Improvement in MonAzPs production and mrPDE expression by exogenous cAMP addition
The effect of exogenous cAMP on M. purpureus HJ11 was evaluated. The MonAzPs production was determined under different concentrations (0, 1.0, 2.0, 3.0 and 4.0 mM) of cAMP in GM medium. In the presence of exogenous cAMP, the strain showed higher MonAzPs yield (Fig. 2). At 2.0 mM of cAMP concentration, maximum MonAzPs yield of 6065 U/g DCW was achieved, compared to the yield of 2606 U/g DCW without cAMP (Fig. 2A). This was consistent with a previous study, in which addition of 1.0 mM exogenous cAMP promoted MonAzPs yield in M. ruber strain M7 [24]. In Fusarium graminearum, mycotoxin deoxynivalenol production was approximately increased by 40-folds in cultures treated with 4 mM cAMP [23]. The yields of red, orange, and yellow MonAzPs were 1812 U/g DCW, 1787 U/g DCW and 2466 U/g DCW, respectively (Fig. 2B, 2C and 2D). Among them, maximum rate of increase (179%) was observed in the yield of yellow MonAzPs (Fig. 2D). These results indicated that the cAMP concentration played an important role in the growth and development of fungi and biosynthesis of secondary metabolites. Moreover, it was ascertained that the MonAzPs yield could be improved by increasing the concentration of cAMP.
When the concentration of cAMP was 2.0 mM, maximum dry cell weight (DCW) of 4.6 g/L was achieved, while maximum DCW without cAMP was only 4.2 g/L (Fig. 2E). Similar results were found in M. ruber strain M7 [24]. We evaluated the effects of exogenous cAMP on the transcription of genes relative in the cAMP-PKA pathway by quantitative reverse transcription-PCR (qRT-PCR) analysis. There were no marked changes in the expression levels of mrpigA and AC genes. The expression of global regulator gene laeA, which can regulate the MonAzPs biosynthetic gene cluster [14], was a little increased. The MonAzPs polyketide synthase gene mrpigA, which was responsible for MonAzPs precursor synthesis [29], showed an obvious increase in expression, explaining the improved production of MonAzPs. Unexpectedly, incubation with cAMP led to a strong induction of expression of a hypothetical PDE gene, named as mrPDE (Fig. 2F and S1). So, we speculated that this gene was responsible for negative regulation of cAMP in M. purpureus HJ11.
Identification Of Mrpde As A Camp Phosphodiesterase
To the best of our knowledge, there was no report about PDE gene in Monascus spp. It was imperative to identify the PDE gene in M. purpureus HJ11. Comparison of the amino acid sequence of MrPDE with known sequences in the NCBI protein databases showed that a hypothetical PDE gene, XP_001264269.1 from A. fischeri, displayed the highest similarity of 64% with MrPDE. To further analyze MrPDE, several known PDEs (4OJV_A from S. cerevisiae, Q5AGE4 from Candida albicans, P12019 from Dictyostelium discoideum, and P36599 from Schizosaccharomyces pombe) were selected. A multiple alignment of 6 sequences mentioned above was also performed. The alignment analysis revealed two highly conserved amino acid motifs, which contained catalytic residues (Fig. 3A). This result indicated that MrPDE may be a fungal PDE.
To verify the function of MrPDE, the enzymatic activity was evaluated through in vitro reaction. MrPDE was heterologously expressed in E. coli BL21(DE3) strain. After induction with 0.5 mM IPTG at 18 oC for 12 h, SDS-PAGE analysis showed that MrPDE was successfully expressed in E. coli under the control of the T7 promoter (Fig. 3B, S lane). The enzyme was purified using Ni affinity chromatography column (Fig. 3B, P lane). The catalytic activity of MrPDE was determined with 3',5'-cAMP as substrate. MrPDE was found to efficiently catalyze the hydrolysis of 3',5'-cAMP (Fig. 3C). The specific activity of purified MrPDE was 15.4 U/mg, which was close to that of PDE1 (20.5 U/mg) from S. cerevisiae S288C [33]. These results indicated that MrPDE was indeed a fungal PDE.
Activation of cAMP signalling pathway by mrPDE knockout
To construct a MonAzPs high-producing strain, a mrPDE gene knockout strain ΔmrPDE and a mrPDE complemented knockout strain CΔmrPDE were successfully engineered through homologous recombination technology. The colony diameter of ΔmrPDE was a little bigger than those of WT and CΔmrPDE, which indicated that the growth of ΔmrPDE strain was not inhibited by mrPDE gene knockout (Fig. 4A). Meanwhile, the morphology of ΔmrPDE colonies grown on GM plate showed more intense color than those of WT and CΔmrPDE, implying that the ΔmrPDE strain might have a high production on MonAzPs (Fig. 4A).
