Alleviation of reactive oxygen species enhances PUFA accumulation in Schizochytrium sp. through regulating genes involved in lipid metabolism

Abstract

The unicellular heterotrophic thraustochytrids are attractive candidates for commercial polyunsaturated fatty acids (PUFA) production. However, the reactive oxygen species (ROS) generated in their aerobic fermentation process often limits their PUFA titer. Yet, the specific mechanisms of ROS involvement in the crosstalk between oxidative stress and intracellular lipid synthesis remain poorly described. Metabolic engineering to improve the PUFA yield in thraustochytrids without compromising growth is an important aspect of economic feasibility. To fill this gap, we overexpressed the antioxidative gene superoxide dismutase (SOD1) by integrating it into the genome of thraustochytrid Schizochytrium sp. PKU#Mn4 using a novel genetic transformation system. This study reports the ROS alleviation, enhanced PUFA production and transcriptome changes resulting from the SOD1 overexpression. SOD1 activity in the recombinant improved by 5.2–71.6% along with 7.8–38.5% decline in ROS during the fermentation process. Interestingly, the total antioxidant capacity in the recombinant remained higher than wild-type and above zero in the entire process. Although lipid profile was similar to that of wild-type, the concentrations of major fatty acids in the recombinant were significantly (p ≤ 0.05) higher. The PUFA titer increased up to 1232 ± 41 mg/L, which was 32.9% higher (p ≤ 0.001) than the wild type. Transcriptome analysis revealed strong downregulation of genes potentially involved in β-oxidation of fatty acids in peroxisome and upregulation of genes catalyzing lipid biosynthesis. Our results enrich the knowledge on stress-induced PUFA biosynthesis and the putative role of ROS in the regulation of lipid metabolism in oleaginous thraustochytrids. This study provides a new and alternate strategy for cost-effective industrial fermentation of PUFA.

The lipid metabolism in thraustochytrids

“…The promoter sequences in the cassette are often those of ubiquitin, α-tubulin or the translation elongation factor EF1α genes. The transferred genetic material can be either a circular [125] or a linearized plasmid [126], or just the amplified cassette with the flanking regions for the homologous recombination [127]. With these methods, overexpressing [128], knocked-in [129], and disrupted [104] mutants have been obtained.…”

“…With these methods, overexpressing [128], knocked-in [129], and disrupted [104] mutants have been obtained. To improve the efficiency of transformation, the cell wall can be weakened by DTT treatments [130], by vortexing cells with zircon beads before resuspension with the amplified DNA construct [127], or by incubation for few hours with a cocktail of various cell-wall degrading enzymes [126]. Some authors also obtained high yield of transformation using commercial electroporation buffers (either Lonza's Nucleofector Solution or Bio-rad's Electroporation Buffer) rather than the 'classical' sorbitol medium [123].…”

“…Alleviation of reactive oxygen species by overexpression of a superoxide dismutase in Schizochytrium sp. results in a down regulation of genes potentially involved in β-oxidation and in a modest but significant (30%) increase of total FAs, including DHA [126]. Another indirect approach attempted to increase the rate of NADPH turnover.…”

Microalgae for the production of lipid and carotenoids: a review with focus on stress regulation and adaptation

“…In a recent study, overexpression of SOD in microalgae Schizochytrium sp. successfully alleviated oxidative stress and increased the lipid content [ 140 ]. It is anticipated that the dramatic progress of innovative technologies will offer more chances for unraveling the regulatory networks responsible for the cellular reactions to oxidative stress, which might offer guidance for the improved microbial lipid and carotenoids overproduction.…”

Enhancing biomass and lipid accumulation in the microalgae Schizochytrium sp. by addition of fulvic acid and EDTA

“…In another study, overexpression of superoxide dismutase (SOD) in Schizochytrium sp. also successfully alleviated oxidative stress and increased the PUFA content by 32.9% (Zhang et al 2018 ). Apart from strain development, the addition of antioxidants offers a simple way to alleviate cellular oxidative damage.…”