Effects of different SAM decarboxylase genes on spermidine production
SAM can be catalyzed to decarboxylated SAM (dcSAM) by SAM decarboxylase, which is a key step for spermidine synthesis. Therefore, efficient SAM decarboxylase genes are believed to enhance the spermidine production. Previously, we constructed a high SAM-producing strain B. amyloliquefaciens HSAM2 , which might provide abundant SAM substrate for spermidine synthesis. Therefore, the HSAM2 was used as the host strain to express SAM decarboxylase genes (speD) from different microorganisms. The speD genes from B. amyloliquefaciens HZ-12, S. cerevisiae CICC 31001 and E. coli DH5α were selected to construct recombinant expression strains, named HSAM2(PHspeD), HSAM2(PSspeD) and HSAM2(PDspeD) respectively. After expressing these genes, the spermidine titers were improved significantly in all recombinant strains (Fig.2a). Among them, the maximum spermidine titer of 91.91 mg/L was obtained in HSAM2(PDspeD), increasing by 8.49-fold compared with the control strain HSAM2(pHY300PLK). These results indicated that the speD gene from E. coli DH5α was the optimal gene for enhanced spermidine production.
Subsequently, the speD gene of E. coli was integrated into the genome of HASM2 by homologous recombination, resulting in the integrated expression strain HSAM2ːːDspeD. After fermentation for 60 h, the spermidine titer reached 33.87 mg/L, which was 2.40-fold higher than that of the control strain HSAM2 (Fig.2b). In comparison, the spermidine titer of the integrated expression strain HSAM2ːːDspeD was much lower than that of plasmid-based expression strain HSAM2(PDspeD). This phenomenon was probably due to the low gene copy number during integration expression, while the pHY300PLK plasmid had the high copy number . Therefore, recombinant plasmid expression was more suitable for the speD gene.
Effects of different spermidine synthase genes on spermidine production
Spermidine synthase catalyzes the transfer of the aminopropyl group from dcSAM to putrescine to form the spermidine , which is considered to be a rate-limiting step in biosynthesis of spermidine . It is particularly essential to exploit efficient spermidine synthase genes (speE). Therefore, different speE genes were evaluated. According to the KEGG database, three spermidine synthase genes from E. coli DH5α, C. glutamicum ATCC13032 and S. cerevisiae CICC31001 were selected and expressed in B. amyloliquefaciens HZMD, resulting in recombinant strains HZMD(PDspeE), HZMD(PGspeE), and HZMD(PSspeE), respectively.
As shown in Fig.3, expressing speE genes from E. coli and S. cerevisiae significantly improved the spermidine production, while the gene from C. glutamicum showed no significant difference. Therein, the maximum spermidine titer reached 49.58 mg/L in HZMD(PSspeE), with a 23% increase than that of the control strain HZMD(pHY300PLK). Previously, overexpression of the native speE gene was confirmed to be efficient for spermidine synthesis in S. cerevisiae. Herein, our results demonstrated that the speE gene from S. cerevisiae also enhanced the spermidine production in B. amyloliquefaciens. It can be inferred that overexpression of this speE gene presumably increased the enzymatic activity of spermidine synthase to promote the spermidine synthesis.
Effect of co-expressing speD and speE genes on spermidine production
Above results showed that genes of speD from E. coli DH5α and speE from S. cerevisiae CICC31001 were efficient to enhance the spermidine production. Therefore, these two genes were ligated into one pHY300PLK plasmid, co-expressed in HSAM2 to generate a recombinant strain HSAM2(PDspeD-SspeE). Then, the control strain HSAM2(pHY300PLK), single gene expression strain HSAM2(PDspeD), and co-expression strain HSAM2(PDspeD-SspeE) were compared after fermentation for 60 h. As shown in Figure 4, the spermidine titer of HSAM2(PDspeD-SspeE) reached 105.24 mg/L at 60 h, further improving by 15% compared with the HSAM2(PDspeD). It indicated that co-expression of speD and speE was effective to increase the spermidine titer, which was probably due to that more upstream substrates of SAM and putrescine were converted to form spermidine.
Optimize fermentation medium
To further improve the spermidine titer of the engineered HSAM2(PDspeD-SspeE), the key components of fermentation medium were optimized, including carbon sources, nitrogen sources and antibiotics. Carbon sources were important for cell growth and metabolites synthesis [32, 33]. Firstly, effects of carbon sources types on spermidine production were investigated. As shown in Fig. 5a, the maximum titer of spermidine was obtained using xylose as the carbon source. Furthermore, the concentration of xylose was optimized (Fig.5b). The maximum titer of spermidine reached 151.79 mg/L when the xylose concentration was 40 g/L, and no significant increase was observed at 60 g/L of xylose.
Corn pulp was a nutrition-rich nitrogen source, and effects of different corn pulp concentrations on spermidine production were investigated. As was indicated in Fig. 5c, the corn pulp concentration significantly affected the spermidine titer, and the maximum spermidine titer was obtained at 20 g/L. Several previous studies investigated the impact of antibiotics on the fermentation process [34-36]. Herein, different concentrations of tetracycline were added into medium. The increased tetracycline concentration resulted in the improved spermidine titer, and no further increase was noted when the tetracycline concentration reached 4 mg/mL (Fig.5d). These results indicated that adding tetracycline could improve spermidine production, which was probably due to that more plasmids could be maintained under the tetracycline stress.
The spermidine production process under the optimized fermentation midium
Under the optimized fermentation medium (40 g/L xylose, 10 g/L peptone, 20 g/L corn pulp, 2 g/L urea, 6.3 g/L (NH4)2SO4, 2.5 g/L NaCl, 3 g/L KH2PO4, and 4.2 g/L MgSO4·7H2O), the spermidine production process was investigated (Fig. 6). The spermidine was synthesized as the cell grew at the early stage of fermentation. The cell growth entered into the stationary phase at about 48 h, while the spermidine was further synthesized until 84 h, indicating that the spermidine synthesis was a partly growth-coupled process. At 84 h, the maximum spermidine titer reached 227.35 mg/L, which was currently the highest titer reported by microbial fermentation. SAM was the key precursor for spermidine synthesis [5, 37], and the SAM concentration was also measured to further understand the fermentation process. In the initial 24 h stage, the SAM was accumulated rapidly to reach the highest point, while the spermidine concentration did not have obvious improvement. When the fermentation time exceeded 24 h, the spermidine production showed a rapid increase accompanied by a sharp drop in the SAM concentration, indicating that the SAM was probably consumed to synthesize spermidine.