Screening of chemical modulators for lipid accumulation in Schizochytrium sp. S31
Graphical abstract
Introduction
Omega-3 long-chain polyunsaturated fatty acids (PUFAs) like eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) are related with the early neural and retinal development and also have beneficial roles in preventing and lowering the risk from some diseases like Alzheimer, inflammation and autoimmune diseases (Morris et al., 2003, Simopoulos, 2002). Among them, DHA is an essential and important compound for brain development in newborns as well as the membranes synthesis of human nervous and retinal tissues (Jiang et al., 1999, Kuratko and Salem, 2013, Liu et al., 2017). Besides the traditional source of DHA from fish oil, production of DHA based on marine microorganisms like Schizochytrium and Crypthecodinium cohnii has attracted more attention in recent years due to its several advantages including no pollution, sustainability and lower purification costs (Hadley et al., 2017, Li et al., 2015, Li et al., 2017, Liu et al., 2017, Sui et al., 2014, Yu et al., 2017).
Heterotrophic genus of Schizochytrium have been taken as a promising fermentative microorganism for DHA production given its fast growth and high yield of DHA accumulation (Raghukumar, 2008). Notably, the security of Schizochytrium has been evaluated systematically including the subacute toxicity test in rats, toxicity test on the reproduction and development of rats, rabbits and pigs as well as mutating test on rats, leading to the validation of its safety (Hammond et al., 2002, Hammond et al., 2001a, Hammond et al., 2001b). In addition, their use in food has been approved by several countries or unions. For example, European Union approved them as novel foods and the oils from Schizochytrium, products derived from Schizochytrium were recommended as a GRAS (generally recognized as safe) by the US FDA (Food and Drug Administration) and they were also taken as a novel food ingredient by New Zealand (García et al., 2017). Generally, DHA could account for 35%–40% of the total fatty acid in Schizochytrium sp. (Ganuza et al., 2008). Nevertheless, to further increase the fatty acids accumulation and the DHA content in Schizochytrium sp., several strategies have been performed. First, optimization of medium components and cultivating conditions has been demonstrated effective for altering DHA production in various marine microalgae (Chaung et al., 2012, Manikan et al., 2015, Zhu et al., 2007). For Schizochytrium genus, optimization of sea salt in the medium have improved the DHA production in Schizochytrium sp. S056 (Chen et al., 2016). Besides, Ling et al. (2015) evaluated the impact of carbon and nitrogen feeding strategy on production of biomass and docosahexaenoic acid DHA by Schizochytrium sp. LU310 (Ling et al., 2015). Moreover, Song et al. (2015) utilized the low-cost substrates including maize starch hydrolysate and soybean meal hydrolysate to produce docosahexaenoic acid (DHA) in Schizochytrium limacinum OUC88, instead of using yeast extract, reaching a higher DHA yields (Song et al., 2015). Second, stress stimulation could also be a feasible strategy. For example, roles of salt and butanol stress in enhancing DHA or oil accumulation have been respectively demonstrated in different Schizochytrium species (Hu et al., 2015, Zhang et al., 2017). Besides, Sun et al. (2016) performed adaptive evolution with high oxygen as a selection pressure in Schizochytrium sp. HX-308, reaching a 32.4% increase in dry weight (Sun et al., 2016). Third, in recent years, a diverse of chemicals like phytohormones and antioxidants have been confirmed to be efficient stimulators to promote oil accumulation of marine microalgae (Bose et al., 2013, Cheng et al., 2012, Yu et al., 2015). For example, 6-benzylaminopurine and gibberellins can stimulate the lipid and DHA accumulation of Aurantiochytrium sp. YLH70 (Yu et al., 2016a, Yu et al., 2016b). Besides, Salama et al. (2014) demonstrated the roles of phytohormones including indole-3-acetic acid and diethyl aminoethyl hexanoate in accelerating microalgal growth and inducing the quality and quantity of fatty acid in Scenedesmus obliquus (Salama et al., 2014). In the previous studies, the lipid accumulation was increased up to ∼8.80% or ∼20% after adding antioxidant butylated hydroxyanisole (BHA) or a combined chemical modulators composed of salicylic acid (SA) and ethanolamine (ETA) as well as naphthoxyacetic acid (BNOA) and ETA in C. cohnii, suggesting the feasibility of this strategy (Li et al., 2015, Sui et al., 2014). For Schizochytrium sp. HX-308, Ren et al. (2017) improved cell dry weight and DHA yield by 16.16% and 30.44% via adding 9 g/L ascorbic acid, respectively (Ren et al., 2017). Nevertheless, few similar studies have been performed in Schizochytrium to identify effective antioxidants or chemical modulators.
In this work, seven chemicals including auxin, signal transducer, amine and antioxidants were first selected for evaluating their effects on lipid accumulation to further improve the lipid and DHA content in Schizochytrium sp. S31. Notably, BNOA and jasmonic acid (JA) showed positive roles on promoting the lipid accumulation. Further, the optimized dose and induction time of BNOA and JA were screened and the synergetic effect of the two chemical modulators was investigated. The work here provided effective strategies for enhancing lipid accumulation in Schizochytrium sp. S31 using chemical modulators.
Section snippets
Chemical modulators
All chemicals used in this study were of analytical pure. BHA, abscisic acid (ABA), 2,4-dichlorophenoxy acetic acid (2,4-D), ETA, and BNOA was purchased from Sigma-Aldrich (St. Louis, USA). JA was purchased from Tokyo Kasei (Tokyo, Japan), and 2,4-epibrassinolide (EBR) was purchased from MedChemExpress (NJ, USA). All the reagents were stored at 4 °C until use. BHA, 2.4-D, JA, EBR and BNOA were dissolved in dimethyl sulfoxide (DMSO) while ABA and ETA were dissolved in double-distilled water (ddH2
Effect of chemical modulators on growth and lipid production in Schizochytrium sp. S31
In this study, based on previous studies of chemical modulators on lipid accumulation in a diverse of organisms (Cheng et al., 2012, Li et al., 2015, Sui et al., 2014, Yu et al., 2016b), seven candidate chemicals from four chemical groups including auxin, signal transducer, amine and antioxidant were selected to investigate their effects on lipid accumulation of Schizochytrium sp. S31. Under the tested condition, the lipid content accounted for 42.62% of the dry cell weight (supplementary data
Discussions
As an endogenous substance of plants, jasmonates and methyl jasmonate (MJ) are always considered as potent lipid regulators that modulate various physiological processes in plants, such as growth, senescence, reproduction and responses to both mechanical trauma and pathogenesis (Liechti and Farmer, 2002, Van der Fits and Memelink, 2000). Up to now, JA or MJ have been successfully applied to induce or increase the biosynthesis of many important secondary metabolites in plants, e.g., ginsenoside
Conclusions
In this study, screening of different chemical modulators and their optimized concentration successfully led to the identification of BNOA and JA, reaching an increased lipid accumulation by 11.16% or 12.71% when adding BNOA or JA with a final concentration of 2 mg/L and 20 mg/L, respectively. In addition, the results showed more increase on lipid when BNOA or JA were added at early lipid accumulation stage (48 h) or used simultaneously, suggesting the effects of BNOA and JA were time-dependent
Acknowledgements
This work was supported by grants from the National Basic Research Program of China (“973” program, project No. 2014CB745101), the Tianjin Municipal Science and Technology Commission (No. 15JCZDJC32500), the National Science Foundation of China, China (No. 21621004), and Zaoneng Biotechnology Inc, Kunming, China.
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These two authors contributed equally to the paper.