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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 four-carbon fatty acids. When the fatty acid is synthesized to 4-carbon, the enzyme will release it from ACP to obtain butyric acid.

Sequence and Features

Experience: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 5000bp, basically conforming to the target band of 4994bp, 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 945bp, and the actual electrophoresis results met the target band size.

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 6xHis tag. The target protein size was 19kDa. Purification results are shown in Figure 22. 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.

3. Functional representation We will convert 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 248nm.

We diluted the sample 800 times and used Kinetex C18 2.6um 100×2.1mm; 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.

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.