Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory

Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter were powerful tools to mediate the protein expression. Moreover, Escherichia coli W3110 strain possesses more advantages than the B strain due to more heat shock proteins and higher tolerance to chemicals. Therefore, implementation of T7-based system in W3110 strain is a conceivable strategy to develop the cell factory. Three novel W3110 strains with chromosome-equipped T7RNAP (i.e W3110:IL5, W3110::L5 and W3110::pI) were engineered to demonstrate the feasibility on protein expression and chemical production. At rst, the LacZ and T7RNAP with IPTG induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green uorescence protein (sfGFP), Cas9, carbonic anhydrase (CA) and lysine decarboxylase (CadA). All the proteins were expressed higher and enzymatic functions were better in W3110::L5 and W3110::pI. Moreover, the highest cadaverine production, lysine consumption and the yield were obtained in W3110::L5(+) strain with pET28a(+)-CadA which reached 32.2 g/L, 45 g/L and 91.7% at 24 h, while the W3110::pI(-) strain with pSU-T7-CadA achieved 36.9 g/L, 43.8 g/L and 103.4% at 12 h which is unnecessary of inducer. Inducer and lacI/lacO regulators on chromosome and plasmid have been in W3110 strains with T7RNAP. The newly engineered W3110::L5 and W3110:pI both possessed similar protein expression compared to commercial BL21(DE3). Furthermore, among all strains, W3110::pI displayed the greatest potential as cell factory


Introduction
It is indispensable to develop the recombinant technology which enables enhanced enzyme expression to endorse high value chemicals production in recent years. The recombinant technology inspired the scienti c studies of enzymes, protein and protein interactions and the in vivo chemical production for industrial applications [1][2][3]. In addition, pharmaceutical and biotechnology companies applied recombinant technology to produce miscellaneous therapeutic proteins with the advantages of high output of the desired proteins and reduced cost due to the simpli ed puri cation processes. For example, recombinant insulin was the rst practical therapeutic protein produced by E. coli and approved by the Food and Drug Administration [4]. Moreover, approximately 30% recombinant proteins without The E. coli B strain and derivatives lacking Lon and OmpT protease empowers producing heterologous proteins without protease attack. One such derivative, BL21(DE3), infected by λDE3 lysogen and exposed to the T7 gene 1, possesses T7RNAP on the chromosome controlled by the P LacUV5 promoter and the nearby lacI/lacO orthogonal regulator [11]. P LacUV5 is a mutant promoter of the native lac promoter, with a low sensitivity to glucose [17]. The expression is regulated by the lacI repressor, which binds to the lac operator (lacO) in the absence of lactose. The repression can be removed by adding an inducer such as lactose or isopropyl-β-D-thiogalactopyranoside (IPTG) [18], resulting in target gene expression by the orthogonal T7 promoter. IPTG is frequently added to the T7 system due to its stability and effectiveness [19]; however, IPTG is expensive and uneconomic for industrial applications of low value-added products [20].
Alternatively, the K-12 strains such as MG1655 and W3110, are commonly used as they express more heat shock genes, are less sensitive to certain stress and also possess higher rates of glucose consumption [21][22]. A comparative proteomics study between BL21 and W3110 manifested that W3110 maintained growth and metabolism at lower oxygen levels, thus enabling foreign protein to be gradually expressed [23]. Therefore, W3110 strain has also been applied to produce various chemicals, including L-methionine [24][25], L-homoserine [26], and L-malate [27].
In this study, a powerful T7 system was equipped in W3110 strain to establish an alternative cell factory as a protein expression platform and microbial chemical production. First, T7RNAPs using different promoters, i.e., P LacUV5 promoter with or without additional LacI repressor and P LacI promoter, were inserted to the W3110 chromosome by a conditional replication, integration, and modular (CRIM) system [28] to develop three engineered W3110 strains. Then, the effect of lacI/lacO was explored by sfGFP as a proof-of-concept. To be a powerful cell factory, new engineered strains were applied to express Cas9 for gene editing, carbonic anhydrase (CA) for capture of carbon dioxide and lysine decarboxylase CadA for production of cadaverine (DAP) as a precursor of bio-nylon materials. We attempted to explore the brighter opportunity of W3110 strain as a new platform to express heterologous protein and produce chemicals.

