Reagents and strain construction
Yeast extract and peptone were purchased from Oxoid (Hampshire, England). BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide) was purchased from Macklin (Shanghai, China). Other chemicals were purchased from the China National Pharmaceutical Group Corporation (Shanghai, China).
All strains and plasmids used in this study are listed in Table 1. The marker-free knockout and knock-in approach has been described in detail before[20, 21]. Briefly, the left flanking region (LF) (∼ 800 bp), target genes (IG), direct repeat (DR) sequence (∼ 500 bp), PC cassette (1900 bp), and right flanking (RF) region (∼ 800 bp) fragments were first amplified using appropriate primers (Table 2), and then fused using overlap-extension PCR in the order LF, DR, PC cassette, and RF or LF, IG, DR, PC cassette, and RF. The resulting purified PCR products LF-DR-PC-RF/ LF-IG-DR-PC-RF were used to transform corresponding competent cells and further selected on chloramphenicol-containing agar plates and MGY-Cl medium. Oligonucleotide primers were synthesized by GenScript (Nanjing, China). The transformation of Bacillus was carried out according to published protocols[22, 23]. B. subtilis BSFX022 was used as the parental strain to construct the strains BSFX023, BSFX024 and BSFX025.
Table 1 Bacterial strains and plasmids used in this study
Strain or plasmid
|
Characteristics
|
Source
|
Plasmids
|
|
|
pTPC
|
pMD19-T harboring the PC cassette
|
[21]
|
pBTE
|
pUC derivative containing cloned BTE
|
[12]
|
Strains
|
|
|
BSFX022
|
B. subtilis 168 derivative,
sfp+yhfl+P43-bte+, ΔfadE
|
[12]
|
BSFX023
|
BSFX022 derivative, Δpps
|
This work
|
BSFX024
|
BSFX023 derivative, Δpks
|
This work
|
BSFX025
|
BSFX024 derivative, insertion of bte under the control of the Pveg promoter, ΔackA
|
This work
|
168 (Pveg-GFP)
|
B. subtilis 168 derivative, insertion of Pveg-gfp cassette at the locus of amyE, ΔamyE
|
This work
|
168 (P43-GFP)
|
B. subtilis 168 derivative, insertion of P43-gfp cassette at the locus of amyE, ΔamyE
|
This work
|
Table 2 Primers used in this study
Primer
|
Sequence
|
PC-F
|
ATTTTTAAAGTATGTATACAAATGA
|
PC-R
|
TTATAAAAGCCAGTCATTAGGCCTA
|
pps-LF-F
|
TTTATTTGAAAGGGAAAGGCGATCC
|
pps-LF-R
|
AATGGCCTCTGTCCGCTAATCCGCTCGGATTCCCTCCAGTTCTCATAATA
|
pps-DR-F
|
TATTATGAGAACTGGAGGGAATCCGAGCGGATTAGCGGACAGAGGCCT
|
pps-DR-R
|
TCATTTGTATACATACTTTAAAAATAATGGCCTCTGTCCGCTAATCCGCT
|
pps-RF-F
|
TAGGCCTAATGACTGGCTTTTATAATTGAGCGAACATACTTATTCTTTAA
|
pps-RF-R
|
CAAGGTGCGCAGCCAGCCGGCTGGC
|
pks-LF-F
|
AGCGTATGTGATGCCAAGTATGGAG
|
pks-LF-R
|
AGAATCGCTTTTCACACTAGTGCCTAGCTTTATTGTAACAAGAAAAAAT
|
pks-DR-F
|
ATTTTTTTCTTGTTACAATAAAGCTAGGCACTAGTGTGAAAAGCGATTCT
|
pks-DR-R
|
TCATTTGTATACATACTTTAAAAATAAAATACTCTCAGAAAACAAATAC
|
pks-RF-F
|
TAGGCCTAATGACTGGCTTTTATAAATGCCAAAACAAATTGACCATGAA
|
pks-RF-R
|
TAATGAGAGTGTGTCAATGCGACTG
|
Pveg-bte-LF-F
|
TTTATGGCGGACAAAAAGGAACTGA
|
Pveg-bte-LF-F
|
AGTGTGATGCTGTGTAAGATAGATCGATTGACGCTCCTTTATACTCTGTA
|
Pveg-F
|
GATCTATCTTACACAGCATCACACT
|
Pveg-R
|
GTTTGTCCTCCTTATTAGTTAATCT
|
bte-F
|
AGATTAACTAATAAGGAGGACAAACATGGCTACAACATCTCTTGCTTCG
|
bte-R
|
ACAACAATATGGCCCGTTTGTTGAATTAAACACGAGGTTCAGCAGGGAA
|
Pveg-bte-DR-F
|
TATCCCTGCTGAACCTCGTGTTTAAATCGCATGAAAGCACATTCTCTTGA
|
Pveg-bte-DR-R
|
TCATTTGTATACATACTTTAAAAATTGAGAAAACAGCGGTATGCTGAAG
|
Pveg-bte-RF-F
|
AAATAACAGATTAAAAAAATTATAAATGTCCAAAATTATTGCAATTAAG
|
Pveg-bte-RF-R
|
TGTTTTCACCGATACCGGCAGTAAA
