Abstract Background Marine protists Aurantiochytrium are recognized as promising sources for commercial lipid production, particularly due to their ability to produce high-value natural compounds like docosahexaenoic acid (DHA). However, wild-type strains isolated from natural environments typically fail to meet commercial demands for DHA yields, partly because they are poorly adapted to the decreased pH conditions encountered during fermentation. Results In this study, we employed a staged acidic adaptive laboratory evolution (ALE) strategy to develop a high DHA-producing strain from Aurantiochytrium sp. PKU#Mn16. By optimizing oxygen and temperature levels under low pH conditions, ALE resulted in a 171.4% increase in DHA concentration in the ALE strain compared to the wild-type strain. Comparative transcriptomics revealed that ALE enhanced the expression of key enzymes in glycolysis and the polyketide synthase (PKS) pathway during both early (metabolic peak) and late (metabolic decline) fermentation stages, promoting growth and polyunsaturated fatty acid synthesis. Additionally, key enzymes in the tricarboxylic acid (TCA) cycle and pentose phosphate (PPP) pathway were upregulated at early and late stages, respectively, suggesting differential ATP/NADPH supply mechanisms that drive DHA accumulation. Notably, the upregulation of glycerol kinase (GK) indicates the potential for using glycerol as an alternative carbon source to further enhance DHA production in our ALE strain. Conclusions In this study, Aurantiochytrium sp. PKU#Mn16 was successfully acclimated using a synergistic approach combining high dissolved oxygen, low temperature, and citric acid-induced acidity. This strategy yielded significant increases of 106.3% in biomass, 243.8% in total fatty acid yield, and 171.4% in DHA yield. Transcriptomic analysis revealed extensive rewiring of central carbon and lipid metabolism, including the upregulation of PKS pathway enzymes and enhanced supply of ATP, NADPH, and acetyl-CoA. Additionally, reduced competing secondary metabolic fluxes optimized substrate allocation. This innovative acclimation strategy not only sheds light on the molecular mechanisms driving efficient fatty acid and DHA production but also lays the groundwork for future comparative genomics and genetic editing efforts aimed at further boosting yields of fatty acids and other natural secondary metabolites in thraustochytrids. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-025-02792-z. Keywords: Adaptive laboratory evolution, Docosahexaenoic acid, Transcriptomics, PKS pathway, Glycolysis Background Docosahexaenoic acid (DHA, C22:6), a long-chain polyunsaturated fatty acid, is highly effective in supporting brain and nervous system development and shows promise in preventing cancer, cardiovascular diseases, and hypertension [[32]1–[33]4]. Consequently, DHA is a highly desirable ingredient in health supplements for all age groups [[34]5]. Traditionally sourced from deep-sea fish oil, DHA derived from this method has drawbacks, including strong odors, unsustainable resource use, and the risk of heavy metal contamination [[35]6, [36]7]. Therefore, there is an urgent need for a sustainable and efficient alternative. Thraustochytrids, unicellular marine protists, are emerging as a promising solution due to their high DHA production capabilities [[37]8]. Cultivating and fermenting thraustochytrids for DHA production offers several advantages: a rapid growth cycle, easily controlled production, and support for sustainable practices [[38]9]. They also have strong adaptability to various carbon sources and environmental conditions, enabling significant productivity improvements after fermentation optimization, with biomass and DHA yields reaching 63–110 g/L and 10–20 g/L, respectively. In addition to thraustochytrids, Crypthecodinium cohnii, produces high-purity DHA but has a lower biomass (20–30 g/L) and DHA yield (1.4–3 g/L), and is highly sensitive to dissolved oxygen (DO), limiting its industrial scale production [[39]10, [40]11]. Some engineered microbes such as Mortierella alpina [[41]12], Yarrowia lipolytica [[42]13], and Shewanella decolorationis [[43]14] can also synthesize DHA but with much lower purity (4%~17% of total fatty acids), and they generally require complex genetic manipulation and costly inducers. Thus, thraustochytrids represent the major microbial group showing naturally high DHA yields and capacity for industrial applications. As a result, the biopharmaceutical