Our team contributed to further investigate the role of the enzyme Tes4 in the biosynthesis of the beneficial metabolite—butyrate. We successfully validated the effectiveness of Tes4 in butyrate production.

As for butyrate sensing, our team designed two butyrate biosensors. One is the pPcha/Lrp system from Escherichia coli strain O157:H7, and the other using pHpdH/HpdR system from Pseudomonas putida (Wang et al., 2023). Both systems are activated in the presence of butyrate. Our team characterized the performance of these two butyrate biosensors in the probiotic E. coli strain Nissile 1917 to demonstrate their potential to be incorporated in our therapeutic platform.

Tes4 characterization improvements

Acyl-ACP thioesterase Tes4 (SZU-China 2021) binds to butyryl-ACP, produced through the native fatty acid biosynthesis (FASII) pathway, to release the tetracarbon acid from ACP. As a result, butyrate is synthesized (Figure 1).

Figure 1. Tes4-mediated butyrate biosynthesis pathway.

Comparing to other butyrate biosynthesis pathways that involve multiple enzymes, expressing Tes4 as the single enzyme likely has a lower gene expression burden for E. coli. Moreover, expressing Tes4 in E. coli BL21 shows that butyrate is produced as the main product (Kallio P. et al., 2014). Hence, we decided to overexpress Tes4 in BL21 to achieve butyrate production.

We utilized the conventional protein expression backbone pET28a(+) for overexpressing Tes4 in BL21(DE3). The plasmid pET28a(+)-Tes4 was sysnthesized by Genscript commercial, and the plasmid was confirmed by full plasmid sequencing. The coding frame of Tes4 was inserted downstream the IPTG inducible T7 promoter (Figure 2).

Figure 2. Plasmid design of pet28a-Tes4. Created by biorender.com.

Tes4 expression was induced by 1 mM IPTG at 37°C for 4 hours and validated by SDS-PAGE (Figure 3). (See Protocol)

Figure 3. SDS-PAGE result of Tes4 production. The band at 19.5kDa indicated Tes4 protein. Lane 1, Protein ladder. Lane 2, Tes4 plasmid with IPTG added. Lane 3, BL21 WT. Lane 4, Tes4 plasmid without IPTG added. Lane 6, Protein ladder.

The butyrate production was validated by GC-MS with uninduced BL21 and butyrate standard as negative and positive controls, respectively. The supernatant of culture media was mixed with acetone (1:1) and injected to GC-MS (1.4ml min^-1 He flow, injector at 250 degree Celcius, 40-250 degree Celcius, 20 dgree Celcius min^-1) Butyrate typically eluted at 11 min. The sample results were confirmed with commercial standards diluted from powder of butyrate ( 1mmol/3mL and 10mmol/3mL) from Sigma-Aldrich). Quantification was done comparing the peak areas (Figure 4). (See protocol)

Figure 4. GC-MS results of butyrate production. Peaks at about 11.4 min represented butyrate. A, pET28a(+)-Tes4 IPTG(-). B, pET28a(+)-Tes4 IPTG(+) without lysis. C, LB blank (Maybe contaminated). D, pET28a(+)-Tes4 IPTG(+) after lysis. E, 1mmol/3mL butyrate standard solution. F, 10mmol/3mL butyrate standard solution.

The peak at 11.4 min indicates the presence of butyrate by comparing the butyrate standard, bacteria culture without lysis, and LB blank sample. For BL21 with Tes4 expression induced, a peak at around 11.4 min was also observed. In contrast, the sample from E. coli without IPTG induction didn't show this characterization peak. Thus, it can be concluded that the expression of Tes4 plays a key role in the production of butyrate.

Our team contributed new data for validating an important pathway for promoting the production of the beneficial metabolite butyrate in E. coli.

PpchA

We wish to build a butyrate biosensor in our project. In our design, leucine-responsive regulatory protein (Lrp) is constitutively expressed using a constitutively expressed promoter, pJ23101, while CI protein is regulated by the butyrate sensor's control and binds to the pLam promoter to inhibit the expression of downstream protein. When butyr.0ate is not present, the expression of CI repression is not activated, and hence the target gene downstream pLam promoter will be expressed. In the presence of butyrate, Lrp forms a complex with butyrate and then binds to the pPcha promoter to activate the expression of the CI repressor, which then inhibits promoter pLam, and stops the expression of the target gene (Figure 5).

Figure 5. Plasmid design of PpchA. Created by biorender.com.

The pPcha/Lrp system has already been developed by previous iGEM teams (iGEM23_NMU-China), which confirmed the induction of gene expression by butyrate. Moreover, given that the pPcha/Lrp system is native to E. coli, we hypothesized that this is likely a promising system for butyrate biosensing in E. coli, the native host.

Characterization of butyrate biosensor using PpchA

The butyrate sensing device includes the pchA promoter and pchA regulator, Lrp, constitutively expressed, located downstream, and CI protein that inhibits promoter Lambda's expression (Figure 6).

Figure 6. Plasmid design of PpchA, Created by biorender.com.

We used plate-reader fluorescence kinetics to quantitatively validate the effectiveness of the above design for butyrate biosensing. A gradient of butyrate concentration was added into E. coli transformed with PpchA, and the value of fluorescence / ABS600 over time was recorded using the plate-reader to represent GFP expression level.

