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The present invention relates to a recombinant plasmid for manufacturing bio-based materials. In particular, it relates to recombinant plasmid including a newly developed promoter system for manufacturing bio-based materials.
A promoter is a cis-acting DNA sequence recognized by RNA polymerase (RNAP) for subsequent transcription. Promoter activity often involves the interaction of the promoter with RNAP and regulatory factors, including regulatory proteins (activator and repressor) and effector molecules (inducer and corepressor). Promoter activity can be quantified by the probability of promoter occupancy by RNA polymerase based on thermodynamic models, and the probability of promoter occupancy by RNAP being proportional to the gene expression is one of the most important assumptions in these models. Transcription regulation can be negative inducible or negative repressible when the repressor protein cooperates with inducers or corepressor, respectively. Additionally, the transcription regulation can be positive inducible or positive repressible when the activator protein cooperates with inducers or corepressors, respectively. Transcriptional regulation usually occurs at the transcription binding and initiation stages.
Bacillus anthracis is a gram-positive, aerobic, and spore-forming bacillus. B. anthracis, which carries two virulence plasmids, pXO1 (182 kb) and pXO2 (95 kb), is a virulent strain. The first mega plasmid pXO1 carries three virulent genes pagA, lef, and cya [encoding cell-binding protective antigen (PA, 85 kDa), lethal factor (LF, 83 kDa), and edema factor (EF, 89 kDa)]. A pair of PA and EF proteins forms an edema toxin and that of PA and LF proteins forms a lethal toxin. The mega plasmid pXO2 carries the capBCADE operon for the production of the antiphagocytic poly-D-glutamic acid capsule, protecting the pathogen from phagocytosis. The secretion of toxins and capsules from B. anthracis is considered critical to protect it against the host immune system and to cause infection. The expression of these virulence genes is highly regulated at the transcriptional initiation level by a class of regulators called phosphoenolpyruvate-dependent phosphotransferase regulation domain containing virulence regulators (PCVRs).
Further, anthrax toxin activator (AtxA, encoded on pXO1) is a major positive regulator of anthrax toxin and capsule expression. Thus, it is a potential target for developing therapeutics for anthrax infection. AtxA is a PCVR that contains two helix-turn-helix (HTH) domains at the N-terminus, two phosphotransferase system regulation domains (PRDs), and one EIIB-like domain at the C-terminus. It has been suggested that the homodimeric state of AtxA is the active structure for regulation, and the dimerization of AtxA is positively promoted by a high CO2/bicarbonate level, dephosphorylation of the EIIB domain and H379 in PRD2. Furthermore, the phosphorylation of H199 in PRD1 is essential for DNA binding. All three functional domains of AtxA are commonly found in PCVR; nevertheless, the molecular mechanisms of AtxA in regulating toxin expression are not fully clear.
The pagA gene has two promoters, P1 and P2. The transcription start sites (TSSs) of P1 and P2 transcripts are −58 and −26 relative to the translation initiation codon site of PA. P1 is regulated by AtxA and the 90 base pair (bp) DNA region upstream of the P1 TSS is sufficient for AtxA-dependent transcription regulation. The sequence of the 90-bp DNA region upstream of the P1 TSS is high in AT % and is predicted to form an intrinsic ATrich curvature structure. The AtxA specific regulation is DNA-structure specific rather than sequence-specific. In some researches, it was further demonstrated that (−105)-(−67) upstream of the P1 TSS is an essential cis-acting site where the region forms a stem loop structure (and thus named as SLII). The specific interaction between AtxA and SLII is thought to be structure-specific rather than sequence-specific. A second curvature is predicted 30 bp downstream of the P1 TSS, and it has been shown that the second curvature is not necessary for AtxA-dependent transcription regulation. Some research have been reported for AtxA-dependent transcription; however, the exact sequence of the P1 promoter has not been fully elucidated. In contrast, P2 is thought to be a constitutive and relatively weak promoter. The −10 and −35 sequences have been suggested, despite the fact that the predicted −35 sequence, TTCCCA, of P2 differs from the consensus sequence, TTGACA and the space of 20 bp between the predicted −10 and −35 sequence is not optimal. A recent RNA-seq analysis disclosed that transcripts starting from P2 are abundant.
