Effect of Na-citrate on biomass and astaxanthin content of X. dendrorhous.
In this study, 2 g/L Na-citrate was added to the medium at several time points (0, 12, 24, 36, 48, and 60 h), and the growth of X. dendrorhous was monitored at 96 and 120 h. Compared with the control group, the Na-citrate increased biomass at 96 and 120 h. Astaxanthin titer increased to a maximum value at 120 h when Na-citrate was added at 24 h (Fig. S1). Several concentrations of Na-citrate (0, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 g/L) were added to the medium at 24 h to explore the influence of Na-citrate on X. dendrorhous. As shown in Fig. S2, the addition of Na-citrate had a significant effect on cell growth and increased the production of carotenoids and astaxanthin. After adding 3 g/L Na-citrate at 24 h, the astaxanthin titer increased to 24.48 mg/L, which was 85.2% higher than that of the control group at 120 h.
The time course of cell growth, residual glucose, carotenoids, and astaxanthin titer under both cultures were monitored after the addition of 3 g/L Na-citrate at 24 h to investigate the effects of Na-citrate on cell growth and astaxanthin biosynthesis during the entire fermentation process. As observed in Fig. 1, Na-citrate treatment achieved higher biomass, carotenoids, and astaxanthin titer compared with the control group. For both cultures, a rapid increase in biomass was observed after 24 h, and the increase rate under Na-citrate treatment was higher than in the control group. The peak biomass with Na-citrate was 6.04 g/L at 120 h, which is 81.93% higher than that in the control group (Fig. 1A). As shown in Fig. 1B, X. dendrorhous cells utilized glucose quickly under Na-citrate conditions after 24 h, which reveals that the addition of Na-citrate can promote glucose utilization when cells enter the logarithmic growth phase, which is beneficial for cells to maintain a faster growth rate. The addition of Na-citrate increased carotenoids and astaxanthin accumulation after the increase of biomass and showed a 2-fold increase in contrast to the control group (Fig. 1C and 1D).
The regulation of Na-citrate to astaxanthin biosynthesis originates from increased cell growth. Na-citrate is a kind of carbon source stimulating cell growth and development (An., 2001; Du et al., 2021). In addition, Na-citrate as an inexpensive chemical that is metabolized by aerobic microorganisms to increase intracellular ATP levels, thereby enhancing the ability of cells to resist acid-stressed environments (Sánchez et al., 2008; Zhou et al., 2011). It can regulate the pH value of the liquid in fermentation, which is more conducive to cell growth and astaxanthin production (Flores-Cotera et al., 2001).
In order to serve the industrial production, the batch fermentation in a 5-L fermentor was processed in two groups. For the control group (Fig. 2A), the cells entered the exponential growth phase after inoculation and reached the stationary phase at 48 h. Glucose concentration decreased before 48 h, which may contribute to the fast cell growth (logarithmic phase) during this period, and glucose consumption was slow after 48 h. At the same time, the carotenoid accumulation in X. dendrorhous increased greatly, and the titer achieved 293.86 mg/L at 120 h. Besides, the astaxanthin production was closely in parallel with the carotenoid accumulation and reached 44.19 mg/L at 120 h. Compared with the control batch fermentation, there was still a slight increase in biomass after rapid growth at 48 h and reached the maximum of 20.15 g/L due to the addition of Na-citrate. This result was also observed from the change of residual glucose, in which the glucose consumption rate of the Na-citrate group was higher than the control group during 48 to 72 h (Fig. 2B). Furthermore, the carotenoids and astaxanthin started to accumulate at 8 h due to the rapid growth of cells. The productivity of carotenoids between 40 and 48 h was 0.38 mg/L/h, which was significantly higher than that of the control group. The biomass, carotenoid, and astaxanthin titer were 1.26-, 1.32- and 2.01-fold higher, respectively, than the values achieved with the control group. The astaxanthin content increased by 48.12% at 120 h after the addition of Na-citrate. The results indicated that the strategy of supplementing Na-citrate could promote the growth of cells and facilitate the synthesis of astaxanthin.
