Production of single cell oil by two novel nonconventional yeast strains of Curvibasidium sp. isolated from medicinal lichen.

Oleaginous yeasts utilize renewable resources to produce lipids, which benefits sustainable development, and it is of great interest to screen robust lipid producers. Curvibasidium sp. belongs to nonconventional yeast that are very limitedly studied. Here, two cold-adaptive strains of Curvibasidium sp., namely, Y230 and Y231, isolated from the medicinal lichen Usnea diffracta were investigated for their potential in lipid production. Genome mining of Curvibasidium sp. Y231 was performed, and the special features related to fatty acid biosynthesis were revealed. Glucose, xylose, and glycerol were tested as sole carbon sources for yeast cell growth and lipid production. The total lipid contents of Curvibasidium sp. Y230 and Y231 range from 38.43% to 54.62% of the cell dry cell weight at 20°C, and glucose is the optimal carbon source. These results indicate that the Curvibasidium sp. strains are promising for sustainable lipid production. Our study provides basis for exploration of lichen-derived strains for biotechnological applications, and also benefits utilization of other nonconventional yeasts for sustainable production based on genome-based studies.

Enhancing the stability of lutein emulsions with a water-soluble antioxidant and a oil-soluble antioxidant.

Lutein is critical for protecting the eye against light damage. The low solubility and high sensitivity of lutein to environmental stresses prevent its further application. The hypothesis is that the combination of one water-soluble antioxidant and one oil-soluble antioxidant will be beneficial to improve the stability of lutein emulsions. A low-energy method was performed to prepare lutein emulsions. The combination of a lipid-soluble antioxidant (propyl gallate or ethylenediaminetetraacetic acid) and a water-soluble antioxidant (tea polyphenol or ascobic acid) were investigated for improving the lutein retention rates. It was shown that the highest lutein retention rate was achieved by using propyl gallate and tea polyphenol, 92.57%, at Day 7. It was proven that the lutein retention rates of emulsions with propyl gallate and tea polyphenol were 89.8%, 73.5% and 55.2% at 4 °C, 25 °C and 37 °C, respectively, at Day 28. The current study is helpful to prepare for the further application of lutein emulsions for ocular delivery.

Engineering the xylose metabolism in Schizochytrium sp. to improve the utilization of lignocellulose.

BACKGROUND: Schizochytrium sp. is a heterotrophic, oil-producing microorganism that can efficiently produce lipids. However, the industrial production of bulk chemicals using Schizochytrium sp. is still not economically viable due to high-cost culture medium. Replacing glucose with cheap and renewable lignocellulose is a highly promising approach to reduce production costs, but Schizochytrium sp. cannot efficiently metabolize xylose, a major pentose in lignocellulosic biomass. RESULTS: In order to improve the utilization of lignocellulose by Schizochytrium sp., we cloned and functionally characterized the genes encoding enzymes involved in the xylose metabolism. The results showed that the endogenous xylose reductase and xylulose kinase genes possess corresponding functional activities. Additionally, attempts were made to construct a strain of Schizochytrium sp. that can effectively use xylose by using genetic engineering techniques to introduce exogenous xylitol dehydrogenase/xylose isomerase; however, the introduction of heterologous xylitol dehydrogenase did not produce a xylose-utilizing engineered strain, whereas the introduction of xylose isomerase did. The results showed that the engineered strain 308-XI with an exogenous xylose isomerase could consume 8.2 g/L xylose over 60 h of cultivation. Xylose consumption was further elevated to 11.1 g/L when heterologous xylose isomerase and xylulose kinase were overexpressed simultaneously. Furthermore, cultivation of 308-XI-XK(S) using lignocellulosic hydrolysates, which contained glucose and xylose, yielded a 22.4 g/L of dry cell weight and 5.3 g/L of total lipid titer, respectively, representing 42.7 and 30.4% increases compared to the wild type. CONCLUSION: This study shows that engineering of Schizochytrium sp. to efficiently utilize xylose is conducive to improve its utilization of lignocellulose, which can reduce the costs of industrial lipid production.

