2.1 Bacterial strains, primers and growth conditions
The B. subtilis SCK6 strain was provided by Daniel R. Zeigler from the Bacillus Genetic Stock Center. Oligonucleotide synthesis (Table 1) and DNA sequencing were performed by Sangon Biotech Co., Ltd. (Shanghai, China). B. subtilis strains were cultivated in Lysogeny Broth (LB) (Bertani et al. 2004) medium, YN medium (Li et al. 2017) or 2×Super-Rich (2×SR) (Song et al. 2017) medium. LB medium consists of 1% tryptone, 0.5% yeast extract and 0.5% NaCl. YN medium is composed of 0.7% yeast extract and 1.8% nutrient broth. 2×SR medium (3% tryptone, 5% yeast extract and 0.6% K2HPO4, pH 7.2) was used for fermentation. Solid medium was obtained by adding 15 g/L agar to the liquid medium. Unless otherwise indicated, the final concentrations of antibiotics were as follows (mg/L): zeocin (Zeo), 20; erythromycin (Em), 5. The inoculums (1%) were transferred into 250 mL flasks containing 30 mL 2×SR medium, and incubated at 37 ℃ with shaking at 200 rpm. Chlorpyrifos (>99%) was purchased from the Macklin Biochemical Technology Co., Ltd. (Shanghai, China).
2.2 Construction of Methyl Parathion Hydrolase (MPH) secretion strain
Four DNA fragments of Pcry3A promoter (Adams et al. 1994), the coding region of signal peptide (SPaprE), MPH-encoding gene mpd (Zhang et al. 2005), and T1T2 transcription terminator (Hartl et al. 2001) were fused by overlap PCR (Shevchuk et al. 2004) using the primers listed in Table 1, generating MPH expression cassette Pcry3A -mpd . The expression cassette was then fused with flanking regions of amyE and Zeo resistant marker (ZeoR), producing insertion construct. Insertion construct was transformed into the competent cells of strain SCK6 and selected by Zeo.Pcry3A -mpd was finally integrated at the locus of amyE of strain SCK6, resulting in strain BPC1.