Microorganism and culture conditions
S. spinosa strain 1733 was used in this study. The solid medium containing 10 g glucose, 1 g beef extract, 5 g yeast extract, 3 g tryptone, 2 g MgSO4, 20 g agar per liter of distilled water (pH was adjusted to 7.4 with 1 N NaOH) was used for plate culture at 28 ℃. The fermentation experiment was carried out as follows: A seed culture was prepared by inoculating S. spinosa into a 250 mL Erlenmeyer flask containing 50 mL of seed medium (glucose 5 g/L, beef extract 1 g/L, yeast extract 5 g/L, tryptone 5 g/L, MgSO4 2 g/L and pH 7.4), which was incubated for 3 d at 28 ℃ with constant shaking at 180 rpm. Next, 8 mL of the seed culture was transferred into the 40ml fermentation medium (glucose 6 g/L, yeast extract 6 g/L, corn steep powder 10 g/L, cake powder 15 g/L, cottonseed meal 10 g/L, FeSO4 0.05 g/L, soybean oil 6 g/L, CaCO3 5 g/L and pH 7.4). The culture (fermentation) was carried out at 28°C and 180 rpm for 10 d. The fermentation medium was supplemented with different concentrations of phosphates (NaH2PO4, Na2HPO4, KH2PO4, and K2HPO4) at different time points to study the effects of phosphates on spinosad production. Each experiment was carried out in triplicates under identical conditions, and standard deviations were calculated.
Growth and spinosad estimation
4 mL of fermentation broth was centrifuged at 4000 rpm for 10 min; the obtained mycelium was re-suspended in 6 mL of 0.1 M HCl with vigorous stirring. Next, this solution was centrifuged at 3000 rpm for 10 min, and dry cell weight (DCW) was measured after drying to a constant weight at 65 ℃. For spinosad extraction, fermentation cultures were mixed with 4× volume of ethanol (28). Then, the mixture was sonicated > 1 h at RT and filtered through a 0.22 µm micro-membrane for HPLC (Waters Symmetry C18 column, 4.6×250 mm, 5 µm) analysis at 254 nm. 10 µL mixtures were separated using buffer A (45% methanol, 45% acetonitrile, 10% 260 mM ammonium acetate) as a mobile phase at a flow rate of 1.0 mL/min. Spinosad was quantified using a regression line generated from a commercially available standard (Nongle Bioproduct Co., Ltd OF Shanghai) dissolved in methanol. Each treatment was performed in triplicates to calculate the standard deviations.
Determination of extra- and intracellular phosphate concentration
1 mL of cell cultures were chilled on ice immediately after collection and then centrifuged at 4000 rpm for 10 min to obtain 700 µL supernatant and 0.1 g cell pellet. The supernatant was filtered by a 0.22 µm aqueous phase filter and stored at − 80°C until used for extracellular phosphate content estimation. The cell pellets were immediately frozen in liquid nitrogen and ground into powder. From these, the intracellular phosphate was extracted using 300 µL 0.2 mol/L HCl and centrifugation at 12000 rpm and 4 ℃ for 10 min. The collected supernatant was filtered by a 0.22 µm aqueous phase filter and the intra- and extracellular phosphate contents were determined using the phosphate content detection kit (Solebul Reagent, China), according to manufacturer’s instructions. The kit is based on the ammonium molybdate spectrophotometry method (22).
RNA extraction and cDNA synthesis
For transcriptional studies, 1 mL of fermentation broth, collected at 120, 144, 168, 192, 216, and 240 h, were centrifuged at 12000 rpm for 10 min to obtain the bacterial cell pellets. These were quickly frozen in liquid nitrogen and ground into powder. Then 0.2 g of bacterial dry powder was incubated with 1 mL Trizol reagent (Thermofid, USA) for 5 min. The mixture was centrifuged at 12000 rpm for 10 min to collect the supernatant, which was mixed with 0.2 mL chloroform. This mixture was centrifuged at 12000 rpm for 10 min and the obtained supernatant was mixed with 0.4 mL isopropanol. The mixture was centrifuged at 12000 rpm for 10 min and the collected precipitation was mixed with 1 mL 75% ethanol. Again, the supernatant was collected after centrifugation at 7500 rpm for 5 min. After drying at RT, the supernatant was dissolved in 50 µL RNase-free water and stored at -80℃ for later use. The concentration, purity, and integrity of RNA were determined by ultraviolet spectrophotometry and gel electrophoresis.