|
amyE-up-F
|
AACCCGACATCCGGCGTTCTCATGG
|
amyE-up-R
|
ACAACAATATGGCCCGTTTGTTGAATCTTGACACTCCTTATTTGATTTTT
|
Te-F
|
AAAAATCAAATAAGGAGTGTCAAGATTCAACAAACGGGCCATATTGTT
|
Te-R
|
ATGTCAAAAGGAGAAGAACTTTTTA
|
gfp-F
|
ATGTCAAAAGGAGAAGAACTTTTTA
|
gfp-R
|
CGGTAAGTCCCGTCTAGCCTTGCCCTTATTTATAAAGTTCGTCCATACCG
|
amyE-down-R
|
AAGGGCAAGGCTAGACGGGACTTACCG
|
amyE-down-F
|
CACCGATGTACACGTCATCTGCAC
|
Pveg -F
|
TAAACATTCTCAAAGGGATTTCTAAGATCTATCTTACACAGCATCACAT
|
Pveg -R
|
TAAAAAGTTCTTCTCCTTTTGACAT TACATTTATTGTACAACACGAGCCC
|
P43-F
|
TAAACATTCTCAAAGGGATTTCTAAGATAGGTGGTATGTTTTCGCTTGAC
|
P43-R
|
TAAAAAGTTCTTCTCCTTTTGACATGTGTACATTCCTCTCTTACCTATAAT
|
Underlined letters represent complementary sequences for overlap-extension PCR.
Culture conditions and surfactin analysis
Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract and 1% NaCl) was used for seed cultures and gene cloning. The fermentation medium contained 6% sucrose, 1% tryptone, 0.6% NaNO3, 0.3% KH2PO4, 1% Na2HPO4, 0.002% FeSO4 and 0.05% MgSO4. The engineered strains were precultured in LB medium and then incubated overnight at 37 °C and 200 rpm. Two milliliters of the resulting seed culture were used to inoculate a 250 ml flask with 50 ml of fermentation medium and cultured at 37 °C with shaking at 200 rpm for 36 h.
The surfactin concentration was determined using high-performance liquid chromatography (HPLC) on a U-3000 instrument (Thermo Fisher Scientific, USA) equipped with an Amethyst C18-P column (4.6 × 250 mm, 5 μm). The mobile phase was composed of 90% (v/v) methanol and 10% (v/v) water, with 0.05% trifluoroacetic acid at a flow rate of 0.8 mL/min. Authentic surfactin (98%) was purchased from Sigma Aldrich (USA). The cell growth was monitored by measuring the optical density at 600 nm (OD600).
Whole cell fluorescence measurements
The coding sequence of green fluorescent protein GFP fused with Pveg promoter or the P43 promoter, was inserted at the amyE locus of the genome of B. subtilis 168 using the primers shown in Table 2. The expression of GFP from the different promoters was monitored by measuring whole-cell fluorescence using a Spectra Max M3 multimode microplate reader (Shanghai Huanxi Medical Equipment Co., Ltd, China). After culturing for 12 hours, 1 ml of fermentation broth was centrifuged at 10,956 × g for 10 minutes, the supernatant was discarded, and the cells were washed 3 times. The washed cells were re-suspended in deionized water to the same optical density (OD600). The excitation wavelength and emission wavelengths were 485 and 525 nm respectively. B. subtilis 168 without the chromosomal gfp expression cassette was used as the negative control. Standard deviations are based on a minimum of three statistically independent experiments.
Isolation, purification and isoform analysis of surfactin
Surfactin was extracted and purified using the acid precipitation method[24]. After fermentation, the cells were removed by centrifugation at 10,956 × g for 10 min. Then, 6 mol/L HCl was added to the supernatant to achieve a pH of 2.0 for acid precipitation, and allowed to settle at 4 °C overnight. The acid precipitate was collected by centrifugation at10,956 × g for 10 min. The final pH was adjusted to 7.0 with 5 mol/L NaOH, and the neutralized precipitate was lyophilized. The dried surfactin components were further extracted with methanol and dried on a rotary evaporator under vacuum.
