Transcription factor-based biosensors - accelerating metabolic and enzyme engineering
An NADPH biosensor based on the transcriptional regulator SoxR of Escherichia coli
Alcohol dehydrogenases are employed in various biotechnological processes, such as for the reduction of ketones to produce enantiopure secondary alcohols. We developed an ultra-high-throughput screening system for NADPH-dependent alcohol dehydrogenases, which is based on the [2Fe−2S] cluster-containing transcriptional regulator SoxR of Escherichia coli. SoxR activates expression of its only target gene soxS in the oxidized but not in the reduced state of the [2Fe−2S] cluster. Importantly, as SoxR is kept in its inactive reduced state by NADPH-dependent reductases, an increased NADPH demand of the cell counteracts SoxR reduction and increases soxS expression. We have taken advantage of these properties to construct the NADPH biosensor pSenSox, in which expression of the eyfp reporter gene is controlled by the soxS promoter (Fig. 1). The functionality of pSenSox was analysed with E. coli cells expressing an NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis (LbAdh), which reduces methyl acetoacetate to (R)-methyl 3-hydroxybutyrate. Under suitable conditions, the specific fluorescence of the cells correlated with the substrate concentration added and with LbAdh enzyme activity, supporting the NADPH responsiveness of the sensor. These properties enabled sorting of single cells harbouring wild-type LbAdh from those with lowered or without LbAdh activity by fluorescence-activated cell sorting (FACS). In a proof-of-principle application, the system was used successfully to screen a mutant LbAdh library for variants showing improved activity with the substrate 4-methyl-2-pentanone (1).
A cAMP biosensor based on the transcriptional regulator GlxR of Corynebacterium glutamicum
Cyclic adenosine monophosphate (cAMP) plays a regulatory role as second messenger in many species. In the industrial model organism Corynebacterium glutamicum, cAMP acts as effector of the global transcriptional regulator GlxR, a homolog of enterobacterial Crp. The cAMP-GlxR complex activates or represses the expression of about 200 target genes. CyaB, a membrane-bound class III adenylate cyclase, synthesizes cAMP from ATP, but another yet unknown cAMP-forming enzyme is likely present in C. glutamicum. Recently, we identified the cAMP phosphodiesterase CpdA, which catalyses the hydrolysis of cAMP to AMP (2). As a tool to search for additional cAMP-forming and degrading enzymes, we constructed a plasmid-based cAMP biosensor by fusing the promoter of cg3195, a gene strongly repressed by GlxR, to the eyfp reporter gene (Fig. 2) (3). In control experiments, the biosensor showed the predicted responses to increased levels of cAMP or GlxR. The biosensor was able to distinguish between C. glutamicum wild type and mutants with defects in cAMP biosynthesis or degradation. Most importantly, the sensor allowed successful sorting of mixtures of wild type and mutant strains by fluorescence activated cell sorting (FACS), thus meeting the requirements for high-throughput screening of libraries for single mutant cells with an altered cAMP level.
References
1. Siedler, S., Schendzielorz, G., Binder, S., Eggeling, L., Bringer, S., and Bott, M. (2014) SoxR as a single-cell biosensor for NADPH-consuming enzymes in Escherichia coli. ACS Synth. Biol. 3, 41-47
2. Schulte, J., Baumgart, M., and Bott, M. (2017) Identification of the cAMP phosphodiesterase CpdA as novel key player in cAMP-dependent regulation in Corynebacterium glutamicum. Mol. Microbiol. 103, 534-552
3. Schulte, J., Baumgart, M., and Bott, M. (2017) Development of a single-cell GlxR-based cAMP biosensor for Corynebacterium glutamicum. J. Biotechnol., In press