Alpha-Tocopherol Significantly Improved Squalene Production Yield of Aurantiochytrium sp. TWZ-97 through Lowering ROS levels and Up-Regulating Key Genes of Central Carbon Metabolism Pathways

Media supplementation has proven to be an effective technique for improving byproduct yield during microbial fermentation. This study explored the impact of different concentrations of bioactive compounds, namely alpha-tocopherol, mannitol, melatonin, sesamol, ascorbic acid, and biotin, on the Aurantiochytrium sp. TWZ-97 culture. Our investigation revealed that alpha-tocopherol was the most effective compound in reducing the reactive oxygen species (ROS) burden, both directly and indirectly. Adding 0.7 g/L of alpha-tocopherol led to an 18% improvement in biomass, from 6.29 g/L to 7.42 g/L. Moreover, the squalene concentration increased from 129.8 mg/L to 240.2 mg/L, indicating an 85% improvement, while the squalene yield increased by 63.2%, from 19.82 mg/g to 32.4 mg/g. Additionally, our comparative transcriptomics analysis suggested that several genes involved in glycolysis, pentose phosphate pathway, TCA cycle, and MVA pathway were overexpressed following alpha-tocopherol supplementation. The alpha-tocopherol supplementation also lowered ROS levels by binding directly to ROS generated in the fermentation medium and indirectly by stimulating genes that encode antioxidative enzymes, thereby decreasing the ROS burden. Our findings suggest that alpha-tocopherol supplementation can be an effective method for improving squalene production in Aurantiochytrium sp. TWZ-97 culture.


Introduction
Squalene is a terpenoid hydrocarbon (C 30 H 50 ) with broad applications in food, medicine, and the cosmetic industry because of its wide range of biological properties. It shows biological activities against microbes (e.g., bacteria and fungi) and viruses and has antioxidant, tumorsuppressing, immunity-enhancing [1][2][3], and cholesterol-lowering properties [4,5]. Humans still mainly rely on deep-sea shark liver oil for squalene since it contains 40-70% of squalene by its dry weight [5]. This leads to the theft of sharks, resulting in massive damage to the marine ecosystem. Other competitive sources, including animals, plants, and microbes, show limited potential due to challenges such as seasons, covered areas, and lower squalene yields [6][7][8]. Because of their low squalene concentration and competition with agricultural land, plant products are widely accepted not to be an alternative resource [5,9]. Interestingly, microbes remain an unexplored source for squalene because of their ease of cultivation and low operational cost [10].

Strain and Culture Condition
Aurantiochytrium sp. TWZ-97 was maintained at room temperature on agar plates containing the growth medium described in our previous study [14]. Seed culture was prepared by inoculating a single colony from the agar plate into a 100 mL Erlenmeyer flask with 50 mL of growth medium and incubating the flask at 28 • C in an orbital shaker set for 24 h at 170 rpm.
To verify shake flask experimental results, batch fermentation was carried out in a 5 L bioreactor (Model: SY9000-V9, Shanghai Dong Ming Industrial Co., Ltd., Shanghai, China) equipped with DO and pH electrodes, a temperature sensor, an impeller, and an air pump. The working volume of the bioreactor was 2.5 L. Fermentation was carried out at 28 • C, 170 r/min for 72 h. At 0 h of fermentation, an appropriate volume of alpha-tocopherol stock was added to the culture to achieve a final concentration of 0.7 g/L.

