Development of a Biosensor for Detection of Benzoic Acid Derivatives in Saccharomyces cerevisiae

Abstract

4-hydroxybenzoic acid (pHBA) is an important industrial precursor of muconic acid and liquid crystal polymers whose production is based on the petrochemical industry. In order to decrease our dependency on fossil fuels and improve sustainability, microbial engineering is a particularly appealing approach for replacing traditional chemical techniques. The optimization of microbial strains, however, is still highly constrained by the screening stage. Biosensors have helped to alleviate this problem by decreasing the screening time as well as enabling higher throughput. In this paper, we constructed a synthetic biosensor, named sBAD, consisting of a fusion of the pHBA-binding domain of HbaR from R. palustris, the LexA DNA binding domain at the N-terminus and the transactivation domain B112 at the C-terminus. The response of sBAD was tested in the presence of different benzoic acid derivatives, with cell fluorescence output measured by flow cytometry. The biosensor was found to be activated by the external addition of pHBA in the culture medium, in addition to other carboxylic acids including p-aminobenzoic acid (pABA), salicylic acid, anthranilic acid, aspirin, and benzoic acid. Furthermore, we were able to show that this biosensor could detect the in vivo production of pHBA in a genetically modified yeast strain. A good linearity was observed between the biosensor fluorescence and pHBA concentration. Thus, this biosensor would be well-suited as a high throughput screening tool to produce, via metabolic engineering, benzoic acid derivatives.

Keywords: biosensor; p-aminobenzoic acid; p-hydroxybenzoic acid; synthetic biology; yeast.

Figure 1

Schematic representation the sHbaR sTF detecting pHBA in vivo in S. cerevisiae.

Figure 2

pHBA activation of sHbaR in S. cerevisiae. mCitrine fluorescence was measured by flow cytometry at late exponential phase. Values are calculated as the mean of three biological replicates.

Figure 3

pABA activates sHbaR. ySCC185-F was grown with YNB containing or not pABA and with the addition, in the medium, of 2 mM of pHBA or pABA. Cells were harvested in late exponential phase. Values are calculated as the mean of three biological replicates.

Figure 4

sBAD activity with different benzoic acid derivatives. mCitrine fluorescence was measured by flow cytometry. Values are calculated as the mean of three biological replicates. The different metabolites are grouped by similarity. (1) mono hydroxybenzoic acids (A pHBA, B 2HBA, C 3HBA), (2) mono aminobenzoic acids (D pABA, E 2NBA, F 3NBA), (3) (G) benzoic acid, (4) (H) aspirin, (5) dihydroxybenzoic acids (I 2,5DHBA, J 3,4DHBA), and (6) longer radical in position 1 of the benzoic ring (K Ferulic acid, L Homovanillic acid, M Vanillic acid). Represented values are the average of the mean fluorescence measured (n = 10). Error bars indicate the standard deviation of the measurements (n = 10). Statistical tests (Dunnett's multiple comparisons test) were performed to calculate differential significance between inducers and the control condition (***p < 0.0001 or **p < 0.001 or *p < 0.01).

Figure 5

Dose-response curves obtained with the strongest benzoic acid derivative effectors of sBAD expressed in S. cerevisiae. mCitrine fluorescence was measured by flow cytometry. The relative fluorescence of mCitrine was measured with cells grown at different concentration of (A) pHBA, (B) pABA, (C) Benzoic acid, and (D) Aspirin is compared with the control condition. In (E), the affinity and dynamic parameters calculated from different the curves dose response are presented. Values are calculated as the mean of three biological replicates.

Figure 6

sBAD functions in an engineered S. cerevisiae strain producing pHBA. (A) pHBA production during 65 h of growth in a medium devoid of pABA. (B) mCitrine fluorescence during culture in strains ySCC185-30 and ySCC185-UbiC. (C) Correlation between the mCitrine fluorescence and pHBA production in ySCC185-F UbiC. (D) Correlation between the ratio of mCitrine over mKATE2 fluorescence with pHBA production. Values are calculated as the mean of three biological replicates.