General
Restriction enzymes, Phusion High-Fidelity DNA polymerase, and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA). Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). DNA sequencing was performed at SeqWright (Houston, TX) or Genewiz (South Plainfield, NJ). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Molecular biology techniques for DNA manipulation were performed according to standard protocols [27] and all cultures were grown in lysogeny broth (LB). Antibiotics and IPTG were prepared as a 1000× stock solution in purified water and sterile filtered with EMD Millipore Millex-GP syringe driven filters (EMD Millipore, Cat. No. SLGP033RS). The modified NBS medium contains MOPS (50 mM, pH 7.4) and per liter: 10 g glycerol; 2.5 g glucose; 5 g yeast extract; 3.4 g KH2PO4; 5.2 g K2HPO4; 3.3 g (NH4)2HPO4, 0.25 g MgSO4·7 H2O, 15 mg CaCl2·2 H2O, 0.5 mg thiamine, and 1 mL of trace metal stock (described by Chin and Cirino [28]). The concentrations of antibiotics used for maintaining the plasmids are as follows: apramycin 50 µg/ml, chloramphenicol 25 µg/ml, ampicillin 100 µg/ml.
Plasmids
Plasmids and strains used in this study are listed in Table 1. All primers used in this study are listed in Additional file 7. Primers pPCC1244-gib-for and pPCC1244-gib-rev were used to amplify lacI-Ptac-araC-SA DNA fragment from pFG29-SA, and the PCR product was assembled with PciI- and XmnI-digested pFG1, resulting in plasmid pPCC1244.
To create plasmid pPCC1250, pZE-EP was double digested with Bsu36I and SphI, and the digested DNA fragment was subjected to a DNA-blunting reaction. The blunt-ended DNA fragment was self-ligated, resulting in plasmid pPCC1250. A DNA fragment containing entC and pchB was amplified from pPCC1250 using primers pPCC1251-gib-for and pPCC1251-gib-rev2, and assembled with XmnI-digested pPCC1244, resulting in plasmid pPCC1251. A DNA fragment containing aroL ppsA, tktA, aroGfbr genes was amplified from pCS-APTA using primers pPCC1252-gib-for and pPCC1251-gib-rev2, and the amplified DNA fragment was assembled with NdeI- and XmnI-digested pPCC1244, resulting in plasmid pPCC1252. Then, a DNA fragment of Ptac-araC-SA-entC-pchB was amplified from pPCC1251 using primers pPCC1253-NheI-for and pPCC1253-NotI-rev, and digested with NheI and NotI. The digested DNA fragment was ligated into pPCC1252 digested with the same restriction enzymes, resulting in plasmid pPCC1253. To remove aroL from plasmid pPCC1253, primers pPCC1251-gib-for and pPCC1253-Ptac-rev were used to amplify the entC-pchB DNA fragment from pPCC1253, and primers pPCC1253-ppsA-for and pPCC1253-tktA-rev were used to amplify the ppsA-tktA section from pPCC1253. The two amplified PCR products were assembled into KpnI/SphI double-digested pPCC1253, resulting in plasmid pPCC1253-aroL.
Construction of pathway RBS library
The RBS calculator [7] was used to design six RBS sequences having different translation initiation rates (TIRs) for each gene (see Additional file 1 for the RBS sequences and calculated TIRs). For each gene, the six designed upstream primers containing different RBS sequences were mixed equimolar, resulting in primer mixtures entC-RBSs-for, pchB-RBSs-for, etc., and each primer mixture was used to construct the RBS library. Primers pFG29_araC_GS_fwd_1 and AraC-gib-rev were used to amplify Ptac-AraC-SA DNA fragment from pPCC1244. Primers entC-RBSs-for and entC-RBS-rev were used to amplify entC from pZE-EP, and primers pchB-RBSs-for and pPCC1251-gib-rev2 were used to amplify pchB from pZE-EP. Plasmid pPCC1244 was double digested by BstAPI and XmnI, and the linearized vector was assembled with the three PCR products, resulting in QSALib1, which contains the RBS libraries for entC and pchB genes.
Primers pFG29_araC_GS_fwd_1 and Ptac-gib-rev were used to amplify a DNA fragment containing promoter Ptac. The aroL gene was amplified from pCS-APTA by primers aroL-RBSs-for and AroL-RBS-rev. Similarly, ppsA was amplified from pCS-APTA using primers ppsA-RBSs-for and ppsA-RBS-rev. Overlap extension PCR (OE-PCR) was performed to assemble these three PCR products using primers pFG29_araC_GS_fwd_1 and QSAlib2-OE123-rev, resulting in DNA fragment QSAlib2-f123. Next, primers tktA-RBSs-for and tktA-RBS-rev were used to amplify tktA from plasmid pCS-APTA, and primers AroG-RBSs-for and pPCC1251-gib-rev2 were used to amplify aroG. tktA and aroG were assembled by OE-PCR using primers QSAlib2-OE45-for and QSAlib2-OE45-rev, resulting in DNA fragment QSAlib2-f45. pPCC1252 was then double digested with BstAPI/BamHI, QSAlib2-f123 was double digested with BstAPI/SpeI, and QSAlib2-f45 was double digested with SpeI/BamHI. Ligation of these three digest fragments resulted in library QSAlib2.