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Bacterial strains and culture conditions The bacterial strains used in this study are listed in Table 1. The E. coli DH5α was used for plasmid construction, and BL21(DE3) was applied for gene expression, while W3110 was engineered to equip with a chromosome-based T7RNAP by HK022 phage attack site. All E. coli strains were cultivated in Luria-Bertani (LB) medium at 37 °C with constant shaking at 200 rpm. Antibiotics were added to the following nal concentrations as needed: ampicillin (100 µg/mL), kanamycin (50 µg/mL), and chloramphenicol (25 µg/mL). The pHK-L5T7-Km was constructed by PCR from pHK-IL5T7-Km with primers EcoRI-P lacUV5 -F and PstI-T7P-R, while plasmid of pHK-pIT7-Km was cloned by PCR from pDS3.0- HsdNC-T7RNAP with primers  EcoRI-PlacI-T7P-F and PstI-PlacI-T7P-R to obtain T7RNAP under different promoters. The primers used  are listed in Table S1.
Engineered Chromosome-based T7RNAP In W3110 Site-speci c recombination was applied to integrate T7RNAP into the W3110 genome. This process requires the CRIM plasmid as mentioned above, which includes an attP phage attachment site complementary to the attB phage attachment site on the genome, and a helper plasmid that includes integrase [28].

Construction Of Expression Vectors
The pET28a(+)-sfGFP plasmid was constructed by amplifying the sfGFP fragment from pSU-placI-sfGFP with primers NdeI-sfGFP-F and XhoI-sfGFP-R, followed by digestion with NdeI and XhoI and cloning into pET28a(+)-RFP, which was digested with the same digestion enzymes in advance. The pET20b(+)-Cas9 was constructed by digestion with XbaI and XhoI from pET21a(+)-Cas9 to obtain the cas9 fragment. The fragment was cloned into pET20b(+), which was digested by XbaI and XhoI in advance. The cadA fragment was ampli ed with HindIII-CadA-F and BglII-CadA-R from E. coli MG1655, digested with HindII and BglII, and inserted into the pSU-T7 backbone, creating the pSU-T7-CadA plasmid. The pSU-P LacUV5 -sfGFP was constructed by replaced the promoter of pSU-T7-sfGFP and the fragment of P LacUV5 was ampli ed from pHK-L5-Km with primers XbaI-L5-F and BamHI-L5-R. The PCR fragment was digested with XbaI and BamHI and cloned into pSU-T7-sfGFP. The pSU-P LacUV5 -NRBS-sfGFP was constructed by replaced the promoter and RBS of pSU-T7-sfGFP. The inserted fragment was ampli ed with primers XbaI-L5-F and HindIII-L5-R from pHK-L5-Km, further digested with XbaI and HindIII and cloned into pSU-T7-sfGFP. The pSU-P LacUV5 -LacZα'-sfGFP was constructed by inserted the fragment of P LacUV5 -LacZα' into the backbone of pSU-T7-sfGFP. The inserted fragment was ampli ed with XbaI-L5-F and HindIII-L5Z-R from pHK-L5-Km. All the plasmids are shown in Table 1.

Culture And Induction
For sfGFP protein and uorescence analysis, the samples were inoculated with 2% (v/v) pre-culture broth in 10-mL glass cultivation tubes and cultivated in a 37 °C incubator. IPTG was added to a nal concentration of 0.1 mM. For protein analysis of Cas9, the samples were inoculated with 2% (v/v) preculture broth in a 100-mL ask with 10 mL LB broth, and the desired antibiotic was added. When the cell density achieved an OD 600 of 0.5 to 0.8, IPTG was added, and transfer to cultivate in a 22 o C incubator for 12 h. The cultivation for the activity measurement of SyCA, 20 mL LB broth was inoculated with 2% (v/v) pre-culture broth in a 100-mL ask. As OD 600 reached 0.6 to 0.8, IPTG was added at three different concentrations of 0.005, 0.01 and 0.1 mM, and 0.5 mM zinc ions (supplied by ZnSO 4 ) were also added.

Measurement Of Cell Growth And Fluorescence Intensity
The cell growth was monitored by absorbance at 600 nm (OD 600 ) and the uorescence intensity was detected by a SpectraMax M2 microreader (Molecular Device, USA) with excitation at 480 nm and emission at 510 nm. All experiments were performed in triplicates.

