Materials and methods

Construction of Plasmids and Strain

The sequences of all the primers used in this study are listed in Table 1. The strains used in this study are listed in Table 2. To construct pHJ5, the vector pET28a was amplified with the primers pET28a-cotA-F and pET28a-cotA-R, and the 5369 bp PCR products were purified on a gel using the QIAquick gel extraction kit (Qiagen, Germany). The cotA gene (BBa_K4309000) was amplified from the genome of the wild-type Bacillus subtilis using primers cotA-pET28a-F and cotA-pET28a-R, which included BamHI and SacI restriction sites, respectively. The cotA gene fragment was cloned into the linearized plasmid pET28a using the ClonExpress II one step cloning kit (Vazyme, China), resulting in the plasmid pHJ5. Recombinant vector pHJ5 was transformed into BL21 (DE3) competent cells, resulting in the AE5 mutant strain.

The lipH8 gene (BBa_K4309001) from Phanerochaete chrysosporium was synthesized after gene optimization by Sangon Biotech (Shanghai, China) and cloned into the vector pET28a, resulting in the plasmid pHJ6. The relE gene (BBa_K4309002) from Escherichia coli was synthesized by Tsingke Biotech (Nanjing, China) and cloned into the vector pBAD43, resulting in the plasmid pHJ9. Recombinant vectors pHJ6 and pHJ9 were transformed into BL21 (DE3) competent cells, resulting in the AE6 and AE9 mutant strains, respectively.

Table 1 Primers used in this study

F, forword; R, reverse. Bold underlined letters indicate restriction sites.

Table 2 Strains used in this study

Growth and induction conditions

All mutant strain cells were first grown in LB medium (10 g/L trypton, 5 g/L yeast extract, 5 g/L NaCl) with corresponding antibiotics for 3-4 h. Afterwards, the cell suspension was diluted to 1:100 in LB medium with 0.1 mM IPTG. Cultivation for CotA and LipH8 expression analysis was conducted under different induction temperatures and times. Then, this preculture was washed once with MSM medium (5g/L NaCl, 1g/L NH4NO3, 1g/L K2HPO4, 1g/L KH2PO4, 0.2g/L MgSO4∙7H2O, 20 mg/L CaCl2∙2H2O, 5 mg/L FeSO4∙7H2O, 20 mg/L Yeast Extract) without carbon source and transferred into fresh minimal medium to an optical density at 600 nm (OD600) of 0.2. Then, the cell suspension was harvested by centrifugation at 6,000 g for 5 min. The harvested cell precipitation was used for protein expression analysis by SDS-PAGE.

Protein gel and SDS-PAGE analysis

30 mL of samples were harvested by centrifugation at 6,000 g for 5 min and resuspended in 5 mL of 40 mM Tris-HCl (pH 8.0) and 1 mM phenylmethylsulfonyl fluoride (PMSF) solution (Beyotime, China). The resuspended cells were broken using sonication cell disrupt (Scientz, China) for 10 min on ice (3 s on, 3 s off). The whole-cell lysate was centrifuged at 12,000 g for 10 min at 4 ℃ to remove the cell debris. Insoluble cellular material was removed by centrifugation at 12,000 g for 10 min at 4 °C to divide the supernatant and pellet fraction. The protein concentration was assayed with a BCA protein quantification kit (Solarbio, China). Five micrograms protein with 1X SDS-PAGE sample loading buffer (Baygene, China) per sample were separated by 10% SDS-PAGE and the gel was either stained with 0.1% Coomassie brilliant blue R250 solution in 25% (v/v) isopropanol and 10% (v/v) glacial acetic acid.

Oxidation of PAHs by recombinant CotA and LipH8

Typical PAH substrates of laccase and lignin peroxidase, phenanthrene were used to investigate PAH oxidation under different conditions, including (1) pET28a only, (2) recombinant CotA only, (3) recombinant LipH8 only, and (4) a combination of recombinant CotA and recombinant LipH8 (0.5:0.5, v/v). For PAHs oxidation analysis, cells were grown in LB medium with 0.1 mM IPTG for 20 h at 16 and 20 ℃ to induce protein CotA and LipH8 expression, respectively. Then, this preculture was washed once with MSM medium without carbon source and transferred into fresh minimal medium containing 10 mg/L phenanthrene to an optical density at 600 nm (OD600) of 0.2. The PAH oxidation reactions were performed in 50 mL Erlenmeyer flasks containing 20 mL of MSM medium with 10 mg/L phenanthrene. The reaction Erlenmeyer flasks were incubated at 20 ℃. After the incubation, the reaction was terminated by adding 50% acetonitrile to a final concentration of 5 mg/L. An aliquot (1 mL) of each sample was centrifuged at 10,000 g for 10 min, and the supernatant was analyzed by high performance liquid chromatography (#LC100, Wufeng Instrument, China). The control strain with pET28a served as the control, and all treatments including the controls were carried out in triplicate.

Statistical analysis

Prism v8.0 (GraphPad Software, USA) was used to process the data, which is the mean ± standard deviation (SD) of three replicates. Statistical differences between the control and treatment groups were determined by one-way ANOVA followed by Dunnett’s test. P < 0.05 indicated statistically significant differences between treatment means and were represented by one asterisk.