Strain, vector, medium, and reagents
Strains and vectors used in this study are listed in Table S1. E. coli BL21(DE3) was purchased from Biomed Co. Ltd. Strains were stored in glycerol-containing Luria-Bertani medium at − 20°C. Prior to culture, they were incubated to test tubes containing 4 mL Luria-Bertani medium and appropriate antibiotics, and shaken at 180 × g and 37°C. For seed culture, E. coli was incubated in M9 minimal medium containing the following components per liter: glucose, 3.1 g; NaCl, 0.5 g; Na2HPO4·7H2O, 12.8 g; KH2PO4, 3.0 g; NH4Cl, 1.0 g; MgSO4, 0.492 g; CaCl2·6H2O, 0.0219 g. One percent (v/v) of seed culture was transferred to a 250-mL flask containing 100 mL M9 minimal broth and appropriate antibiotics, and cultured in a shaker at 150×g and 30 °C. The concentrations of kanamycin and chloramphenicol were 50 μg/mL and 34 μg/mL, respectively. Vectors pKD46, pKD13, and pCP20 were used for gene knockout (Datsenko and Wanner, 2000). Other vectors used in this study were derived from vector pACYC-Duet1 or pET-28a. Restriction endonucleases and DNA polymerase were purchased from TaKaRa (Dalian, China). Kits for ligation were purchased from Thermo (Beijing, China). Kits for plasmid isolation and DNA purification were purchased from Biomed (Beijing, China). DNA marker and other reagents were purchased from Bioroyee (Beijing, China). Gene synthesis and DNA sequencing were accomplished by BGI (Beijing, China) and Beijing Syngentech Co., Ltd. Standards for metabolite assay were products of Sigma (Beijing, China).
Gene knockout
The E. coli ldhA gene was knocked out using the reported method (Datsenko and Wanner, 2000). To obtain cassette 52nt-FRT-Kan-FRT-50nt, PCR primers were designed according to the 52-nt upstream and 50-nt downstream sequence immediately flanking the ldhA gene (Table S2). The kanamycin resistance gene (KanR) and two FRT (FLP recognition target) sites were cloned by PCR from vector pKD13. The 50-μL mixture for PCR reaction included 25 μL of Premix PrimeSTAR® HS, 1 ng of pKD13, 0.4 mM of forward, and reverse primers of pKD13. The protocol for PCR reaction includes initial denaturation at 98°C for 2 min, followed by 30 cycles of 98 °C for 10 s, 60 °C for 50 s, 72 °C for 110 s, and 72°C for 10 min. The Dpn I endonuclease was used to digest vector pKD13.
The competent E. coli BL21 cells were electro-transformed with vector pKD46 and incubated in Luria-Bertani medium at 30 °C for 1 h. Positive recombinants were screened by Luria-Bertani ampicillin plates. After overnight cultivation, positive recombinants were confirmed by colony PCR. The strains harboring vector pKD46 were grown in Luria-Bertani medium to reach OD600 of 0.12–0.2. Next, 100 mM L-arabinose was used to induce recombinase expression at 37°C for 1.5 h. The strains were concentrated by 30-fold at 4 °C and washed twice with cold distilled water. Electroporation was performed in Bio-rad Micropulser with 100 μL competent cells and 5 μL PCR products added in a 0.2-cm cuvette. The transformed cells were recovered in 890 μL Luria-Bertani medium at 30°C for 2 h, and then grown in kanamycin Luria-Bertani plate at 30 °C for 72 h. Positive clones were confirmed by colony PCR and DNA sequencing.
Vector pCP20 harbors ampicillin and chloramphenicol resistance genes (AmpR and CmR), temperature-sensitive replication, and FLP sequence for acting on two FRT sites. After pCP20 was transformed into competent E. coli cells, the kanamycin resistance gene on E. coli chromosome was eliminated upon cultivation at 30°C for 24 h. The resulting KanR mutant was continuously grown in an antibiotics-free Luria-Bertani plate at 42°C for 3–5 generations of subculture, and then grew in Luria-Bertani plate containing ampicillin and chloramphenicol to examine whether the strains still harbored vector pCP20. Mutant E. coliΔldhA was confirmed by colony PCR and DNA sequencing.
