Difference between revisions of "Part:BBa K3784006"

Revision as of 01:30, 18 October 2021

Fld

Fld consists of TAA1 (BBa_K3784004), fldH (BBa_K3784003), fldB (BBa_K3784001), fldC(BBa_K3784002) and acdA(BBa_K3784000), aiming to build a pathway in the engineered bacteria to metabolize tryptophan to produce indolepropionic acid, which can consume the remaining tryptophan and strengthen the intestinal mucosal barrier.

Usage and Biology

We reconstructed a five-step reaction. We cloned its tryptophan aminotransferase(TAA1) from Arabidopsis thaliana, which can catalyze the production of indole pyruvate from tryptophan. Then cloned from Clostridium sporogenes four enzymes -- fldB, fldC, fldH, and acdA in the fld gene cluster. fldB and fldC encode the 2-hydroxyacyl-CoA dehydratase protein family. fldH encodes phenyllactate dehydratase. acdA encodes 3-(aryl) acrylate reductase acdA. Tryptophan is first catalyzed by tryptophan aminotransferase(TAA1) to produce indolepyruvate, and then indolepyruvate is catalyzed by phenyllactate dehydratase (fldH) to produce indole lactic acid. This enzyme is also the rate-limiting enzyme of the entire reaction[1]. Then indole lactic acid is catalyzed by the 2-hydroxyacyl-CoA dehydratase protein family encoded by fldBC to produce indole acrylic acid, and finally indole acrylic acid is catalyzed by the 3-(aryl) acrylate reductase acdA encoded by acdA to produce indole propionic acid. In this way, our engineered bacteria can metabolize tryptophan to produce indolepropionic acid and repair the intestinal mucosal barrier.

Characterization

Figure 1. Gene expression level verification

We successfully constructed plasmids contained TAA1, fldH, fldB, fldC, and acdA. TAA1, fldH and fldBC are each initiated by a T7 promoter. acdA is transcribed by a self-contained promoter cloned from Clostridium sporogenes.

We transformed the plasmid into E. coli BL21(DE3) and induced it to express. After extracting total RNA, we obtained its cDNA by reverse transcription. We used cDNA as a template to amplify each fragment, then checked it by agarose gel electrophoresis. We chose rsmA (16S rRNA m(6)2A1518, m(6)2A1519 dimethyltransferase) as the internal reference gene and set a negative control. Five genes in the IPA metabolic pathway had been well expressed(Figure 1) and the corresponding mRNAs have been successfully transcribed. The expression levels of internal reference genes in the experimental group (rsmAE) and the control group (rsmAC) were consistent.

Figure 2. Determination of indole propionic acid in supernatant by HPLC

To test whether our engineered bacteria can metabolize tryptophan to produce indolepropionic acid, we used HPLC (High performance liquid chromatography) to detect the content of HPLC in the fermentation supernatant.

We set up three biological replicates, namely IPA1, IPA2 and IPA3, as well as a control and a standard BZP1. We can learn that the experimental group and the standard have a peak at the same retention time(Figure 2), which proves the presence of Indole-3-propionic acid in the experimental group. Meanwhile, there was no peak in control group, which proved that E. coli could not metabolize tryptophan to produce indole propionic acid.

Sequence and Features

Reference

[1] Dodd D, Spitzer MH, Van Treuren W, Merrill BD, Hryckowian AJ, Higginbottom SK, Le A, Cowan TM, Nolan GP, Fischbach MA, Sonnenburg JL. A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature. 2017.