GPCR-Based Chemical Biosensors for Medium-Chain Fatty Acids

Abstract

A key limitation to engineering microbes for chemical production is a reliance on low-throughput chromatography-based screens for chemical detection. While colorimetric chemicals are amenable to high-throughput screens, many value-added chemicals are not colorimetric and require sensors for high-throughput screening. Here, we use G-protein coupled receptors (GPCRs) known to bind medium-chain fatty acids in mammalian cells to rapidly construct chemical sensors in yeast. Medium-chain fatty acids are immediate precursors to the advanced biofuel fatty acid methyl esters, which can serve as a "drop-in" replacement for D2 diesel. One of the sensors detects even-chain C8-C12 fatty acids with a 13- to 17-fold increase in signal after activation, with linear ranges up to 250 μM. Introduction of a synthetic response unit alters both dynamic and linear range, improving the sensor response to decanoic acid to a 30-fold increase in signal after activation, with a linear range up to 500 μM. To our knowledge, this is the first report of a whole-cell medium-chain fatty acid biosensor, which we envision could be applied to the evolutionary engineering of fatty acid-producing microbes. Given the affinity of GPCRs for a wide range of chemicals, it should be possible to rapidly assemble new biosensors by simply swapping the GPCR sensing unit. These sensors should be amenable to a variety of applications that require different dynamic and linear ranges, by introducing different response units.

Engineering a model cell for rational tuning of GPCR signaling

“…As we have demonstrated here, our model strain also offers immediate applications for engineering yeast as biosensors, whether as single strains or as part of engineered consortia. Already efforts in synthetic biology have used engineered yeast biosensors as medical diagnostics 57 , for pathogen detection 58 , and as a tool for accelerating metabolic engineering 22,59 . In all these applications, it is desirable for the user to have control over the response to input and the magnitude of gene expression it triggers.…”

“…Core to this pathway is an extensivelystudied mitogen-activated protein kinase (MAPK) signaling cascade that functions with its own intrinsic feedback to maintain a robust input-output relationship in varying conditions 21 . As this MAPK cascade can be considered as a black-box processing unit in transduction through the pathway 11,22 , we chose to make this natural system the core from which we build and tune GPCR signaling pathways.…”

“…Increasing the expression levels of the Ste12 transcription factor was not possible due to toxicity issues (Supplementary Figure S7), so we kept the expression of all transcription factor variants fixed at initial conditions. By fusing the pheromone-responsive domain of Ste12 (PRD; 216-688) to the full-length LexA bacterial repressor protein 22,31 we generated a synthetic transcription factor (LexA-PRD) able to target the pathway output to a library of modular synthetic promoters containing LexA operator sequences (LexO). These promoters were designed to direct different levels of expression by having different numbers of upstream transcription factor binding sites or different core promoter regions 30 .…”

Applications and advances of metabolite biosensors for metabolic engineering

“…A recent study used Gprotein coupled receptors (GPCRs) to sense medium-chain fatty acid and coupled it via the yeast mating pathway to a specific transcription factor, which activates the transcription of a GFP, resulting in sensors that detect even-chain C8-C12 fatty acids with dynamic range up to 17-fold in Saccharomyces cerevisiae (Mukherjee et al, 2015). Similar sensors can be potentially created by engineering other signaling pathways.…”

Biofuel metabolic engineering with biosensors

“…An alternative to cytosolic TFs is GPCR-based biosensors expressed on the cell surface [33–35]. For these biosensors, the binding of an extracellular metabolite to a GPCR results in signal transduction and, ultimately, changes in gene expression (Fig.…”