Construction of recombinants
The AHL synthesis gene cluster luxI-luxR from V. fischeri ES114 (Papenfort and Bassler 2016) was cloned by PCR and inserted into vector pACYCDuet1 at BamH I/EcoR I, leading to vector pluxIR which served as the common vector for all recombinant strains (Fig.1). To construct an AHL-responsive vector, the original T7 promoter in vector pET-28a(+) was replaced by an AHL-inducible luxi promoter, resulting in vector pET-Pluxi. To determine the influence of AHL on lactic acid production, a set of vectors were constructed. The genes—egfp (followed by ssrA degradation tag: GCTGCTAACGACGAAAACTACGCTCTGGCTGCT), ldhA, and aiiA—were independently inserted into vector pET-Pluxi at EcoRI/Hind III, BamHI/EcoRI, and HindIII/XhoI sites, resulting in vectors pET-Pluxi:egfp, pET-Pluxi:ldhA, and pET-Pluxi:aiiA, respectively. The aiiA gene tailored for AHL degradation was cloned from B. thuringiensis.
To determine the effects of AiiA on egfp expression and lactic acid production, its coding gene aiiA was equipped with a moderate strength of RBS (AAGTTAAGAGGCAAGA) and then inserted into vectors pET-Pluxi:egfp and pET-Pluxi:ldhA at Hind III/Xho I sites, resulting in vectors pET-Pluxi:egfp-aiiA and pET-Pluxi:ldhA-aiiA, respectively. To vary aiiA expression, the aiiA gene with weak or strong RBS (weak: CACCATACACTG; strong: AAGGAGGTTTGGA) was independently inserted into vector pET-Pluxi:ldhA at Hind III/Xho I sites, leading to vector pET-Pluxi:ldhA-aiiA(weak) and pET-Pluxi:ldhA-aiiA(strong), respectively. After the above vectors were independently transformed into competent E. coliΔldhA cells, we obtained seven recombinant strains named Q01, Q02, Q03, Q04, Q05, Q06, and Q07 (Table S1). To exploit AHL for coordinated gene expression, the vector pluxIR was transformed into above seven recombinant strains, resulting in recombinant strains Q11, Q12, Q13, Q14, Q15, Q16, and Q17, respectively. The control strains were constructed using empty pET- and pACYC-series vectors, including C01, C02, C03, C04, and C05. We expected that the strains overexpressing AHL synthesis gene exhibited “ON” state, while the strain coexpressing AHL synthesis and degradation gene manifested “ON to semi-OFF” state. All primers are listed in Table S2.
SDS-PAGE analysis
To determine whether the engineered QS cassette functioned properly in E. coli, the strains were independently inoculated in 250 mL shake-flasks, each containing 100 mL Luria-Bertani medium and antibiotics as appropriate. After 3 h cultivation, 0.5 mM IPTG was added to induce protein expression. After 16 h shaking cultivation at 150×g and 37 °C, cells were harvested by centrifugation at 7379×g for 10 min, and then mixed with 4×SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and boiled for 5 min. Protein expression was analyzed by 12% (w/v) SDS-PAGE with cell-free extract under denaturing conditions. Mini-Protein III Electrophoresis System (Bio-Rad, USA) was used to perform this operation. Coomassie Brilliant Blue R-250 (0.2%, w/v) was used to stain proteins on the gel, and protein concentration was measured by Bradford method with bovine serum albumin (BSA) as standard protein (Table S3).
Determination of relative fluorescence
To investigate eGFP expression, strains were grown in shake-flasks at 37°C. Samples were taken every 2 h from 0 to 46 h, and 200 μL fermentation broth was added into a 96-well microtiter plate (Corning, NY, USA). To avoid fluorescence quenching, samples were stored in the dark at 4°C before the instrument was ready. Fluorescence intensity was measured using the Enspira microplate reader (PerkinElmer, Singapore) at 485 nm excitation wavelength and 9.5 mm measurement height. Relative fluorescence intensity was defined as the fluorescence value divided by OD600.