Analytical Methods
The intracellular ROS levels and total antioxidant capacity (T-AOC) were measured according to the procedures described in our previous study [29]. The TAO-C was calculated by Ferric Reducing Ability of Plasma (FRAP) assay [30,31]. In this assay, the reduction of ferric to ferrous ions at low pH yields a colored ferrous-tripyridyltriazine complex using the T-AOC assay kit (Solarbio, Beijing, China). In brief, microbial cells were collected every 12 h interval and pelleted by centrifuging at 4 • C, 4000 rpm for 5 min. The collected pellet was washed briefly with deionized water. The resulting cell pellet was transferred into a mortar and crushed in liquid nitrogen with a pestle. The resulting cell powder was suspended in the extraction buffer supplied in the kit. Then, the suspension solution was centrifuged at 4 • C, 10,000 rpm for 10 min. The supernatant was mixed with three reagent solutions (7:1:1) provided in the kit. The absorbance of the reaction solution was recorded at 593 nm, and the total antioxidant capacity (U/mL) was calculated by following the manufacturer's instructions. Cellular ROS were detected using the Reactive Oxygen Species Assay Kit (Meilun, Shenzhen, China), which contains DCFH-DA (2,7-Dichlorodi-hydrofluorescein diacetate) [32] a non-fluorescence dye to pass over the cell membrane. This probe does not disrupt the cell layers and simply labels the ROS with an illumination. The cells were collected every 12 h and washed with distilled water; the DI-water-washed cells initially were treated with DTT (Sigma-Aldrich, St. Louis, MO, USA) snailase (Solarbio, Beijing, China) to soften cell wall and then incubated with DCFH-DA diluted to 10 µM with 10 mM PBS buffer and were directly treated with the dye and incubated at 37 • C for 40 min in dark. After removing extra dye in the reaction with 10 mM PBS, the excitation wavelength was carried out at 488 nm and emission at 525 nm at 450 V gain on fluorescence spectrophotometer F97 Pro (Lengguang, Shanghai, China).
Residual glucose levels were estimated following the methods mentioned in our previous study [17]. Briefly, 1 mL fermentation broth was centrifuged for 10 min at 10,000 rpm and 4 • C. The supernatant was transferred and diluted to 10× with distilled water in a new tube for glucose concentration analysis using the Glu Kit (Biosino Bio-Technology and Science inc., Beijing, China). The intensity of the red-colored products from the kit assay was recorded at the wavelength of 505 nm using the spectrophotometer manufactured by (Multiskan GO, Thermo Scientific, Waltham, MA, USA).
The dry cell weight (DCW) and squalene concentration were quantified according to the methods described elsewhere [14,33].

RNA Sequencing and Bioinformatics Analysis
To probe the effect of alpha-tocopherol on the transcriptional regulation of squalene biosynthesis, the transcriptome of TWZ-97 strain was analyzed with (test) and without (control) the supplementation. Triplicate culture samples from the control and test groups were collected at 42 h of fermentation for RNA sequencing (RNA-Seq). Each sample was centrifuged at 12,000 rpm for 5 min at 4 • C; the pellets were frozen directly in liquid nitrogen and then stored at −80 • C.
Salmon [37] was used to quantify the expression of transcripts/unigenes by calculating TPM [38]. This efficient tool was used to calculate transcript expressions in RNA-seq data providing accurate and fast results by removing fragment wise GC content bias. It links modern double-phase models, i.e., parallel inference algorithm and feature-rich bias. The differentially expressed unigenes were selected with log2 (fold change) > 1 or log2 (fold change) < −1 and with statistical significance (p value < 0.05) by R package edgeR [39].

Quantitative PCR
The total RNA was extracted from control and test (supplemented with alpha-tocopherol) samples using E.Z.N.A. plant RNA kit (Omega Bio-tek, Inc., Norcross, GA, USA). cDNA was synthesized using random primers with SPARKscript 1st Strand cDNA Synthesis Kit (with gDNA Eraser) (SparkJade, China). Gene-specific primers were designed for glucose-6phosphate isomerase, squalene synthase, and glucose-6-phosphate dehydrogenase (reference gene) ( Table 1). To confirm the primers and cDNA, PCR was conducted in a 25 µL reaction volume, containing 12.5 µL 2X Taq pol PCR master mix, 1 µL of each primer (10 µM), 1 µL cDNA, and 9.5 µL nuclease-free water. The PCR program was set to 95 • C for 3 min, 34 cycles of 95 • C for 30 s, 55 • C (59 • C for SQS) for 30 s, 73 • C for 30 s, and 10 min for final elongation. The size of the PCR product was checked by 2% agarose gel electrophoresis. Quantitative PCR (qPCR) assays were performed in triplicate on a CFX Connect™ Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with ChamQ™ SYBR qPCR Master Mix (Vazyme, Nanjing, China). QPCR was performed in a 10 µL reaction volume, containing 5 µL qPCR master mix, 0.3 µL of each primer (10 µM), 0.8 µL cDNA, and 3.6 µL nuclease-free water. The PCR program was set to 95 • C for 3 min, followed by 39 cycles of 95 • C for 10 s, 55 • C (59 • C for SQS) for 30 s, and then 72 • C for 20 s. The expression levels of genes (SQS and GPI) with reference to the G6PDH gene were calculated following the 2 −∆∆CT method [39]. The melt curve analysis showed a single peak for each gene.

