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.
Highlights
Synthetic biology proposes cutting-edge methodologies for using natural resources (Jullesson et al, 2015; Smanski et al, 2016; Le Feuvre and Scrutton, 2018; Schindler et al, 2018), understanding basic cellular functions (Metzger et al, 2018; Toda et al, 2018) or reprogramming cell fate (Black and Gersbach, 2018)
Notwithstanding the fact that a previous in vitro study described a synthetic transcription factor (sTF) activated by 4-hydroxybenzoic acid (pHBA) (Yao et al, 2018), to our knowledge, sHbaR is the first biosensor that can be activated by pHBA in vivo, in S. cerevisiae
This work has presented the successful design, construction and characterization of an orthogonal benzoic acid derivative biosensor in S. cerevisiae using the binding domain of the bacterial HbaR from R. palustris linked to the LexA protein as a DNA binding domain and B112 as a transactivation domain
Summary
Synthetic biology proposes cutting-edge methodologies for using natural resources (Jullesson et al, 2015; Smanski et al, 2016; Le Feuvre and Scrutton, 2018; Schindler et al, 2018), understanding basic cellular functions (Metzger et al, 2018; Toda et al, 2018) or reprogramming cell fate (Black and Gersbach, 2018). The clear bottleneck in metabolic engineering lays in the low throughput of analytical techniques used to determine products yields compared to the rate of the construction of new engineered microorganisms (Rogers and Church, 2016). To overcome this limitation, natural or synthetic biosensors are potential tools capable of correlating the concentration of a target chemical within the cell to an monitored output signal, such as fluorescence (Farmer and Liao, 2000; Schulman and Heyman, 2004; Dietrich et al, 2010; Liu et al, 2017; Mannan et al, 2017). The simple transfer of a tetracycline resistance gene circuit from yeast to mammalian cells required extensive optimizations such as the translation of the reporter, the DNA sequences of the heterologous proteins, the nuclear localization signal of the transcription factor and the design of the promoter (Nevozhay et al, 2013)
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