and nutraceutical industries are increasingly interested in thraustochytrids as a viable alternative to fish oil. However, the DHA yield from thraustochytrids often falls short of commercial needs due to limited adaptability to fermentation conditions. Bioprocessing and metabolic engineering approaches to develop strains with enhanced resilience and productivity are crucial to maximizing DHA yields [[44]6]. Adaptive laboratory evolution (ALE) is a powerful technique for developing strains with desired traits. It involves subjecting cells to specific or gradually increasing environmental pressures during continuous culture, ultimately selecting strains that thrive under those conditions [[45]15]. In thraustochytrid fermentation, key environmental factors like temperature, DO, and pH significantly impact cell growth and fermentation efficiency [[46]16–[47]18]. ALE has been successfully employed to generate robust thraustochytrids capable of withstanding diverse stressors while exhibiting improved biomass and lipid production [[48]17]. No matter the temperature [[49]14–[50]17], DO [[51]11, [52]16, [53]18], or NaCl stress [[54]15] conditions, through the assistance of ALE, thraustochytrids can resist these stress factors and improve their biomass and the production of unsaturated fatty acids. For instance, researchers have used high-temperature adaptation to create strains with enhanced heat tolerance, leading to efficient DHA production at elevated temperatures [[55]19]. Similarly, adaptation to high-DO conditions has significantly increased dry cell weight (DCW) and DHA production [[56]16]. Besides applying single-stress acclimatization, recent research has explored the synergistic effects of acclimatization under low-temperature and high-salt stresses [[57]20]. The synergistic effects of acclimatization under more than 2 stressors remains to be explored. Thraustochytrid batch fermentation often experiences a pH drop (e.g., from 7 to below 6) likely due to accumulation of organic acid byproducts, impairing growth and DHA production [[58]21]. Neutralizing with alkalis is costly, making ALE targeting low pH a practical alternative, as it enhances acid tolerance, stabilizing enzyme activity and membrane integrity critical for DHA synthesis [[59]21]. From an industrial perspective, acid-adapted strains that can grow and accumulate lipids under low pH conditions help minimize the need for continuous pH adjustment using buffering agents or alkali supplements, thus reducing fermentation costs and process complexity. Furthermore, low-temperature ALE up-regulates cold-resistance genes like ATP-dependent RNA helicase, boosting energy and substrate availability for fatty acid production [[60]22], while high-DO ALE triggers antioxidant defenses, optimizing lipid composition under oxidative stress [[61]4, [62]23]. Potential synergistic effects of low pH, low temperature, and high DO as evolutionary parameters could amplify DHA yields by coordinating metabolic efficiency, stress resilience, and lipid synthesis, offering a promising but unexplored strategy for industrial-scale production. Previous studies have revealed several molecular mechanisms underlying high DHA production in thraustochytrids. Firstly, the polyketide synthase (PKS) pathway is more efficient than the traditional fatty acid synthase (FAS) pathway, producing fewer intermediate products (e.g., docosapentaenoic acid) and directly synthesizing DHA. Secondly, key enzymes such as ATP-citrate lyase, acetyl-CoA carboxylase, malonyl-CoA: ACP transacylase, glucose-6-phosphate dehydrogenase, malic enzyme, and omega-3 fatty acid desaturase can be regulated to enhance DHA production by increasing precursor molecule supply, regulating desaturation and elongation, and suppressing competing pathways [[63]24–[64]29]. Furthermore, optimizing the metabolic network through strategies like gene overexpression [[65]30], adaptive laboratory evolution [[66]20], and nitrogen/phosphorus limitation induction [[67]26, [68]31] can further boost DHA yields. Thus, we hypothesize multi-factor acclimation for DHA improvements would involve complex metabolic rewiring that awaits dissection. To this end, this study investigates a co-ALE approach using combined low-pH, low-temperature, and high-DO stresses to improve acid adaptability simultaneously and DHA production in a promising Aurantiochytrium sp. PKU#Mn16 strain. We aim to elucidate the underlying metabolic shifts responsible for enhanced DHA yields through comparative