Surfactin components were analyzed by reverse-phase UPLC–MS (UPLC, Agilent, 1290) coupled with a single quadrupole MS (Q-TOF, Agilent, 6550) on an extend C18 column (2.1×50mm 1.7µm; Agilent) using a method based on a acetonitrile/water (acidified with 0.1% formic acid) gradient that allowed the simultaneous detection of all three lipopeptide families. Elution was started at 10% acetonitrile at a flow rate of 0.50 mL/min. After 7 min, the percentage of acetonitrile was increased to 95% and held until 5 min. Then, the column was re-equilibrated with 10% acetonitrile for 1 min. The compounds produced by BSFX024 and BSFX025 were compared. Ionization and source conditions were set as follows: source temperature, 150˚C; desolvation temperature, 350˚C; nitrogen flow, 15 L/min; voltage, 4000V.
Fatty acid side-chain analysis
The fatty acid side-chains were analyzed according to the method reported by Mu et al. [25]. Briefly, 10mg of the purified surfactin was hydrolyzed with 6 mol/L HCl in an ampoule at 90°C for 20 hours, and the solvent was subsequently removed by blowing air at 60 °C. Then, 500 μL acetonitrile-BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide) (3 : 2 by vol) was added to the sample, and reacted at 60 °C for 20 min. Subsequently, the samples were analyzed by GC–MS. GC-MS analysis was performed on a 6890-5975 C gas chromatography-mass spectrometry instrument (GC-MS; Agilent, USA) equipped with an HP-5 MS capillary silica column (60 m × 0.25 mm × 0.25 μm, Agilent, USA). The ions were obtained by electron ionization EI at 70 eV using a source temperature of 230°C. The column oven temperature was kept initially at 60°C for 3 min, increased to 250°C at a rate of 10 °C/min, and held for 5 min. The other conditions were as follows: helium carrier gas, 99.999%; flow rate, 1.0 mL/min; injector temperature, 250°C; injector volume, 1 μL; split ratio, 20:1.
Critical micelle concentration (CMC) of the biosurfactant
As the concentration increases, the decreasing rate of surface tension will suddenly change at CMC[24]. The surfactin samples obtained from recombinant strains BSFX024 and BSFX025 were dissolved in distilled water at different concentrations (0-100 mg/L). Then, the surface tension was measured using the platinum plate method on an automated tensiometer (BZY-3B; Shanghai Automation Instrumentation Sales Center, China) at 25°C. The CMC was determined based on the inflection point of surface tension versus concentration.
Measurement of Emulsification Activity
For the measurement of emulsification activity, 2 ml of a solution containing 200 mg/L surfactin obtained from recombinant strain BSFX024 or BSFX025 and 2 ml of different hydrocarbons (dodecane, tetradecane, hexadecane, octadecane, p-xylene and liquid paraffin) were mixed in cylindrical glass vials, respectively. The mixtures were vortexed at maximum speed (QT-2; Qite Corp., Shanghai, China), and then incubated at 25°C for 24 h. The emulsification activity was calculated using the emulsification index (EI24) formula[4]:
Oil washing efficiency
The standard oil sand for measuring the oil-washing efficiency was prepared according to a reported method [11, 26]. Briefly, 170 g of quartz sand, 4 g of artificial crude oil (Shengli Oilfield, China) and 10 ml of petroleum ether were mixed. The mixtures were heated at 80 ℃ for 1 h to remove the petroleum ether and then aged at 60 ℃ for 7 days. Subsequently, 2 g of the aged oil sand was placed into a flask with 20 ml of different surfactin preparations with different concentrations (0.05 g/L, 0.1 g/L, 0.15 g/L and 0.2 g/L). Then, the flasks were shaken at 90 rpm and 70 ℃ for 12 h. The sand containing residual oil was dried at 80 ℃ for 12h and the removed oil remaining in the solvent fraction was further extracted with petroleum ether. The absorbance at 225 nm was measured (722S UV–vis spectrometer; Shanghai Precision Instrument Co., China) to calculate the oil washing efficiency of the surfactin preparations using the formula:
Contact Angle Measurements
Wettability was determined by measuring the contact angle of the aqueous surfactant solution on an oil film. The oil film was prepared by painting a thin layer of crude oil onto a glass slide, which was then aged at 80 ℃ for 7 days. The contact angle of the surfactant solutions with different concentrations (0.05 g/L, 0.1 g/L, 0.15 g/L and 0.2 g/L) on the oil film was measured using a drop-shape analyzer (DropMeter A100P; Ningbo Ouyi Testing Instrument Co., Ltd, China).