Engineered E. coli W3110 equipped with T7RNAP
Three different designs of T7RNAP cassettes were constructed and integrated into the W3110 chromosome by traditional site-specific recombination at the attB sites of lambdoid coliphage HK022 (Fig. 1a). The first strain, denoted as W3110::IL5 and similar to BL21(DE3), contained a T7 gene 1 driven by PLacUV5 with lacI/lacO and an additional lacI genes located upstream of the promoter. Second strain was W3110::L5, in which additional lacI at upstream of the PLacUV5 promoter was not presented. The third strain, W3110::pI, was significantly different from the previous two strains because the T7 gene1 was driven by PLacI, which is widely used in iGEM (https://parts.igem.org/Part:BBa_R0010) (see detail sequence in Table S2). The newly created strains were verified by amplifying a characteristic fragment in each strain (Fig. 1b), and the results showed that all strains were successfully constructed.
The cell growth (Fig. 2a), LacZ and T7RNAP protein expressions (Fig. 2b) were compared among three engineered W3110 strains equipped with the T7RNAP. The biomass at the log-phase was slightly different between the condition with or without IPTG induction, while, at 12 h, the biomass was similar at the OD ranging from 2.2 to 2.5. Furthermore, to confirm the protein expression, SDS-PAGE was performed (Fig. 2b) and showed there were two distinct bands as comparing between the condition with or without IPTG. The identity of distinct proteins was detected by LC-MS/MS and shown in the Table 2, where a band near 100 kDa corresponded to T7RNAP, and the band between 100 to 135 kDa was identified as LacZ. Besides, the quantification was performed based on the ImageLab and shown in Table 3. Interestingly, LacZ expression was much higher in the W3110 than that in BL21(DE3) (Fig. 2b and Fig. S1). The enhancements were observed as 2.63-fold for W3110, 3.41-fold for W3110::IL5, 5.21-fold for W3110::L5, and 4.61-fold for W3110::pI (Table 3). Furthermore, the T7RNAP expression also reached up to 8.72-fold for W3110::IL5, 6.72-fold for W3110::L5 and 11.92-fold for W3110::pI, implying the promoters of PLacUV5 was stronger in W3110 than that in BL21(DE3) and the PLacI even the strongest promoter in W3110. Except for the promoter effect, the inserted locus was supposed to be the reason that T7RNAP expression was extremely higher than that in BL21(DE3) because the locus was at HK022 attB site in engineered W3110 strains from 1053856–1057711 bp, while that of BL21(DE3) was around the lac operon site (i.e., 360473–365652 bp) (Fig. 1a). With IPTG induction, maximum T7RNAP was observed in W3110::pI while the lowest one was occurred in the W3110::L5 (Table 3, T7RNAP). The feasibility of three strains used in protein expression and chemical production was further verified in the following.
Table 2
MS/MS analysis of T7 RNA polymerase and lacZ gene from engineered E. coli W3110.
No. | Protein | Accession no. | Score | Molecular weight (Mw, Da) | Isoelectric point (pI) |
1 | T7-like RNA polymerase | WP_001092355 | 955 | 98.79 | 6.77 |
2 | LacZ beta-galactosidase | WP_096183704 | 1036 | 116.42 | 5.28 |
Table 3
Quantification of relative protein of LacZ and T7RNAP by IPTG induction in BL21(DE3), W3110, W3110::IL5, W3110::L5, W3110::pI.
Relative protein amounts | BL21(DE3)* | W3110 | W3110::IL5 | W3110::L5 | W3110::pI |
LacZ | 1.0 | 2.63 | 3.41 | 5.21 | 4.61 |
T7RNAP | 1.0 | - | 8.72 | 6.72 | 11.92 |
*LacZ and T7RNAP in BL21(DE3) with IPTG is defined as the reference of W derivatives. |
Demonstrate lacI/lacO regulation in the new strains by sfGFP
Two expression vectors, the pET28a(+) plasmid with the orthogonal lacI/lacO repressor (Fig. 3a) and the pSU-T7 plasmid without repressor were used for evaluation (Fig. 3b). At first, lacI was included in the system to verify the lacI/lacO effect of engineered strains. As shown in Fig. 3c, there was no obvious florescence intensity in all strains without IPTG. Besides, when IPTG was induced, it displayed the similar level of sfGFP protein with the average fluorescence of 47,000 a.u. in three engineered W3110 strains, but lower expression in BL21(DE3) (i.e. 31000 a.u.). The sfGFP expression by the pSU-T7-sfGFP plasmid which lacked of lacI/lacO, the highest fluorescence intensity of 18,000 a.u. in W3110::pI without IPTG and the similar florescence intensity at 11,000 a.u. was observed in other strains (Fig. 3d). Interestingly, when IPTG was added, the specific fluorescence intensity of W3110::L5 was the highest with enhancement of 46%, 75% and 77% as compared to the BL21(DE3), W3110::IL5 and W3110::pI, respectively (Fig. 3d). As a result, lacI/lacO was the key component to regulate protein expression in W strains under IPTG induction.