Using nanomaterials to increase the efficiency of chemical production in microbial cell factories: A comprehensive review.

Microbes have proven to be robust workhorses for the large-scale production of many chemicals. Especially, high-value biochemicals (e.g., natural pigments, unsaturated fatty acids) that cannot be derived from fossil fuels, can be produced by engineered microbes. There is a growing interest in both academia and industry to find new technologies that can enhance the efficiencies of microbial cell factories and boost the circular bioeconomy. Rapid technological innovations, such as microbial genome editing and synthetic biology, have greatly advanced the production of chemicals in engineered microbes. Nanomaterial-based technologies that exploit the unique physiochemical properties of nano-scale materials (e.g., large surface area, excellent catalytic activity, tunable optical and electrical performance) have demonstrated great potential and attracted increasing attention. There are many studies showing that nanomaterials can assist microbes in the synthesis of chemicals by providing micronutrients, inducing anti-ROS responses, promoting gas-liquid mass transfer, immobilizing microbial cells and promoting electron transfer in electrosynthesis. Furthermore, the latest studies demonstrate that nanomaterials can be used to construct photocatalyst-microbe hybrids and achieve solar driven chemical production. In this review, we comprehensively summarize these advances and discuss the current gaps as well as future perspectives. With the rapid development of synthetic biology and nanotechnology, we believe more nanomaterial-based technologies will be developed and used to improve the productivity of microbial cell factories.

Exploring kinetics, removal mechanism and possible transformation products of tigecycline by Chlorella pyrenoidosa.

The accumulation of antibiotics in wastewater leads to broad antibiotic resistance, threating human health. Microalgae have been receiving attention due to their ability to remove antibiotics from wastewater. Tigecycline (TGC) is a broad-spectrum glycylcycline antibiotic. It has not been investigated for removal by microalgae. The removal kinetics of TGC by Chlorella pyrenoidosa were evaluated under different initial dry cell densities, TGC concentrations, temperatures and light intensity conditions. Approximately 90% of TGC could be removed when the TGC concentration was 10 mg∙L-1 and the initial dry cell density was more than 0.2 g∙L-1. A low value of TGC per g dry cell weight ratio led to a high removal efficiency of TGC. The initial dry cell density of microalgae was also critical for the removal of TGC. A high initial dry cell density is better than a low initial dry cell density to remove TGC when the ratio of the TGC concentration to dry cell weight are the same at the beginning of the cultivation. The removal mechanisms were investigated. Photolysis was a slow process that did not lead to removal at the beginning. Adsorption, hydrolysis, photolysis and biodegradation by microalgae were the main contributors to the removal of TGC. TGC was easily hydrolyzed under high -temperature conditions. Three transformation products of TGC by microalgae were identified. The stability of TGC was evaluated in water and salt solutions of citric acid, K2HPO4·3H2O and ferric ammonium citrate. TGC was stable in ultrapure water and citric acid solution. TGC was hydrolyzed in K2HPO4·3H2O and ferric ammonium citrate solutions.

Identification dehydratase domains from Schizochytrium sp. and Shewanella sp. and distinct functions in biosynthesis of fatty acids.