The above information disclosed in this section is only for enhancement of understanding of the background of the described technology and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art.
In the present invention, a 509-bp DNA fragment [(−508)-(+1) relative to the P2 TSS), and termed 509 sequence in the present invention], containing P1 and P2, was cloned upstream of rbs-GFPuv to elucidate the AtxA-based transcription regulation. First, the 509 sequence was dissected into five genetic elements: −10 sequence, −35 sequence, Airier, tract, SLI/SLII, and the upstream site (
To achieve aforementioned effect, the present invention provides a recombinant plasmid for manufacturing bio-based materials, comprising: a promoter system; and a rbs′-synthetic gene cluster, wherein the promoter system comprises a Pribnow box and a transcriptional activator.
In one embodiment of the present invention, the method of producing the promoter system comprises: (a) inserting a 509seq (SEQ ID NO: 29) into pET29a-EGFP to form a plasmid pTOL02A; (b) replacing EGFP gene of pTOL02A with the GFPuv gene to form the pTOL02B; (c) deleting a SLI/SLII from the plasmid pTOL02B to form a plasmid pTOL02C; (d) subjecting the plasmid pTOL02C to site-directed mutagenesis deletion to form a plasmid pTOL02F; and (e) inserting the transcriptional activator into the plasmid pTOL02F to form a plasmid pTOL03F.
In one embodiment of the present invention, comprising replacing an rbs-GFPuv of the plasmid pTOL03F with the rbs′-synthetic gene cluster, and the rbs′-synthetic gene cluster comprises a rbs′-phaCAB gene cluster.
In one embodiment of the present invention, the site-directed mutagenesis deletion of the step (d) deletes −35, ATrich tract, and an upstream site of the plasmid pTOL02C.
In one embodiment of the present invention, the transcriptional activator is an AtxA operon.
In one embodiment of the present invention, the AtxA operon is a PT7-rbs-atxA-T7 terminator.
In one embodiment of the present invention, the bio-based materials comprises polyhydroxybutyrate.
In one embodiment of the present invention, an activity of the promoter system is regulated by the transcriptional activator, a Pribnow box, and a cognate RNAP.
Many of the attendant features and advantages of the present invention will become better understood with reference to the following detailed description considered in connection with the accompanying drawings.
The detailed structure, operating principle and effects of the present invention will now be described in more details hereinafter with reference to the accompanying drawings that show various embodiments of the present invention as follows.
Reference will now be made in detail to the exemplary embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Therefore, it is to be understood that the foregoing is illustrative of exemplary embodiments and is not to be construed as limited to the specific embodiments disclosed, and that modifications to the disclosed exemplary embodiments, as well as other exemplary embodiments, are intended to be included within the scope of the appended claims. These embodiments are provided so that this invention will be thorough and complete, and will fully convey the inventive concept to those skilled in the art.
For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this invention belongs.
Various embodiments will now be described more fully with reference to the accompanying drawings, in which illustrative embodiments are shown. The inventive concept, however, may be embodied in various different forms, and should not be construed as being limited only to the illustrated embodiments. Rather, these embodiments are provided as examples, to convey the inventive concept to one skilled in the art. Accordingly, known processes, components, and techniques are not described with respect to some of the embodiments.
The singular forms “a”, “and”, and “the” are used herein to include plural referents unless the context clearly dictates otherwise.
The biological materials used in the present invention need not be deposited according to 37 CFR 1.802 since those biological materials are known and available to the public, or can be made, or isolated without undue experimentation.
It should be noted that, the product in the present invention includes, but not limited to polyhydroxybutyrate. Any bio-based materials that can be theoretically produced by the process of the present invention can be the products in the present invention. Similarly, rbs′-phaCAB gene cluster used in the present invention is merely one kind of the gene cluster to replace an rbs-GFPuv, and any other synthetic gene cluster that is suitable for the process of the present invention can replace phaCAB in the present invention.