Protein Expression, Quanti cation And Identi cation Analysis
The recombinant protein expression was analyzed by 8%, 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After a 12-h culture, the cells were harvested by centrifugation (12000 × g, 3 min). The cell concentration was adjusted to OD 4, and the cells were suspended in dH 2 O. The supernatant and cell debris were separated by centrifugation after the whole cells were degraded by a One Shot instrument (Constant Systems, UK). 1D electrophoretic protein separation was achieved by following a standard SDS-PAGE protocol (80 V, 30 min; 120 V, 70 min). Afterwards, the gel was stained with Coomassie brilliant blue G-250 dye for visualization. ImageLab® (Bio-rad) adopted from a Gaussian model was used in nding differentially expressed proteins of SDS-PAGE [30]. The target protein was sent for LC-Q-TOF mass spectrometer (Applied Biosystems, Lincoln, In vitro assay of Cas9-ribonucleoprotein (Cas9-RNP) Double-stranded DNA sgRNA was synthesized via overlap PCR to combine the crRNA fragments and the scaffold. The sgRNA was synthesized by in vitro transcription with double-stranded DNA sgRNA as the template, 10x transcription buffer, rNTP solution mixture, and T7 RNA polymerase. The crude sgRNA was puri ed by the phenol-chloroform method. Crude Cas9 protein was puri ed by His-trap metal-ion a nity chromatography with an AKTA™ start machine (GE Healthcare, UK). Imidazole was removed from the puri ed Cas9 protein by Amicon®100-kDa ultra ltration (Merck, USA). The Cas9-RNP was formed by mixing equal molar ratios of puri ed Cas9 and sgRNA in 5x reaction buffer (100 mM HEPES, pH 7.5,  Three different designs of T7RNAP cassettes were constructed and integrated into the W3110 chromosome by traditional site-speci c recombination at the attB sites of lambdoid coliphage HK022 (Fig. 1a). The rst strain, denoted as W3110::IL5 and similar to BL21(DE3), contained a T7 gene 1 driven by P LacUV5 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 P LacUV5 promoter was not presented. The third strain, W3110::pI, was signi cantly different from the previous two strains because the T7 gene1 was driven by P LacI , which is widely used in iGEM (https://parts.igem.org/Part:BBa_R0010) (see detail sequence in Table S2). The newly created strains were veri ed 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 con rm 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 identi ed as LacZ. Besides, the quanti cation 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 P LacUV5 was stronger in W3110 than that in BL21(DE3) and the P LacI 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 veri ed in the following. 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 rst, lacI was included in the system to verify the lacI/lacO effect of engineered strains. As shown in Fig. 3c, there was no obvious orescence intensity in all strains without IPTG. Besides, when IPTG was induced, it displayed the similar level of sfGFP protein with the average uorescence 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 uorescence intensity of 18,000 a.u. in W3110::pI without IPTG and the similar orescence intensity at 11,000 a.u. was observed in other strains (Fig. 3d). Interestingly, when IPTG was added, the speci c uorescence 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 rst, 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 de ned 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 overexpressed 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 e ciently 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. 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 P LacI promoter was effective. For the pSU-T7-CadAharbored 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). 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.

