GPCR-Based Chemical Biosensors for Medium-Chain Fatty Acids

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

Medium-Throughput Screen of Microbially Produced Serotonin via a G‑Protein-Coupled Receptor-Based Sensor

Chemical biosensors, for which chemical detection triggers a fluorescent signal, have the potential to accelerate the screening of noncolorimetric chemicals produced by microbes, enabling the high-throughput engineering of enzymes and metabolic pathways. Here, we engineer a G-protein-coupled receptor (GPCR)-based sensor to detect serotonin produced by a producer microbe in the producer microbe’s supernatant. Detecting a chemical in the producer microbe’s supernatant is nontrivial because of the number of other metabolites and proteins present that could interfere with sensor performance. We validate the two-cell screening system for medium-throughput applications, opening the door to the rapid engineering of microbes for the increased production of serotonin. We focus on serotonin detection as serotonin levels limit the microbial production of hydroxystrictosidine, a modified alkaloid that could accelerate the semisynthesis of camptothecin-derived anticancer pharmaceuticals. This work shows the ease of generating GPCR-based chemical sensors and their ability to detect specific chemicals in complex aqueous solutions, such as microbial spent medium. In addition, this work sets the stage for the rapid engineering of serotonin-producing microbes

A Heritable Recombination System for Synthetic Darwinian Evolution in Yeast

Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism <i>in vitro</i> are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis <i>via</i> homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in <i>Saccharomyces cerevisiae</i>. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10¹³. Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology

Matching Protein Interfaces for Improved Medium-Chain Fatty Acid Production

Medium-chain fatty acids (MCFAs) are key intermediates in the synthesis of medium-chain chemicals including α-olefins and dicarboxylic acids. In bacteria, microbial production of MCFAs is limited by the activity and product profile of fatty acyl-ACP thioesterases. Here, we engineer a heterologous bacterial medium-chain fatty acyl-ACP thioesterase for improved MCFA production in <i>Escherichia coli</i>. Electrostatically matching the interface between the heterologous medium-chain <i>Acinetobacter baylyi</i> fatty acyl-ACP thioesterase (AbTE) and the endogenous <i>E. coli</i> fatty acid ACP (<i>E. coli</i> AcpP) by replacing small nonpolar amino acids on the AbTE surface for positively charged ones increased secreted MCFA titers more than 3-fold. Nuclear magnetic resonance titration of <i>E. coli</i> ¹⁵N-octanoyl-AcpP with a single AbTE point mutant and the best double mutant showed a progressive and significant increase in the number of interactions when compared to AbTE wildtype. The best AbTE mutant produced 131 mg/L of MCFAs, with MCFAs being 80% of all secreted fatty acid chain lengths after 72 h. To enable the future screening of larger numbers of AbTE variants to further improve MCFA titers, we show that a previously developed G-protein coupled receptor (GPCR)-based MCFA sensor differentially detects MCFAs secreted by <i>E. coli</i> expressing different AbTE variants. This work demonstrates that engineering the interface of heterologous enzymes to better couple with endogenous host proteins is a useful strategy to increase the titers of microbially produced chemicals. Further, this work shows that GPCR-based sensors are producer microbe agnostic and can detect chemicals directly in the producer microbe supernatant, setting the stage for the sensor-guided engineering of MCFA producing microbes

Microbial Synthesis of Pinene

The volumetric heating values of today’s biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered <i>Escherichia coli</i> for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ∼28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The <i>Abies grandis</i> GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered <i>E. coli</i>. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio