SoxR as a Single-Cell Biosensor for NADPH-Consuming Enzymes in Escherichia coli

Abstract

An ultra-high-throughput screening system for NADPH-dependent enzymes, such as stereospecific alcohol dehydrogenases, was established. It is based on the [2Fe-2S] cluster-containing transcriptional regulator SoxR of Escherichia coli that activates expression of soxS in the oxidized but not in the reduced state of the cluster. As SoxR is kept in its 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 by placing the eyfp gene under the control of the soxS promoter and analyzed the response of 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 harboring 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.

Principles of genetic circuit design

“…As circuits become more advanced, entire algorithms from control theory could be applied to improve biochemical production (Fig. 1a) 7–16 . Synthetic regulation is also an important tool for the discovery of natural products, including pharmaceuticals, insecticides, and entirely new classes of chemicals.…”

13. Reactivity of iron-sulfur clusters with nitric oxide

“…Moreover, changes in the intracellular NAD(P)H/NAD(P) ratio also result in the activation of SoxRS [196][197][198]. We note that the solvent exposed nature of [2Fe-2S] cluster likely enables rapid electron transfer to and from the cluster.…”

Development of biosensors and their application in metabolic engineering

“…Hence, it is not surprising that one straightforward approach to exploit TFs for high-throughput screening is to hack into the host transcription system and employ a synthetic or native condition-specific promoter to drive the expression of a reporter gene. For instance, Siedler et al developed an NADPH/NADP + redox sensor in Escherichia coli using its native redox sensitive TF SoxR [27 ]. Similarly, Knudsen et al constructed a yeast NADH/NAD + redox sensor by expressing GFP under the control of GPD2 promoter, which is known to be activated by an increased NADH/NAD + ratio [28].…”