Bacterial strains, plasmids and materials
The bacterial strains and plasmids used in this study are listed in Table 1. E. coli DH5α was used in plasmids construction and amplification. B. sutilis 168 were used in the determinations of plasmids properties. B. subtilis WB600 was employed in gene expressions. The sequences of the alkaline pectate lyase from Paenibacillus sp. 0602 (pelN, GenBank: KC351190.1), the alkaline protease gene spro1 from alkaliphilic Bacillus sp. 221 (spro1, Sequence ID: D13157.1) and the pullulanase gene pulA11 from Anoxybacillus sp.LM18-11 (GeneBnak ID: HQ844266.1) were deposited in NCBI. The restriction endonucleases and DNA polymerase were commercially supplied by Thermo Fisher Scientific Co., Ltd. All other enzymes, chemicals and reagents were purchased from TaKaRa Biotechnology (Dalian, China) Co., Ltd.
Cultivation conditions
All cells were routinely grown at 37 oC and 200 rpm in Luria-Bertani (LB) medium or fermentation medium. LB medium (1 L) consisted of tryptone 10 g, yeast extract 5 g and NaCl 5 g. 1 L of fermentation medium consisted of tryptone 16 g, soluble starch 37 g, CaCl2 1.5 g, NaCl 2 g, MgSO4·7H2O 0.2 g, KH2PO4 1 g, FeSO4·7H2O 0.05 g and (NH4)2SO4 1.5 g. Different antibiotics (50 μg/mL kanamycin and 25 μg/mL kanamycin) were added in the medium for the relevant recombinant strains.
B. subtilis WB600 strains containing recombinant plasmids were cultured in Shake flask (SHUNIU, GG-17, Sichuan SHUBO Co., LTD, China) or in a 10-L fermenter (5BG, bxbio, China) to produce foreign proteins. The shake-flask culturing was performed as followed. A 5 mL aliquot of LB medium supplemented with 25 μg/mL kanamycin was inoculated with a frozen glycerol stock of recombinant strain (20μL), and then incubated for up to 14 h at 37 oC and 200 rpm in a rotary shaker (ZQWY-200G, Shanghai Zhichu Instruments Co., Ltd., China). An aliquot of this preculture (0.5 mL) was transferred into a 250 mL shake-flask that was loaded with 50 mL of LB medium supplemented with 25 μg/mL kanamycin, which was then shaken in the rotary shaker (200rpm) at 37 oC. After culturing for 48 h, samples of each culture were collected and analyzed for enzyme activities. The culture was harvested by centrifugation at 12,000×g for 10 min at 4 °C to obtain the culture supernatant. Enzyme activities in the supernatant were determined with corresponding assays. Then the culture supernatant was analyzed with SDS-PAGE. The percentages of the produced extracellular proteins were calculated by a software Gel-Pro analyzer 4.0 (Media Cybernetics, CA, USA ).
Fermentation of the recombinant B. subtilis WB600 strain was performed in a 10-L fomenter. The seed culture was prepared as described in the above section and then used to inoculate an initial batch of fermentation medium supplemented with 25 μg/mL kanamycin. The biomass and enzyme activities were analyzed at designated time intervals. The cell density was determined by measuring the OD600nm with a UV-1800/PC spectrophotometer (Epoch 2, BioTek, USA). To determine the wet cell weight (WCW), a 2-mL sample of the culture broth were centrifuged at 12,000×g for 10 min at 4 °C. The resulting pellet was collected and weighed. The enzyme activities in the culture supernatant were further determined with corresponding methods. The whole fermentation process was stopped and finished when continuous reductions of biomass and enzyme activities were observed.
