Screening chemical modulators and optimization of fermentation conditions

The effect of five selected chemical modulators on lipid accumulation in Schizochytrium sp. were studied (Additional file 1: Table S1). For the evaluation of their performance, lipid yield, GLCR and fatty acids content were used as indicators. After 3 days of fermentation, the addition of vorasidenib and terbinafine slightly promoted lipid accumulation (37.13 and 37.8 g/L vs 37.01 g/L), which is in line with our expectations, but the GLCR did not improve (Fig. 1). Interestingly, the addition of vorasidenib led to an increase in the proportion of PUFAs to 64.58%, in which the proportion of EPA was 1.24 times that of the control, but the addition of terbinafine had a limited effect on fatty acid content. In contrast, the addition of quinoxaline and hexaconazole negatively affected the lipid accumulation and GLCR, but the percentage of SFAs was increased by 17.32% and 15.27%, respectively. Unexpectedly, the lipase inhibitor orlistat produced different results: the lipid yield and GLCR were significantly higher than the control, and encouraged Schizochytrium sp. to accumulate more PUFAs (Additional file 1: Fig. S1).

Fig. 1
figure1

The influence of different chemical modulators on the lipid yield, GLCR (A) and fatty acids content (B) of Schizochytrium sp. HX-308

The screening results for the optimal concentration of orlistat are shown in Fig. 2. It was obvious that the lipid production and CDW of Schizochytrium sp. significantly increased with the increase of orlistat concentration (0–1000 mg/L). After 120 h of fermentation, 87.89 g/L lipid production and 138.83 g/L CDW were obtained at a concentration of 1000 mg/L orlistat, which were 1.34 and 1.25 times higher than that of the control, respectively. Furthermore, the addition of lipase inhibitors also had a significant impact on the fatty acid composition of Schizochytrium sp. In the concentration range of 0–5 mg/L, the proportion of SFA decreased with an increase in orlistat concentration. Moreover, the proportion of SFA gradually increased when the concentration of orlistat was higher than 5 mg/L, and the proportion of SFA accounted for 39.71% of the total fatty acids (TFAs) with 1000 mg/L orlistat. In addition, the promotion of lipid yield in Schizochytrium sp. gradually decreased when the concentration of orlistat exceeded 100 mg/L (Fig. 2A). It illustrates that the GLCR increased as the concentration of orlistat increased, and up to 22.69% at 1000 mg/L orlistat, which was 9.45% higher than that of the control. Not surprisingly, the addition of orlistat accelerated the consumption of glucose by Schizochytrium sp., and the total glucose consumption was 1.23 times higher than that of the control when orlistat concentration was 1000 mg/L (Fig. 2B).

Fig. 2
figure2

The effect of different lipase inhibitor concentration on the fermentation of Schizochytrium sp. HX-308. A Changes in Lipid yield, CDW, SFA, PUFA, GLCR and B glucose consumption under fermentation conditions in the presence of different concentrations of lipase inhibitors

The addition of chemical regulators at different time points may yield different results [22]. Therefore, 0 h (fermentation start), 12 h (exponential growth period), 24 h (late growth period), and 36 h (rapid lipid accumulation period) were chosen to add the lipase inhibitor with concentrations of 5 mg/L and 1000 mg/L. After 5 days of fermentation, the addition of orlistat at different stages resulted in different changes in lipid productivity (Fig. 3). In the 5 mg/L orlistat treatments, adding orlistat at 24 h yielded 69.24 g/L lipid production, which was 5.94% higher than the control. Additionally, the proportion of PUFAs increased to 68.41%, while that of the control group was only 58.8% (Additional file 1: Fig. S2). The lipid yield in the 1000 mg/L orlistat treatments decreased with the delay in the orlistat supplementation time, and a lipid yield of 87.89 g/L was obtained at 0 h, which was 34% higher than that of the control (Fig. 3A). It was speculated that after low concentrations of orlistat are metabolized by Schizochytrium sp., lipase will have a rapid consumption period of lipids, and adding inhibitors at 24 h can delay the rapid consumption of lipids to the later stages of fermentation. Recently, Chang et al. found that TAG lipase may be involved in the preferential hydrolysis of SFAs in the process of lipid turnover [23]. The changes in the ratio of fatty acids during the non-feeding fermentation process also support this conclusion (Additional file 1: Figs. S3, S4), which may explain the experimental phenomenon was observed. Therefore, adding 1000 mg/L lipase inhibitor at 0 h improved lipid accumulation, and adding 5 mg/L lipase inhibitor at 24 h resulted in a higher proportion of PUFAs.