transcriptomic analysis of wild-type and ALE strains. The findings from this research offer valuable insights and strategies for optimizing large-scale DHA production in thraustochytrids. Materials and methods Strain and culture medium In this study, Aurantiochytrium sp. PKU#Mn16 (CGMCC 8095) [[69]32], previously isolated from coastal mangrove habitats in the Pearl River Delta, was used as the ALE starting strain (the wild-type). The strain was maintained on MV solid medium (glucose 20 g/L, peptone 1.5 g/L, yeast extract 1 g/L, sea salt 33 g/L, agar 20 g/L) at 28℃. To prepare the seed culture for ALE or fermentation tests, we inoculated the PKU#Mn16 strain into the liquid M4 medium (glucose 20 g/L, peptone 1.5 g/L, yeast extract 1 g/L, KH[2]PO[4] 0.25 g/L, sea salt 33 g/L, initial pH 6.5) and incubated at 28 °C with shaking at 170 rpm for 24 h. Subsequently, the seed culture after 24 h of incubation was transferred (10% v/v) to 300 mL of M4 medium and incubated on a shaker at 28 °C and 170 rpm for another 24 h. Subsequent experiments used seed cultures generated in this process as inoculum (10% v/v). Adaptive laboratory evolution (ALE) Previous studies and our preliminary data suggest that a low temperature at 16 °C [[70]33, [71]34] and a high DO induced by 230 rpm shaking [[72]35] lead to significantly increased DHA production in thraustochytrids compared to the standard 28 °C and 170 rpm shaking conditions. In order to optimize the experimental process and workload, we used orthogonal experimental design to combine two levels of temperature (16 °C and 28 °C), two levels of DO indicated by the shaking speed (170 rpm and 230 rpm), and three acid types (citric acid, acetic acid, and hydrochloric acid) to obtain 12 combinations of ALE conditions (Additional file 1: Table [73]S1). During ALE, the wild-type and evolved strains were incubated in four isothermal shakers with different settings (Table [74]1), i.e., normal (28 °C, 170 rpm), low temperature (16 °C, 170 rpm), high DO (28 °C, 230 rpm), and low temperature with high DO (16 °C, 230 rpm). Table 1. Adaptive laboratory evolution process Stage pH Times Days 1 5.5 15 30 2 5.0 15 30 3 4.5 15 30 4 4.0 15 30 5 3.5 15 30 Total 75 150 [75]Open in a new tab The staged method was used for the acid ALE, which involved transferring 3 mL of the fermentation broth to fresh acidic medium and incubating at different settings for 48 h. This constituted one generation, and after 15 passages, the first ALE stage at pH 5.5 is completed. The above steps were repeated to complete the second to fifth stages of ALE experiments at gradually decreased pH until 3.5 (Table [76]1). The entire ALE process involves 75 passages over a period of 150 days. Every month during the ALE experiments, glycerol preservation was performed for the acclimated strains in triplicate. Biochemical analysis To investigate the effects of long-term acclimation under low pH induced by different acids, low temperature, and/or high DO level on the biomass and fatty acid production capacity in thraustochytrids, the wild-type strain of Aurantiochytrium sp. PKU#Mn16 and 12 ALE strains (Additional file 1: Table [77]S1) were grown in triplicate under a normal fermentation condition (28 ℃, 170 rpm, M4 medium without acid supplement) for 72 h before DCW, total fatty acid (TFA), and DHA quantification. The measurement process involved centrifuging the cultures at 8000 rpm at 4 °C for 10 min to collect cell pellets, washing the pellets twice with sterile distilled water, freeze-drying the washed cells for 48 h using a freeze dryer (Christ, Germany), determining the DCW using a gravimetric method, and preparing methyl ester fatty acids through direct transesterification [[78]36]. The methyl ester fatty acid composition and content in the samples were analyzed using gas chromatography following established protocols [[79]37]. Transcriptome analysis Comparative transcriptomics were conducted between the wild-type strain and the ALE strain (ALE_AC_11, Additional file 1: Table [80]S1) under normal fermentation conditions (28 °C, 170 rpm, M4 medium without acid supplement). Triplicate samples for each strain were collected at 36 h and 48 h of fermentation, during which period the growth started to slow down, suggesting a potential transition from the metabolic peak stage to the metabolic decline stage (Additional file 1: Fig. [81]S1). For each sample, 15 mL of culture was centrifuged at 12,000 rpm for 5 min at 4 °C to obtain cell pellets. Subsequently, the cell