Statistical Analysis
The data are expressed as a mean ± standard deviation (SD). The significance test (one-way ANOVA) was performed in Origin Pro software (student version).

Effect of Supplementation on Squalene Fermentation
This study evaluated various bioactive compounds for their effects on squalene fermentation by the TWZ-97 strain. The results showed that melatonin (at concentrations of 0.25 g/L and 0.30 g/L), sesamol (at concentrations of 87.5 mg/L and 105 mg/L), ascorbic acid (at a concentration of 9 g/L), and biotin (at concentrations of 0.01, 0.05, and 0.10 mg/L) all had a significant positive impact on the biomass of the TWZ-97 strain ( Table 2). However, in the case of squalene production, our study found that alpha-tocopherol (at concentrations of 0.5 to 0.8 g/L), mannitol (at a concentration of 1.0 g/L), sesamol (at concentrations of 87.5 g/L and 105 g/L), and ascorbic acid (at concentrations of 6 g/L and 9 g/L) all had a significant positive impact. Among these supplements, alpha-tocopherol at a concentration of 0.7 g/L was the most effective, with the highest squalene production (170.36 ± 1.7 mg/L) and yield (27.2 ± 2.8 mg/g). This resulted in an increase of 31.2% in squalene production and 37.8% in yield.
The results of this study revealed that the residual glucose content of the alphatocopherol-supplemented culture was significantly lower during the fermentation period (i.e., 12-60 h) when compared to the culture without alpha-tocopherol supplementation ( Figure 1). This observation indicated that adding alpha-tocopherol improved glucose uptake in the TWZ-97 strain. Similar effects have been reported with flaxseed oil supplementation [20] and the addition of ascorbic acid [18]. These findings suggest that media supplementation with bioactive compounds can improve biomass and squalene production by increasing glucose uptake into the cells.
(i.e., 12 h-60 h) when compared to the culture without alpha-tocopherol supplementation ( Figure 1). This observation indicated that adding alpha-tocopherol improved glucose up take in the TWZ-97 strain. Similar effects have been reported with flaxseed oil supplemen tation [20] and the addition of ascorbic acid [18]. These findings suggest that media sup plementation with bioactive compounds can improve biomass and squalene production by increasing glucose uptake into the cells. We performed a 5 L batch fermentation experiment to evaluate the effectiveness o supplementation using 0.7 g/L alpha-tocopherol. Our results showed that the squalene concentration and yield reached 240.3 ± 0.9 mg/L and 32.5 ± 2.0 mg/g, respectively, which were 41.1% and 19.5% higher than the results (170.3 mg/L and 27.21 mg/g) obtained from the 100 mL flask culture and 27.4% and 72% higher than previously reported values o 188.6 mg/L and 18.83 mg/g for this strain. In addition, the biomass increased from 6.29 g/L to 7.42 g/L, which was 18% higher compared to the results from the 100 mL flask culture These results supported the efficacy of alpha-tocopherol in improving squalene and bio mass production. More importantly, this study provides the first evidence that alpha-to copherol supplementation can increase biomass and squalene yield in thraustochytrids.

Effect of Alpha-Tocopherol on Intracellular ROS Level and T-AOC
To further understand the biological effects on the TWZ-97 strain, we investigated the antioxidant properties of alpha-tocopherol by comparing the levels of intracellular ROS and T-AOC in control and supplemented TWZ-97 cultures. The results showed tha alpha-tocopherol supplementation lowered ROS levels throughout fermentation ( Figure  2). These findings suggest that alpha-tocopherol can effectively protect TWZ-97 cells from We performed a 5 L batch fermentation experiment to evaluate the effectiveness of supplementation using 0.7 g/L alpha-tocopherol. Our results showed that the squalene concentration and yield reached 240.3 ± 0.9 mg/L and 32.5 ± 2.0 mg/g, respectively, which were 41.1% and 19.5% higher than the results (170.3 mg/L and 27.21 mg/g) obtained from the 100 mL flask culture and 27.4% and 72% higher than previously reported values of 188.6 mg/L and 18.83 mg/g for this strain. In addition, the biomass increased from 6.29 g/L to 7.42 g/L, which was 18% higher compared to the results from the 100 mL flask culture. These results supported the efficacy of alpha-tocopherol in improving squalene and biomass production. More importantly, this study provides the first evidence that alpha-tocopherol supplementation can increase biomass and squalene yield in thraustochytrids.

