A genome-wide approach for identification and characterisation of metabolite-inducible systems

Abstract

Inducible gene expression systems are vital tools for the advancement of synthetic biology. Their application as genetically encoded biosensors has the potential to contribute to diagnostics and to revolutionise the field of microbial cell factory development. Currently, the number of compounds of biological interest by far exceeds the number of available biosensors. Here, we address this limitation by developing a generic genome-wide approach to identify transcription factor-based inducible gene expression systems. We construct and validate 15 functional biosensors, provide a characterisation workflow to facilitate forward engineering efforts, exemplify their broad-host-range applicability, and demonstrate their utility in enzyme screening. Previously uncharacterised interactions between sensors and compounds of biological relevance are identified by employing the largest reported library of metabolite-responsive biosensors in an automated high-throughput screen. With the rapidly growing genomic data these innovative capabilities offer a platform to vastly increase the number of biologically detectable molecules.

Strategies for Improving Small-Molecule Biosensors in Bacteria

“…The efficacy of this approach has benefited greatly from the advent of next-generation sequencing technologies, as the increasing number of available microbial genome sequences enables the rapid identification of numerous testable homologs [ 113 , 114 ]. Genomic mining has proven remarkably effective at identifying new biosensors in nature, enabling the development of sensors for stilbenes [ 115 ], β-alanine [ 116 ], and progesterone [ 117 ]. Bioinformatic approaches have also been used to construct chimeras to combine properties amongst biosensors within related families, with this approach having been applied to both two-component systems [ 118 , 119 ] and allosteric transcription factors [ 120 ].…”

Single-cell image analysis to explore cell-to-cell heterogeneity in isogenic populations

“…Several novel experimental tools have recently been developed to measure levels of metabolites in single cells, many of which make use of metabolite-binding transcription factors (Hanko et al, 2020). A step beyond quantifying metabolite concentration is measuring flux, the relevant functional output of metabolic activity.…”

Impaired purine homeostasis plays a primary role in trimethoprim‐mediated induction of virulence genes in Burkholderia thailandensis

“…The Xdh inhibitor allopurinol elicited a similar effect as trimethoprim in terms of upregulation of malR and malA (Figures 1e and 3d) with corresponding increases in intracellular xanthine concentrations (Figure 3c). In some bacterial species, xanthine has been reported to induce an increase in expression of the genes encoding Xdh (Hanko et al, 2020;Self, 2002), so we wanted to ascertain if growth with allopurinol or trimethoprim led to changes in xdh expression. Both allopurinol and trimethoprim caused modestly (<2-fold) increased expression of xdh a change in expression also seen on addition of xanthine (Figure 5a), further indicating that trimethoprim and allopurinol elicit similar changes in purine metabolism.…”