Cas9 Expression And Characterization In Engineered W3110
Cas9 is a heterologous toxic protein derived from Streptococcus pyogenes and plays an important role in the type II CRISPR/Cas system. Two pET systems were used to express Cas9 protein: the pET21a(+) system, which includes an orthogonal lacI/lacO repressor (Fig. 4a), and the pET20b(+) system, which lacked both sequences (Fig. 4b). The protein analysis results for pET21a(+)-Cas9 showed that the protein was only produced in the presence of an inducer, and the order of protein content was BL21(DE3) ~ W3110::pI > W3110::L5 > W3110::IL5 (Fig. 4c). For pET20b(+)-Cas9, when the IPTG was absent, there were equal levels of protein expression in BL21(DE3), W3110::IL5, and W3110::L5, while that in W3110::pI was 2-fold higher than other three strains. After IPTG induction, the protein expression was increased or kept at similar level in all strains except for W3110::IL5. Among all combination of strains and conditions, W3110::pI with or without IPTG produced highest Cas9 protein, similar to those of BL21(DE3) and W3110::L5 with induction, (Fig. 4d).
To evaluate the functionality of the Cas9 protein produced from our engineered W3110 strain, an in vitro Cas9-RNP assay was applied. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, rbcL) was selected as our targeted template (i.e. 1.5 k) for digestion and the sgRNA was designed to slice the DNA into two fragments (i.e. 0.6 k and 0.9 k). The results of in vitro cleavage showed that the rbcL fragment was successfully divided into two desired fragments (Fig. 4e), demonstrating that all three engineered strains produced functional Cas9 successfully.
Carbonic Anhydrase Expression And Activity In Engineered W3110 Strains
In our previous report, BL21(DE3) overexpressing carbonic anhydrase (CA) showed the arrested cell growth as compared to that without CA expression [32]. Therefore, we considered whether W3110 strain would tolerate CA overexpression via pET32a-SyCA. At first, the result of biomass in BL21(DE3) has 30% reduction at 12 h which showed the toxicity to the cell; however, newly engineered W3110 could tolerate to the CA more robustly because the reduced biomass was only 25%, 2% and 18% for W3110::IL5, W3110::L5 and W3110::pI, respectively (Fig. 5a). Due to lacI/lacO regulation in the plasmid, IPTG induction was required for CA production. The CA activity was shown in Fig. 5b. In BL21(DE3), the CA activity increased from 803 WAU to 1100 WAU as IPTG increased from 0.005 mM to 0.01 mM and kept the similar level with 0.1 mM IPTG. The CA activity of W3110:IL5 was 1400 WAU with 0.005 mM IPTG and sharply decreased to 400 WAU at that with 0.1 mM IPTG. However, the W3110::L5 and W3110:pI expressed highest CA activity with 1710 WAU at 0.01 mM IPTG and 1795 WAU at 0.005 and 0.01 mM IPTG, respectively. Therefore, the best strain for production of CA is W3110::pI because it maintains the biomass and possesses the highest CA activity with extremely low IPTG.
Conversion of lysine to cadaverine by the engineered W3110 strains
It has been reported that the W3110 strain could tolerant to 20 g/L diaminopentane (DAP) [33]. Herein, the DAP toxicity test was performed in 4 strains by the ratio of viable cell which defined as the ratio of biomass between DAP addition and without addition. It showed that the cell would dramatically reduce after the DAP of 10 g/L was added, but W3110 possess higher survival rate than BL21(DE3), in which the viable cell percent was 32.1%, 34.0%, 42.8% and 36.6% for BL21(DE3), W3110:IL5, W3110::L5 and W3110::pI (Fig. S2). Afterwards, two CadA-containing plasmids with or without lacI/lacO (i.e. pET28a(+)-CadA and pSU-T7-CadA) were transformed into four strains and the in vivo DAP production was conducted to verify the function of lysine decarboxylase.