The following descriptions are provided to elucidate a method of creating a recombinant microorganism for manufacturing a fermentation product and to aid it of skilled in the art in practicing this invention. These embodiments are merely exemplary embodiments and in no way to be considered to limit the scope of the invention in any manner.
Material and Method
Construction of pTOLAtxA, pCDFDuet-533seq, and pTOL02A
The method of constructing pTOLAtxA, pCDFDuet-533seq, and pTOL02A in the present invention is as follows. The recombinant pTOLAtxA was constructed by inserting the PCR-generated atxA fragment (NC_007322.2) into pCDFDuet-1 at Ndel and Xhol sites. The primers dlc-F-AtxA-01 (SEQ ID NO: 1) and dlc-R-AtxA-01 (SEQ ID NO: 2) were used for generating atxA fragment. The sequences of primers used in the present invention are shown in Table 2. The recombinant pCDFDuet-533seq was constructed by inserting the PCR-generated 533-bp DNA fragment [(−508)-(+25) relative to the P2 TSS)] (SEQ ID NO: 28) to pCDFDuet-1 at Ndel and Xhol sites. The primers dlc-F-cisacting-01 (SEQ ID NO: 3) and dlc-R-cisacting-01 (SEQ ID NO: 4) were used for generating the 533-bp DNA fragment. The recombinant pTOL02A was constructed by inserting the PCR-generated 509-bp DNA fragment [(−508)-(+1) relative to the P2 TSS)] (SEQ ID NO: 29) into pET29a-EGFP backbone. The vector backbone was generated by PCR using pET29a-EGFP as the template and primers of SLIC-F-pET29a-GFP-01 (SEQ ID NO: 5) and SLIC-R-pET29a-GFP-01 (SEQ ID NO: 6). The insert 509-bp DNA fragment was generated by PCR using pCDFDuet-533seq as the template and primers of SLIC-F-cisacting-02 (SEQ ID NO: 7) and SLIC-R-cisacting-02 (SEQ ID NO: 8). The sequences of aforementioned primers are list in Table 2.
Construction of pTOL02 and pTOL03 Series
To construct pTOL02B, the DNA fragment encoding full-length GFPuv was amplified from pDSKGFP. with primers SLIC-F-GFP.-01 (SEQ ID NO: 9) and SLIC-R-GFP.-01 (SEQ ID NO: 10), and the vector fragment was amplified from pTOL02A, a pET29a-based backbone, with primers SLIC-F-pTOL02A-01 (SEQ ID NO: 11) and SLIC-R-pTOL02A-01 (SEQ ID NO: 12). The assembly of two fragments was achieved by one-step sequence- and ligation-independent cloning (SLIC), which is a conventional technology. In brief, the vector and insert fragments in deionized water were mixed at molar ratios of 1:1. One microliter of T4 DNA polymerase (New England BioLabs Inc., MA, USA) was added to 10 μL of the vector/insert mixture and incubated at room temperature for 2.5 min. The reaction mixture was immediately placed in an ice bath for 10 min to inhibit the nuclease reaction while facilitating annealing. This mixture was directly used for the bacterial transformation. pTOL02C was obtained by deleting SLI/SLII from pTOL02B using the Q5 ® Site-Directed Mutagenesis Kit (New England BioLabs Inc., MA, USA) with primers DL-F-SLI/II-01 (SEQ ID NO: 13) and DL-R-SLI/II-01 (SEQ ID NO: 14). The linearized pTOL02B fragment containing the SLI/SLII deletion mutation was generated by polymerase chain reaction (PCR) with pTOL02B as the template and DL-F-SLI/II-01 (SEQ ID NO: 13) and DL-R-SLI/II-01 (SEQ ID NO: 14) as the primer set. Then, 1 μL of PCR product was mixed with the kinase, ligase, and Dpnl (KLD) enzyme mixture (New England BioLabs Inc., MA, USA) to circulize the linearized DNA fragment while removing the pTOL02B template.
