Overview
Clostridium tyrobutyricum (C. tyrobutyricum) is an anaerobic probiotics, and the butyric acid produced by bacteria is good for both biological intestinal tract and alkaline soil. The main purpose of this project is to make C. tyrobutyricum facultative anaerobic, so that it can function well under anaerobic condition in gastrointestinal tract as a probiotics and under aerobic condition in alkaline soil as soil-improving bacteria. This goal can be achieved by introducing the dps and aceE genes into C. tyrobutyricum. dps protein can enhance the viability of the bacteria in aerobic environment mainly by protecting DNA. PDH expressed by aceE can substitute the main metabolic enzyme PFOR in the bacteria which is inactivated in the presence of oxygen.
To realize the above aim, we successfully constructed 7 parts as listed in Table 1. Among them, two important devices, BBa_K4407012 and BBa_K4407013 , were successfully established. We used these devices to construct facultative anaerobic C. tyrobutyricum. SDS-PAGE was used to verify the protein expression in the recombinant bacteria and cell growth was monitored by measuring the optical density at 600 nm (OD600) with a spectrophotometer under aerobic and anaerobic conditions.
Table 1 Part list
| No. | Name | Type | Description | Length |
|---|---|---|---|---|
| 1 | BBa_K4407001 | coding | dps,DNA protection against oxidative damage | 645 bp |
| 2 | BBa_K4407002 | coding | aceE,Alternate metabolic pathway | 2264 bp |
| 3 | BBa_K4407010 | device | Pthl-dps,Expression of dps with Pthl promoter | 1312 bp |
| 4 | BBa_K4407011 | device | Pthl-aceE,Expression of aceE with Pthl promoter | 3331bp |
| 5 | BBa_K4407012 | device | Plac-dps,Expression of dps in response to lactose | 1905 bp |
| 6 | BBa_K4407013 | device | Plac-aceE,Expression of aceE in response to lactose | 3924 bp |
| 7 | BBa_K4407014 | device | Plac-dps-aceE,Coexpression of dps and aceE in response to lactose | 4575 bp |
| 8 | BBa_K4407015 | device | Pthl-dps-aceE,Coexpression of dps and aceE with Pthl promoter | 3982 bp |
| 9 | BBa_K4407016 | regulatory | vgb promoter with another FNR binding site inserted, microaerobic | 149 bp |
Target 1：Expression of dps in C. tyrobutyricum
We first constructed a plasmid pMTL-Pthl-dps to express dps with Pthl promoter, and transformed it into C. tyrobutyricum. The effect of improving the oxygen tolerance of the recombinant strain was tested. The effect of polyacrylamide gel electrophoresis (SDS-PAGE) experiment verification and growth performance tests were not ideal, so we considered replacing the thiolase promoter Pthl with the lactose induced promoter Plac, which was expected to have better effect.
Eventually, we successfully constructed C. tyrobutyricum transformed with pMTL-Plac-dps [Ct (Plac-dps) strain], confirmed the expression of dps protein in this strain, and verified that the growth of the engineered bacteria was enhanced under aerobic condition compared to the control strain.
1.C. tyrobutyricum with pMTL-Pthl-dps plasmid
1)Plasmid construction: pMTL-Pthl-dps
We amplified a plasmid pMTL-Pthl-BS2 offered by team NJTech_China to obtain a linearized pMTL-Pthl vector. Then, we obtained the fragment of dps gene from the genome of D. wulumuqiensis R12 by PCR. The dps gene fragment and linearized pMTL-Pthl vector were ligated by the Gibson assembly method. Then, we ran a colony PCR for the transformed bacterial colony (E .coli JM109), inoculated the positive bacterial colonies, and extracted the plasmids, which were then confirmed to be pMTL-Pthl-dps by gene sequencing.
2) C. tyrobutyricum transformed with pMTL-Pthl-dps
After the recombinant plasmid pMTL-Pthl-dps was conjugated into C. tyrobutyricum using E. coli CA434 as the donor strain. The effect of polyacrylamide gel electrophoresis (SDS-PAGE) experiment verification and growth performance tests were not ideal. So we considered replacing the thiolase promoter Pthl with the lactose induced promoter Plac, which was expected to have better effect.
2.C. tyrobutyricum with pMTL-Plac-dps plasmid
1)Plasmid construction: pMTL-Plac-dps
Using the recombinant plasmid pMTL-Pthl-dps, we amplified and obtained a linearized pMTL-dps vector. Then, using the pMTL-perR-HR-tetR plasmid as template, we obtained the Plac promoter fragment. The Plac promotor fragment and linearized pMTL-dps vector were ligated by the Gibson assembly method. Then, we ran a colony PCR for the transformed bacterial colony (E .coli JM109), inoculated the positive bacterial colonies, and extracted the plasmids, which were then confirmed to be pMTL-Plac-dps by gel electrophoresis and gene sequencing (Figure 2).
