New insights into phenotypic heterogeneity for the distinct lipid accumulation of Schizochytrium sp. H016

Abstract

Background: Schizochytrium sp. is a marine heterotrophic protist and an important sustainable resource for high value-added docosahexaenoic acid in the future. The production of different phenotypes during the continuous subculture of Schizochytrium sp. results in a serious reduction in lipid yield and complicates the used of this strain in scientific research and industrial production. Hence, obtaining an improved understanding of the phenotypic differences and molecular mechanisms underlying the cell-to-cell heterogeneity of Schizochytrium sp. is necessary.

Results: After continuous culture passage, Schizochytrium sp. H016 differentiated into two subpopulations with different morphologies and showed decreased capacity for lipid production. The presence of cell subpopulations with degraded lipid droplets led to a substantial decrease in overall lipid yield. Here, a rapid screening strategy based on fluorescence-activated cell sorting was proposed to classify and isolate subpopulations quickly in accordance with their lipid-producing capability. The final biomass and lipid yield of the subpopulation with high cell lipid content (i.e., H016-H) were 38.83 and 17.22 g/L, respectively, which were 2.07- and 5.38-fold higher than those of the subpopulation with low lipid content (i.e., H016-L), respectively. Subsequently, time‑resolved transcriptome analysis was performed to elucidate the mechanism of phenotypic heterogeneity in different subpopulations. Results showed that the expression of genes related to the cell cycle and lipid degradation was significantly upregulated in H016-L, whereas the metabolic pathways related to fatty acid synthesis and glyceride accumulation were remarkably upregulated in H016-H.

Conclusion: This study innovatively used flow cytometry combined with transcriptome technology to provide new insights into the phenotypic heterogeneity of different cell subpopulations of Schizochytrium sp. Furthermore, these results lay a strong foundation for guiding the breeding of oleaginous microorganisms with high lipid contents.

Keywords: Flow cytometry; Lipid biosynthesis; Phenotypic heterogeneity; Schizochytrium sp.; Transcriptome analysis.

Fig. 1

Comparative analysis of the fermentation performances and morphological characteristics of Schizochytrium sp. H016 after consecutive subculturing. a Biomasses and lipid yields of Schizochytrium sp. H016 during subculture. b Observation of the 10th generation strain under optical microscopy. c Flow cytometry analysis. The histogram shows the distribution of Nile red fluorescence intensity. FITC (x-axis) reflects the fluorescence intensity, and Count (y-axis) is the number of cells. d Dot blots of Schizochytrium sp. H016 subpopulations. The red dots are H016-H, and the green dots are H016-L. e Biomass, lipid content and lipid yield of the 10th generation strain and sorted strains

Fig. 2

Microscopic morphology of H016-H and H016-L. Optical microscopy, SEM, TEM, and LSCM observations of cell morphology

Fig. 3

Growth kinetics analyses and fatty acid composition of H016-H and H016-L. a Growth curves based on glucose residues, biomasses, and lipid yields. b TLC analysis of extracted lipids from H016-H (left lane) and H016-L (right lane). c TAG concentration of H016-H and H016-L during fermentation. d FFA concentration of H016-H and H016-L during fermentation

Fig. 4

Overview of transcriptome analysis during the fermentation of the two cell subpopulations. a PCA plot shows the clustering between transcriptome samples. b DEG number of upregulated and downregulated genes in H016-H and H016-L at the four fermentation stages. c KEGG pathway enrichment analysis of all DEGs

Fig. 5

Differential expression of representative genes involved in various aspects of the cell cycle, including DNA replication, cell division, tubulin, and cell cycle regulation

Fig. 6

Overview of the transcriptional reprogramming of the central carbon metabolic and lipid-related pathways in H016-H and H016-L at 24, 48, 96, and 144 h. The heatmaps show transcriptional differences in log₂ (fold change) in H016-H relative to those in H016-L