Fig. 3
figure3

Effect of 5 mg/L and 1000 mg/L lipase inhibitor addition time for A Lipid, CDW and B total glucose consumption and C lipid productivity and D GLCR

Figure 3B shows that the addition of orlistat led to a significantly higher consumption of glucose by Schizochytrium sp. than the control. Moreover, as shown in Fig. 3C, the lipid productivity increased with culture time in the three groups, with the 1000 mg/L orlistat treatment reaching its maximum at 60–72 h, whereas the maximum lipid productivity of the control was at 72–84 h. Compared with the control, the orlistat treatments exhibited an overall higher lipid productivity during the entire fermentation period. Even at the end of fermentation, the lipid productivity reached 0.49 g/L/h in the 1000 mg/L orlistat treatment, which was about 1.3-fold higher than in the control. In addition, orlistat resulted in a change in GLCR, which increased by 21.88% compared with the control at a concentration of 1000 mg/L orlistat in the middle stage of fermentation (60–96 h). In a word, by adding the lipase inhibitor orlistat, the lipid productivity and GLCR of Schizochytrium sp. are improved, which greatly saves the substrate cost and time cost of producing microbial lipids (Table 1).

Table 1 Some methods to improve the productivity and GLCR of Thraustochytrids

Effects of lipase inhibitors on cellular antioxidant activity

It was observed that the addition of orlistat caused a significant increase of ROS in Schizochytrium sp. (Fig. 4A). In the 1000 mg/L orlistat group, the level of ROS in the cells of the 24 h was 6.1 times that of the control, and at 24–96 h, the ROS in the 1000 mg/L orlistat group was much higher than that of the control. When 5 mg/L orlistat was added after 24 h of fermentation, the ROS increased significantly at 48 h, reaching 3.3 times that of the control group, and then dropped rapidly. The T-AOC of Schizochytrium sp. under normal fermentation conditions gradually increased during the fermentation process, and reached the maximum at 96 h (Fig. 4B). Interestingly, the T-AOC of the 5 mg/L orlistat addition group also gradually increased, but the T-AOC level exceeded the control group at 72 h, and reached the maximum at 120 h. Meanwhile, the T-AOC of the 1000 mg/L orlistat added group was at a low level from 24 to 96 h, but increased rapidly at 120 h, and almost three times that of the control group. NADPH is an essential reducing power for fatty acid synthesis. It was observed that the NADPH level of the control group was always higher than the orlistat addition group from 24 to 120 h (Fig. 4C).

Fig. 4
figure4

Comparison of the ROS, T-AOC and NADPH content of Schizochytrium sp. HS-308 in the presence of orlistat and normal conditions

Dynamic expression of genes associated with the lipase inhibitor

During the oscillation, significant changes were detected in the expression of genes in the orlistat treatment groups, particularly, the transcription of genes responding to fatty acid metabolism at 72 h (Fig. 5). As shown in Fig. 5A, approximately 7900 genes from Schizochytrium sp. were included in the statistical analysis. Regarding DEGs, there were 170 and 145 upregulated genes, and 16 and 13 downregulated genes in the 5 mg/L and 1000 mg/L orlistat treatment groups compared with the control, respectively. Orlistat addition induced modulations of 264 significant DEGs, of which 80 showed overlap in the 5 mg/L and 1000 mg/L orlistat treatments. In contrast, 106 and 78 DEGs were expressed exclusively in the 5 mg/L and 1000 mg/L orlistat treatments, respectively (Fig. 5B), suggesting drastic metabolic reorganization to combat the stress of orlistat addition and enhance the chance of survival.

Fig. 5
figure5

A Volcano plots showing p-values (− log10) vs. feature ratio of (log2), 5 mg/L vs. Control and 1000 mg/L vs. Control. B Venn diagram showing the unique and overlapping differentially abundant features in different orlistat concentration. C Functional analysis (including KEGG pathways and GO classes for gene classification) of gene with significantly changed induced by orlistat under 5 mg/L and 1000 mg/L. D Analysis of the transcription level of key enzyme genes of Schizochytrium in orlistat and normal conditions at 72 h. The three subunits of the PKS gene, include PfaA, PfaB and PfaC; DGAT gene: diacylglycerol acyltransferase gene; SOD gene: superoxide dismutase gene; CAT gene: catalase gene; APX gene: ascorbate peroxidase gene