pellet was rapidly frozen in liquid nitrogen and stored in a -80 °C freezer. The total RNA extraction and transcriptome sequencing service was provided by Hangzhou Lianchuan Biotechnology Co. Ltd. The experimental process included library preparation and sequencing. The total RNA was extracted from frozen samples using an RNA kit (containing Trizol reagent). The quality and integrity of the extracted RNA were assessed using Nanodrop (Thermo Scientific™ brand) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA), while the RNA concentration was accurately determined using the Qubit RNA Assay Kit (Life Invitrogen, USA). For high-quality RNA samples, 2 µg of total RNA was used to construct an RNA library, and deep sequencing analysis was performed on the Illumina HiSeq 2500 sequencing platform. Sequencing data were subjected to strict quality control, including removal of repetitive sequences, adapter sequences, and low-quality reads. Since a suitable reference genome is not available for the Aurantiochytrium sp. PKU#Mn16 strain used in this study, a reference-free (de novo) transcriptome assembly strategy was employed. Specifically, Trinity software was used to assemble the filtered reads de novo into contigs [[82]38], which were subsequently processed and concatenated to form unigene sequences. BLASTx between the unigenes and databases of Gene Ontology (GO), Kyoto Encyclopedia of Genes (KEGG), NCBI non-redundant protein (Nr), Protein Families (Pfam), Swiss Protein Knowledgebase (Swiss-Prot) and eggNOG was conducted using a threshold of E-value ≤ 10^− 5. Multi-stage fermentation The experiments were carried out in a 5-L fermenter (model: SY-9000-V9, Shanghai Dongming Industrial Co. Ltd., China) equipped with DO and pH electrodes, temperature sensors, impellers and an air pump. The initial mixed carbon source concentration was 60 g/L [[83]38], and when the carbon source concentration was lower than 20 g/L, the fermentation process was replenished to maintain the carbon source concentration between 20 and 40 g/L to ensure a sufficient carbon source for DHA production. After 84 h of fermentation, the carbon source and defoamer were not replenished. During the cultivation period, the following fermentation strategy was adopted: 0 to 24 h, 32 °C and 50% oxygen, aiming at rapid biomass accumulation; 24 to 48 h, 28 °C and 50% oxygen, aiming at sustained accumulation of biomass as well as the increase of the TFA content in the biomass; 48 to 84 h, 28 °C and 10% oxygen, ensuring a continuous increase in TFA concentration; 84 to 144 h, 32 °C and 50% oxygen, to increase the DHA content in TFA. After the fermentation was completed, a triplicate of 5 mL samples was taken every 12 h to analyze the DCW and fatty acid production. Results and discussion Triple-stress adaptive evolution synergistically enhances biomass and lipid productivity Previous research results have shown that low-temperature acclimation can increase the content of DHA/TFA of cells [[84]22], and high-DO acclimation can enhance the antioxidant capacity of cells [[85]39]. In addition, low-pH acclimation could improve strains’ tolerance to acid accumulation during fermentation, activate oxidative stress defense systems, and redirect carbon fluxes to achieve enhanced DHA production [[86]40, [87]41]. Therefore, acidic pH values were combined with low temperature and high DO in a synergistic manner, with the expectation of obtaining high-DHA-producing strains. To further investigate the impact of different acclimation conditions on the DCW, TFA and DHA accumulation ability of Aurantiochytrium sp. PKU#Mn16, the wild-type strain and 12 ALE endpoint strains (Additional file 1: Table [88]S1) were cultured under standard fermentation conditions (28 °C, 170 rpm, M4 medium) in triplicate. After 72 h of cultivation, these three indicators were measured. Strains acclimated with citric acid generally showed higher biomass than those acclimated with other acids, with only exception of slightly higher biomass in the strain acclimated with acetic acid than the one acclimated with citric acid at 16 ℃ and 170 rpm (Fig. [89]1A). The biomass of all acclimated strains was higher than that of the wild-type strain. Moreover, the biomass of strains acclimated with citric acid or acetic acid was higher than that of strains acclimated with hydrochloric acid (Fig. [90]1A). This indicates that adaptability to specific acid types, rather than just a low pH value, contributes to improving biomass yield, suggesting a role of