Development of a Transcription Factor-Based Diamine Biosensor in Corynebacterium glutamicum

Abstract

Diamines serve as major platform chemicals that can be employed to a variety of industrial scenarios, particularly as monomers for polymer synthesis. High-throughput sensors for diamine biosynthesis can greatly improve the biological production of diamines. Here, we identified and characterized a transcription factor-driven biosensor for putrescine and cadaverine in Corynebacterium glutamicum. The transcriptional TetR-family regulatory protein CgmR (CGL2612) is used for the specific detection of diamine compounds. This study also improved the dynamic range and the sensitivity to putrescine by systematically optimizing genetic components of pSenPut. By a single cell-based screening strategy for a library of CgmR with random mutations, this study obtained the most sensitive variant CgmRI152T, which possessed an experimentally determined limit of detection (LoD) of ≤0.2 mM, a K of 11.4 mM, and a utility of 720. Using this highly sensitive putrescine biosensor pSenPutI152T, we demonstrated that CgmRI152T can be used as a sensor to detect putrescine produced biologically in a C. glutamicum system. This high sensitivity and the range of CgmR will be an influential tool for rewiring metabolic circuits and facilitating the directed evolution of recombinant strains toward the biological synthesis of diamine compounds.

A Synthetic Biosensor for Detecting Putrescine in Beef Samples

“…There have been two reports , describing the development of synthetic promoters for detecting putrescine. In particular, in Chen et al, they modified the Tac (TacR) and the wild-type promoter (PuuAp) to monitor intracellular putrescine production.…”

“…To engineer our library of synthetic promoters, we used two commonly used strategies: (1) modify the −35 and −10 consensus sequence and (2) add transcription factor binding regions at different locations within the operator . As an initial design, we used the two highest dynamic range promoters, TacR(2) and LR2, from Chen et al and generated a hybrid promoter consisting of the “–10” site from the LR2 promoter and the “–35” site from TacR promoter because hybrid promoters are known to be highly efficient when directing transcription compared to nonhybrid promoters. , Next, we obtained sequences from PuuA and PuuD genes, , which are known PuuR binding regions, GTGG­TCATT­ATAT­TTT­ACGC (we call “A”), ATGT­TCAA­TATT­TTTT­CAAT (B), and GTGG­ACT­AAAT­TAT­CGC­CAT (C), and placed these binding regions within (W) and/or downstream (D) of the −35 and −10 sites to create different combinations of binding sites as it has been shown that mixing binding sites can lead to changes in the sensing and regulation. − Using these two strategies, we used 4 promoters from Chen et al as controls and created 7 hybrid promoters (Figure B).…”

“…As far as we are aware, there are only two reports (by Chen et al 12 and Zhao et al 13 ) that have developed a biosensor for BA detection. These works describe whole-cell biosensors that use cell-based transcriptional circuits to monitor concentrations of putrescine.…”

“…These works describe whole-cell biosensors that use cell-based transcriptional circuits to monitor concentrations of putrescine. Our work builds on the work by Chen et al 12 and Zhao et al 13 by investigating new synthetic promoters to improve on the limit of detection, dynamic range, sensitivity, and specificity. In addition, we integrated the biosensor with a cell-free expression system 16 to lower the cost and to adapt this platform for use in restaurants, supermarkets, butcher shops and at home.…”

Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor

“…A biosensor is an ideal connector between enzyme activity and the cellular phenotype, allowing direct in vivo ultrahigh-throughput characterization of enzyme activity . Transcription factors (TFs) regulate gene expression over a greater dynamic range, and they have been increasingly developed for protein engineering, metabolic engineering, and synthetic biology. − However, limited by the inherent shortcomings of natural TFs, they often need to be adaptively evolved before meeting application needs . For instance, natural TFs are susceptible to full activation by a trace concentration of inducers (usually less than 1 mM), but the progressive reporter gene expression is difficult to achieve. − Accordingly, in some cases, natural TFs need to be adaptively evolved for wider operating ranges because the strong correlation between the ligand concentration and the signal output is vital for enzyme screening.…”