Polyunsaturated fatty acid (PUFA) synthase is a special and effective enzyme for PUFA synthesis, and dehydratase (DH) domain played a crucial role in it. In this work, we compared four different DH domains from different strains (Schizochytrium sp. HX-308 and Shewanella sp. BR-2) and different gene clusters. First bioinformatics analysis showed that DH1, 2 and DH3 were similar to FabA and PKS-DH, respectively, and all of them got a hot-dog structure. Second, four DH domains were expressed in Escherichia coli that increased biomass. Especially, Schi-DH1,2 presented the highest dry cell weight of 2.3 g/L which was 1.62 times of that of control. Fatty acids profile analysis showed that DH1,2 could enhance the percentage of unsaturated fatty acids, especially DH1,2 from Schizochytrium sp., while DH3 benefited for the saturated fatty acid biosynthesis. Furthermore, five kinds of fatty acids were added to the medium to study the substrate preferences. Results revealed that DH1,2 domain preferred to acting on C16:0, while DH3 domain trended acting on C14:0 and C15:0, which illustrated DH from different clusters do have specific substrate preference. Besides, DH expression could save the cell growth inhibition by mid-chain fatty acids. This study provided more information about the catalysis mechanism of polyunsaturated fatty acid synthase and might promote the modification study based on this enzyme.

Growth Performance and Antioxidative Response of Chlorella pyrenoidesa, Dunaliella salina, and Anabaena cylindrica to Four Kinds of Ionic Liquids.

Ionic liquids are widely used for lipid and pigment extractions from microalgae. It is possible that ionic liquids are discharged into environments. The evaluation of growth performance and antioxidative response of ionic liquids to microalgae is helpful to explore the stress regulation mechanism and investigate possible environmental risk. Ionic liquids induce production of reactive oxygen species (ROS) to microalgae. These oxidative stresses are possible from cations, anions, and salinity. In this study, the growth inhibitions of [BMIM]Br, [BMIM]Cl, [EMIM]Cl, and [EMIM]EtOSO3 to Anabaena cylindrica, Chlorella pyrenoidesa, and Dunaliella salina were evaluated. It was interesting that Br- and two kinds of cations, [BMIM] and [EMIM], had significant effects on growth inhibitions of these microalgae. IC50 values of these ionic liquids for A. cylindrica, C. pyrenoidesa, and D. salina were also estimated based on the results of growth inhibitions. It was proved that [EMIM]Cl is relatively harmless to C. pyrenoidesa and D. salina, and [EMIM]EtOSO3 is relatively or practically harmless to C. pyrenoidesa. [BMIM]Br and [BMIM]Cl are practically harmless to A. cylindrica and C. pyrenoidesa, and relatively harmless to D. salina. More than 0.8 g/L [EMIM]EtOSO3 led to bleaching of both A. cylindrica and D. salina at 48 h which was shown that the anion, EtOSO3-, had higher inhibition to A. cylindrica and D. salina than Cl-. In addition, high concentration of ionic liquids led to reductions of chlorophyll content in these three kinds of microalgae, increase of ROS levels and malondialdehyde contents for most of the cases. High concentration of ionic liquids also increased the activities of superoxide dismutase in three kinds of microalgae. There were positive correlations between ROS levels or MDA content, and inhibitions ratios of these ionic liquids to microalgae except [EMIM]Cl to A. cylindrica. These antioxidant enzymes were beneficial for reducing the ROS induced by ionic liquids.

Effects of three antibiotics on growth and antioxidant response of Chlorella pyrenoidosa and Anabaena cylindrica.

Antibiotics are essential for treatments of bacterial infection and play important roles in the fields of aquaculture and animal husbandry. Antibiotics are accumulated in water and soil due to the excessive consumption and incomplete treatment of antibiotic wastewater. The accumulation of antibiotics in ecological systems leads to global environmental risks. The toxic effects of spiramycin (SPI), tigecycline (TGC), and amoxicillin (AMX) on Chlorella pyrenoidesa and Anabaena cylindrica were evaluated based on growth inhibition experiments, and determinations of ROS production and antioxidant enzyme activities (catalase, superoxide dismutase, and malondialdehyde). Half maximal effective concentrations (EC50) of TGC, SPI, and AMX for A. cylindrica were 62.52 µg/L, 38.40 µg/L, and 7.66 mg/L, respectively. Those were 6.20 mg/L, 4.58 mg/L, and > 2 g/L for C. pyrenoidesa, respectively. It was shown that A. cylindrica was much more sensitive to these antibiotics than C. pyrenoidesa. In addition, EC50 values of SPI and TGC were lower than that of AMX. It was indicated that SPI and TGC had higher toxic than AMX to C. pyrenoidesa and A. cylindrica. The current study is helpful to evaluating possible ecological risks of TGC, SPI, and AMX by green microalgae and cyanobacteria.