pTOL02D and pTOL02E were constructed by substitution mutations of pTOL02B and pTOL02C, respectively, using the Q5 ® Site-Directed Mutagenesis Kit (New England BioLabs Inc., MA, USA) as described above. The primer sets of SS-F-ATGC average-01 (SEQ ID NO: 15)/SS-R-ATGC average-01 (SEQ ID NO: 16) and SS-F-ATGC average-01 (SEQ ID NO: 15)/SS-R-ATGC average-02 (SEQ ID NO: 17) were used for pTOL02D and pTOL02E constructions, respectively. The ATrich tract (ATTATCTCTTTTTATTTATATTATA, 92% AT) in pTOL02B and pTOL02C was replaced with a random-sequence DNA fragment, ATCGavg (AAGCTTAGAGGATCGAGATCGATCT, 56% AT).
The recombinant plasmid pTOL02F was prepared by subjecting pTOL02E to site-directed mutagenesis deletion (Q5 ® Site-Directed Mutagenesis Kit, New England BioLabs Inc., MA, USA) with primers DL-F- −10 upstream-01 (SEQ ID NO: 18)/DL-R- −10 upstream-01 (SEQ ID NO: 19), so that −35 and the upstream site was removed. The resulting pTOL02F contained 4% of the 509 sequence and only the −10 sequence was retained.
The pTOL03 series was obtained by combining the synthetic atxA operon (PT7-rbs-atxA-T7 terminator) in the pTOLAtxA with pTOL02 series. The synthetic atxA operon (1,953 bp) was obtained by PCR using primers HF-F-AtxA-01 (SEQ ID NO: 20)/HF-R-AtxA-01 (SEQ ID NO: 21), and the pTOL02 backbone was obtained by PCR with primers HF-F-pTOL02system-01 (SEQ ID NO: 22)/HF-R-pTOL02system-01 (SEQ ID NO: 23). The synthetic atxA operon and the pTOL02 backbone were assembled using the NEBuilder HiFi DNA Assembly Kit (New England BioLabs Inc., MA, USA).
pTOL03FphaCAB was obtained by replacing the rbs-GFPuv in pTOL03F with rbs′-phaCAB gene cluster of C. manganoxidans. The rbs′-phaCAB gene cluster (3,725 bp) was obtained by PCR using primers HF-F-phaCAB-01 (SEQ ID NO: 26)/HF-R-phaCAB01 (SEQ ID NO: 27), and the pTOL03F backbone was obtained by PCR with primers HF-F-pTOL03system-01 (SEQ ID NO: 24)/HF-R-pTOL03system-01 (SEQ ID NO: 25). The rbs' phaCAB gene cluster and the pTOL03F backbone were assembled using the NEBuilder HiFi DNA Assembly Kit (New England BioLabs Inc., MA, USA). The rbs' sequence is the native rbs sequence of phaCAB operon in C. manganoxidans.
Recombinant plasmids were constructed and maintained in E. coli DH5α. Plasmid DNA was isolated from E. coli was by using the Miniprep Purification Kit (QIAGEN, Hilden, Germany) Electroporation (Bio-Rad Gene Pulser, Bio-Rad, California, USA) was used for transformation.
All the plasmids used in the present invention, and the sources are listed in Table 1 as follows.
Bacillus anthracis) under the control of T7 promoter,
Bacillus anthracis], where the 533-bp DNA fragment
One milliliter of bacterial solution was collected and centrifuged at 17,000×g for 3 min at 4° C. The supernatant was decanted and the bacterial pellet was washed three times with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·2H2O, 2 mM KH2PO4) at 4° C. Finally, bacterial pellet was resuspended in 1-mL of iced-cold PBS for subsequent fluorescence measurements (F-2500, HITACHI, Tokyo, Japan). The excitation, emission, excitation slit, emission slit, and photomultiplier voltage were set at 395 nm, 507 nm, 5 nm, 5 nm, and 700 V, respectively. 1 mm fluorescence cuvette was used.