Finally, primers pPCC1253-NheI-for and pPCC1253-NotI-rev were used to amplify Ptac-araC-SA-RBS-entC-RBS-pchB from QSAlib1. The PCR product was digested with NheI and NotI, and ligated with QSAlib3 digested with the same enzymes, resulting in QSAlib3. QSAlib3 contains the RBS libraries for all six genes. Sanger sequencing of QSAlib3 confirmed proper library construction.
Strain construction
The strains used in this study are listed in Table 1. Plasmid pPCC1155-5 was integrated into the chromosome of QH4 at site HK022 as described [29]. Removal of FRT-flanked apramycin resistance cassette resulted in strain SQ18. A Phage λ Red disruption method [30] was used to delete lacZ from the lac operon of strain SQ18, resulting in strain SQ22. Deletion of rnd in strains QH4 and SQ22 was similarly performed, resulting in strains QH4∆rnd and SQ22∆rnd, respectively.
Sensor-reporter fluorescence assays
Essentially as described [20], 500 μl LB + apramycin in 2-ml-well, 96-well plate was inoculated with strain HF19 harboring pFG29-SA. These starter cultures were incubated for 6 h at 37 °C, 900 rpm, then diluted to OD595 = 0.05 in 500 μl “biosensor medium” containing different concentrations of the compound of interest. After 6 h, the cultures were pelleted and washed with an equal volume of phosphate-buffered saline, before measuring OD595 and fluorescence (400 nm excitation, 510 nm emission) using plate readers.
Salicylate production in baffled flasks
A colony of the salicylate-producing strain was used to inoculate 3 ml LB + apramycin, and grown in a test tube for 8 h at 37 °C and 250 rpm. This seed culture was then diluted to OD595 = 0.05 into 25 ml modified NBS medium containing apramycin and 250 mM IPTG, in 125 ml baffled flasks. The flasks were shaken at 37 °C and 250 rpm for 48 h, at which time OD595 values were measured and salicylate concentrations were analyzed by HPLC.
Screening the RBS library for improved salicylate producers
Strain SQ22 was transformed with QSAlib3. The outgrowth was transferred into LB + apramycin, and grown at 37 °C, 250 rpm for 12 h. The resulting culture was diluted and spread onto large plates containing modified NBS-agar with IPTG (250 µM), X-Gal (40 µg/ml), and apramycin. After 24 h of incubation, the top 5 blue colonies from each screening plate were picked and streaked onto fresh LB plates supplied with apramycin. The resulting colonies were tested for salicylate production in liquid culture.
Construction and screening of SQ22 transposon insertion library
Strain SQ22 harboring the highest-producing salicylate plasmid (pQSA-50) was transformed with 1 µg of plasmid pPCC507, and the outgrowth was grown in 1 ml SOB medium supplied with 20 µM IPTG at 37 °C for 1 h. The outgrowth was transferred into 500 ml LB + apramycin and 12.5 µg/ml chloramphenicol, and grown at 37 °C for 12 h. The resulting culture was diluted and plated on modified NBS-agar plates containing IPTG (250 µM), X-Gal (40 µg/ml), apramycin, and 12.5 μg/ml chloramphenicol. Totally 70,000 colonies were screened on 10 screening plates. After 24 h of incubation, the top 5 blue colonies from each screening plate were picked and streaked onto fresh LB plates containing apramycin and 12.5 µg/ml chloramphenicol. The resulting colonies were tested for salicylate production in liquid culture.
Salicylate quantification by HPLC
500 µl of cell culture was centrifuged at 17,900×g, and the supernatant was filtered through a 0.45 µm filter. The salicylate concentration in the filtrate was determined by reverse-phase HPLC using a C18 column on a Shimadzu LC-20AD HPLC system (Kyoto, Japan) equipped with a UV monitor. The elution profile was as follows: Solvent A, 1% (v/v) acetic acid in water; solvent B, 1% (v/v) acetic acid in acetonitrile; gradient: 5–95% B (0–15 min), 95–5% B (15–17 min), 5% B (17–20 min). The column temperature was set to 50 °C. Salicylate eluted around 11.2 min at a flow rate of 1 ml/min. Elution absorbance at 310 nm was monitored and peak areas were converted to sample concentrations based on calibration with pure salicylate.