Strains and plasmids
Monascus purpureus strain HJ11 was isolated from rice wine starter and preserved in our laboratory. Escherichia coli BL21 (DE3) was used as gene expression host. M. purpureus HJ11 strain was co-incubated with Agrobacterium tumefaciens EHA105, containing recombinant pCAMBIA3300, to obtain deletion mutants. pCAMBIA3300 vector was used as deletion vector and pET30a(+) was used as heterologous gene expression in E. coli BL21 (DE3).
Flask cultivation
M. purpureus strains were pre-cultured in 10 mL of glucose mineral salt (GM) medium at 30 oC, 120 rpm for 5 days to obtain the seed culture (GM medium in g/L: glucose 30, (NH4)2SO4 5, KH2PO4 5, Na2HPO4 3, MgSO4 0.1, CaCl2 0.1, ZnSO4•7H2O 0.1, FeSO4•7H2O 0.1, CoSO4•7H2O 0.05, CuSO4•5H2O 0.02, and MnSO4•H2O 0.01). The seed culture was inoculated in 50 mL of GM medium in 250-mL flasks and incubated under same condition for 10 days. When needed, sterile cAMP powder was added to culture medium. The sterile AMP was prepared as following: the cAMP powder (Sigma, Shanghai, China) was dissolved in dd H2O to 25 mM, and filtered with sterile 0.22 μm membrane filter for removing microorganism, and then the resulting solution was lyophilized to obtain sterile cAMP powder. The collected mycelia were dried to a constant weight at 80 °C to determine DCW.
The fermentation in rice medium was performed as following: 20 g rice (pre-immersed for 2 h in water at 30 oC), and 50 mL water were put into a 250-mL Erlenmeyer flask, and mixed well. After sterilization at 121 oC for 20 min, 3 mL spore solution (2Í105/mL) was inoculated, and then incubated at 30 oC for 5-7 days. The spore solution was prepared by washing the mycelium, pre-cultivated on agar for 7 days, with Tween-80 solution (0.5 %). The fermented rice was dried to a constant weight at 80 oC and analyzed for pigments.
Heterologous expression and purification of MrPDE
RNA extraction and cDNA synthesis were performed according to our previous study [29]. The mrPDE gene fragment, Monpu1|448456|e_gw1.142.20.1 (ID number of the JGI database), was cloned from cDNA with specificd esigned primers using PCR reaction. The mrPDE DNA fragment and vector pET30a(+) were digested by endonucleases EcoRI and HindIII, respectively. The recombinant vector pET30a(+)-mrPDE was constructed with T4 DNA ligase and used for expression of mrPDE. All sequences were confirmed by DNA sequencing. The recombinant vector pET30a(+)-mrPDE was transformed into E. coli BL21(DE3) for expression. The resultant E. coli strain was inoculated in 100 mL LB medium, containing 50 μg/mL kanamycin, in 500-mL flask and incubated at 37 oC and 200 rpm. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the medium at a final concentration of 0.5 mM, when the optical density of culture reached 0.6-1.0 at 600 nm. Then, the culture was incubated at 18 oC and 200 rpm for 16 h. The His-tagged mrPDE protein was purified using Nickel-NTA Agarose (Qiagen, Valencia, CA, USA), as described in our previous study and was examined on 12% (w/v) SDS-PAGE gel [30]. Protein concentration was estimated by the Bradford method using bovine serum albumin as standard.
PDE activity detection
Phosphodiesterase activity was determined using a PDE activity assay kit (Colorimetric) following the manufacturer’s instructions (Abcam, Cambridge, United Kingdom) with slight modification [31]. The PDE activities were determined in purified mrPDE and recombinant E. coli lysates. The lysates were obtained by disrupting the cells using sonication (W140D, Heat System-Ultrasonics, Inc., NY), followed by centrifugation at 12,000 × g for 10 min to remove cell debris. One unit of PDE activity for 3’5’-cAMP was defined as the amount of enzyme required to release 1.0 μmol 5’-AMP from 3’5’-cAMP per minute at 30 oC.