Adaptive Evolution Improves Algal Strains for Environmental Remediation.

Microalgae have potential for environmental remediation, but they must better tolerate stress. Adaptive laboratory evolution (ALE) is effective to construct evolved strains, but its efficiency is low. Highly efficient ALE relies on selecting suitable environmental stress, original strain selection, and optimizing initial cell density and stress strategy.

Exergy analysis for docosahexaenoic acid production by fermentation and strain improvement by adaptive laboratory evolution for Schizochytrium sp.

Exergy analysis is powerful tool for process optimization and mechanism analysis. In this study, exergy analysis was performed for docosahexaenoic acid (DHA) fermentation process. More than 86% of input exergy was contributed by glucose. The exergy of biomass was about 64.66% of the total output exergy when the phosphate concentration was 4 g L-1. The exergy efficiencies of DHA (ηDHA) for the starting strains and the evolved strains under high oxygen concentration, low temperature, and two-factor conditions were also investigated. The ηDHA in the collected experimental data was not more than 20.9%. It was proved that there was a positive correlation between ηDHA and the biomass yield. It was indicated that adaptive laboratory evolution (ALE) improved biomass yield which had the most important effect on enhancing ηDHA and DHA yield (or DHA productivity). It is necessary to improve ηDHA through process optimization and ALE in future.

Kinetic model for effects of simulated flue gas onto growth profiles of Chlorella sp. AE10 and Chlorella sp. Cv.

Microalgae are potential candidate for biofuel production as alternative one for fossil fuels. CO2 in flue gas is available carbon source to support microalgae growth. In this study, the effects of different concentrations of the simulated flue gas onto algal growth and photosynthetic activity were evaluated for both Chlorella sp. AE10 and Chlorella sp. Cv. The growth profiles were correlated by a simple kinetic model. It was indicated that the simulated flue gas led to low pH and the photosynthetic activity was partially destroyed. Chlorella sp. Cv can tolerate full simulated flue gas, 10% CO2  + 200 ppm NOx  + 100 ppm SOx . The pH in medium maintained at 6 and the photosynthetic activity was more than 0.6 at the first 6 days. If the concentration of NOx was more 100 ppm and that of SOx was more than 50 ppm, the pH was declined to 4 at day 2 or 3 for Chlorella sp. AE10. At the same time, the related photosynthetic activities of Chlorella sp. AE10 were less than 0.4, which was not suitable for algal growth. It was shown that Chlorella sp. Cv could be used for CO2 fixation from the simulated flue gas.

Application of chemicals for enhancing lipid production in microalgae-a short review.

Microalgae have attracted great attention as a promising sustainable resource for biofuel production. In studies aiming to improve lipid accumulation, many key enzymes involved in lipid biosynthesis were identified and confirmed, but genetic engineering remains a challenge in most species of microalgae. In an alternative approach, various chemical modulators can be used to directly regulate the lipid biosynthesis pathway, with similar effects to gene overexpression and interference approaches, including improving the precursor supply and blocking competing pathways. The produced lipid can be protected from being converted into other metabolites by the chemicals such as lipase inhibitors. In addition, a few chemicals were also demonstrated to greatly influence cell growth and lipid accumulation by indirect regulation of the lipid biosynthesis pathway, such as increasing cell permeability or regulating oxidative stress. Thus, adding chemical modulators can be a useful alternative strategy for improving lipid accumulation in large-scale cultivation of microalgae.

Effect of light quality on growth rate, carbohydrate accumulation, fatty acid profile and lutein biosynthesis of Chlorella sp. AE10.