Effect of Alpha-Tocopherol on Intracellular ROS Level and T-AOC
To further understand the biological effects on the TWZ-97 strain, we investigated the antioxidant properties of alpha-tocopherol by comparing the levels of intracellular ROS and T-AOC in control and supplemented TWZ-97 cultures. The results showed that alpha-tocopherol supplementation lowered ROS levels throughout fermentation (Figure 2). These findings suggest that alpha-tocopherol can effectively protect TWZ-97 cells from oxidative damage caused by ROS during fermentation. The high ROS levels at the start of fermentation can be attributed to the seed culture, as reported in previous studies [40,41]. Furthermore, the lowest ROS level was detected at 48 h of fermentation in both groups, possibly due to the intracellular accumulation of squalene and carotenoids, as explained in some previous studies [18,40,41]. After 48 h of fermentation, the ROS level increased in both the non-supplemented and supplemented cultures. The lower ROS levels throughout the fermentation in the supplemented group can be attributed to alpha-tocopherol's indirect and direct antioxidative effects on the TWZ-97 strain. The defense system against oxidative stress exists in two different mechanisms: direct and indirect antioxidant effects [19,42].
In the direct antioxidant effect, the ROS are directly adsorbed to the antioxidants and detoxified, whereas in the indirect method, the expressions of genes for antioxidant enzymes (such as superoxide dismutase (SOD) and catalase (CAT)) are involved to reduce the oxidative burden.
possibly due to the intracellular accumulation of squalene and carotenoids, as explained in some previous studies [18,40,41]. After 48 h of fermentation, the ROS level increased in both the non-supplemented and supplemented cultures. The lower ROS levels throughout the fermentation in the supplemented group can be attributed to alpha-tocopherol's indirect and direct antioxidative effects on the TWZ-97 strain. The defense system against oxidative stress exists in two different mechanisms: direct and indirect antioxidant effects [19,42]. In the direct antioxidant effect, the ROS are directly adsorbed to the antioxidants and detoxified, whereas in the indirect method, the expressions of genes for antioxidant enzymes (such as superoxide dismutase (SOD) and catalase (CAT)) are involved to reduce the oxidative burden. Our study examined the impact of alpha-tocopherol supplementation on the T-AOC of TWZ-97 culture throughout the fermentation process ( Figure 3). The results revealed that while T-AOC initially remained low in non-supplemented and supplemented cultures, it increased during the 12 h and 48 h fermentation periods. However, a decline in T-AOC was observed after 48 h in both cultures. This decline in T-AOC may be linked to the high levels of ROS produced during fermentation. Further research is needed to understand the underlying mechanisms behind this decline in T-AOC. Our study examined the impact of alpha-tocopherol supplementation on the T-AOC of TWZ-97 culture throughout the fermentation process ( Figure 3). The results revealed that while T-AOC initially remained low in non-supplemented and supplemented cultures, it increased during the 12 h and 48 h fermentation periods. However, a decline in T-AOC was observed after 48 h in both cultures. This decline in T-AOC may be linked to the high levels of ROS produced during fermentation. Further research is needed to understand the underlying mechanisms behind this decline in T-AOC. Some research has found that alpha-tocopherol can promote the growth of certain microorganisms, such as lactic acid bacteria [43]. This biological activity is likely due to its antioxidant properties, which can help protect the microbe from the harmful effects of ROS. However, in other studies, researchers have found that alpha-tocopherol can inhibit the growth of certain microorganisms, such as pathogenic bacteria [44], because alpha-tocopherol can disrupt the membrane structure of the microorganism, thus making it difficult for them to survive. It should be noted that the effect of alpha-tocopherol on microorganisms can vary depending on the species, study conditions, and alpha-tocopherol concentration. More research is needed to fully understand the effects of alpha-tocopherol on different microorganisms. Antioxidants 2023, 12, 1034 8 of 14 Some research has found that alpha-tocopherol can promote the growth of certain microorganisms, such as lactic acid bacteria [43]. This biological activity is likely due to its antioxidant properties, which can help protect the microbe from the harmful effects of ROS. However, in other studies, researchers have found that alpha-tocopherol can inhibit the growth of certain microorganisms, such as pathogenic bacteria [44], because alphatocopherol can disrupt the membrane structure of the microorganism, thus making it difficult for them to survive. It should be noted that the effect of alpha-tocopherol on microorganisms can vary depending on the species, study conditions, and alpha-tocopherol concentration. More research is needed to fully understand the effects of alpha-tocopherol on different microorganisms.