Table 1 presents the comparison between the fermentation results of our study and other studies, excluding the results of genetic modification fermentation. The astaxanthin content of our study was higher than that of previously published reports except for the result by de la Fuente et al. (de la Fuente et al., 2010), who obtained 4.77 mg/g by culturing P. rhodozyma VKPM 2476 in a 10 L fermenter. The increased content of astaxanthin in a unit cell indicated that the enhanced astaxanthin could regulate cell growth of X. dendrorhous because of Na-citrate and involved the induction of crucial metabolic pathways related to astaxanthin biosynthesis. Based on this phenomenon, metabolome analysis was carried out to find changes caused by Na-citrate at the metabolite level in X. dendrorhous.
Metabolic changes of X. dendrorhous cultured with and without Na-citrate
Metabolic profiles of X. dendrorhous were analyzed by a partial least squares-discriminant analysis (PLS-DA) score plot (Fig. 3). Analysis of the score plots revealed that the metabolomic profiles of the control and the Na-citrate treatment groups were separated at all four-time points. As shown in Tables 2 and 3, a total of 34 chemically classified metabolites with a very important variable of projection (VIP) value greater than 1 and P values less than 0.05, including organic acids, amino acids, carbohydrates, fatty acids, and ergosterol were analyzed. Most of them were involved in the TCA cycle, carbohydrate metabolism, fatty acid synthesis, and amino acid metabolism. The functional classification is shown in the heat map in Fig. 4.
Influence of Na-citrate on glycolysis pathway in X. dendrorhous
For the intracellular glycolysis pathway, a clear metabolite tendency was observed. In Na-citrate cultures, the glycolysis pathway was significantly upregulated (Tables 2 and 3) because of the rapid absorption of glucose from the medium induced by Na-citrate addition. The concentrations of intracellular glucose in cells were increased by 107.25% (P < 0.05) at 48 h and 151.57% (P < 0.05) at 72 h, respectively (Fig. 1B). After 96 h, although the Na-citrate group consumed more glucose, the intracellular glucose in the Na-citrate group was kept at a similar level to the control group, suggesting that the addition of Na-citrate led to an accelerated rate of glucose uptake for cell growth at the early and middle stages of the fermentation. In contrast, the absorbed glucose at the later stage was utilized for its metabolic activities.
In addition, as a product of yeast anaerobic fermentation, the content of ethanol in the Na-citrate group was obviously lower than that in the control group (Table 2 and 3), indicaing that a large part of the pyruvate generated by the glycolysis pathway was directly converted into acetyl-CoA, rather than entering the anaerobic fermentation to accumulate ethanol. As a key substrate of various cellular processes, the increase of acetyl-CoA may make the carbon metabolism flow more to the fatty acid synthesis pathway and astaxanthin synthesis pathway. It is worth noting that, the addition of Na-citrate resulted in a significant decrease of the intracellular phosphate concentration (Tables 2). Flores-Cotera et al. (Flores-Cotera et al., 2001) found that under phosphate-limiting conditions, RNA synthesis was affected, which influences DNA replication rate and protein biosynthesis, making key substrates and coenzymes (acetyl-CoA, ATP, and NADPH), available for other metabolisms, including the TCA cycle, fatty acid synthesis, and carotenoid synthesis. This is consistent with the results of our study.
Influence of Na-citrate on the TCA cycle in X. dendrorhous
During the cultivation period, most intermediates of the TCA cycle were dramatically decreased with Na-citrate treatment in addition to citric acid and malic acid (Tables 2 and 3), indicating that the addition of Na-citrate reduced the metabolic flux toward the TCA cycle. The addition of Na-citrate enhanced the concentration of citric acid, thereby inhibiting the catalytic activity of citrate synthase from synthesizing citric acid and TCA cycle weakening. In the oleaginous yeasts and fungi, low TCA activity may induce citric acid accumulation in the mitochondria, which is transported into the cytoplasm. In the cytoplasm, citric acid is degraded into acetyl-CoA and oxaloacetate under the catalysis of ATP citrate lyase (ACL). In X. dendrorhous, the production of fatty acids and carotenoids can be promoted (Venkateshwaran et al., 2015). Thus, citrate is considered the precursor of acetyl-CoA for fatty acid and astaxanthin synthesis (Chavez-Cabrera et al., 2010; Ratledge & Wynn, 2002). In the cytoplasm, oxaloacetate can be converted into malic acid by malate dehydrogenase (NADP+) used for pyruvate and NADPH production (Chen et al., 2009). Pyruvate can be converted into acetyl-CoA through the pyruvate dehydrogenase complex, and the NADPH can be used for fatty acid synthesis. Exogenous Na-citrate may provide more acetyl-CoA by cleaving citrate to produce acetyl-CoA and reduce the consumption of acetyl-CoA by the TCA cycle, promoting astaxanthin biosynthesis.