2) C. tyrobutyricum conjugated with pMTL-Plac-dps
Using E. coli CA434 as the donor strain, the recombinant plasmid pMTL-Plac-dps was conjugated into C. tyrobutyricum to construct the recombinant bacteria [Ct (Plac-dps) strain], and the empty plasmid pMTL82151 was conjugated into C. tyrobutyricum to construct a control strain, Ct(control). The expression of dps protein in the Ct (Plac-dps) strain was confirmed by SDS-PAGE (Figure 3).
[(Ct (Plac-Dps): Ct conjugated with pMTL-Plac-dps; Ct(control): Ct conjugated with empty plasmid pMTL82151]
To determine how dps overexpression in C. tyrobutyricum affected the viability of the bacteria under aerobic and anaerobic conditions. We measured the OD600 values to compare the growth of the Ct (Plac-dps) and Ct(control) strains under these two conditions, respectively.
Under anaerobic condition, the maximum biomass (OD600) of the Ct (Plac-dps) strain was similar to that of the Ct(control) strain (9.67 vs 9.18), with overlapping error bars (Figure 4). Therefore, dps expression in the Ct (Plac-dps) strain made no significant difference in the maximum growth rates.
[(Ct (Plac-dps): Ct conjugated with pMTL-Plac-dps; Ct(control): Ct conjugated with empty plasmid pMTL82151]
Under aerobic condition (100 rpm), the growth of the Ct(control) strain was affected, with a longer lag phase (Figure 5). Moreover, although there was no significant difference between the maximum biomasses of the two strains, the growth rate of the Ct (Plac-dps) strain was greater than the control strain. Therefore, overexpressing dps protein could improve the growth of C. tyrobutyricum under aerobic conditions.
[(Ct (Plac-dps): Ct conjugated with pMTL-Plac-dps; Ct(control): Ct conjugated with empty plasmid pMTL82151]
Target 2：Expression of aceE in C. tyrobutyricum
We first constructed a plasmid pMTL-Pthl-aceE to express aceE with Pthl promoter, and transformed it into C. tyrobutyricum. The effect of improving the oxygen tolerance of the recombinant strain was tested. The effect of polyacrylamide gel electrophoresis (SDS-PAGE) experiment verification and growth performance tests were not ideal, so we considered replacing the thiolase promoter Pthl with the lactose induced promoter Plac, which was expected to have better effect.
Eventually, we successfully constructed C. tyrobutyricum transformed with pMTL-Plac-aceE [Ct (Plac-aceE) strain], confirmed the expression of aceE protein in this strain, and verified that the growth of the engineered bacteria was enhanced under aerobic condition compared to the control strain.
1.C. tyrobutyricum with pMTL-Pthl-aceE plasmid
1)Plasmid construction: pMTL-Pthl-aceE
Using the plasmid pMTL-Pthl-BS2 provided by NJTech_China team as the template, the linearized vector pMTL-Pthl was amplified. The fragment of aceE gene was amplified from the genome of Escherichia coli DH5α. Gibson assembly method was used to connect aceE gene fragment with the linearized pMTL-Pthl vector. Then, we ran a colony PCR for the transformed bacterial colony (E .coli JM109), inoculated the positive bacterial colonies, and extracted the plasmids, which were then confirmed to be pMTL-Pthl-aceE by gel electrophoresis and gene sequencing.
2) C. tyrobutyricum transformed with pMTL-Pthl-aceE
After the recombinant plasmid pMTL-Pthl-aceE was conjugated into C. tyrobutyricum using E. coli CA434 as the donor strain. The effect of improving the oxygen tolerance of the recombinant strain was tested. The effect of polyacrylamide gel electrophoresis (SDS-PAGE) experiment verification and growth performance tests were not ideal, so we considered replacing the thiolase promoter Pthl with the lactose induced promoter Plac, which was expected to have better effect.
2.C. tyrobutyricum with pMTL-Plac-aceE plasmid
1）Plasmid construction: pMTL-Plac-aceE
Using the recombinant plasmid pMTL-Pthl-aceE, we amplified and obtained a linearized pMTL-dps vector. Then, using the pMTL-perR-HR-tetR plasmid as template, we obtained the Plac promoter fragment. The Plac promotor fragment and linearized pMTL-aceE vector were ligated by the Gibson assembly method. Then, we ran a colony PCR for the transformed bacterial colony (E. coli JM109), inoculated the positive bacterial colonies, and extracted the plasmids, which were then confirmed to be pMTL-Plac-aceE by gel electrophoresis and gene sequencing (Figure 7).