organic acid tolerance and/or re-mobilization in biomass accumulation during microbial fermentation. The mechanism could be partly attributed to multifaceted cellular adaptations that enable microorganisms to maintain metabolic activity under acidic stress, as previously reported in acid-tolerant microbes [[91]42]: increased expression of proton extrusion systems can facilitate proton export and help sustain pH homeostasis, preserving enzymatic activity and metabolic flux; acid-induced remodeling of the membrane can reduce proton influx and stabilize the cell envelope, enhancing nutrient uptake efficiency; up-regulation of purine biosynthesis and translation machinery under acidic stress can elevate demand for DNA repair and protein synthesis, which are essential for continued growth and cell division. These changes allow acid-adapted strains to sustain active biosynthesis and rapid growth under decreased pH during fermentation. Although the potential re-mobilization of citric acid (into TCA cycle) and acetate acid (into acetyl-CoA synthesis) require future investigation through metabolic flux analysis or isotopic labeling techniques, our preliminary work suggest neither the wild type nor the evolved strain shows growth advantage in the medium supplemented with citric acid, indicating a limited utilization of citric acid as an external carbon source (Fig. [92]S2). Fig. 1. [93]Fig. 1 [94]Open in a new tab Biomass and fatty acid yields in the wild-type and ALE strains acclimated under different conditions. Measurements were taken at 72 h of fermentation under the normal growth condition (28 ℃, 170 rpm, M4 medium without acid supplement). Error bars represent the standard deviations of biological triplicates. (A) Dry cell weight (DCW), total fatty acids (TFA) and DHA. (B) DHA faction in TFA. The TFA color block in the legend represents the production of TFA except DHA. The asterisk indicates the ALE_AC_11 strain, which exhibited the highest biomass and fatty acid production. Compared with using citric acid alone as the acclimation condition, the addition of low-temperature or high-DO stress alone cannot further enhance the biomass accumulation ability. This result differs from the previous finding that when the Schizochytrium sp. HX-308 strain was acclimated under a high-DO environment as the selective pressure, its DCW was significantly increased by 19.10% [[95]16]. However, when acclimated under the triple stresses of citric acid, low temperature, and high DO, the ALE_AC_11 strain showed the highest biomass accumulation (12.4 g/L) after 72 h of fermentation under normal conditions. This was 106.7% higher than that of the wild-type strain (6.0 g/L) (Fig. [96]1A). Compared with other single-factor or dual-factor acclimation experiments [[97]19, [98]20], the increase in biomass in this study has obvious advantages. Overall, our data strongly indicate that long-term synergistic acclimation under multiple stresses induced by citric acid or acetic acid, low temperature, and high DO can increase the biomass yield of thraustochytrids. Similarly, we compared the TFA and DHA differences between the ALE strain and the wild-type strain to investigate which acclimation conditions lead to the development of high DHA-producing strains. Compared to the wild-type strain, all the ALE strain exhibited higher TFA concentration in 72 h of fermentation under normal growth conditions (Fig. [99]1A), suggesting the effectiveness of acidic acclimation in enhancing the capacity of TFA production. When compared to acclimation using citric acid, acetic acid, or hydrochloric acid alone under normal temperature and oxygen conditions, applying low-temperature or high-DO stress alone significantly increased the TFA concentration in the ALE strain (Fig. [100]1A). This outcome starkly contrasts with the previous biomass results, further confirming that low-temperature and high-DO conditions enhance fatty acid production by maintaining normal cellular physiology and energy metabolism [[101]43]. Among all acclimated strains, the ALE_AC_11 strain obtained through multi-stress acclimation with citric acid under low-temperature and high-DO conditions achieved the highest TFA concentration (5.5 g/L) after 72-hour fermentation, representing a 243.8% increase compared to the wild-type strain (1.6 g/L) (Fig. [102]1A). This improvement surpasses the enhancement observed in Schizochytrium sp. CCTCC M209059 under high-temperature acclimation [[103]19], strongly validating the scientific rationale