For cDNA synthesis, the extracted RNA (described above) was reverse transcribed using HiScript III 1st Strand cDNA Synthesis Kit (Novezan, China) in a total volume of 20 µl containing 2 µl of 10 × RT buffer, 2 µl HiScript III Enzyme Mix, 10 µl total RNA and 6 µl RNase-free H2O. After evenly mixing, the reactions were performed as follows: incubation for 15 min at 37℃, and then reaction termination by heating at 85 ℃ for 5 sec. The products of reverse transcription reaction were stored at − 80℃ until used.
Quantitative reverse transcriptase PCR (RT-qPCR)
Triplicate independent RT-qPCR assays were performed using the LightCycler 480 (Roche Diagnostics GmbH, Germany). ACEQ Universal SYBR qPCR Master Mix Kit (Novizan, China) was used to monitor the real-time amplification of the qPCR product following the manufacturer’s protocol. The reaction was performed in 20 µl containing 10 µl of 2× qPCR Master Mix, 0.4 µl each primer, 1 µl cDNA and 8.2 µl RNase-free ddH2O. After gentle mixing, the PCR reactions were performed as follows: initial denaturation at 95 ℃ for 5 min; 40 cycles of 10 s denaturation at 95 ℃; 30 s annealing, and final extension at 72 ℃. The target genes spnA, spnJ, spnG, spnO, spnP, gtt, fadA, fadE, fadG and fadI were amplified accordingly. The used primer sequences are listed in Table 1 (23). CT values of the target genes were normalized to 16S rRNA of S. spinosa, and the results were analyzed using the 2 − CT method.
Extraction and HPLC analysis of intracellular acetyl-CoA
Intracellular acetyl-CoA were extracted as described previously with slight modifications (2). Briefly, 1 mL cell cultures were collected at different fermentation stages and centrifuged at 12000 rpm for 10 min to collect the bacterial pellets. These were quickly frozen in liquid nitrogen and ground into powder. 0.2 g of precisely weighed dry powder was stored at -80 ℃ for analysis. 1 mL of 10% (m/v) precooled trichloroacetic acid was added to the dry powder, swirled for 30 s, and then centrifuged at 12,000 rpm at 4℃ for 10 min. The collected supernatant was filtered by a 0.22 µm aqueous phase filter for analysis. The standard for analysis was prepared by dissolving 1.0 mM acetyl-CoA in 100 mM sodium acetate (pH 4.0), which was stored at -80 ℃ until use.
HPLC method was employed to estimate the acetyl-CoA content as described previously with slight modifications (5). The samples and standards were filtered through 0.22 µm micro-membrane filters and analyzed using HPLC (Plastisil ODS C18 column, 4.6×250 mm, 5 µm) at 254 nm. Buffers A (0.2 M sodium dihydrogen phosphate) and B (0.25 M sodium dihydrogen phosphate and 20% acetonitrile) were used as the mobile phase solvents. 10 µL samples were separated on the HPLC column with a flow rate of 1.0 mL/min. The analytes were eluted with a gradient as follows: 90% buffer A (10% buffer B) for 5 min; 90–60% buffer A (40% buffer B) in 15 min, and then 60–50% buffer A (50% buffer B) in 20 min. Standards of acetyl-CoA were used to find its retention time (17.5 min).
Statistical analyses were performed using GraphPad Prism version 8, t-test Statistical significance was accepted for p < 0.05. All experiments were conducted in triplicates. Data are presented as mean values with standard deviations (SD).