Shuttle plasmids construction
Construction of shuttle plasmids is schematically presented in Fig.1. All plasmids were constructed through the method of MEGAWHOP [45]. All of the specific primers used for PCR amplification were synthesized by the Beijing Genomics Institute (BGI) and listed in Table 2 and the isolation and manipulation of recombinant DNA were performed according to the previously published protocol[46]. 1.5 mL of culture was poured into a 2 mL EP tube and centrifuged at 12,000×g for 10 min at 4 °C. The supernatant was discarded and the bacterial pellet was collected. The bacterial samples were treated with solution I (100 mL of solution I consisted of 1 M pH 8.0 Tris-HCl, 2.5 mL; 0.5 M EDTA 2 mL; ddH2O 91 mL; 20% sterile glucose 4.5 mL), solution II (100 mL of solution II consisted of 10% SDS 50 mL; 2 M NaOH 50 mL), solution III (500 mL of solution III consisted of KAc 147 g; HAc 57.5 mL, ddH2O was added to 500 mL), mixture of phenol: chloroform (volume ration 1:1) and 70% ethanol successively according to the protocol. The DNA sample was dissolved with 80 μL ddH2O and stored in -20 °C for further experiments.
See Table 2
According to the sequencing map, 169 bp of the membrane-biding region BA3 (515 bp) originated from pUB110 was deleted during the construction of pWB980. In order to be mentioned conveniently, the remaining 346 bp fragment of BA3 was named BA3-1 in this work as shown in Figure 1. pWB980 is consist of five parts: BA3-1, P43 promoter cohered with a signal-peptide sequence, the replicase-coding gene rep in B. subtilis ,the kanamycin-resistance gene kanR and the bleomycin gene bleoR gene (Fig 1). In this work, the bleoR was deleted with primers DB-1/DB-2. The plasmid replication origin ori (888 bp) from pUC19 was inserted at the four sites up- and down-stream BA3-1 region and rep gene in pWB980-DB (3388 bp) separately. With primer pairs P1-S/P1-A and P2-S/P2-A, ori integrated into the site (659 bp) upstream the BA3-1 with forward and reverse directions. The recombinant plasmids were named as pUC980-1 and pUC980-2 respectively. At the site (169 bp) downstream the BA3-1, primers P3-S/P3-A and P4-S/P4-A were used in ori-insertions, resulting plasmids pUC980-3 and pUC980-4 in forward and reverse directions. Upstream the rep gene, the ori was inserted at the 3118 site using primers P5-S/P5-A and P6-S/P6-A. The recombinant plasmids were named as pUC980-5 and pUC980-6 with forward and reverse ori-insertions respectively. Ori was put into the 1722 site downstream the rep by using primers P7-S/P7-A and P8-S/P8-A. The plasmid with forward ori insertion was pUC980-7 and the other one was pUC980-8.
With primer pair 62-S/62-A, the alkaline pectate lyase gene pelN was inserted into the sites after the P43 promoter of plasmids pWB980, pWB980-DB and all pUC980-serial plasmids except pUC980-8. The recombinant plasmids were pWB980-pelN, pWB980-DB-pelN, pUC980-1-pelN, pUC980-2-pelN, pUC980-3-pelN, pUC980-4-pelN, pUC980-5-pelN, pUC980-6-pelN and pUC980-7-pelN correspondently.
Determination of plasmid copy number by quantitative real time PCR (qPCR)
The quantitative real-time PCR (qPCR) was performed using iQ™ SYBR® Green Supermix (Bio-Rad) as described by Turgeon [47]. The dnaN and kanR genes were chosen as a single copy reference gene on the B. subtilis chromosome and the test gene in plasmids correspondingly [48-50]. Specific primer pairs dnaNS/dnaNA and kanS/kanA (Table 2) were designed to generate products of approximately 150 bp. The standard curves were prepared based on the pMD-18-T vector. The PCR mixtures (total volume 20 μL) contained 10 μL 2xTakara SYBR Green Real-Time PCR Master Mix, 1.5μL forward and reverse primers (10 μM) and 1 μL diluted (10−1 to 10−4) sample DNA. The reaction condition was as followed: 95 oC for 10 min, 45 cycles of 95 oC for 10 s, 62 oC for 20 s, and 72 oC for 30 s, followed by a gradient temperature from 55 oC to 95 oC. The calculated mean of the kanR gene in each compared plasmid was divided by the dnaN gene (single-copy reference), resulting the copy number. All experiments were performed with three independent biological replicates.