Transcriptional Regulation of Metabolism
Alpha-tocopherol supplementation impacted multiple metabolic pathways significantly compared to the control group. Our analysis revealed that a total of 3557 genes were significantly overexpressed (FDA ≤ 0.05), and 1001 genes were down-regulated (Table 3). Our findings indicate that the genes predominantly involved in pathways such as glycolysis, gluconeogenesis, the pentose phosphate pathway (PPP), the fructose mannose pathway, the tricarboxylic acid (TCA) cycle, and free radical exchange pathways were among those overexpressed (Table 4, Figures S1 and S2). The genes encoding key enzymes in the gluconeogenesis pathway, including hexokinase, glucose 6-phosphate isomerase (GPI), 6-phosphate fructokinase, fructose 1,6-bisphosphate, triose phosphate isomerase, phosphoglycerate kinase, enolase, and pyruvate carboxylase, were significantly overexpressed in glycolysis. The overexpression of these genes suggested increased carbon flow in the cell, as described in a previous study [45]. Moreover, it has been reported that increased acetyl CoA production can boost squalene production [46].

Transcriptional Regulation of Metabolism
Alpha-tocopherol supplementation impacted multiple metabolic pathways significantly compared to the control group. Our analysis revealed that a total of 3557 genes were significantly overexpressed (FDA ≤ 0.05), and 1001 genes were down-regulated (Table 3). Our findings indicate that the genes predominantly involved in pathways such as glycolysis, gluconeogenesis, the pentose phosphate pathway (PPP), the fructose mannose pathway, the tricarboxylic acid (TCA) cycle, and free radical exchange pathways were among those overexpressed (Table 4, Figures S1 and S2). The genes encoding key enzymes in the gluconeogenesis pathway, including hexokinase, glucose 6-phosphate isomerase (GPI), 6-phosphate fructokinase, fructose 1,6-bisphosphate, triose phosphate isomerase, phosphoglycerate kinase, enolase, and pyruvate carboxylase, were significantly overexpressed in glycolysis. The overexpression of these genes suggested increased carbon flow in the cell, as described in a previous study [45]. Moreover, it has been reported that increased acetyl CoA production can boost squalene production [46].  We found that PPP, an NADPH generation pathway linked to glycolysis at the initial stage, was also overexpressed (Table 4). It has been reported that elevated NADPH production can increase squalene production [47]. NADPH acts as a cofactor for the key enzyme SQS in the mevalonate pathway [48]. In PPP, the overexpressed genes encode 6-phosphogluconate dehydrogenase, 6-phosphogluconolactonase, trans-aldolase, fructose 1,6-bisphosphatase I, 6-phosphofructokinase, transketolase, fructose 6-bisphosphate aldolase, and ribose-phosphate pyrophosphokinase. These enzymes can result in an elevated NADPH [49] and an enhancement in squalene production [50,51].
Alongside glycolysis and PPP, the galactose metabolism pathway was also overexpressed. Genes encoding enzymes, such as UTP-glucose-1-phosphate uridyl-transferase, UDP-glucose 4-epimerase, hexokinase, alpha-galactosidase, and maltase-glucoamylase, were overexpressed (Table 4). These enzymes regenerate glucose, fructose, and galactose molecules. Therefore, galactose was likely recycled in the process to provide a continuous supply, while glucose entered the glycolysis pathway and fructose was metabolized in the fructose mannose pathway (FMP). In FMP, the overexpressed genes encoding enzymes that included hexokinase, mannose-6-phosphate isomerase, phospho-mannomutase, fructose 1,6-bisphosphatase I, 6-phosphofructokinase, GDP mannose 4,6-dehydratase, and GDP-Lfucose synthase. Overall, our results suggest that the significant enrichment of the central metabolic pathways ( Figure S2) resulted in an ample flow of energy to the TCA, the optimal consumption of glucose in the cell, and the production of a substantial amount of NADPH.
The formation of acetyl CoA is a crucial step that fuels the TCA cycle and provides the necessary building units for the biosynthesis of fatty acids and isoprenoids [52,53]. In the present study, genes encoding enzymes involved in the TCA cycle, such as citrate synthase, isocitrate dehydrogenase, aconitate hydratase, succinyl CoA synthetase alpha subunit, succinate dehydrogenate, fumarate hydratase, and malate dehydrogenase, were significantly overexpressed. Furthermore, the interconversion step of acetaldehyde and alcohol by alcohol dehydrogenase was also overexpressed ( Table 4). The overexpression of these genes possibly fueled the energy generation inside cells, which enhanced the energy flow towards the mevalonate (MVA) pathway, resulting in a significant increase in squalene production. In the MVA pathway, the essential genes involved in squalene biosynthesis, including acetoacetyl CoA synthetase, hydroxymethylglutaryl CoA synthase, and farnesyldiphosphate farnesyltransferase (SQS), were overexpressed. In contrast, the gene encoding for the enzyme responsible for converting squalene to sterol, sterol 1-4 alpha-demethylase, was significantly down-regulated. These findings provide the mechanisms for the increased production of squalene in the supplemented culture.