Microalgae are feedstocks for multiple product development based on algal biorefinery concept. The effects of light quality (white, red and blue light emitting diodes) and macro-element starvations on Chlorella sp. AE10 were investigated under 20% CO2 and 850 µmol m-2 d-1. Nitrogen and phosphorus starvations had negative effects on its growth rate. The biomass productivities were decreased from day 1 and the highest one was 1.90 g L-1 d-1 under white light conditions. Phosphorus starvation promoted carbohydrate accumulation under three LED light sources conditions and the highest carbohydrate content was 75.9% using red light. Blue light increased lutein content to 9.58 mg g-1. The content of saturated fatty acids was significantly increased from 37.51% under blue light and full culture medium conditions to 77.44% under blue light and nitrogen starvation conditions. Chlorella sp. AE10 was a good candidate for carbohydrate and lutein productions.

Application of the CRISPR/Cas system for genome editing in microalgae.

Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.

Current states and challenges of salt-affected soil remediation by cyanobacteria.

Natural and human activities lead to soil degradation and soil salinization. The decrease of farmlands threatens food security. There are approximately 1 billion ha salt-affected soils all over of world, which can be made available resources after chemical, physical and biological remediation. Nostoc, Anabaena and other cyanobacterial species have outstanding capabilities, such as the ability to fix nitrogen from the air, produce an extracellular matrix and produce compatible solutes. The remediation of salt-affected soil is a complex and difficult task. During the past years, much new research has been conducted that shows that cyanobacteria are effective for salt-affected soil remediation in laboratory studies and field trials. The related mechanisms for both salt tolerance and salt-affected soil remediation were also evaluated from the perspective of biochemistry, molecular biology and systems biology. The effect of cyanobacteria on salt-affected soil is related to nitrogen fixation and other mechanisms. There are complicated interactions among cyanobacteria, bacteria, fungi and the soil. The interaction between cyanobacteria and salt-tolerant plants should be considered if the cyanobacterium is utilized to improve the soil fertility in addition to performing soil remediation. It is critical to re-establish the micro-ecology in salt-affected soils and improve the salt affected soil remediation efficiency. The first challenge is the selection of suitable cyanobacterial strain. The co-culture of cyanobacteria and bacteria is also potential approach. The cultivation of cyanobacteria on a large scale should be optimized to improve productivity and decrease cost. The development of bio-remediating agents for salt-affected soil remediation also relies on other technical problems, such as harvesting and contamination control. The application of cyanobacteria in salt-affected soil remediation will reconstruct green agriculture and promote the sustainable development of human society.

Enhancement of lipid accumulation in microalgae by metabolic engineering.

Microalgal lipids have drawn great attention as a promising sustainable resource for biodiesel or food supplement production. The development of high-performance strains of microalgae by metabolic engineering is invaluable for increasing the quantity or quality of desired lipids. The synthesis routes of lipids used as biodiesel in microalgae are based on fatty acid synthase (FAS) and triacylglycerols (TAG) biosynthesis pathway. Polyunsaturated fatty acids (PUFAs), including ω-6 and ω-3 fatty acids, are essential nutrients for humans. Notably, microalgae possess two distinct pathways for polyunsaturated fatty acids (PUFAs) biosynthesis, including the desaturase/elongase pathway and the polyketide synthase (PKS) pathway. Thus, it is necessary to identify which biosynthetic pathways are responsible for PUFA synthesis in particular microalgae species. In recent years, various key enzymes and functional domains involved in fatty acid and TAG biosynthesis pathway were identified and potentially regulated by genetic engineering approaches to elevate specific lipids content. In addition, other studies have reported the implementation of strategies to increase lipid accumulation based on increasing acetyl-CoA/NADPH supply, enhancing photosynthetic efficiency, or blocking competing pathways. Furthermore, other efforts have used transcription factor engineering to simultaneously regulate multiple genes related to lipid accumulation. This review summarizes recent research about a variety of microalgae lipid biosynthesis pathways, and discusses multiple gene manipulation strategies that have been employed for specific lipid overproduction in industrial microalgae.

