Transporter-Driven Engineering of a Genetic Biosensor for the Detection and Production of Short-Branched Chain Fatty Acids in Saccharomyces cerevisiae

Abstract

Biosensors can be used for real-time monitoring of metabolites and high-throughput screening of producer strains. Use of biosensors has facilitated strain engineering to efficiently produce value-added compounds. Following our recent work on the production of short branched-chain fatty acids (SBCFAs) in engineered Saccharomyces cerevisiae, here we harnessed a weak organic acid transporter Pdr12p, engineered a whole-cell biosensor to detect exogenous and intracellular SBCFAs and optimized the biosensor’s performance by varying PDR12 expression. We firstly constructed the biosensor and evaluated its response to a range of short-chain carboxylic acids. Next, we optimized its sensitivity and operational range by deletion and overexpression of PDR12. We found that the biosensor responded to exogenous SBCFAs including isovaleric acid, isobutyric acid and 2-methylbutanoic acid. PDR12 deletion enhanced the biosensor’s sensitivity to isovaleric acid at a low concentration and PDR12 overexpression shifted the operational range towards a higher concentration. Lastly, the deletion of PDR12 improved the biosensor’s sensitivity to the SBCFAs produced in our previously engineered SBCFA-overproducing strain. To our knowledge, our work represents the first study on employing an ATP-binding-cassette transporter to engineer a transcription-factor-based genetic biosensor for sensing SBCFAs in S. cerevisiae. Our findings provide useful insights into SBCFA detection by a genetic biosensor that will facilitate the screening of SBCFA-overproducing strains.

In Vivo Detection of Low Molecular Weight Platform Chemicals and Environmental Contaminants by Genetically Encoded Biosensors

“…So far, TFs have been successfully identified by combined transcriptome and proteome analyses after exposure of (microbial) cells to the desired small molecule as well as the computer-assisted mining of databases. Furthermore, (prokaryotic) TFs can be responsive to different but structurally related compounds (e.g., War1 for SMCFAs, , BmoR, and AlkS , for SMCAHs, or CadC for Cd 2+ , Pb 2+ , and other HM ions). This ligand binding promiscuity facilitated the random mutagenesis by epPCR and the rational engineering of various LBDs and DBDs by well-established protein engineering techniques. ,,,,, A complementary approach for the identification of new biosensors was employed by Zhang et al, who scouted chemicals with molecular shapes similar to the target small molecule.…”

“…Similarly, Miyake et al enhanced the intracellular concentration of SMCFAs by adjusting expression levels of PDR12, a transporter for organic acids. This strategy increased the sensitivity and the operational window of their SMCFA-responsive biosensor ( P PDR12 /War1 coupled to GFP) . Complementary, export proteins can be used to reduce intracellular concentrations of potentially interfering chemical entities.…”

“…29 Similarly, Miyake et al utilized P PDR12 /War1 and GFP to detect the branched SMCFAs isobutyric acid (C 4 ; 88.11 g mol −1 ), 2-methylbutanoic acid and 3-methylbutanoic acid (both C 5 ; 102.13 g mol −1 ), as well as unsaturated methacrylic acid (C 4 ; 86.06 g mol −1 ), and interestingly, benzoic acid (C 7 ; 122.12 g mol −1 ). 30 The authors also altered biosensor performance (e.g., sensitivity and operational range) by varying the expression levels of the weak organic acid transporter PDR12 and suggested the implementation of War1 mutants, displaying different binding affinities of the LBD and the DBD for branched SMCFAs and cognate operator sequences, respectively. 31 For the detection of the corresponding (branched-chain) SMCAHs, Zhang et al developed a TF-based biosensor system in S. cerevisiae.…”

“…This strategy increased the sensitivity and the operational window of their SMCFA-responsive biosensor ( P PDR12 /War1 coupled to GFP). 30 Complementary, export proteins can be used to reduce intracellular concentrations of potentially interfering chemical entities. One example from above featured the reduction of intracellular Zn 2+ ions by the overexpression of the ZitB metal transporter to minimize interference with the CadR-based detection of Cd 2+ ions in E. coli .…”