PHB Production in E. coli BL21P/pTOL03FphaCAB
25 ml of fresh LB media in a 250 ml glass flask was inoculated with an overnight culture of E. coli BL21P/pTOL03FphaCAB to reach an initial 0.D. 600 of 0.05. After 6-h cultivation, sterilized glucose and IPTG were added to the final concentration of 10 g/L and 15 nM, respectively. The pH of bacterial culture was measured at 12, 24,36, and 48 h and adjusted to 7 with 2 N HCl or 2 N NaOH when needed. Note that the genotype of E. coli BL21P is E. coli BL21(DE3)ΔptsG.
The gas chromatography (GC) method was used for PHB quantification. Briefly, freeze-dried cell (ca. 20 mg), obtained from the bacterial culture solution, was transferred to a clean spiral test tube and 1 mL of chloroform, 0.85 mL of methanol, and 0.15 mL of sulfuric acid were added. The tube was incubated in a water bath for 140 min at 80° C. and cooled down to room temperature. Then 1 mL of DI H2O was mixed well by vortexing. After standing and layering, the organic phase was removed and filtered through a 0.2-nm PVDF filter and then analyzed by GC. The temperatures of the injector and detector were 230° C. and 275° C. respectively. The temperature of the column was set at 100° C. and increased to 200° C. at a rate of 10° C./min and maintained at 200° C. for 2 min. The PHB standard (363502) was purchased from Sigma-Aldrich. The PHB content was calculated as follows: PHB concentration (g/L)/biomass concentration (g/L)(%), where the biomass concentration was calculated by dividing freeze-dried cell weight by the culture volume for preparing freeze-dried cell.
AtxA is a Positive Regulatory Activator of E. coli Transcription
To examine the effect of AtxA on GFPuv expression in E. coli, pTOL02B and pTOLAtxA were co-introduced into E. coli BL21(DE3). As shown in
First, SLI and SLII were removed from pTOL02B to obtain pTOL02C. The Airier, tract in pTOL02B was replaced with a DNA fragment (ATCGavg) that had the same length as a random sequence (56% AT, 14/25 bp,
As shown in
Removal of the Upstream Site and −35 Sequence Revealed that AtxA Regulated Transcription E. coli is Independent of P1 and P2
The effect of the upstream site and −35 sequence on AtxA regulated transcription was investigated by constructing pTOL02F. As shown in
The strong expression of GFPuv in BL21(DE3)/pTOL02F+pTOLAtxA indicated that the AtxA regulated transcription involved three basic elements, which were AtxA, −10 sequence (TATA box), and E. coli cognate RNAP. The present invention further constructed pTOL03F by inserting the PT7-rbs-AtxA-ter operon into pTOL02F to verify the minimal elements for AtxA-regulated transcription in E. coli.
AtxA-Regulated Transcription in E. coli is Stationary-Phase Specific
The cis-acting element required for AtxA-dependent GFPuv expression simply consists of a −10 sequence, but no −35 sequence in E. coli. It can be concluded that AtxA-regulated transcription is independent of housekeeping σ70. In fact, the −10 sequence (TATACT) exactly matched that of as-dependent promoter, also known as a type III promoter, which requires only the −10 sequence (TATACT) for transcription. The sigma factor σs is the starvation/stationary-phase sigma factor, and it is also known as a global regulator for responding to stress conditions, such as hyperosmolarity. The present invention demonstrated that AtxA-regulated transcription was stationary-phase specific and was suggested to be as-dependent.
