Results

We characterized the expression of the oleic acid promoter by placing the mRFP fluorescent protein under the control of the FadR promoter. The engineered bacteria transformed with oleic acid promoter gene were cultured under two environmental conditions, aerobic and anaerobic, respectively, and under two media conditions with or without oleic acid. The results showed that the oleic acid inducer was effectively triggered to produce a fluorescent signal after induction with oleic acid. In addition, the oleic acid promoter performed better under anaerobic conditions, indicating that the oleic acid promoter is well adapted to the intestinal anaerobic environment (Figure 1).

In addition, we built models to predict the number of operators matching the induction thresholds of different oleic acid concentrations (Figure 2) (due to the difficulty of gene fragment integration, we will not be able to complete the synthesis of oleic acid promoters with different operator numbers before October, so we cannot conduct wet experiments for realistic verification). By adjusting the number of operators, We can effectively activate gene expression by controlling the concentration of oleic acid, which is perfectly consistent with the process of high-oil diet mentioned in our daily life. When the concentration of oleic acid in the diet reaches the defined limit of high-oil diet, the oleic acid promoter is switched on. When the concentration of oleic acid in the diet is below this limit, the whole system is silenced without affecting the absorption of proper nutrients during the normal diet. Moreover, due to the specific constitution of different people, the concept of high-fat diet is not fixed, and we can also adjust the number of operators to perfectly match this dynamic situation.

In order to obtain the optimal combination of genotypes for cholesterol degradation, the IsmA, BSH and BCoAT genes were arranged and combined to obtain the following six genotypes: IsmA, BSH, BCoAT, isma-bsh, ISma-Bcoat, ISma-Bsh-Bcoat. Its ability to down-regulate cholesterol content was tested by different methods.

The cholesterol-lowering effect of IsmA gene was characterized by measuring the cholesterol content of the medium by OPA method after the strain containing IsmA gene was cultured in basal cholesterol medium for 48 hours. The results are shown in Figure 3. The efficiency of cholesterol degradation was up to 30%.

In addition, for the BSH gene, we evaluated the effect of different gene combinations on bile saline hydrolysis. In this process, all the combinations of effect genes showed good bile saline hydrolysis effect, and obvious precipitation rings appeared around the filter paper on the medium (Figure 4). In the dynamic observation of the whole process of precipitation ring formation, we found that the activity performance of the BSH gene was generally better under hypoxic conditions than under aerobic conditions, and in the triplet strain (IsmA-BCoAT-BSH), the activity performance of BSH was higher than that of the BSH monogenic strain (Figure 4).

In the detection process of the above two rounds of experiments, the recombined genes showed different expression advantages compared with their own genes. Therefore, we continued to detect the expression of SCFA module for BCoAT gene. In the evaluation of SCFA production, we detected three indicators: butyrate, acetate, and propionic acid.
In the butyrate assay, all effector gene combinations showed butyric acid production (Figure 5). However, in the statistical analysis, only the butyric acid production of the BCoAT single gene showed significant difference compared with the control group, and also showed significant difference compared with other combinations of effector genes (Figure 5).

In acetic acid assays, the BCoAT single gene showed acetic acid production, but it was not statistically significant (Figure 6)." However, other functional gene combinations showed little ability to produce acetic acid (Figure 6). In the functional genome comparison, the BCoAT gene was significantly different from the triplet (IsmA-BSH-BCoAT) gene in acetate production capacity (Figure 6). This part shows the outstanding acetic acid production ability of the BCoAT single gene, but it is not completely convincing.

In the detection of propionic acid, the BCoAT gene showed good propionic acid production characteristics with statistical significance compared with the control group, and in the comparison between effector genomes, it showed significant difference with the triplet (IsmA-BSH-BCoAT). "Other effector gene composition types, however, do not reflect this propionic acidogenic property (Figure 7).

In summary, it seems that in the targeted evolution of cholesterol down-regulation, different genotypes still show differences and complexity in their ability to regulate cholesterol levels. Although the combination of the three genes in tandem gave strong performance in the direct cholesterol degradation by IsmA and the down-regulation of cholesterol by bile-salt hydrolases secreted by the BSH genes, this does not mean that they can perform equally well in the regulation through short-chain fatty acids (SFAs). The optimal evolutionary pathway still needs to be evaluated and determined, and perhaps a more precise answer can be obtained in subsequent in vivo experiments.