Part:BBa_K3838613

TES4

Butyric acid promotes the repair of intestinal epithelial cells, strengthens the intestinal mucosal barrier, and can inhibit multiple inflammatory pathways. Butyric acid is also the main energy source of colon epithelial mucosa. The proportion of patients with intestinal butyric acid bacteria decreased, and butyric acid can not be taken directly by mouth, only enema, etc., more painful. Engineered microorganisms enable sustained, long-term drug delivery. Solution: We use the fatty acid synthesis pathway of lactic acid bacteria and Escherichia coli and introduce exogenous TES4 gene can achieve the production of butyric acid with glucose as substrate. Tes4 enzyme has some specificity for tetracarbon fatty acids. When the fatty acid is synthesized to tetracarbon, the enzyme will release it from ACP to obtain butyric acid.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 259
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 259
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 288
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 259
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 259
  
• 1000
  COMPATIBLE WITH RFC[1000]

Data:SZU-China 2021 TEAM

1. The DNA level

The plasmid PJT was selected for validation and transformed into DH5α (DJT) and Nissle 1917(NJT). Plasmids were transformed into DH5a for plasmid running gel validation. As can be seen from figure 1, the sample band was about 5000 bp, basically conforming to the target band of 4994 bp, which proved that our plasmid transformation was successful. As the plasmid concentration extracted by Nissle 1917 was too low, PCR validation was carried out on the plasmids in NJT, as shown in figure 1. The target band size was 945 bp, and the actual electrophoresis results met the target band size.

Fig.1 A Plasmid was extracted from transformed DH5α and gel electrophoresis was performed. The target band size was 5477bp. B PCR was performed on plasmids extracted from transformed Nissle 1917.

2. Protein level

We then verified the engineering bacteria at the protein level. Intracellular protein was extracted from the cultured bacteria and purified by affinity with 6x His tag. The target protein size was 19 kDa. Purification results are shown in figure 2. In the purification, Tes4 protein was not fully combined, and the presence of hybrid proteins with the same size of Tes4 protein led to the existence of bands with the same size in the eluent, which was expected by us. However, due to time constraints, we did not have time to explore the details of elution conditions. Therefore, due to problems in elution gradient, concentration control and sample grafting in the experimental process, there were other hybrid proteins in the eluent, but the purified band of Tes4 protein was clearly visible, which effectively indicated that the engineered bacteria successfully expressed BSH protein. For Nissle 1917, SDS-PAGE also shows clearly visible bands of expression, as shown in red box in figure 2.

Fig.2 A SDS-PAGE of affinity purification of transformed DH5α intracellular protein. B SDS-PAGE electrophoretic diagram of Nissle 1917 intracellular protein.

3. Functional representation

We converted the pJTes4 plasmid DH5a and Nissle 1917 cells broken overnight after the training, the supernatant were collected for detection of butyric acid.In order to be more efficient to test whether our engineering bacteria produced butyric acid, the samples were collected for the derivatization of short-chain fatty acids, as the following figure. The benzene ring is introduced, in order to increase its color, which is easier to be detected at 248 nm.

We diluted the sample 800 times and used Kinetex C18 2.6μm 100×2.1 mm; Mobile phase: A:0.1% phosphoric acid-water, B:0.1% phosphoric acid-acetonitrile; Flow rate: 0.5 mL/min; Injection volume: 1 μL; Column temperature: 35℃; The mobile phase gradient was carried out in the following table.

As shown in the figure, we selected butyric acid standard as positive control, LB medium as negative control, WT as wild-type strain Nissle 1917 blank control. Our engineered bacteria samples D (DH5α -JTES4) and NJT(Nissle 1917-JTES4) showed the same retention time as the butyric acid sample. Compared with the negative control and blank control, it can be considered that our engineered bacteria successfully expressed the target protein and produced butyric acid.

Fig.3 Butyric acid detection chromatogram.

However, the results of wild-type Nissle 1917 chromatogram in the above method showed the shortcomings of this method, which could not completely separate different substances. We hoped to further optimize this method to completely separate substances, but due to time constraints, we failed to optimize the chromatographic conditions, and the chromatogram produced was not ideal.Therefore, we searched for more literature materials to try, and finally we used the following method to separate different short-chain fatty acids, so as to better detect butyric acid.

In the following equation, o-benzyl hydroxylamine was replaced with 3-nitrophenylhydrazine for derivatization, so as to detect the derivatized substance at 355 nm.

As shown in the figure, we compared the mixed acid (butyric acid + isobutyric acid),butyric acid, Nissle 1917, NJT(Nissle 1917-JTES4) and DJT(DH5α -JTes4). The retention time of butyric acid and butyric acid in the mixed acid is exactly the same, which proves that this chromatographic condition has a good separation effect. At the same time, there are peaks similar to butyric acid in our sample. Although both NJT(Nissle 1917-JTES4) and Nissle1917 detected only trace amounts of the substance.

Fig.4 Comparison of chromatograms of different samples.

To further confirm the presence of butyric acid in our sample, we mixed DJT(DH5α -Jtes4) with butyric acid sample and detected whether their peaks overlapped with each other. The results are as follows. The retention time of DJT(DH5α -JTes4) mixed with butyric acid is the same, and the peak area increases, indicating that there is a certain amount of butyric acid in our sample, but the yield is still low.