To explore the effects of hyperosmotic conditions on the growth behavior and FI/OD600 of E. coli, the growth behavior and FI/OD600 of E. coli BL21(DE3)/pTOL03D were investigated in presence of 0.17, 0.30, and 1.00 M NaCl. As shown in
The stationary-phase specific protein expression was supported by investigating the AtxA induction time. When the supplementation of 0.03 mM IPTG for AtxA expression was supplemented at 2 and 3h, which is the time when E. coli BL21(DE3)/pTOL02D+pTOLAtxA reached the late-log-phase/stationary-phase transition stage (
AtxA-regulated transcription was subsequently tested for its background expression by cultivating E. coli BL21(DE3)/pTOL03E and E. coli BL21(DE3)/pTOL03F without IPTG supplementation. Table 4 shows that both pTOL03E and pTOL03F had low FI/OD600 of 155±1 and 333±10, respectively. Thereafter, the FI/OD600 of both strains were investigated to determine the dependence on IPTG concentration. The FI/OD600 of pTOL03E had a linear relationship with the IPTG concentration and reached a saturation value of 44,121±247 at 200 μM (
E. coli BL21(DE3)/pTOL03F, and E. coli BL21(DE3)/pDKS-GFPuv.
333 ± 10
98 ± 2
22,878 ± 3,742
PHB production in E. coli BL21P/pTOL03Fpha CAB
To achieve the stationary-phase PHB production, E. coli BL21P/pTOL03phaFCAB was first aerobically cultivated in LB for 6 h, followed by the supplementation of glucose and IPTG to reach the concentrations of 10 g/L and 15 μM, respectively. It was shown in
In the present invention, a 509-base pair DNA fragment [termed 509sequence, (−508)-(+1) relative to the P2 transcription start site] was cloned upstream of rbs-GFPuv as pTOL02B to elucidate the AtxA regulated transcription. The 509sequence was dissected into the −10 sequence, −35 sequence, ATrich tract, SLI/SLII, and the upstream site. In conjunction with the heterologous co-expression of AtxA (under the control of the T7 promoter), the −10 sequence (TATACT) was sufficient for AtxA-regulated transcription. Integration of pTOL02F+pTOLAtxA as pTOL03F showed that the AtxA-regulated transcription exhibited a strong specific fluorescence intensity (FI)/0D600 of 40,597±446 and an induction/repression ratio of 122. An improved induction/repression ratio of 276 was achieved by cultivating Escherichia coli/pTOL03F in M9 minimal medium. The newly developed promoter system termed PAtxA consists of AtxA, the −10 sequence, and Escherichia RNA polymerase (RNAP). These three elements synergistically and cooperatively formed a previously undiscovered transcription system termed the promoter three-hybrid (P3H−10, AtxA), which exhibited a tight-control, high-level, modulable, and stationary-phase specific transcription. The PAtxA was used for phaCAB expression for the stationary-phase polyhydroxybutarate production and the results showed that a PHB yield, content, and titer of 0.20±0.27 g/g-glucose, 68±11%, and 1.5±0.4 g/L can be obtained. The positive inducible PAtxA, in contrast to negative inducible, should be a useful tool to diversify the gene information flow in synthetic biology.
It's widely known that the transcription of pagA (encoding PA toxin protein) in B. anthracis has two promoters, P1 and P2. It has been considered that the P1 promoter is subjected to AtxA regulation while the P2 promoter is constitutive. Some related arts have been conducted to elucidate the AtxA regulation mechanism, including biochemical studies of AtxA and identification of sequence- and/or structure-specific cis acting sites. Nevertheless, the complexity of pagA transcription makes results among literature inconsistent.
To avoid the complex and hierarchical regulation of pagA transcription in B. anthracis, trans-acting AtxA and the cis-acting 509 sequence, containing P1 and P2, were reconstituted in E. coli to study AtxA-regulated transcription and seek practical applications. In the present invention, AtxA was found to be a transcriptional activator in E. coli that actively facilitated transcription (as shown in pTOL02F+pTOLAtxA and pTOL03F).
The minimal AtxA-regulated promoter contains SLII, and SLII and AtxA binding is specific; however, the present invention showed that SLI/SLII removal increased the strength of the AtxA-regulated transcription. Those data suggested that SLII/AtxA binding actively downregulated the transcription activity in E. coli. This also suggested that the interaction between AtxA and Escherichia RNAP determines the AtxA-regulated transcription.