behind the experimental design. DHA production in all the ALE strains was also improved compared to the wild type, despite significant differences among these strains (Fig. [104]1A). In terms of the effects of different acid types, the DHA concentration in the strains acclimated with hydrochloric acid increased the least while it generally increased the most with citric acid. This may verify that the adaptability induced by biological acids has an especially promoting effect on enhancing the DHA production in thraustochytrids. Compared to acetate acid, citric acid can promote better acclimation effects, which is most likely because it plays a role in the TCA cycle, which is crucial for intracellular metabolic processes in thraustochytrids [[105]44]. In contrast, while acetic acid contributes to acetyl-CoA synthesis, its lower dissociation at pH 4 allows it to enter cells in molecular form [[106]45], which is membrane-permeable and can diffuse into cells, potentially disrupting intracellular pH balance and metabolism. This can cause intracellular damage, disrupt metabolic activities, and potentially explain the poor acclimation effect observed with acetic acid. Furthermore, compared to the two-carbon molecule acetic acid, the six-carbon structure of citric acid makes it a more promising “alternative carbon source” for the strain, potentially providing additional energy during the fermentation process [[107]46]. When compared to acidic acclimation using citric acid, acetic acid, or hydrochloric acid alone under normal temperature and oxygen conditions, triple-stress acclimation under low-temperature and high-DO conditions yielded the optimal DHA production. On one hand, low temperature typically reduces membrane fluidity and thereby impacts cellular material and energy exchange. However, polyunsaturated fatty acids (PUFAs) such as DHA maintain liquidity better than saturated fatty acids (SFAs) at low temperatures, prompting cells to increase DHA content to preserve membrane fluidity [[108]33, [109]47, [110]48]. On the other hand, prior studies have demonstrated that sustained oxygen supply maintaining a high-DO transfer coefficient in the fermentation system can also enhance DHA production in thraustochytrids [[111]49]. In this study, citric acid acclimation coupled with low temperature and high DO led to a strain (ALE_AC_11) with the highest DHA concentration (1.9 g/L), which is 46.2% higher than the simple acidic acclimation strain and 171.4% higher than the wild-type strain (0.7 g/L) under identical culture conditions (Fig. [112]1A). The stability of the phenotype was validated by measurements across six successive generations (maintained under non-stress conditions) (Fig. [113]S3). Additionally, other acclimation conditions such as combinations of citric acid with low temperature or high DO, as well as hydrochloric acid with low temperature, also conferred relatively high DHA production capabilities to endpoint strains (Fig. [114]1A). Studies conducted previously have proven that the initial pH of the culture medium significantly influences the growth and lipid production of thraustochytrids [[115]21]. When cultivating Schizochytrium limacinum SR21 with acetic acid, the TFA content was highest at pH 7.0, but TFA production decreased by 40% at pH 6.0 [[116]50]. When the pH value is reduced from 7.0 to 4.0, the lipid content of Schizochytrium sp. AB-610 is increased from 37.88% to 42.01%. This strain has the highest biomass of 62.63 g/L when the pH value is 7.0. The biomass is slightly reduced when the pH value is 5.0, and the biomass is sharply reduced when the pH value is 4.0, which in turn reduces the lipid and DHA production [[117]21]. In our study, acclimation under low pH (especially induced by citric acid), low temperature, and high DO significantly improved the capacity of Aurantiochytrium sp. PKU#Mn16 in biomass, TFA, and DHA production, providing a valuable reference and a promising strategy for optimization of thraustochytrids and other lipid-producing microorganisms. But we also noted that, despite significant increase in both TFA and DHA production in the ALE_AC_11 strain, the normalized TFA/DCW and DHA/DCW values were slightly lower than those of the wild-type strain, suggesting that the increased fatty acid production is largely driven by enhanced biomass growth. In addition, considering from the perspective of molecular mechanism, under high-DO conditions, the expression of genes related to glycolysis and pentose phosphate pathway