Adaptive evolution and carbon dioxide fixation of Chlorella sp. in simulated flue gas.

Carbon dioxide and other greenhouse gas emissions leads to global warming. Biological capture through microalgae is a potential approach for solving this environmental problem. It is still a technical challenge to enhance the tolerance of microalgae to flue gas if CO2 is fixed from flue gas directly. A new strain, Chlorella sp. Cv was obtained through adaptive evolution (46 cycles) against simulated flue gas (10% CO2, 200 ppm NOx and 100 ppm SOx). It was confirmed that Chlorella sp. Cv could tolerate simulated flue gas conditions and the maximum CO2 fixation rate was 1.2 g L-1 d-1. In a two-stage process, the biomass concentration was 2.7 g L-1 and the carbohydrate content was 68.4%. Comparative transcriptomic analysis was performed for Chlorella sp. Cv under simulated flue gas and control conditions (10% CO2). These responses against simulated flue gas uncovered the significant difference between the evolved strain and the original strain. The metabolic responses to flue gas were explored with focus on various specific genes. Upregulation of several genes related to photosynthesis, oxidative phosphorylation, CO2 fixation, sulfur metabolism and nitrogen metabolism was beneficial for the evolved strain to tolerate the simulated flue gas. The upregulation of genes related to extracellular sulfur transport and nitrate reductase was essential to utilize the sulfate and nitrate from dissolved SOx and NOx. The results in this study are helpful to establish a new process for CO2 capture directly from industrial flue gas.

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

Microalgae have drawn great attention as promising sustainable source of lipids and carotenoids. Their lipid and carotenoids accumulation machinery can be trigged by the stress conditions such as nutrient limitation or exposure to the damaging physical factors. However, stressful conditions often adversely affect microalgal growth and cause oxidative damage to the cells, which can eventually reduce the yield of the desired products. To overcome these limitations, two-stage cultivation strategies and supplementation of growth-promoting agents have traditionally been utilized, but developing new highly adapted strains is theoretically the simplest strategy. In addition to genetic engineering, adaptive laboratory evolution (ALE) is frequently used to develop beneficial phenotypes in industrial microorganisms during long-term selection under specific stress conditions. In recent years, many studies have gradually introduced ALE as a powerful tool to improve the biological properties of microalgae, especially for improving the production of lipid and carotenoids. In this review, strategies for the manipulation of stress in microalgal lipids and carotenoids production are summarized and discussed. Furthermore, this review summarizes the overall state of ALE technology, including available selection pressures, methods, and their applications in microalgae for the improved production of lipids and carotenoids.

Adaptive evolution of microalgae Schizochytrium sp. under high salinity stress to alleviate oxidative damage and improve lipid biosynthesis.

Lipid accumulation of Schizochytrium sp. can be induced by stress condition, but this stress-induction usually reduce cell growth and cause oxidative damage, which can eventually lower the lipid yield. Here, adaptive laboratory evolution (ALE) combined high salinity was performed to enhance the antioxidant system and lipid accumulation. The final strain ALE150, which was obtained after 150 days, showed a maximal cell dry weight (CDW) of 134.5 g/L and lipid yield of 80.14 g/L, representing a 32.7 and 53.31% increase over the starting strain, respectively. Moreover, ALE150 exhibited an overall higher total antioxidant capacity (T-AOC) and lower reactive oxygen species (ROS) levels than the starting strain. Furthermore, the regulatory mechanisms responsible for the improved performance of ALE150 were analyzed by transcriptomic analysis. Genes related to the antioxidant enzymes and central carbon metabolism were up-regulation. Moreover, the metabolic fluxes towards the fatty acid synthase (FAS) and polyketide synthase (PKS) pathways were also changed.

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

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.