Under the two orlistat treatments, the average transcriptional level of ABC transporters that require ATP consumption increased by 3.85 and 4.35 times, respectively. At the same time, electron carrier activity and oxidoreductase activity-related gene transcription levels were also significantly upregulated (Fig. 5C). In addition, five key genes related to fatty acid synthesis and lipid accumulation (i.e., FAS, PfaA, PfaB, PfaC and DGAT), two key genes related to the accumulation and utilization of acetyl-CoA (i.e., ACC and ACL), two key genes related to NADPH supply (i.e., ME and G6PDH), one key gene involved in glycolysis pathway (i.e., PK), one gene related to TAG hydrolysis (i.e., TAGL), and three key genes related to the oxidative defense system (i.e., SOD, CAT and APX) were analyzed by the transcription levels at 72 h. Compared with normal culture, the transcription levels of PK and TAGL genes were significantly upregulated in the orlistat addition group. However, the presence of high concentrations of orlistat inhibited TAGL from functioning, thus leading to the accumulation of lipids. Interestingly, five genes related to fatty acid biosynthesis (FAS, PfaA, PfaB, PfaC and DGAT) and the two main genes produced by NADPH (G6PDH and ME) were no significant change, which also verified that the accumulation of lipids was originated from the inhibitory effect of orlistat on lipase.

Metabolite profiling of Schizochytrium sp. HX-308 with lipase inhibitor

Compared with the control treatment, glucose consumption increased over time in the 1000 mg/L orlistat treatment group, and the fructose, mannitol, and xylitol concentrations also increased (Fig. 6). In addition, the galactose and sorbitol concentrations increased. The changes in the fructose, mannitol, and galactose concentrations indicated that the addition of orlistat can accelerate the rate of glucose metabolism and change intracellular glucose metabolism to prevent and deal with the possible external adverse environment. Additionally, glycine concentration converted from 3-phosphoglycerate and alanine converted from pyruvate changed significantly compared with the control. The amino acid content remained high at 48 h and then declined in the later fermentation stages (Additional file 1: Fig. S5). The TCA cycle is the main source of ATP for lipid accumulation in Schizochytrium sp., and succinic acid is the main metabolite in the TCA cycle. It showed that the succinic acid content in the orlistat treatment groups was higher than that in the control group. The increase in the succinic acid content indicates that the carbon metabolic flow of the TCA cycle was active during the fermentation process, which implied that the addition of orlistat might cause the migration of the metabolic flow, resulting in a large amount of acetyl-CoA entering the TCA cycle to produce more ATP for the growth of Schizochytrium sp. and fatty acid accumulation, which was consistent with the transcriptome analysis. At the same time, the proline and 4-aminobutyric acid concentrations increased compared with the control, especially 4-aminobutyric acid, which was low before 48 h. Upon entering the lipid accumulation period, the concentration of 4-aminobutyric acid increased rapidly. The control reached its peak at 96 h, which was 1.75 times higher than the initial intracellular concentration, while the peak in the 1000 mg/L orlistat treatment group was earlier (Additional file 1: Fig. S5).

Fig. 6
figure6

Heat map of the significantly changed metabolites in Schizochytrium sp. HX-308 induced by orlistat under 5 mg/L and 1000 mg/L

The addition of orlistat reduced the phosphoric acid concentration in the metabolites of Schizochytrium sp., but the concentration of inositol increased significantly. When Schizochytrium sp. entered the period of lipid accumulation, the enhancement of the GABA metabolic pathway could supplement the deficiency of NADH and maintain intracellular reactive oxygen species levels, which is beneficial for lipid accumulation, while proline and myo-inositol played an important biological function for cells to adapt to environmental pressure. During the fermentation process, the phosphoric acid concentration also changed significantly.

In addition, the fermentation conditions of orlistat were tested in Thraustochytrid Aurantiochytrium. Unlike Schizochytrium sp., 5 mg/L orlistat obviously promoted Aurantiochytrium sp. to accumulate saturated fatty acids, while 1000 mg/L orlistat had no obvious effect on the fatty acid composition of Aurantiochytrium sp., which might be the different types of lipase in Aurantiochytrium sp. and Schizochytrium sp. (Additional file 1: Figs. S6, S7). After the 120 h fermentation, Aurantiochytrium sp. lipid productivity and GLCR increased by 11.18% and 4.59%, respectively (Additional file 1: Fig. S8). The result proved that the applicability of orlistat fermentation conditions for the production of microbial lipids by thraustochytrids.

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