metabolism is significantly up-regulated, leading to more NADPH and acetyl-CoA being used for cell growth and lipid synthesis [[118]43]. At the same time, this process is accompanied by the down-regulation of the transcriptional levels of superoxide dismutase (SOD) and ascorbate peroxidase (APX), which may be the reason for the reduction in the proportion of DHA in TFA. Most current studies focus on the lipid induction effects of single or dual stress factors (e.g., temperature or/and oxygen) [[119]51, [120]52]. Existing strategies primarily aim to maintain phenotypic stability of acclimated strains through intensive exogenous nutrient or environmental condition inputs. This not only increases fermentation substrate costs but may also trigger metabolic flux redistribution due to byproduct accumulation (e.g., acetic acid or reactive oxygen species), thereby inhibiting sustained efficient operation of product biosynthesis pathways. Additionally, the transcriptional regulatory mechanisms underlying the effects of multiple stresses on acclimated strains remain understudied. Acid-adaptive acclimation shifts lipid biosynthesis toward total fatty acid accumulation Thraustochytrids can produce various types of fatty acids. In addition to PUFAs such as DHA, long-chain saturated fatty acids, such as stearic acid and palmitic acid [[121]8], also account for a large proportion. From the perspective of being beneficial to human health, the utilization value of PUFAs is significantly higher than that of SFAs [[122]3]. Therefore, while paying attention to whether the concentration of TFA increases, we should also pay attention to the types of fatty acids that increase. In this study, our focus is on exploring the proportion of DHA in TFA. We found that the DHA/TFA fraction in most ALE strains decreased compared to that in the wild-type strain (42.3%), and increased in only two strains that were obtained through the citric acid (47.0%) and acetic acid (51.6%) acclimation at the normal temperature and oxygen level, representing a fraction 11.1% and 22.0% higher than that in the wild-type strain respectively (Fig. [123]1B). This finding is consistent with previous research demonstrating that high dissolved oxygen levels promote rapid growth and lipid accumulation in thraustochytrids, but result in reduced DHA content within TFA [[124]43, [125]53]. Given that high DO is associated with oxidative stress, and DHA is peroxidation-sensitive, the decreased DHA/TFA fraction in high-DO strains could be partly attributed to oxidative degradation. Combining TFA and DHA data, we concluded that acid acclimation under low-temperature and/or high-DO conditions resulted in a greater fold increase in TFA concentration than in DHA concentration, indicating that these synergistic acclimation conditions might be more effective in improving TFA production rather than DHA production. Future studies should, therefore, be directed towards enhancing the proportion of DHA. While this study focuses on TFA and DHA as key lipid indicators, future investigations on SFA/PUFA ratio and compositional lipid profiles would provide more comprehensive understanding on the cellular responses to the multi-stress ALE processes and the underlying metabolic trade-offs between growth, total lipid production, and PUFA enrichment, which is important in guiding bioprocess optimization for biomanufacturing. Comparative transcriptomic profiling between the ALE strain and wild-type strain To provide an integrative understanding on how the metabolic processes in the optimal ALE strain ALE_AC_11 were rewired to support enhanced growth and fatty acid production, we grew the ALE_AC_11 strain and the wild-type strain in a normal fermentation condition (28 °C, 170 rpm, M4 media without acid supplement, initial pH 6.5) and collected triplicate samples at 36 h and 48 h, during which the growth of both strains started to slow down typically indicating metabolic transition from active growth to lipid accumulation (Additional file 1: Fig. [126]S1), for transcriptomic analysis. The raw transcriptomic sequencing data of all samples demonstrated good quality and reliability (Additional file 1: Table [127]S2). After assembling the resulting sequences, we obtained a total of 72,080 transcripts and 56,711 unigenes (Additional file 1: Table [128]S3). While the Aurantiochytrium sp. PKU#Mn16 genome has not been sequenced, genes of other strains provide annotation references based on sequence homology. We selected six databases (GO,