The TCA cycle is associated with the production of reactive oxygen species (ROS), which can stimulate the massive accumulation of astaxanthin in X. dendrorhous (Du et al., 2019; Zhang et al., 2019). The TCA cycle was inhibited by the addition of Na-citrate, and the inhibitory effect was weakened with the depletion of Na-citrate. These results indicated that the addition of Na-citrate could regulate the metabolic flux from the TCA cycle to carotenoid biosynthesis and regulate the citric acid-pyruvate cycle, which provides a large amount of substrate and energy for cell growth and astaxanthin accumulation and generates a large amount of ROS to enhance astaxanthin synthesis.
Influence of Na-citrate on amino acid metabolism in X. dendrorhous
Amino acid content affects protein synthesis, which is closely related to the growth and reproduction of yeast. As observed Table 2, the content of amino acids was significantly increased with Na-citrate treatment in addition to aspartic acid. The content of two amino acids, namely alanine and serine, which are derived from pyruvate, increased in the Na-citrate group at different time points (Table 3). The contents of four amino acids, namely L-5-oxoproline, leucine, L-phenylalanine, and valine derived from intermediates of the TCA cycle, also increased in the Na-citrate group. The addition of Na-citrate resulted in a significant decrease in intracellular aspartic acid content. Aspartic acid was closely related to the TCA cycle. It could be oxidatively deaminated to generate oxaloacetate since the addition of Na-citrate could regulate the TCA cycle, which was more active after 72 h. Thus, more aspartic acid needs to be converted into substances in the TCA cycle for supplementation, which reduces the content of aspartic acid.
At the same time, as shown in Table 3, compared with the control group, many amino acid metabolites were much higher in the Na-citrate group. It was strange that, the protein expressed (Fig. S3) by X. dendrorhous in the control group was shown higher than the Na-citrate group. It was indicated that the higher-level amino acids were not used to synthesize the specific protein but to response to the stress. This is just as Zhang et al. was reported that Mortierella alpina could adjust the metabolic state to rapidly change the pathways and adapt themselves to the new environments (Zhang et al., 2017). Furthermore, the amino acids showed higher abundance in Na-citrate cultures, which suggests that the protein synthesis is restricted in X. dendrorhous. Acetyl-CoA is a key intermediate used in both primary and secondary metabolic pathways. The astaxanthin and fatty acid biosyntheses need acetyl-CoA, ATP, and NADPH as substrates. So, substrates entered into these pathways must be regulated. The availability of acetyl-CoA, ATP, and NADPH may be key factors for switching the carbon flux from TCA-respiratory to astaxanthin biosynthesis during the restriction of protein synthesis in X. dendrorhous, increasing the accumulation of astaxanthin.
