Transcriptome analysis of malate-induced Schizochytrium sp. FJU-512 reveals a novel pathway for biosynthesis of docosahexaenoic acid with enhanced expression of genes responsible for acetyl-CoA and NADPH accumulation

Abstract

Schizochytrium is one of the few oleaginous microalgae that produce docosahexaenoic acid (DHA)-rich lipids. In this study, global changes in gene expression levels of Schizochytrium sp. FJU-512 cultured with malate in a 15 l-bioreactor was analyzed using comparative transcriptomics. The changes were found mainly in the genes involved in oxidative phosphorylation, β-oxidation, and pentose phosphate pathways. Consequently, the global changes in genes associated with the pathways could lead to an increase in the influx throughputs of pyruvate, branched-chain amino acids, fatty acids, and vitamin B6. Our transcriptome analysis indicated pyruvate dehydrogenase E2 component and acetolactate synthase I/II/III large subunit as major contributors to acetyl-CoA biosynthesis, whereas glucose-6-phosphate dehydrogenase was indicated as the major contributor to the biosynthesis of NADPH. An increase in DHA titer of up to 22% was achieved with the addition of malate to the fed-batch culture of Schizochytrium sp. FJU-512. This study provides an alternate method to enhance DHA production in Schizochytrium sp. FJU-512 through malate induced upregulation of genes responsible for acetyl-CoA and NADPH biosynthesis.

Keywords: acetyl-CoA; comparative transcriptomics; docosahexaenoic acid; fatty acid metabolism; malate.

Figure 1

Comparison of biomass, FAME, DHA content, and DHA yield by Schizochytrium sp. FJU-512 shaking cultivated under different malate conditions. Each point is the mean ± SD of three independent experimental replicates. Error bars represent the standard error of means. CK: control group (no malate added); M36, M60, M84: malate added at 36, 60, and 84 h, respectively; −1, −2, −3: 1 g/l, 3 g/l, and 5 g/l of malate, respectively. (A) Biomass, FAME (%), and DHA (%) with respect to malate addition and time points. (B) DHA (g/L) with respect to malate addition and time points.

Figure 2

Time course (growth characteristics) of changes in DHA production in Schizochytrium sp. FJU-512 grown with or without malate. Each point is the mean ± SD of three independent experiment replicates. Error bars represent the standard error of the means. CK: control group (no malate addition); Malate: 3 g/l malate added at 60 h. (A) Growth curve. (B) FAME (%) was calculated at different time intervals. (C) DHA concentration (g/L) at different time intervals.

Figure 3

Volcano map and Venn diagram of differentially expressed genes (DEGs). Volcano map of DEGs of (A) 72 h samples and (B) 86 h samples. The transverse coordinates represent multiple changes in genes expression in different samples, while the longitudinal coordinates represent the significant variations in gene expression; the DEGs are represented as red dots (upregulation) and green dots (downregulation), while blue dots (unchanged). (C) Venn diagram showing DEGs among three experimental groups (A–C).

Figure 4

Reconstruction and illustration of the key genes with highly enhanced transcriptional levels and their metabolic processes. The metabolic processes of the TCA cycle, glycolysis pathway, PPP, fatty acid metabolism, β-oxidation, amino acid biosynthesis and degradation, oxidative phosphorylation, nitrogen metabolism, and vitamin B6 synthesis are associated with the fermentation of Schizochytrium sp. FJU-512 when malate is added. Red (upregulated), black (no distinct disparities), and green (downregulated); arrows indicate transcriptional regulation of genes: solid line arrows represent a one-step reaction, while dashed ones represent a multi-step reaction. The abbreviations of all genes are detailed in Supplementary Table 2.

Figure 5

Validation of the gene expression profiles of the ilvB, ilvG, ilvI, g6pd, pdhe2, fabF, acsl, and idh genes by quantitative reverse-transcription PCR. Light blue represents the control group at 72 h, which is the expression level of 18 s rDNA; At 86 h, Dark blue represents the control group. While dark green indicates each gene’s expression at 86 h, light green indicates each gene’s expression at 72 h.

Figure 6

Effect of additives on growth, FAME, DHA content, and DHA yield of Schizochytrium sp. FJU-512 when shake culture at 72 h. Each point is the mean ± SD of three independent experimental replicates. Error bars represent standard errors of the means. CK: control group (no additive added); P-1, P-2, P-3: addition of 1, 2.5 and 4 mmol/l sodium pyruvate, respectively; B6-1, B6-2, B6-3: 0.05, 0.25, and 0.45 mg/ml of Vitamin B6, respectively; V-1, V-2, V-3: 0.06, 0.30, and 0.54 mg/ml of Val, respectively; L-1, L-2, L-3: 0.06, 0.30, and 0.54 mg/ml of Leu, respectively; I-1, I-2, I-3: 0.06, 0.30, and 0.54 mg/ml of Ile, respectively. (A) Concentration of DHA (g/L). (B) Concentration of biomass, FAME (%), and DHA (%).