Plasmid stability assay
Plasmid segregational stability in B. subtilis 168 was detected according to the method of Bron and Luxen [38] with minor modifications. Single colonies on kanamycin selective (25 μg/mL) LB agar plate were used to inoculate 5 mL kanamycin selective (25μg/mL) LB medium. After 24 h culturing at 37 oC, the cultures were diluted 1:1000 in fresh LB media without antibiotics and incubated at 37 oC. This iterative subculturing process was repeated every 24 h for 30 consecutive days. Samples taken at appropriate intervals were checked for the fraction of plasmid-containing cells by replica plating on LB agar plates containing 25μg/mL kanamycin.
N: Total amount of colonies on the selectable plate
Plasmids were extracted from host cells after 30-days continuously culturing and detected the structural stability by EcoRI/HindIII enzyme-digestion verifications.
Alkaline pectate lyase assay
The pectate lyase activity was measured using the method described previously by Wang et al. [51]. 20μL of the diluted enzyme solutions were added to 400μL of 0.2% pectin in 50 mmol/L glycine-NaOH buffer (with 50μmol/L CaCl2 ) at pH 9.0. The reaction mixture was incubated at 65 oC for 10 min, and the reaction was terminated by adding 580μL of 30 mmol/L H3PO4. The product yields were detected using a spectrophotometer at 235 nm (Epoch 2, BioTek, USA). One standard enzyme unit was defined as the yield of 1μmol unsaturated pectin per minute under the above-mentioned conditions.
Alkaline protease assay
The alkaline protease activity was measured using the modified method described by Tjalsma et al.[27] One standard enzyme unit was defined as the as the amount of enzyme that hydrolyzes 1 μg casein minute under the situation of pH 9.0, 50℃. 250μL of the diluted enzyme solution were mixed with 250 μL solution containing 2% casein in pH 9.0 buffer. The mixture was incubated at 50 oC for 10 min, and the reaction was terminated by adding 500μL 0.4 M TCA. The productions were detected using a spectrophotometer (Epoch 2, BioTek, USA) at 280 nm. Casein with concentrations from 0 to 500 μg/mL were used as standard.
Pullulanase assay
Pullulanase activity was measured using the modified dinitrosalicylic acid (DNS) method[52]. One standard enzyme unit was defined as the amount of enzyme that releases 1 μmol reducing sugar per minute under the assay conditions. Glucose with concentrations from 0 to 400 mg/mL were used as standard. 50 μL of the diluted enzyme solutions were added into the 450 μL reaction mixture containing 5% pullulan solutions and the pH6.0 buffer at the volume ratio of 1:8. After incubations at pH 6.0, 60℃ for 30 min, 500 μL DNS were used to stop the reactions. The productions were detected using a spectrophotometer (Epoch 2, BioTek, USA) at 540 nm. The amount of reducing sugar released during incubations were tested as the enzyme-index.
SDS-PAGE
B. subtilis WB600 transformant cells harboring recombinant plasmids were cultured in LB medium containing 25 μg/ml kanamycin at 37 oC for 48 h. The cultures (0.5 mL respectively) were centrifuged at 5000 rpm, 4 oC for 10 min and the supernatant solutions were reserved. The cell pellets were re-suspended in 0.5 mL ddH2O, followed by sonication to release intracellular content. Extracellular proteins were precipitated with 13% (w/v) TCA for 30 min on ice, then the precipitates which were collected by 10 min centrifugation at 4 oC and 13,000×g, were carefully washed with ice-cold acetone and dried under vacuum. The collective proteins were dissolved in 20μL urea (6M) and mixed with appropriate volume of 4× SDS loading buffer. Samples were then heated in boiling water bath for 10 min and centrifuged at 13,000×g for 10 s. 12 μL of the samples were loaded onto 10% SDS-PAGE and run at 20 mA for around 1h. Gel was stained using Coomassie brilliant blue dye.