PKA activity determination
Suspended cells were lysed by a high-pressure homogenizer. Cell lysates were incubated at 30 °C as specified by the protocol of PKA (Protein Kinase A) Colorimetric Activity Kit (Thermo Fisher Scientific). The PKA kinase activity was measured by the colorimetric method at 450 nm on a spectrophotometer. The PKA kinase activity was calculated using a calibration curve, which was constructed using five PKA standard solutions.
qRT-PCR
RNA extraction, cDNA synthesis, and qRT-PCR were performed as previous work described [29]. Briefly, total RNA was extracted from mycelial samples, and then checked for purity and integrity. cDNA synthesis was performed using the PrimeScript RT master mix (TaKaRa) according to the manufacturer instruction. qRT-PCR was conducted with a two-step thermal producure (step 1, 95°C for 10 s, and step 2, 40 cycles of 95°C for 3 s and 60°C for 25 s) on a 7500 Fast real-time PCR system (Applied Biosystems). The cycle number was used for the quantitation of the expression level. Relative expression level of target cDNA was obtained by the 2-ΔΔCT method via normalization to β-actin (GenBank accession no. AJ417880).
cAMP concentration measurement
The cAMP concentration was determined according to the previous work with modification [32]. M. purpureus HJ11 strains were cultivated in GM medium at 30oC. Fresh mycelia were harvested, frozen and lyophilized and ground into powder using liquid nitrogen. The powder sample was kept in chilled 6% (w/v) trichloro acetic acid (TCA) solution and incubated for for 20 min. After centrifugation of the mixture at 3500 × g at 4 oC for 20 min, the supernatant was extracted 3 times with 10 volumes of diethyl ether to remove TCA residues. The resulting extract was dried before further analysis. The cAMP levels were quantified using cAMP Enzyme Immunoassay Kit, Direct (Sigma-Aldrich, St. Louis, MO) following the manufacturer's instructions. In total, assay was repeated three times independently with three biological replicates for each strain.
NADPH/NADP+ ratio determination
M. purpureus HJ11 strains were cultivated in GM medium at 30 oC. Mycelia were collected after centrifugation and washed in PBS (50 mM, pH 7.0). Mycelia cells were lysed in base solution containing 1% (w/v) dodecyl (trimethyl)azanium bromide. Then, NADP+ and NADPH were individually detected according to NADP/NADPH-Glo™ Assay (Promega, Southampton, UK) following the manufacturer’s instructions.
Batch and fed-batch fermentation
The batch fermentation medium was as follows (g/L): glucose 100, (NH4)2SO4 30, KH2PO4 10, Na2HPO4 5, MgSO4 0.5, CaCl2 0.5, ZnSO4•7H2O 0.3, FeSO4•7H2O 0.3, CoSO4•7H2O 0.2, CuSO4•5H2O 0.2, and MnSO4•H2O 0.1; pH 4.0. The seed culture was prepared by inoculating 10 mL of 5-days preculture into 100 mL of GM medium in a 500-mL flask and incubated at 30 oC and 120 rpm for 7 days. The seed culture (300 mL) was transferred to a 10-L stirred tank bioreactor (Baoxing Bioengineering Equipment Co. Ltd, Shanghai, China) containing 6 L of fermentation medium. Fermentation was performed at 30 oC with an agitation speed of 50 to 200 rpm and airflow rate of 1 to 2 vvm. The pH was maintained at 4.0 by automatic addition of 1.0 M NaOH solution.
Similarly, fed-batch fermentation medium was as follows (g/L): glucose 100, (NH4)2SO4 50, KH2PO4 30, Na2HPO4 10, MgSO4 1.0, CaCl2 1.0, ZnSO4•7H2O 1.0, FeSO4•7H2O 0.5, CoSO4•7H2O 0.5, CuSO4•5H2O 0.3, and MnSO4•H2O 0.1. The seed culture and inoculation were performed similar to the batch fermentation, in a 10-L stirredtank bioreactor (Baoxing Bioengineering Equipment Co. Ltd, Shanghai, China) containing 6 L fermentation media. Fermentation was conducted at 30 oC with an agitation speed of 300 to 800 rpm and an airflow rate of 1.5 to 2.5 vvm. pH was controlled at 4.0 by automatic addition of NaOH solution. After 4 days of fermentation, the residual glucose in medium was determined. When the residual glucose was below 5 g/L, a certain amount (about 300 mL) of feed medium, containing 500 g/L glucose, was added to the 10-L stirred tank bioreactor to maintain glucose concentration at 30 g/L in the fermentation medium, and same amount of culture was withdrawn to analyze residual glucose concentration, biomass and MonAzPs concentration.
MonAzPs analysis
Intercellular MonAzPs concentration was estimated following our previous method [29]. In this study, the concentration of MonAzPs was measured using UV-1700 spectrophotometer (Shimadzu, Tokyo, Japan) at specific wavelength of 410, 470, and 510 nm that corresponded to the characteristic absorbance of yellow, orange and red pigments, respectively. Total MonAzPs was calculated as the sum of yellow, orange, and red pigments. Statistical analysis was performed using Student's t-test [29].
Gene knockout in M. purpureus HJ11
Targeted gene knockout and complementation of mrPDE in M. purpureus HJ11 was performed as described in our previous study [29].
Data availability
Sequences of the genes mentioned in this article are available in GenBank [29].