Microscopic observation of fluorescence
To directly view eGFP expression, strains were independently cultivated in shake-flasks, and fermentation broth was sampled at 8 h, 20 h, and 32 h and stored in the dark at – 20 °C before the microscope was ready. For microscopic observation, 30 μL sample was added on a microscope slide (Thermo Fisher Scientific, MA, USA), and an equal volume of a mixture containing glycerol and phosphate buffer saline (PBS) at pH 7.4 was used as a sealant, which was under a cover glass without air bubble. Leica SP8 laser scanning confocal microscopes using Leica DMC2900 imaging system (Leica, Wetzlar, Germany) was set to eGFP mode with 40× objective and scale bar of 20 μM.
Enzyme activity assay
To measure the LdhA activity of Q12, Q15, Q16, and Q17, broths were sampled at different time points (8 h, 14 h, 20 h, 32 h, and 48 h), and then centrifuged at 7379×g for 10 min. The harvested cells were washed twice using 100 mM PBS (pH 6.5), and then sonicated and centrifuged at 10,625×g for 10 min. Proteins were quantified using Bicinchoninic acid (BCA) assay kit (Bioroyee, Beijing, China). 20 μL supernatant was mixed with 200 μL BCA assay solution and incubated at 60°C for 30 min. When the mixture was cooled to room temperature, absorbance at 570 nm was measured using a microplate reader. The LdhA activity was determined by lactate dehydrogenase (LDH) kit (Beijing Xinhualvyuan Science and Technology, Beijing, China), which can spectrophotometrically analyze the decrease rate of absorbance at 340 nm corresponding to NADH oxidation at 37°C. This decrease rate is named as ∆A/min. The reaction mixture consisted of 0.05 mL supernatant, 2.0 mL LDH assay solution, and 0.2 mL pyruvate solution. After pyruvate solution was added into the reaction mixture, enzyme activity was immediately measured. The specific activity formula is LdhA(U/mg)=∆A/min*(106/6220)*(2.25/0.05)/protein concentration=∆A/min*7235/protein concentration, and the molar extinction coefficient is 6220 M-1 cm-1 at 340 nm.
Determination of relative transcriptional levels
To clarify how the “ON” mode of LdhA expression affected glycolysis and glucose phosphotransferase system (PTS) and to confirm the AiiA expression under different strength of RBS, qRT-PCR was performed. The strain Q12 and the reference strains C01 and C02 were sampled at different time points and harvested by centrifugation. RNA was isolated using QIAamp RNA Blood Mini Kit (Qiagen, Duesseldorf, Germany). The same RNA extraction was also performed for Q15, Q16, and Q17. After RNase-free DNase was added, cDNA was synthesized through SuperScript III Reverse Transcriptase (Invitrogen, CA, USA). Next, qRT-PCR analysis was carried out using the resultant cDNA by ABI ViiA 7 Real-Time PCR System (Thermo Fisher Scientific, MA, USA). The 16S rRNA coding gene was used as the control to determine the relative expression levels of genes ldhA, gapA, gapC, ptsH, ptsI, pykF, and aiiA.
Analytical methods
Cell concentration was determined by microplate reader at 600 nm with 200 μL fermentation broth added in a cuvette. The fermentation broth was centrifuged at 10,625×g for 10 min, and the supernatant was filtered through a 0.22-μM PES membrane. Metabolites including lactic acid and acetic acid were measured by a high-performance liquid chromatography (HPLC) system (Shimazu, Kyoto, Japan) equipped with a C18 column and an SPD-20A UV detector at 210 nm. Column temperature was maintained at 25 °C, and the mobile phase was 0.05% phosphoric acid at a flow rate of 0.8 mL min−1. Injection volume was 20 μL. Glucose and L-lactic acid were measured by SBA-40E biosensor (Shandong, China). The reaction and cleaning time were 20 s and 10 s, respectively. The injection volume was 25 μL. D-lactic acid is the total lactic acid minus L-lactic acid.
