpyruvate carboxylase (PEPC) genes under nutritional deprivation conditions. N, nitrogen; P, phosphorus; Fe, iron.

Figure 4

Hydroxide radical (OH) and malondialdehyde (MDA) content, and the peroxidase (POD) activity and superoxide dismutase (SOD) activity of Chlorella pyrenoidosa under different nutrient limitations. (a)⋅OH, (b) MDA, (c) POD, (d) SOD. N, nitrogen; P, phosphorus; Fe, iron.

Figure 5

Expression level of acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DGAT) genes under nutritional deprivation conditions. N, nitrogen; P, phosphorus; Fe, iron; bccp, biotin carboxyl carrier protein.