Discussion
Tunable protein expression is crucial for synthetic and system biology. One of the powerful tools is the T7RNAP and its orthogonal T7 promoter. T7RNAP originating from the bacteriophage T7 elongates the RNA at a rate approximately 5-fold faster than that of E. coli native RNA polymerase with speci c recognition for the T7 promoter [34]. Therefore, the T7RNAP-mediated protein expression system was rst applied in BL21(DE3) and showed that the chromosome-based T7 gene 1 is more suitable for toxic gene expression from plasmids by preventing instability from the strong orthogonality of T7 system when compared to the plasmid-driven T7 gene 1 [35]. W3110 is one of the eminent E. coli strain and has been reported with high capability of withstanding different toxic chemicals [21][22]. Consequently, W3110 is a suitable strain to be equipped with the T7RNAP onto the chromosome. For examples, Liu and his colleagues has constructed the W3110(DE3), which entirely encompasses the same genetic design of BL21(DE3), to produce D-xylonic acid [36]. In this study, we provide three new W3110 strains equipped with different cassette of the T7RNAP as W3110::IL5, W3110::L5 and W3110::pI, which is achieved by the HK022 site-speci c recombination, and displays its feasibility to produce heterologous protein and chemical production. Besides, the IPTG and lacO/lacI effect was further been elucidated.
From our results, W3110 showed a strong lac operon because the lacZ was overexpressed than that in BL21(DE3) when IPTG was expressed (Table 3), which was the rst observation in our best knowledge.
After addition of the T7RNAP cassette, the LacZ was further increased in expression, which was supposed that the partition of lacI from the lacZ to T7RNAP enhanced the lacZ expression. LacI partition has already been proven to occur and applied in different applications. Cranenburgh and his coworkers developed a plasmid stabilization system, in which the plasmid containing multiple lacO sequence must be maintained in the cell to compete the lacI binding to prevent the binding to an essential gene [37]. On the other hand, in our design, the T7RNAP was immensely overexpressed in W3110 than that in BL21(DE3), which was elucidated by the insertion locus. Actually, the expression of insertion gene will be in uenced by the surrounding DNA context which may contain a promoter, RBS or even a terminator to in uence the down-stream gene expression [38]. In such context, it has been supposed that the wield behavior of W3110::IL5 with higher T7RNAP than that in W3110::L5 was also contributed by the surrounding DNA context to in uence the lacI expression, which further affect the T7RNAP expression. Moreover, recently, the chromosome-base gene expression has been detailed elucidated in the aspect of the gene locus and it shown that the gene expression level must be determined by the easiness to relocate the gene to the nucleoid periphery [39], which further supported our explanation.
For the different proteins expression, by using a lacI/lacO containing plasmid, it shows that all the expression could only occur as IPTG exists, while, as the plasmid without lacI/lacO was utilized, the expression could both be observed in absence or presence of IPTG. Additional lacI in plasmid (i.e. pET plasmid, except for pET20b) could repress the T7RNAP expression tightly to reduce the leakage expression [6]. As the lacI/lacO was not encompassed in the plasmid (i.e. pSU or pET20b), the leakage expression is observed, but the protein expression with induction was still higher than that without induction in BL21(DE3), W3110::IL5 and W3110::L5, which is reasonable because the lacI on the chromosome could still su ciently repress the P LacUV5 with native lac RBS to express T7RNAP.
Intriguingly, in pSU plasmid series, W3110::pI could express similar amount of heterologous protein with or without IPTG, which was attributed to the highly leakage level of T7RNAP. Actually, to establish a tightly regulated chromosome-based gene expression is intricate due to the di culty to well-balance the promoter strength, lacI and lacO amount [40]. Besides, with the similar promoter strength of P LacI and P LacUV5 [6], the strength of RBS dominates the leakage expression of T7RNAP, where the B0034 RBS was extremely stronger than the lac native RBS (Fig. S4).The in vivo DAP production was also affected and displayed higher DAP yield in a short-term transformation (i.e. 6 and 12 h) as the plasmid without lacI/lacO was used, while the time used for highest production by plasmid with lacI/lacO must be extended to 24 h.
IPTG dose response is another point that have to be considered in an inducible system as pET with lacI/lacO was used [41]. In our results, W3110::IL5 must at least be induced by 0.1 mM to obtain the enough recombinant protein, while W3110::L5 and W3110:pI only used 0.01 mM to induce su cient protein amount. Even with the similarly high expression of recombinant protein, the solubility would further affect the function of the recombinant protein [42], supporting the CA result where the optimal activity was occurred with 0.01 mM IPTG in the three engineered W3110 strains. Furthermore, the high orthogonality of T7 system was reported as an energy-intensive process [35], leading to less energy in folding the recombinant protein [43]. The in vivo production of DAP by W3110::pI harboring pSU-T7-CadA produced higher DAP without induction, primarily due to the cellular energy was more concentrated for lysine and DAP transportation as well as PLP regeneration [44][45].

Conclusion
In this study, the characterization of three engineered W3110-equipped with T7RNAP was accomplished as well as the difference of the lacI/lacO regulation between W3110 and BL21(DE3) strains was proposed. Among all, the most robust one, W3110::pI strain with a T7RNAP driven by the promoter P LacI , enables effective, stable and constitutive production of several recombinant proteins and chemicals while strain W3110::L5 showed the similar capacity to produce the recombinant protein, but higher capacity to produce chemical as compared to the commercial BL21(DE3). Even though the W3110::IL5 does not behave e ciently as the other strains, it provides the new insights into the difference of Lac operon between B and W derivatives. Therefore, we not only provided a cost-effective, robust and novel engineered W3110 strain as a cell factory for recombinant technology, but also presented more understandings of different E. coli strains.
Declarations Figure 1 Scheme diagram of the engineered W3110 strain with chromosome-equipped T7RNAP (a) genetic design of engineered T7RNAP in E. coli W3110. Integration was achieved by site-speci c recombination at the HK022 attB site. Three engineered T7RNAP expression strains were created. The strain W3110::IL5 has a

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