Influence of Na-citrate on fatty acid and sterol metabolism in X. dendrorhous
In X. dendrorhous, carotenoid biosynthesis is closely connected with fatty acid metabolism by the same precursor acetyl-CoA (Du et al., 2019; Zhang et al., 2019). The contents of seven fatty acids in the Na-citrate groups were higher than those in the control group (Tables 2 and 3). The content of oleic acid, linoleic acid, and linoelaidic acid (unsaturated fatty acids) showed a significant increase (1.6-16.9 folds) in Na-citrate treatment groups at different culture stages. The content of hexadecanoic acid increased by 38.2-fold at 72 h in the Na-citrate treatment group, which suggested that Na-citrate facilitated the accumulation of fatty acids in X. dendrorhous, and the increase in fatty acids, especially unsaturated fatty acids. Unsaturated fatty acids enhanced the fluidity and permeability of cell membranes (Los et al., 2013). We found that the fatty acids increased before 72 h and showed a decreasing trend after 72 h. In the Na-citrate treatment groups, the high biomass may cause insufficient nutrients in the medium. Therefore, it is necessary to use a part of fatty acids for energy supply. Fatty acids may be used as a carbon source to enrich the acetyl-CoA supply for carotenoid biosynthesis. Fatty acids were induced by Na-citrate to increase the fluidity and permeability of the cell membrane. The uptake of the substrate of glucose and nitrogen in the medium into the cell might be accelerated, and metabolism might be promoted.
Metabolomics analysis showed that the addition of Na-citrate significantly changed the content of sterols (Tables 2 and 3). The content of ergosterol and ergosta-7, 22-dien-3-ol in cells with Na-citrate treatment was 1.3 and 3.7-fold of that in the control group at 48 h. However, these two substances content decreased after 72 h. Ergosta-7, 22-dien-3-ol, and ergosterol play an important role in ensuring the integrity of the cell membrane, the activity of membrane-bound enzymes, membrane fluidity, cell viability, and cellular substance transport (Veen & Lang, 2004). Therefore, in the early stage of fermentation, high content of sterols can make cells better absorb nutrients in the medium, providing conditions for cell growth and accumulation of metabolites. Ergosterol can also compete with astaxanthin at the same time, as they have the same precursor named farnesyl pyrophosphate (FPP) (Misawa, 2011). Carotenoid and fatty acid syntheses share several common features with sterol synthesis, including the substrates of acetyl-CoA, ATP, and NADPH. Therefore, the content of ergosterol decreased in the later stage of fermentation culture. The content of ergosterol can regulate the expression of 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in the mevalonate pathway. A high content of ergosterol can inhibit the expression of HMGR and HMGS. Therefore, in the later phase, the addition of Na-citrate can lead to a significant downregulation of ergosterol, thereby releasing the inhibition of key rate-limiting enzymes in the mevalonate pathway and making the FPP in the metabolic pathway more directed towards the astaxanthin synthesis.
Influence of Na-citrate on ROS level
Astaxanthin is a scavenger of free radicals, a chain-breaking antioxidant, and a potent quencher of ROS, such as singlet oxygen, superoxide ion, and hydrogen peroxide (Alesci et al., 2015; Martinez-Cardenas et al., 2018). The presence of astaxanthin means a higher survival ability of the cells since it enhances the resistance of the cell to oxidative stress. Thus, the biosynthesis of astaxanthin serves as a survival strategy under oxidative stress for X. dendrorhous (Cuellar-Bermudez et al., 2015; Gessler et al., 2007). The ROS levels of both control and Na-citrate groups increased, peaked at 72 h, and then decreased to basal level at 120 h (Fig. 5). The production of ROS gradually increases with the enhanced metabolic activity of yeast cells. Subsequently, with the production of astaxanthin, which can scavenge ROS, the level of intracellular ROS is gradually reduced. As presented in Fig. 5, the ROS level was higher from 48 to 96 h in the Na-citrate group than in the control group, suggesting that Na-citrate induced ROS accumulation, increased redox signaling, and induced synthesis of astaxanthin in X. dendrorhous.
In addition, the content of myo-inositol, a carbohydrate metabolism intermediate, in response to environmental stress was significantly upregulated before 72 h (Tables 2 and 3). Myo-inositol is a growth factor for yeast and contributes to responses to environmental factors, such as oxygen and osmotic pressure in Aurantiochytrium sp. and Schizochytrium sp. strains (Jakobsen et al., 2007; Yu et al., 2016b). In the present study, adding Na-citrate could cause a significant increase in myo-inositol (2.1-3.9 folds) before 72 h, which might be a key stress indicator in response to Na-citrate treatment. Thus, further investigation is needed to determine the relationship between Na-citrate addition and myo-inositol metabolism.
