Development of a cooperative two-factor adaptive-evolution method to enhance lipid production and prevent lipid peroxidation in Schizochytrium sp

Abstract

Background: Schizochytrium sp. is a marine microalga with great potential as a promising sustainable source of lipids rich in docosahexaenoic acid (DHA). This organism's lipid accumulation machinery can be induced by various stress conditions, but this stress induction usually comes at the expense of lower biomass in industrial fermentations. Moreover, oxidative damage induced by various environmental stresses can result in the peroxidation of lipids, and especially polyunsaturated fatty acids, which causes unstable DHA production, but is often ignored in fermentation processes. Therefore, it is urgent to develop new production strains that not only have a high DHA production capacity, but also possess strong antioxidant defenses.

Results: Adaptive laboratory evolution (ALE) is an effective method for the development of beneficial phenotypes in industrial microorganisms. Here, a novel cooperative two-factor ALE strategy based on concomitant low temperature and high salinity was applied to improve the production capacity of Schizochytrium sp. Low-temperature conditions were used to improve the DHA content, and high salinity was applied to stimulate lipid accumulation and enhance the antioxidative defense systems of Schizochytrium sp. After 30 adaptation cycles, a maximal cell dry weight of 126.4 g/L and DHA yield of 38.12 g/L were obtained in the endpoint strain ALE-TF30, which was 27.42 and 57.52% higher than parental strain, respectively. Moreover, the fact that ALE-TF30 had the lowest concentrations of reactive oxygen species and malondialdehyde among all strains indicated that lipid peroxidation was greatly suppressed by the evolutionary process. Accordingly, the ALE-TF30 strain exhibited an overall increase of gene expression levels of antioxidant enzymes and polyketide synthases compared to the parental strain.

Conclusion: This study provides important clues on how to overcome the negative effects of lipid peroxidation on DHA production in Schizochytrium sp. Taken together, the cooperative two-factor ALE process can not only increase the accumulation of lipids rich in DHA, but also prevent the loss of produced lipid caused by lipid peroxidation. The strategy proposed here may provide a new and alternative direction for the industrial cultivation of oil-producing microalgae.

Keywords: Adaptive evolution; Antioxidant enzyme; Docosahexaenoic acid; Lipid peroxidation; Schizochytrium sp..

Fig. 1

Cell dry weight (CDW) and fatty acid content (as % of TFAs) of different evolved strains during adaptive laboratory evolution. Values and error bars represent the means and the standard deviations of triplicate experiments. a Low-temperature ALE, b high-salinity ALE, c cooperative two-factor ALE

Fig. 2

Fatty acid content (%TFAs) of the starting strain and evolved strains (ALE-LT30, ALE-HS30 and ALE-TF30) at the end of fermentation. a C14:0 percentage (% TFAs). b C14:0 percentage (% TFAs). c EPA percentage (% TFAs), d DPA percentage (% TFAs)SFA, e DHA percentage (% TFAs) content, f SFA represents the summation of C14:0 and C16:0. PUFA represents the summation of EPA, DPA and DHA. Values and error bars represent the means and the standard deviations of triplicate experiments

Fig. 3

Determination of cell morphology and ROS levels using DCFH-DA by confocal laser microscopy. A The parental strain cultured in normal medium for 1 cycle. B The parental strain cultured under low-temperature and high-salinity conditions for 1 cycle. C The evolved strain ALE-TF30 cultured in normal medium for 1 cycle

Fig. 4

Comparison between the parental strain and the endpoint strain ALE-TF30 for substrate consumption (a), CDW and lipids (b), SFA and PUFA percentage in TFAs (c), as well as DHA percentage in TFAs and DHA yield (d) in a 5-L bioreactor. Values and error bars represent the means and the standard deviations of triplicate experiments

Fig. 5

Comparison between the parental strain and the endpoint strain ALE-TF30 for ROS (a), T-AOC (b), and MDA (c) in a 5-L bioreactor. Values and error bars represent the means and the standard deviations of triplicate experiments

Fig. 6

Real-time quantitative PCR results for the SOD, CAT, APX, FAS, ORFA, ORFB, and ORFC genes from the parental strain and endpoint strain ALE-TF30. Values and error bars represent the means and the standard deviations of triplicate experiments

Fig. 7

Schematic diagram of the experimental process of the two different adaptive laboratory evolution (ALE) strategies. Yellow represents normal medium and blue represents high-salinity medium