Fig.5 Mixed sample comparison.

References

[1] Wss A , Hgp B , Smk B , et al. Chemical derivatization-based LC–MS/MS method for quantitation of gut microbial short-chain fatty acids - ScienceDirect[J]. Journal of Industrial and Engineering Chemistry, 2020, 83:297-302.

[2]Simultaneous determination of short‐chain fatty acids in human feces by HPLC with ultraviolet detection following chemical derivatization and solid‐phase extraction segmental elution[J]. Journal of Separation Science, 2019, 42(15):2500-2509.

Supplementary part description

From the literature, we learned that butyrate not only has anti-inflammatory effects in the intestine, but also enhances the intestinal barrier, reduces intestinal permeability, circulating LPS, and systemic inflammation, thereby indirectly inhibiting the formation of atherosclerosis^[1]. The thioesterase expressed by the Tes4 gene hydrolyzes the thioester bond of the fatty acyl-S-acyl proteins, promoting the release of short-chain fatty acids, including butyric acid^[2].

Design and Expectation

We hope to induce E.coli BL21(DE3)(with His-tag)to produce thioesterase by IPTG and detect our butyric acid by gas chromatography^[3].

Materials and Methods

1. Plasmids Construction

Tes4 is constructed using primers by overlap extension PCR. The constructed fragment is ligated to the target site in pET28 by PCR and homologous recombination. The correct construction is confirmed by sequencing.

2. Expression and Purification

Plasmid pET-28a(+)-Tes4 (with His-tag) is transformed to Escherichia coli BL21(DE3). The E.coli strain is cultured in the LB medium containing 10 μg/mL kanamycin. When the optical density of the cultured bacteria was between 0.6 and 0.8, IPTG was added to the final concentration of 1mM, and the bacteria were induced at 18℃ overnight. The harvested bacteria are resuspended with 1xPBS and binding buffer, and then the bacteria are lysed by a Freezing low-temperature pressure crusher. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China).

3. Validation of the target product

We learned that some samples needed to be processed before HPLC could be used to detect butyric acid, and that the instrument was more complicated to use, so we chose to use gas chromatography which is relatively clean, qualitative, quantitative, and more operationally convenient.

Results and analysis

We verified the production of our target protein by SDS-PAGE gel electrophoresis, and the expected result was a target band between 15 kDa-25 kDa. However, two consecutive results showed two bands. We speculated that the thioesterase expressed by the Tes4 might be linked to the coenzyme, or the protein was modified by different proteins. Based on the size of the target protein (19kDa), we thought that the brighter band above was our target protein(Figure 1).

Figure 1.

To further verify that thioesterase was present and functional, the supernatant was subjected to gas chromatography, which confirmed the production of butyric acid. Explanation of two peaks in the second picture: we suspect that Escherichia coliBL21(DE3) (with His-tag) itself will produce a substance with properties similar to butyric acid. According to the gas chromatography results of the butyric acid standard solution, it can be proved that the peak time located between 9.6min and 9.7min shows our target product, butyric acid(Figure 2).

Figure 2. Results of gas chromatography for butyric acid.
A: The peak time of n-butyric acid was between 9.6min and 9.7min in 0.03g/L n-butyric acid solution.
B: The difference value between the butyric acid content induced by IPTG and that induced without IPTG.

Reference

[1] Kasahara K, Krautkramer KA, Org E, Romano KA, Kerby RL, Vivas EI, Mehrabian M, Denu JM, Bäckhed F, Lusis AJ, Rey FE. Interactions between Roseburia intestinalis and diet modulate atherogenesis in a murine model. Nat Microbiol.2018, 3(12):1461-1471

[2] Jing F, Cantu DC, Tvaruzkova J, Chipman JP, Nikolau BJ, Yandeau-Nelson MD, Reilly PJ. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity. BMC Biochemistry, 2011, 12(1):1-16

[3] 魏琦麟, 向 蓉, 袁明贵,等. 顶空-气相色谱法测定丁酸梭菌发酵液中短链脂肪酸含量[J]. 中国油脂, 2019.

Team: BNDS-China 2024

Verification of butyrate production by Tes4

In our plasmid design of Tes4, IPTG was added to induce the expression of Tes4 (Figure 1). The Tes4 DNA sequence and the vector pET-28a(+) were synthesized and obtained from Genscript.

Figure 1. Plasmid design of Tes4. Created by biorender.com.

To verify the successful expression of Tes4 and its effectiveness to produce butyrate, we performed SDS-PAGE to the proteome of E. coli expressing Tes4. The band at 19.5kDa represented the successful expression of Tes4 (Figure 2).

Figure 2. SDS-PAGE result of Tes4 production. 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.

We also performed GC-MS to verify our system's ability to produce butyrate by Tes4. The peak at 11.39 min in (D) suggested the successful production of butyrate (Figure 3).

Figure 3. 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 peaks at 11.25-11.45 min indicated the presence of butyrate.

Reference

Kallio, P., Pásztor, A., Thiel, K., Akhtar, M. K. & Jones, P. R. (2014). An engineered pathway for the biosynthesis of renewable propane. Nature Communications, 5(1), 4731. https://doi.org/10.1038/ncomms5731