The IPTG concentration was varied in the four strains harboring pET28a(+)-CadA (Fig. 6). Similar levels of CadA proteins were observed for BL21(DE3), W3110::L5 and W3110::pI, in which the CadA was expressed at IPTG concentrations from 0.1 to 0.01 mM. More CadA was expressed with 0.1 mM IPTG than that with 0.01 mM IPTG in BL21(DE3) (Fig. 6b). However, CadA in W3110::IL5 was only over-expressed with 0.1 mM IPTG, and expressed with a critical low amount of protein by 0.01 mM IPTG (Fig. 6c). Furthermore, CadA could be expressed in a critical low IPTG (i.e., 0.001 mM) in W3110::L5 (Fig. 6d), and the highest expression occurred in W3110::pI with 0.01 mM IPTG (Fig. 6e). For the in vivo lysine production, lysine, IPTG, and PLP were added at 0.35 M, 0.1 mM, and 0.01 mM during the initial exponential phase (i.e., 3 h). All the strain possessed highest DAP production at 24 h as pET28a(+)-CadA was used. The highest lysine consumption for in BL21(DE3)(+) and W3110::IL5(+) was only 21.6 g/L and 18.13 g/L, respectively. However, the lysine could be more efficiently utilized in the W3110::L5 and W3110::pI with a 45 g/L and 32.41 g/L consumption of lysine. Therefore, the highest DAP production were obtained from W3110::L5(+) with 45.01 g/L lysine consumption, 32.2 g/L DAP, 1.34 g-DAP/L/h productivity and 91.73% yield at 24 h (Table 4).
Table 4
Biomass, lysine consumption, DAP titer, DAP productivity, yield of in vivo time-course with pET28a(+)-CadA plasmid and 50 g/L lysine in BL21(DE3), W3110::IL5, W3110::L5 and W3110::pI, respectively.
Strain* | Time (h) | Lysine consumption (g/L) | DAP Titer (g/L) | Productivity (g-DAP/L/h) | Yield (g/g, %) |
BL21(DE3)(+) | 6 | 9.07 ± 1.5 | 2.5 ± 0.1 | 0.42 ± 0.07 | 6.78 ± 1.17 |
12 | 9.40 ± 1.7 | 5.2 ± 1.0 | 0.43 ± 0.08 | 13.80 ± 2.50 |
24 | 21.60 ± 1.4 | 11.3 ± 1.3 | 0.47 ± 0.06 | 30.05 ± 3.95 |
W3110::IL5(+) | 6 | 9.14 ± 2.4 | 4.0 ± 1.5 | 0.67 ± 0.25 | 11.59 ± 2.09 |
12 | 16.67 ± 3.7 | 10.4 ± 1.9 | 0.87 ± 0.16 | 29.38 ± 1.80 |
24 | 18.13 ± 0.3 | 13.6 ± 1.6 | 0.57 ± 0.07 | 38.12 ± 5.89 |
W3110::L5(+) | 6 | 17.13 ± 1.8 | 11.8 ± 0.1 | 1.97 ± 0.02 | 33.62 ± 4.45 |
12 | 40.90 ± 2.8 | 29.0 ± 2.0 | 2.42 ± 0.16 | 82.46 ± 7.62 |
24 | 45.01 ± 1.2 | 32.2 ± 0.2 | 1.34 ± 0.01 | 91.73 ± 8.07 |
W3110::pI (+) | 6 | 14.50 ± 3.7 | 10.12 ± 1.1 | 1.69 ± 0.18 | 29.05 ± 2.51 |
12 | 28.45 ± 3.0 | 19.85 ± 2.1 | 1.65 ± 0.17 | 56.96 ± 3.05 |
24 | 32.41 ± 2.0 | 22.61 ± 2.4 | 0.94 ± 0.10 | 64.89 ± 1.01 |
*(+) means with IPTG induction. The errors represent the standard derivation of 3 independent experiments (n = 3). |
