Oleaginicity of the yeast strain Saccharomyces cerevisiae D5A

Abstract

Background: The model yeast, Saccharomyces cerevisiae, is not known to be oleaginous. However, an industrial wild-type strain, D5A, was shown to accumulate over 20% storage lipids from glucose when growth is nitrogen-limited compared to no more than 7% lipid accumulation without nitrogen stress.

Methods and results: To elucidate the mechanisms of S. cerevisiae D5A oleaginicity, we compared physiological and metabolic changes; as well as the transcriptional profiles of the oleaginous industrial strain, D5A, and a non-oleaginous laboratory strain, BY4741, under normal and nitrogen-limited conditions using analytic techniques and next-generation sequencing-based RNA-Seq transcriptomics. Transcriptional levels for genes associated with fatty acid biosynthesis, nitrogen metabolism, amino acid catabolism, as well as the pentose phosphate pathway and ethanol oxidation in central carbon (C) metabolism, were up-regulated in D5A during nitrogen deprivation. Despite increased carbon flux to lipids, most gene-encoding enzymes involved in triacylglycerol (TAG) assembly were expressed at similar levels regardless of the varying nitrogen concentrations in the growth media and strain backgrounds. Phospholipid turnover also contributed to TAG accumulation through increased precursor production with the down-regulation of subsequent phospholipid synthesis steps. Our results also demonstrated that nitrogen assimilation via the glutamate-glutamine pathway and amino acid metabolism, as well as the fluxes of carbon and reductants from central C metabolism, are integral to the general oleaginicity of D5A, which resulted in the enhanced lipid storage during nitrogen deprivation.

Conclusion: This work demonstrated the disequilibrium and rebalance of carbon and nitrogen contribution to the accumulation of lipids in the oleaginous yeast S. cerevisiae D5A. Rather than TAG assembly from acyl groups, the major switches for the enhanced lipid accumulation of D5A (i.e., fatty acid biosynthesis) are the increases of cytosolic pools of acetyl-CoA and NADPH, as well as alternative nitrogen assimilation.

Keywords: Lipid accumulation; Nitrogen assimilation; Oleaginous yeast; RNA-Seq; Saccharomyces cerevisiae; Transcriptomics; Triacylglycerol (TAG).

Fig. 1

Growth (a) and FAME content (b) in strains D5A and BY4741. Red arrows indicate samples used for transcriptomics as they clearly show the induction of lipid production from T1 to T2 and T3 in D5A. Data shown as the mean ± standard deviation of duplicate OD₆₀₀ samples and triplicate FAME samples

Fig. 2

Transcript levels of genes in metabolic pathways of fatty acid biosynthesis (a), nitrogen metabolism (b), and amino acid metabolism (c) in D5A during N deprivation [5 mM (NH₄)₂SO₄, − N] compared to N replete [35 mM (NH₄)₂SO₄, + N]. The numbers in the shaded rectangles next to blue gene names indicate the log₂-transformed fold changes in time points T1, T2, and T3, from left to right, respectively. Shades of green indicate the respective degree of which the gene is down-regulated and shades of red indicate the respective degree of which the gene is up-regulated. All transcriptional differences are shown as the mean of duplicate log₂-based values

Fig. 3

Transcript levels of genes in central carbon metabolism in D5A during N deprivation [5 mM (NH₄)₂SO₄, − N] compared to N replete [35 mM (NH₄)₂SO₄, + N]. The numbers in the shaded rectangles next to blue gene names indicate the log₂-transformed fold changes in time points T1, T2, and T3, from left to right, respectively. Shades of green indicate the respective degree of which the gene is down-regulated and shades of red indicate the respective degree of which the gene is up-regulated. All transcriptional differences are shown as the mean of duplicate log₂-based values

Fig. 4

Transcript levels of genes in the metabolic pathways of fatty acid biosynthesis (a), nitrogen metabolism (b), and amino acid metabolism (c) in D5A compared to BY4741. Up-regulated or down-regulated levels are indicated by log₂-based values in shaded rectangles with red and blue arrows, respectively. Time series changes (T1, T2, and T3) of D5A compared to BY4741 during N deprivation are indicated by shaded rectangle boxes with the log₂-transformed values from left to right. Shades of green indicate the respective degree of which the gene is down-regulated and shades of red indicate the respective degree of which the gene is up-regulated. All transcriptional differences are shown as the mean of duplicate log₂-based values

Fig. 5

Transcript levels for transcriptional regulators related to carbon and nitrogen metabolism. The numbers in the two rows of shaded rectangles next to orange circled gene names indicate the log₂-transformed fold changes in time points T1, T2, and T3, from left to right, respectively. The top row represents transcriptional differences in D5A during N deprivation [5 mM (NH₄)₂SO₄, − N] compared to N replete [35 mM (NH₄)₂SO₄, + N]. The bottom row represents transcriptional differences between the D5A and BY4741 strains. All transcriptional differences are shown as the mean of duplicate log₂-based values. The gene names in the boxes show the pathway genes affected by the differentially expressed regulators

Fig. 6

Transcript levels of genes in central carbon metabolism in D5A compared to BY4741. Up-regulated or down-regulated levels are indicated by log₂-based values in shaded rectangles with red and blue arrows, respectively. Time series changes (T1, T2, and T3) of D5A compared to BY4741 during N deprivation are indicated by shaded rectangle boxes with the log₂-transformed values from left to right. Shades of green indicate the respective degree of which the gene is down-regulated and shades of red indicate the respective degree of which the gene is up-regulated. All transcriptional differences are shown as the mean of duplicate log₂-based values