The cAMP concentration was determined in each strain at different times. Notably, the cAMP concentration in ΔmrPDE strain was significantly higher than those of WT and CΔmrPDE strains, attaining a maximum of 11145 pmol/g on sixth day (Fig. 4B). The cAMP concentration in WT strain increased to 3219 pmol/g at the fourth day before gradually decreasing (Fig. 4B). Similar cAMP trend was observed in CΔmrPDE strain. This result suggested that knockout of mrPDE could inhibit the cAMP degradation, which led to a higher intracellular cAMP concentration. It has been reported that deletion of PDE gene in Ustilaginoidea virens and Magnaporthe oryzae both resulted in double-fold increase in cAMP concentration [21, 34]. Thus, it was ascertained that deletion of PDE gene could efficiently increase cAMP concentration in M. purpureus HJ11.
In cAMP signalling pathway, PKA activity is essential for regulation of primary and secondary metabolism [35, 36]. Our data showed that knockout of mrPDE could induce PKA kinase activity (Fig. 4C), suggesting the increase of cAMP concentration triggered PKA activation [37]. The DCW of ΔmrPDE strain reached 5.1 g/L, which was higher than those of WT (4.2 g/L) and CΔmrPDE strains (4.2 g/L) (Fig. 4D). These results indicated that the cAMP signalling pathway was activated by knockout of mrPDE.
Improvement of MonAzPs yield in Δ mrPDE strain
For further confirmation of the MonAzPs production in ΔmrPDE strain, shake-flask fermentation was performed. After fermentation for 10 days, the MonAzPs yield of ΔmrPDE strain reached 8563 U/g DCW, which was 2.3-fold higher than that of WT strain (Fig. 5). The yields of red, orange, and yellow pigments of ΔmrPDE strain were 2377 U/g DCW, 2245 U/g DCW, and 3941 U/g DCW, respectively (Fig. 5B, 5C and 5D). These yields were 1.58-times, 1.80-times, and 3.46-times higher than those of WT strain, respectively. Similar study has been reported in F. graminearum, the knockout of PDE gene pde1 resulted in increased production of secondary metabolite deoxynivalenol [23]. The deletion of PDE gene pdeH from A. flavus led to an increased production of aflatoxin to 110 mg/mL from 48 mg/mL [38].
To explain the reason of increasing MonAzPs yield, the expression of each genes from Monascus azaphilone pigments biosynthetic gene cluster (MPBGC) was determined by quantitative reverse transcription-PCR (qRT-PCR) analysis. All MPBGC genes were expressed in the WT and ΔmrPDE strains. Unexpectedly, as shown by the ΔmrPDE strain, transcriptional activation of large parts of the genes in the MABGC was achieved, apart from mrpigH, mrpigI, mrpigL and mrpigP, which didn’t participate in MonAzP biosynthesis (Fig. S2) [29, 39]. The downstream targets of PKA include transcriptional regulators and other effectors to control gene transcriptional expression [40]. These suggested that PKA, activated by cAMP, improved the transcriptional expression of MPBGC genes through indirect activation [41].
It was worth noting that the increased proportion of yellow MonAzPs yield was significantly higher than that of total MonAzPs yield in ΔmrPDE strain (Figs. 2 and 5). In our previous study, orange MonAzPs were found to be converted into yellow MonAzPs in the presence of adequate NADPH [42]. So, it was speculated that the increased cAMP concentration led to higher rate of NADPH/NADP+. There is little knowledge about the influence of cAMP on NADPH/NADP+ rate in microorganism. Herein, it was found that the ratio in ΔmrPDE strain was 0.91, which are much higher than those of WT and CΔmrPDE strains (0.55 and 0.54, respectively) (Fig. S3). This might be the main reason for the significantly higher yield of yellow MonAzPs than those of red and orange MonAzPs.
High-density Fermentation For High Monazps Production
In order to enhance the production of MonAzPs, batch fermentation of ΔmrPDE strain was performed in a medium, containing initial glucose concentration of 100 g/L. After 10 days of cultivation, total MonAzPs production reached a maximum of 158.9 U/mL (Fig. 6A), and corresponding total MonAzPs yield was 6782 U/g (Fig. 6B). However, this yield was lower than that in shake flask (8563 U/g). This might be attributed to the insufficient glucose supply (< 5 g/L) in the middle and later stages of fermentation, where mycelia accumulated the main part of MonAzPs (Fig. 6A).
For improvement of glucose supply, fed-batch fermentation was also performed in a 10-L stirred fermenter [43]. The ΔmrPDE strain displayed rapid growth, and DCW reached 38.1 g/L, which was significantly higher than that of batch fermentation (Fig. 6C). In another study, fed-batch fermentation of Monascus anka strain GIM 3.592 achieved a DCW of 39.77 g/L [44]. Subsequently, a significant increase in MonAzPs production was observed, reaching a maximum of 332.1 U/mL. The total MonAzPs yield reached 8739 U/g DCW, slightly higher than that in shake flask fermentation (Fig. 6D). It has been reported that a mutant M. purpureus strain M183 produced MonAzPs at 211.6 U/mL [15]. To the best of our knowledge, this study achieved highest MonAzPs yield and production.