Construction of Glucose-6-Phosphate Dehydrogenase Overexpression Strain of Schizochytrium sp. H016 to Improve Docosahexaenoic Acid Production.

Docosahexaenoic acid (DHA) is an important omega-3 polyunsaturated fatty acid (PUFA) that plays a critical physiological role in human health. Schizochytrium sp. is considered an excellent strain for DHA production, but the synthesis of DHA is limited by the availability of nicotinamide adenine dinucleotide phosphate (NADPH). In this study, the endogenous glucose-6-phosphate dehydrogenase (G6PD) gene was overexpressed in Schizochytrium sp. H016. Results demonstrated that G6PD overexpression increased the availability of NADPH, which ultimately altered the fatty acid profile, resulting in a 1.91-fold increase in DHA yield (8.81 g/L) and increased carbon flux by shifting it from carbohydrate and protein synthesis to lipid production. Thus, G6PD played a vital role in primary metabolism. In addition, G6PD significantly increased DHA content and lipid accumulation by 31.47% and 40.29%, respectively. The fed-batch fermentation experiment results showed that DHA production reached 17.01 g/L in the overexpressing G6PD strain. These results elucidated the beneficial effects of NADPH on the synthesis of PUFA in Schizochytrium sp. H016, which may be a potential target for metabolic engineering. Furthermore, this study provides a promising regulatory strategy for the large-scale production of DHA in Schizochytrium sp.

Identification and characterization of a novel diacylglycerol acyltransferase gene from Mortierella alpina.

OBJECTIVES: To clone and express a diacylglycerol acyltransferase (DGAT) gene from Mortierella alpina in Saccharomyces cerevisiae and characterize oil production and fatty acid composition of the resulting recombinant RESULTS: A new, full-length cDNA, putatively encoding a DGAT, was cloned from M. alpina. We subsequently cloned the gene, except the transmembrane-encoding region, termed MaDGAT, its molecular mass was 31.3 kDa. MaDGAT shares 75% identity with a DGAT from Mortierella verticillata NRRL 6337. A recombinant vector expressing MaDGAT, pYES2-DGAT, was constructed and transformed into S. cerevisiae H1246, a neutral, lipid-deficient quadruple mutant. TLC analysis showed that the recombinant vector restored triacylglycerol biosynthesis and its content in the recombinant strain was 3.9%. CONCLUSION: MaDGAT is a novel DGAT gene and could increase TAG biosynthesis in M. alpina or other filamentous fungi, thereby promoting the synthesis of polyunsaturated fatty acids.

Improvement in the docosahexaenoic acid production of Schizochytrium sp. S056 by replacement of sea salt.

Schizochytrium is a marine microalga that requires high concentrations of sea salt for growth, although problems arise with significant amounts of chloride ions in the culture medium, which corrodes the fermenters. In this work, we evaluated that cell growth and docosahexaenoic acid (DHA) production can be improved when using 1 % (w/v) sodium sulfate instead of 2 % (w/v) sea salt in the culture medium for Schizochytrium sp. S056. In practice, the use of sodium sulfate as the sodium salt led to chloride ion levels in the medium that can be completely removed, thus avoiding fermenter corrosion during Schizochytrium sp. S056 growth, reducing cost and increasing DHA production, and simplifying the disposal of fermentation wastewater. Additionally, we demonstrated that the osmolality of growth media did not play a crucial role in the production of DHA. These findings may be significantly important to companies involved in production of PUFAs by marine microbes.

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

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, time­resolved 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.

Enhancement of lipid accumulation and docosahexaenoic acid synthesis in Schizochytrium sp. H016 by exogenous supplementation of sesamol.

Schizochytrium sp. is one of the most promising marine oleaginous microorganisms for industrial production of docosahexaenoic acid (DHA). In this study, the exogenous supplementation of 1 mM sesamol to the fermentation medium effectively prevented the peroxidation of polyunsaturated fatty acids in the fermentation process, which thereby significantly increasing the lipid and DHA yield by 53.52% and 78.30%, respectively. The addition of sesamol also increased the total antioxidant capacity of cells and induce the gene expression of polyketide synthase and antioxidant enzyme system. Moreover, the supply of nicotinamide adenine dinucleotide phosphate was regulated by sesamol by inhibiting the malic enzyme activity and promoting the glucose-6-phosphate dehydrogenase activity. Finally, fed-batch fermentation showed that the addition of sesamol significantly enhanced the DHA yield by 90.76%. This study provides an important reference for enhancing the DHA productivity of Schizochytrium sp. in industrial fermentation.

High-value utilization of the waste hydrolysate of Dioscorea zingiberensis for docosahexaenoic acid production in Schizochytrium sp.

The starch saccharification liquid of Dioscorea zingiberensis tubers (SSLD) is a glucose-rich agro-industrial waste. Herein, SSLD was used as a novel potential carbon source for the biosynthesis of docosahexaenoic acid (DHA) in Schizochytrium sp. to achieve waste recycling and high-value utilization. Component analysis showed that SSLD contains abundant nutrients, such as glucose, amino acids, phenolics and flavonoids. When the total sugar concentration in SSLD was optimized to 90 g/L, the biomass and DHA yield reached 44.85 and 6.60 g/L, respectively, which were 32.1% and 36.92% higher than that at pure glucose culture condition. Fermentation characteristics and gene expression analysis showed that SSLD could remarkably improve cell antioxidant capacity, which is beneficial to scavenge intracellular reactive oxygen species and increase the gene expression of antioxidant enzymes in Schizochytrium sp. Hence, SSLD is an effective and economic carbon source for DHA production in Schizochytrium sp.