In the present invention, the promoters in pTOL02B and pTOL03B did not contain the downstream ATrich tract; therefore, the effect of the downstream ATrich tract on promoter activity could not be concluded. However, the present invention was the first to prove that the first ATrich tract, more specifically, the 25-bp sequence upstream of the −35 box, is the most critical repressor operator. Removal of the first ATrich tract from the 509 sequence greatly increased the AtxA-regulated transcription activity in E. coli (
In the present invention, the promoter, RNAP, and regulation factor were three typical elements that constitute the promoter system, the −10 sequence (TATACT), cognate RNAP, and AtxA exhibited a new transcription mechanism, where the synergistic and cooperative talks of three provided a tight-control, high-level, modulable, and stationary-phase specific expression promoter. The present invention hypothesizes a possible mechanism which involves two interactions. The first was that between Escherichia RNAP and −10 sequence, where the −10 sequence (TATACT) from Bacillus could barely be recognized by Escherichia RNAP. This became the basis for the tight-control of AtxA regulated transcription. The second is a possible interaction between Bacillus AtxA and Escherichia RNAP, in which AtxA acted as an activator for the resulting strong promoter activity. More specifically, the employment of the −10 sequence resulted in strong promoter activity specifically at the stationary phase. With the simplicity of the PAtxA, which involves three transcriptional elements, the employment of PAtxA in different hosts, following the central dogma of biology, is expected to have minimum toxicity and can be decoupled from the potential regulation restriction. Interestingly, most promoters used in synthetic biology are negative inducible, the positive inducible PAtxA should be a useful tool to diversify the gene information flow in synthetic biology. While the orthogonality of hybridized holo-RNAP has been demonstrated that determines the initiation of the promoter activity at the certain cellular state, attention to the future development of PAtxA can be genetically and biochemically focused on the activation mechanism of AtxA, or other PCVRs.
Another interesting DNA fragment of 509 sequence was the upstream site/−35 sequence, as seen in pTOL03E. The role of the upstream site/−35 sequence in AtxA-regulated transcription is clear, which titrates the AtxA activation activity.
Compared to pTOL03F, pTOL03E was less sensitive to the AtxA induction (
Stationary phase protein expression is of interest in engineering applications, especially for toxic protein overexpression. However, promoters that can be used for stationary phase protein production often suffer from the low activity. Promoter activity at the stationary phase can be improved by random mutagenesis of the promoter region to isolate the mutant and the promoter can be increased by 16 fold to reach approximately 3,500 fluorescence intensity (GFPuv)/0D600. The development of PAtxA is one way to achieve an accurate and strong protein expression in the stationary phase while inflicting the minimum adverse effect on bacterial growth. For example, a high FI/OD600 of 40,688±446 for E. coli BL21(DE3)/pTOL03F (
In summary, a promoter three-hybrid has been developed, involving the −10 sequence (TATACT), Bacillus AtxA, and Escherichia RNAP. The promoter three-hybrid provides a tight-control, high-level, modulable, and stationary-phase specific transcription activity and was decoupled from the complex regulation restriction in B. anthracis. PAtxA requires 15 uM IPTG for full promoter activity. A high induction/repression ratio of 230 was achieved when M9 minimal medium with g/L glucose was used. The 25-bp ATrich tract was a strong negative operator for PAtxA and SLI/SLII was a minor negative operator. The upstream site/−35 sequence may act as a decoy and titrate AtxA activity as a transcription activator.
In addition to the recombinant protein expression, the capability of PAtxA was investigated in the application of metabolic engineering, as shown in
It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples, and data provide a complete description of the present invention and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations or modifications to the disclosed embodiments without departing from the spirit or scope of this invention.
This application claims the benefit under 35 U.S.C. § 119(e) to the U.S. provisional patent application having the Ser. No. 63/351,824 filed on Jun. 14, 2022, the entirety of which is incorporated herein by reference.
Number | Date | Country | |
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63351824 | Jun 2022 | US |