Transcriptional responses of genes involved in astaxanthin biosynthesis
A real-time PCR technique was used to detect the gene expression of the astaxanthin biosynthesis pathway to explore the molecular mechanisms underlying the higher astaxanthin accumulation induced by Na-citrate. We analyzed six key genes, including ICL (encoding isocitrate lyase), HMGS (encoding HMG-CoA synthase), crtE (encoding GGPP synthase), crtYB (encoding phytoene synthase/lycopene cyclase), crtI (encoding phytoene dehydrogenase) and crtS (encoding astaxanthin synthase). As shown in Fig. 6, the transcription of these key genes was elevated by Na-citrate during the cultivation period. ICL is a key enzyme in the glyoxylate cycle to split isocitrate into glyoxylate and succinate. Glyoxylate is combined with a molecular of acetyl-CoA to form malate. Compared with the control group, transcription of ICL in the Na-citrate group was increased at 36 h (2.51-fold), 60 h (3.14-fold), and 84 h (2.15-fold), respectively, which indicates that cells can convert substances, such as fatty acids into carbohydrates, promoting cell growth and product accumulation. The increased HMGS transcription in Na-citrate treatment suggested that the mevalonate pathway was elevated with Na-citrate treatment. The enhanced transcript level of crtE, crtYB, crtI, and crtS encoding the key enzymes for controlling the biosynthesis of astaxanthin in Na-citrate treatment during the entire cultivation period indicated that astaxanthin biosynthesis in X. dendrorhous was strengthened by Na-citrate.
Possible regulatory mechanism for X. dendrorhous in response to Na-citrate treatment
Increased biomass and astaxanthin accumulation were observed in X. dendrorhous under Na-citrate treatment in this study. The comparison of the metabolites of the Na-citrate and control groups showed that metabolites content involved in the glycolysis pathway, amino acid metabolism, TCA cycle, and lipid and sterol biosyntheses were changed substantially in response to Na-citrate in X. dendrorhous. The metabolic mechanism of Na-citrate regulating cell growth and astaxanthin accumulation is observed in Fig. 7.
The addition of Na-citrate can promote the assimilation of glucose from the medium by cells. During the cultivation period, the consumption rate of intracellular glucose in the Na-citrate group was higher than that of the control group, indicating that the glycolysis flux was induced by Na-citrate so that intracellular glucose could generate more pyruvate. The increased glycolytic flux might suggest that more glucose went through the pentose phosphate pathway to supply the increased demand of NADPH required for lipid synthesis and ROS increase. In addition, the flux of pyruvate to ethanol and lactic acid through anaerobic fermentation was weakened, allowing more pyruvate to be converted to acetyl-CoA for astaxanthin synthesis. Acetyl-CoA has several metabolic pathways. It can participate in the TCA cycle, and astaxanthin, fatty acid, protein and sterol syntheses. In the current study, the addition of Na-citrate increased the content of intracellular citric acid, and the concentration of citric acid in the TCA cycle increased, inhibiting the catalytic activity of citrate synthase and weakening the reaction rate of oxaloacetate synthesis of citric acid, while the remaining Na-citrate in the mitochondria entered the cytoplasm and was cleaved into acetyl-CoA. The significant increase in ICL transcription also suggested that the cytoplasm acetyl-CoA was increased, thereby providing a large number of substrates for the production of astaxanthin in X. dendrorhous.
Intracellular ROS was significantly increased with Na-citrate treatment in our study. The accumulated astaxanthin increases the resistance of X. dendrorhous to Na-citrate stress by removing ROS species due to its strong antioxidant activity, which increases redox signaling and induces the synthesis of astaxanthin in X. dendrorhous. Furthermore, the supply of Na-citrate significantly upregulated the expression of the other five key genes involved in carotenogenesis. Astaxanthin is synthesized in X. dendrorhous via the mevalonate pathway, in which HMGS is a rate-limiting enzyme. HMGS catalyzes the formation of HMG-CoA. In the present study, the significant increase in HMGS transcription suggested that the mevalonate pathway was increased, which is consistent with the enhancement of astaxanthin accumulation in X. dendrorhous.