The protein expression in pSU-T7-CadA without lacI/lacO (Fig. 6f) was analyzed in four different strains. The results were similar to the Cas9 protein results, except for W3110::IL5; in addition, the leakage was lower in W3110::L5 than in BL21(DE3) and W3110::IL5 (Fig. 6g). The CadA expressions in W3110::pI(-) and (+) are similar, which indicated constitutive PLacI promoter was effective. For the pSU-T7-CadA-harbored strains as shown in Table 5, in vivo production of DAP by lysine consumption of all strains (except for W3110::IL5) reached 80% yield at 12 h, while DAP yield decreased until 24 h, mainly due to DAP would be further utilized in the metabolic pathway. The best condition was used W3110::pI(-) to obtain 36.9 g/L DAP, 3.08 g-DAP/L/h productivity and 103.4% yield by pSU-T7-CadA. We found out that it is reasonable that the yield value was higher than 100%. Because the lysine concentration in LB medium was approximately 1.61 g/L by HPLC analysis from the retention time (Fig S3a) and calibration curve (Fig. S3b). As comparing the results between pSU-T7-CadA and pET28a(+)-CadA, a significant enhancement in the lysine consumption rate existed by pSU-T7-CadA. The strains harboring the pSU-T7-CadA consumed up to 41.59, 37.35, 41.98 and 42.09 g/L lysine for BL21(DE3)(+), W3110::IL5(+), W3110::L5(+) and W3110::pI(+) at 24 h, while the lysine consumption of strains harboring the pET28(+)-CadA was only 21.6, 18.13, 45.01 and 32.41 g/ in the same strains. This manifested a more feasible strategy to apply the constitutive system (i.e. without the lacI/lacO regulation) in W3110 for chemical production due to higher chemical production rate and precursor consumption rate.
Table 5
Biomass, lysine consumption, DAP titer, DAP productivity, yield of in vivo time-course with pSU-T7-CadA plasmid and 50 g/L lysine in BL21(DE3), W3110::IL5, W3110::L5 and W3110::pI, respectively.
Strain | Time (h) | Lysine consumption (g/L) | DAP Titer (g/L) | Productivity (g DAP/L/h) | Yield (g/g, %) |
BL21(DE3)(+) | 6 | 24.21 ± 1.3 | 18.3 ± 1.2 | 3.05 ± 0.05 | 51.3 ± 1.2 |
12 | 38.94 ± 2.2 | 32.4 ± 2.5 | 2.70 ± 0.02 | 90.7 ± 3.2 |
24 | 41.59 ± 1.9 | 28.0 ± 1.5 | 1.17 ± 0.01 | 78.4 ± 4.3 |
W3110::IL5(+) | 6 | 16.56 ± 0.6 | 12.8 ± 0.5 | 2.13 ± 0.03 | 35.9 ± 2.9 |
12 | 31.14 ± 1.1 | 22.4 ± 1.1 | 2.60 ± 0.04 | 62.7 ± 1.6 |
24 | 37.35 ± 1.6 | 23.9 ± 0.9 | 1.00 ± 0.01 | 66.9 ± 1.8 |
W3110::L5(+) | 6 | 19.92 ± 1.0 | 15.4 ± 0.3 | 2.56 ± 0.02 | 43.1 ± 1.9 |
12 | 37.50 ± 2.4 | 29.3 ± 0.5 | 2.44 ± 0.05 | 82.1 ± 1.7 |
24 | 41.98 ± 1.4 | 24.9 ± 0.8 | 1.04 ± 0.01 | 69.7 ± 12.5 |
W3110::pI (+) | 6 | 32.51 ± 1.1 | 25.4 ± 1.6 | 4.24 ± 0.07 | 71.1 ± 3.2 |
12 | 40.22 ± 1.7 | 31.9 ± 2.0 | 2.66 ± 0.05 | 89.4 ± 3.5 |
24 | 42.09 ± 1.9 | 26.3 ± 1.9 | 1.10 ± 0.02 | 73.7 ± 4.2 |
W3110::pI (-) | 6 | 41.25 ± 1.1 | 36.7 ± 2.0 | 6.11 ± 0.11 | 102.8 ± 2.7 |
12 | 43.85 ± 1.5 | 36.9 ± 1.3 | 3.08 ± 0.09 | 103.4 ± 3.0 |
24 | 44.69 ± 0.9 | 31.4 ± 2.8 | 1.31 ± 0.02 | 88.0 ± 2.2 |
*(+) means with IPTG induction; (-) means without IPTG induction. The errors represent the standard derivation of 3 independent experiments (n = 3). |