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.

D-Xylose Sensing in Saccharomyces cerevisiae: Insights from D-Glucose Signaling and Native D-Xylose Utilizers

“…As a parallel strategy to modifying the endogenous signaling pathways, an increasing number of studies have used synthetic biology approaches to construct artificial regulatory and signaling circuits that render microbes sensitive to non-native substrates [297][298][299][300]. Synthetic signaling networks are defined as either: (i) a cascade of novel signaling events, or (ii) a set of exogenous or engineered TFs with new specificities and signals.…”

Emerging biosensors in detection of natural products

“…Castaño-Cerezo et al proposed a fluorescence-based synthetic biosensor that could detect the production of benzoic acid in a genetically modified Saccharomyces cerevisiae both in vitro and in vivo . The sensor is composed of a pHBA (4-Hydroxybenzoic Acid)-binding domain of HbaR from Rhodopseudomonas palustris , the transactivation domain B112 and the LexA DNA binding domain at the C-terminus and N-terminus, respectively [ 89 ]. González-Andrade et al developed a fluorescent-biosensor based on human fluorophore-labeled Calmodulin (CaM) for the detection of potential CaM inhibitors such as malbrancheamide and tajixanthone.…”

Toward Multiplexed Optogenetic Circuits

“…To achieve this real-time tuning, optogenetic actuators could be coupled in bioreactors with biosensors and enable in vivo metabolite sensing. Even if the type of biosensor needs to be diversified, recent studies have broadened the biosensor library, specific to the metabolic pathway of interest, such as monoterpenes ( d’Oelsnitz et al, 2021 ) or phenol derivatives ( Castaño-Cerezo et al, 2020 ). Connecting optogenetic systems to metabolite sensing has opened up cybergenetic applications [reviewed in Carrasco-López et al (2020) ].…”