To analyze AHL and its derivatives, cell-free supernatant was extracted with acidified ethyl acetate (by 0.05% formic acid). The extraction was performed in triplicate, and mixture was evaporated to dryness. The extract was dissolved in methanol, filtered through a 0.45-μm organic membrane and followed by UPLC-MS/MS analysis (Waters ACQUITY UPLC/Quattro Premier, USA, Massachusetts). A Waters ACQUITY SDS C18 column was utilized as the solid phase and maintained at 45°C. Mobile phase consists of 0.1% acetic acid (A) and acetonitrile (B). Gradient elution was conducted with the profile of 99% A at the first 1 min, 40% A from 2 to 4 min, 100% B from 5 to 8 min, 100% B from 9 to 11 min, and 99% A from 12 to 13 min. Flow rate was 0.3 mL/min. Mass detection was performed in ES+ ionization mode and data were analyzed by Masslynx V4.1 software.
Characterization of recombinants
In this study, QS was exploited to coordinate cell growth and lactic acid production in E. coli. In doing so, we constructed two vectors carrying different resistance genes and replicons. The medium-copy vector, pluxIR, carries luxI-luxR gene cluster to automatically synthesize AHL and constitutively express AHL-binding protein LuxR (Fig.1B). The high-copy vector pET-Pluxi harbors a Pluxi promoter and a pET-28a(+) backbone, where gene expression is controlled by QS. Due to this architecture, the vector pluxIR could express LuxR-AHL, which in turn induced the expression of gene on vector pET-Pluxi. With the increase of cell density, the transcriptional regulator LuxR-AHL would bind Pluxi promoter and elicit the transcription of downstream gene. Apart from this inducible expression, the QS-based “ON to semi-OFF” expression was realized by using pluxIR and pET-Pluxi:aiiA, where the aiiA gene was inserted downstream of the Pluxi promoter on vector pET-Pluxi. Since pluxIR was used to express the LuxR-AHL complex which in turn initiated the transcription of aiiA on vector pET-Pluxi:aiiA, the resulting AiiA would repress the transcription of Pluxi promoter-driven luxi and aiiA due to AiiA-mediated AHL degradation, therefore leading to “OFF” state. Notably, this “OFF” state was transient because AiiA expression was rapidly turned off. When AiiA concentration decreased to a threshold at which AHL could not be completely degraded, QS was then turned on and gene expression was restarted. The above architecture was designated as “ON to semi-OFF”. In such an architecture, the LuxR-AHL complex activated gene transcription and thereby led to constant “ON” state (Fig. 1). By contrast, the combination of LuxR-AHL and AiiA triggered not only AHL synthesis but also its degradation, leading to “ON to semi-OFF” state (Fig. 1).
This engineered system would dynamically control the expression of gene downstream of Pluxi promoter on vector pET-Pluxi or the gene between Pluxi promoter and aiiA gene on vector pET-Pluxi:aiiA where the transcription initiation of target gene is earlier than aiiA gene. RBS was inserted upstream of aiiA to allow aiiA and target gene sharing a cassette. To test the two engineered QS circuits, eGFP was employed as the reporter (Fig. 1, strain A and B). To confirm the QS circuits, egfp was replaced by ldhA (Fig. 1, strain C and D). To exclude the influence of native ldhA gene on QS circuits, it was deleted from E. coli genome through λ Red recombination method. To do so, three vectors, viz, pkD13, pkD46, and pCP20 were used (Table S1). The vector, pKD13, was used as PCR template including FRT, kanamycin resistance gene, and homology arms of ldhA gene. After pkD46 and PCR products were electro-transformed into strains, the resulting colonies were screened by kanamycin plates. Next, vector pCP20 was transformed into positive clones, and kanamycin resistance gene was eliminated. Positive clones were screened by colony PCR, and ldhA mutant was acquired (Fig. 2B). All plasmids and mutants were confirmed by DNA sequencing. The primers used for PCR amplification are shown in Table S2.