Transcriptional Regulation of Antioxidative Pathways
Alpha-tocopherol has been shown to be a potent antioxidant, and thus, the genes related to pathways involved in scavenging ROS were analyzed in this study. The analysis revealed that genes involved in ROS scavenging, such as superoxide dismutase, catalase, gamma-glutamyl cysteine synthase, and glutathione peroxidase, were significantly overexpressed (Table 4, Figure S3). These results indicated that reduced ROS levels and increased T-AOC can enhance biomass and squalene production.
The results obtained through the transcriptomic analysis were further validated using the qPCR method. The reference gene, G6PDH, was used as a normalizer, and the expression levels of SQS and GPI were quantified. The results revealed that SQS and GPI genes showed 5.49-and 3.89-fold higher expression levels in the supplemented culture, respectively, compared to the non-supplemented culture.

Conclusions
The addition of alpha-tocopherol to the culture of Aurantiochytrium sp. TWZ-97 had significant positive effects on both its growth and squalene production. Adding 0.7 g/L of alpha-tocopherol led to a reduction in the burden of ROS and an improvement in biomass yield and squalene content. These effects were mediated by the overexpression of genes involved in glucose uptake, including those related to glycolysis, the PPP, the galactose pathway, the fructose-mannose pathway, and the TCA cycle. The higher energy flow resulting from the overexpression of these central metabolic pathways possibly led to the upregulation of genes involved in squalene biosyntheses, such as HMG-CoA, acetoacetyl CoA synthetase, and SQS. These findings suggest that adding alpha-tocopherol could be a valuable strategy for increasing thraustochytrids' biomass yield and squalene content in various biotechnological applications.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox12051034/s1, Figure S1: Detailed graphical representation of genes expressed in multiple induced pathways and involved in higher biomass, squalene production in supplemented sample. Glyceraldehyde 3-phosphate (G3P) phosphoglycerate kinase (PKG) Glyceraldehyde 3-phosphate (G3PDH), fructose bisphosphate aldolase (FBAL/ALD0); multiple red arrows show higher energy glow in system, and octagonal structure shows alpha-tocopherol.; Figure S2: KEGG enriched pathways between supplemented and non-supplemented groups; Figure S3: Graphical representation of ROS types, generation sites, damage sites and genes responsible for neutralization of ROS in biological system. Red box shows up-regulated genes and blue box shows down-regulated genes.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. The author Guangyi Wang has been involved as a an expert witness in the Qingdao Institute for Ocean Technology of Tianjin University Co., Ltd., which belongs to Tianjin University.