The plasmid synthesized by the company contained a point mutation on the ribosome binding site (RBS), resulting a low translational rate for Lrp. To address this, we utilized the promoter calculator function by De novo DNA (LaFleur et al., 2022). The results confirmed that Lrp expression was significantly below our expectations (Figure 7).

Figure 7. Promoter calculator function with mutated and fixed RBS. A, the translation rate of our design without the RBS point mutation. B, the decreased translation rate affected by the RBS point mutation.

In the wet lab, we performed a kinetics analysis of this plasmid, which revealed slight differences and a correct trend as the gradient of inducer was added, though the dynamic range was suboptimal (Figure 8).

Figure 8. Kinetics of PpchA before fixing with multiple butyrate concentrations over 18 hours. Fluorescence / ABS was used to represent GFP expression; higher fluorescence represented lower PpchA activity. The butyrate concentration ranges from 0mM to 70mM.

We wish to fix this RBS point mutation to gain better results. The two fragments for goldengate is based on the sysnthesized plasmid of pUC57-PpchA-CI. Goldengate Assembly was used to construct the plasmid (Figure 9). Sequencing verified the construct as correct.

Figure 9. The AGE results of the PCR products of PpchA construction. A, materials to construct PpchA. B, Goldengate assembly result of PpchA construction. The band at 5278bp in (B) indicated the success in plasmid construction.

Following the successful construction of the PpchA-RBS-Fixed plasmid, we conducted a kinetics analysis to evaluate its performance (Figure 10).

Figure 10. Kinetics of PpchA after RBS was fixed with multiple butyrate concentrations over 16.7 hours. Normative fluorescence / ABS600 values were used to represent GFP expression; higher fluorescence represented lower PpchA activity. The butyrate concentration ranges from 0 to 100 mM.

The overall performance of the PpchA-Lrp sensor is sufficient to demonstrate its ability to detect and respond to the presence of butyrate accurately at most butyrate concentrations. However, the sensor exhibits low sensitivity in distinguishing between small variations in butyrate levels; furthermore, the dynamic range of this detection system was only about 2-fold. This is likely attributed to the inherent limitation of this system, as a similar noisy butyrate induction pattern of PpchA-Lrp has been previously reported in literature (Serebrinsky-Duek et al., 2023). To address those limitations, we developed an alternative sensor, pHdpH, for further investigation.

pHpdH

Considering that the result of PpchA is not significant, the pHpdH/HpdR system from Pseudomonas putida, we adopted an alternative butyrate biosensor. It has been used by the previous iGEM team in Bacillus subtilis and Vibrio nitriegens (iGEM2018 UIOWA and iGEM2018 Marburg), and shown to have up to 41-fold induction with the best response to butyrate (Wang et al., 2023). When butyrate is present, it binds to HpdR, changing its conformation so it can bind and activate pHpdH, resulting in the expression of downstream genes.

We firstly characterized the butyrate induction by constitutively expressing HpdR driven by Plpp1 promoter and placing GFP downstream. (Figure 11) (Wang et al., 2021).

Figure 11. Plasmid design of pHpdH. Created by biorender.com.

Characterization of butyrate biosensor using pHpdH

We used kinetics to test the effectiveness of this butyrate detection system quantitatively. A gradient of butyrate concentration was added into E. coli transformed with the plasmid, and the value of fluorescence / ABS600 over time was detected using plate-reader to represent GFP expression (Figure 12).

Figure 12. Kinetics of GFP expression over 16.7 hours. Fluorescence / ABS was used to represent GFP expression. Black, no butyrate added. Purple, 0.3mM butyrate. Blue, 0.6mM butyrate. Cyan, 1.2mM butyrate. Green, 2.4mM butyrate. Yellow, 4.8mM butyrate. Orange, 9.8mM butyrate. Red, 20mM butyrate. Pink, 40mM butyrate. Gray, 86mM butyrate.

The hpdR, phpdH, and phpdR were synthesized from Gensript. We used Golden Gate Assembly to construct pHdpH. PCR and Gel Electrophoresis were performed to verify the success in constructing the fragment and backbone of the overall pHdpH plasmid (Figure 13).

Figure 13. The AGE result of the PCR products of pHdpH construction. A, materials to construct pHdpH. B, golden gate assembly result of pHdpH construction. The band at 4555bp in (B) indicated the success in plasmid construction.

When the butyrate concentration was below 20mM, our system showed the desired trend, as fluorescence increases proportionally with butyrate concentration. Above this point, however, the system failed to distinguish between 20mM, 40mM, and 86mM butyrate. This might be because 20mM butyrate reached the saturation concentration of this biosensor. Since our goal was to detect the shortage of butyrate in the gut system, the sensitivity of our biosensor at low butyrate concentrations is sufficient.

In conclusion, we contributed to the iGEM part registry by validating the Tes4-mediated butyrate production and characterizing two butyrate biosensors. The experimental data has been updated at the iGEM part page (BBa_K3838613 for Tes4, BBa_K4442001 for PpchA, and BBa_K2560304 for pHpdH/HpdR). These validation works can provide further guidance for future iGEM teams working on projects with similar topics.

Reference

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