Strains, plasmids and reagents
E. coli GB05-dir was derived from strain GB2005 (DH10B, fhuA::IS2, ΔybcC, ΔrecET) by integrating an arabinose-inducible ETγA operon (full-length recE, recT, redγ and recA) at the ybcC locus [26, 31]. GB08-red was derived from GB2005 by integrating an arabinose-inducible γβαA operon (redα, redβ, redγ and recA) at the ybcC locus . GB05-dir and GB08-red were used for direct cloning and recombineering experiments and grown in LB medium (10 g tryptone, 5 g yeast extract, 5 g NaCl per liter, pH 7.0) at 37 °C or 30 °C. B. glumae PG1 wild-type was cultivated in LB medium at 30 °C and used as a host for homologous expression of the lipAB genes. For production of lipase, B. glumae strains were cultivated in PG medium , which contains 6 g (NH4)2SO4, 3.5 g KH2PO4, 3.5 g K2HPO4, 0.02 g CaCl2, 1 g MgSO4.7H2O, 2 g yeast extract per liter (pH 6.5) with 1% (vol/vol) olive oil or 0.5% (wt/vol) glucose as a carbon source.
Plasmid p15A-cm-tetO-tetR-hyg-ccdB contains a p15A origin, a hyg-ccdB cassette, a tetracycline-inducible promoter PtetO, and conveys chloramphenicol resistance . Plasmid pRk2-km-apra carries the broad-host-range Rk2 origin and conveys kanamycin and apramycin resistance. Plasmid pBBR1-km has the broad-host-range pBBR1 origin and conveys kanamycin resistance. Appropriate antibiotics were added when necessary at the following concentrations: for E. coli strains, kanamycin, chloramphenicol and apramycin were each used at 15 µg/ml; for B. glumae strains, kanamycin and apramycin were each used at 30 µg/ml. The concentration of L-arabinose used for induction was 1.4 mg/ml. Plasmids used in this work are listed in Table S1.
Direct cloning of the lipAB operon
The p15A-cm vector was amplified from p15A-cm-tetO-tetR-hyg-ccdB with PCR using the PAGE-purified oligonucleotides PG1-lipAB-A and PG1-lipAB-S, which contain 80 nt homology arms to the target DNA sequence, and 20 nt standard PCR primers at the 3’ end. DNA oligonucleotides are listed in Table S2. The template plasmid and PCR products were purified with the Qiagen plasmid mini kit and eluted from the column with autoclaved ddH2O. The genomic DNA of B. glumae PG1 was prepared from the lysate by phenol-chloroform-isoamyl alcohol extraction (25:24:1, pH 8.0) and ethanol precipitation, and digested with EcoR V and EcoR I to expose the terminal homology arms . Ten micrograms of digested genomic DNA and 200 ng of p15A-cm linear vectors were co-electroporated into arabinose-induced, RecET-expressing GB05-dir cells for direct cloning of the lipAB operon, which was expressed under the control of its native promoter, PlipAB. Transformants were selected after growth on LB agar plates containing chloramphenicol at 37 °C overnight, and correct recombinants were identified by restriction analysis. The final construct, p15A-cm-PlipAB-lipAB, was electroporated into GB08-red cells for further engineering.
Subcloning the lipAB operon into a broad-host-range vector
Subcloning vectors pBBR1-km and pRk2-km were prepared by PCR amplification from plasmids pBBR1-km and pRk2-km-apra with the pRlmer pairs RK2-lipAB-A/S and BBR1-lipAB-A/S, respectively. PCR products were purified using the Qiagen plasmid mini kit. The pBBR1-km and pRk2-km cassettes, flanked by the 80 nt homology arms, contain a broad-host-range origin and a kanamycin-selection marker and were electroporated into arabinose-induced, redαβ-expressing GB08-red cells harboring p15A-cm-PlipAB-lipAB to subclone the lipAB genes. Transformants were selected with kanamycin, and correct recombinants were identified by restriction digestion. The plasmid-borne lipase was expressed under an native promoter. The final constructs, pBl and pRl, were electroporated into B. glumae PG1 for further studies.
Promoter exchange of the lipAB genes
The promoter of the lipAB was exchanged with the constitutive promoter Papra via Redαβ recombineering. Papra segments, flanked by homology arms, were amplified with PCR from the template plasmid pRK2-km-apra using the 100 nt primers Papra-A and Papra-S. Purified PCR products were recombined with plasmids pBl or pRl in arabinose-induced, Redαβ-expressing GB08-red cells to exchange the PlipAB promoter. Following confirmation by restriction analysis, the final constructs, pBa and pRa, were electroporated into B. glumae PG1 to examine lipase activity.
Electroporation of the expression plasmid into B. glumae PG1
For B. glumae PG1, overnight cultures were diluted into 1.3 ml fresh LB medium without any antibiotics (OD600 = 0.1) and incubated at 30 °C, 950 rpm, until the OD600 was around 0.6. Cells were pelleted by centrifugation at 9500 rpm for 1 min at room temperature, washed, and then 10 µg of resuspended plasmid was added to the electrocompetent cells. Electroporation was conducted using an Eppendorf 2510 electroporator at 1250 V with a 1 mm electroporation cuvette. The cells were resuspended in 1 ml LB medium and transferred into a 1.5 ml punctured micro-centrifuge tube. The culture was incubated at 30 °C, 950 rpm, for 2 h before plating on LB plates containing kanamycin (30 µg/ml) followed by incubation at 30 °C for 48 h. LB agar plates contained 1.2% (wt/vol) of agar. Recombineering in E. coli was performed as previously described .
Lipase activity assay
Lipase activity in culture supernatants was assayed with para-nitrophenylpalrnitate (pNPP) as the substrate . First, 100 µl acetonitrile containing 0.9 mg pNPP was mixed with 900 µl of 50 mM Tris-HCl buffer (pH 8.0). After 5 min of prewarming at 37 °C, freshly prepared substrate solution was mixed with 50 µl of 5000-fold dilutions of supernatant, and then incubated at 37 °C for 5 min. Next, 200 µl of the complex was taken to measure the OD410 against an enzyme-free control using a spectrophotometer. Relative lipase activity was calculated as units/ml. One unit (U) was defined as the amount of lipase that liberates 1 mmol of para-nitrophenol per minute.
Lipase proteins in whole-cell extracts and supernatants were analyzed with SDS-PAGE . Culture supernatants obtained by centrifugation (4 °C, 8000 rpm, 1 min) were mixed with SDS sample buffer (5×), and then 20 µl was subjected to SDS-PAGE. Cell pellets were resuspended by sonication, and 20 µl cell suspension was mixed with SDS sample buffer for analysis. SDS-PAGE was carried out using 12% polyacrylamide gels, and proteins were stained with Coomassie brilliant blue R-250.
Quantitative real-time PCR
The analysis of transcriptional levels of lipA was performed with real-time qPCR in an ABI PRISM 7500 System (Applied Biosystems, USA) using SYBR green. Primers used for qPCR are listed in Table S2. The constitutively expressed gene rpoD was used as the internal reference. The amount of PCR product was calculated using the ΔΔCT-method, as previously described .