2) C. tyrobutyricum conjugated with pMTL-Plac-aceE
Using E. coli CA434 as the donor strain, the recombinant plasmid pMTL-Plac-aceE was conjugated into C. tyrobutyricum to construct the recombinant bacteria [Ct (Plac-aceE) strain], and the empty plasmid pMTL82151 was conjugated into C. tyrobutyricum to construct a control strain, Ct(control). The expression of aceE protein (99.7 kDa) in the Ct (Plac-aceE) strain was confirmed by SDS-PAGE (Figure 8).
[(Ct (Plac-aceE): Ct conjugated with pMTL-Plac-aceE; Ct(control): Ct conjugated with empty plasmid pMTL82151]
To determine how aceE overexpression in C. tyrobutyricum affected the viability of the bacteria under aerobic and anaerobic conditions. We used OD600 to compare the growth of the Ct (Plac-aceE) and Ct(control) strains under these two conditions, respectively.
Under anaerobic condition, the maximum biomass (OD600) of the Ct (Plac-aceE) strain was lower than that of the control strain (Figure 9). It is speculated that under anaerobic conditions, the aerobic and anaerobic pathways from pyruvate to acetyl CoA in the recombinant strain may compete, which has a partial impact on the growth of the strain.
[(Ct (Plac-aceE): Ct conjugated with pMTL-Plac-aceE; Ct(control): Ct conjugated with empty plasmid pMTL82151]
Under aerobic condition (100 rpm), the growth of the control strain was affected, and its delay period was longer than that of the Ct (Plac-aceE) strain (Figure 10). In addition, the growth rate of the Ct (Plac-aceE) strain was significantly higher than that of the control strain.
[(Ct (Plac-aceE): Ct conjugated with pMTL-Plac-aceE; Ct(control): Ct conjugated with empty plasmid pMTL82151]
Target 3：Co-expression of dps and aceE in C. tyrobutyricum
1.Plasmid construction: pMTL-Pthl-dps-aceE
pUsing pMTL-Pthl-dps constructed previously as the template, pMTL-Pthl-dps linearization vector was amplified via PCR. Using pMTL-Pthl-aceE as the template, aceE fragments with ribosome binding site (RBS) were amplified, too. Gibson assembly method was carried out to link the aceE fragment (with RBS) to pMTL-Pthl-dps vector. Then colony PCR was performed on the transformed colonies to verify the correct ones (Figure 11), which would be further cultured for the extraction of the plasmid. The recombinant plasmid pMTL-Pthl-dps-aceE was obtained after sequencing verification, and be transferred into E. coli CA434 for the conjugation with C. tyrobutyricum.
2.Plasmid construction: pMTL-Plac-dps-aceE
In the early stage, the constructed pMTL-Pthl-aceE recombinant plasmid and pMTL-Pthl-dps recombinant plasmid were transformed into C. tyrobutyricum respectively through combination, and the effect of improving the oxygen tolerance of the recombinant strain was tested. The effect of polyacrylamide gel electrophoresis (SDS-PAGE) experiment verification and growth performance tests were not ideal, so we considered replacing the thiolase promoter Pthl with the lactose induced promoter Plac, which was expected to have better effect.
Using pMTL-Plac-dps constructed previously as the template to amplify pMTL-Plac-dps linearization vector. Plasmid pMTL-Plac-aceE was used as the template for the amplification of aceE fragments with ribosome binding sites (RBS). The aceE fragment with RBS was linked to the linearization vector by Gibson assembly method. Then colony PCR was performed on the transformed colonies to verify the correct ones (Figure 12), which would be further cultured for the extraction of the plasmid. The recombinant plasmid pMTL-Plac-dps-aceE was obtained after sequencing verification, and be transferred into E. coli CA434 for the conjugation with C. tyrobutyricum.
Due to temporary close down of laboratory during COVID-19 pandemic, transformation of the plasmids into C. tyrobutyricum and functional verification experiments cannot be done within the time frame of this project and need to be pursued in the future.
Summary
We successfully constructed two strains of C. tyrobutyricum using plasmids pMTL- Plac-dps and pMTL-Plac-aceE. The adaptation of the engineered C. tyrobutyricum to aerobic conditions was significantly improved.
Future work
In the future, pMTL-Plac-dps-aceE plasmid can be transformed into C. tyrobutyricum and its effect should be evaluated. This plasmid can co-express dps and aceE genes, and has the potential to greatly improve the resistance to oxidative stress of the bacteria.
