Evolving a generalist biosensor for bicyclic monoterpenes

Prokaryotic transcription factors can be repurposed as analytical and synthetic tools for precise chemical measurement and regulation. Monoterpenes encompass a broad chemical family that are commercially valuable as flavors, cosmetics, and fragrances, but have proven difficult to measure, especially in cells. Herein, we develop genetically-encoded, generalist monoterpene biosensors by using directed evolution to expand the effector specificity of the camphor-responsive TetR-family regulator CamR from Pseudomonas putida. Using a novel negative selection coupled with a high-throughput positive screen (Seamless Enrichment of Ligand-Inducible Sensors, SELIS), we evolve CamR biosensors that can recognize four distinct monoterpenes: borneol, fenchol, eucalyptol, and camphene. Different evolutionary trajectories surprisingly yielded common mutations, emphasizing the utility of CamR as a platform for creating generalist biosensors. Systematic promoter optimization driving the reporter increased the system's signal-to-noise ratio to 150-fold. These sensors can serve as a starting point for the high-throughput screening and dynamic regulation of bicyclic monoterpene production strains.

DNA Binding and Sensor Specificity of FarR, a Novel TetR Family Regulator Required for Induction of the Fatty Acid Efflux Pump FarE in Staphylococcus aureus

ABSTRACT Divergent genes in Staphylococcus aureus USA300 encode the efflux pump FarE and TetR family regulator FarR, which confer resistance to antimicrobial unsaturated fatty acids. To study their regulation, we constructed USA300 ΔfarER, which exhibited a 2-fold reduction in MIC of linoleic acid. farE expressed from its native promoter on pLIfarE conferred increased resistance to USA300 but not USA300 ΔfarER. Complementation of USA300 ΔfarER with pLIfarR also had no effect, whereas resistance was restored with pLIfarER or through ectopic expression of farE. In electrophoretic mobility shift assays, FarR bound to three different oligonucleotide probes that each contained a TAGWTTA motif, occurring as (i) a singular motif overlapping the −10 element of the PfarR promoter, (ii) in palindrome PAL1 immediately in the 3′ direction of PfarR, or (iii) within PAL2 upstream of the predicted PfarE promoter. FarR autorepressed its expression through cooperative binding to PAL1 and the adjacent TAGWTTA motif in PfarR. Consistent with reports that S. aureus does not metabolize fatty acids through acyl coenzyme A (acyl-CoA) intermediates, DNA binding activity of FarR was not affected by linoleoyl-CoA. Conversely, induction of farE required fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of unsaturated fatty acids into phospholipid. We conclude that FarR is needed to promote the expression of farE while strongly autorepressing its own expression, and our data are consistent with a model whereby FarR interacts with a FakA-dependent product of exogenous fatty acid metabolism to ensure that efflux only occurs when the metabolic capacity for incorporation of fatty acid into phospholipid is exceeded. IMPORTANCE Here, we describe the DNA binding and sensor specificity of FarR, a novel TetR family regulator (TFR) in Staphylococcus aureus. Unlike the majority of TFRs that have been characterized, which function to repress a divergently transcribed gene, we find that FarR is needed to promote expression of the divergently transcribed farE gene, encoding a resistance-nodulation-division (RND) family efflux pump that is induced in response to antimicrobial unsaturated fatty acids. Induction of farE was dependent on the function of the fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of exogenous unsaturated fatty acids into phospholipid. This represents a novel example of TFR function.

Involvement of the TetR-Type Regulator PaaR in the Regulation of Pristinamycin I Biosynthesis through an Effect on Precursor Supply in Streptomyces pristinaespiralis

ABSTRACTPristinamycin I (PI), produced byStreptomyces pristinaespiralis, is a streptogramin type B antibiotic, which contains two proteinogenic and five aproteinogenic amino acid precursors. PI is coproduced with pristinamycin II (PII), a member of streptogramin type A antibiotics. The PI biosynthetic gene cluster has been cloned and characterized. However, thus far little is understood about the regulation of PI biosynthesis. In this study, a TetR family regulator (encoded bySSDG_03033) was identified as playing a positive role in PI biosynthesis. Its homologue, PaaR, fromCorynebacterium glutamicumserves as a transcriptional repressor of thepaagenes involved in phenylacetic acid (PAA) catabolism. Herein, we also designated the identified regulator as PaaR. Deletion ofpaaRled to an approximately 70% decrease in PI production but had little effect on PII biosynthesis. Identical to the function of its homologue fromC. glutamicum, PaaR is also involved in the suppression ofpaaexpression. Given that phenylacetyl coenzyme A (PA-CoA) is the common intermediate of the PAA catabolic pathway and the biosynthetic pathway ofl-phenylglycine (l-Phg), the last amino acid precursor for PI biosynthesis, we proposed that derepression of the transcription ofpaagenes in a ΔpaaRmutant possibly diverts more PA-CoA to the PAA catabolic pathway, thereby with less PA-CoA metabolic flux towardl-Phg formation, thus resulting in lower PI titers. This hypothesis was verified by the observations that PI production of a ΔpaaRmutant was restored byl-Phg supplementation as well as by deletion of thepaaABCDEoperon in the ΔpaaRmutant. Altogether, this study provides new insights into the regulation of PI biosynthesis byS. pristinaespiralis.IMPORTANCEA better understanding of the regulation mechanisms for antibiotic biosynthesis will provide valuable clues forStreptomycesstrain improvement. Herein, a TetR family regulator PaaR, which serves as the repressor of the transcription ofpaagenes involved in phenylacetic acid (PAA) catabolism, was identified as playing a positive role in the regulation of pristinamycin I (PI) by affecting the supply of one of seven amino acid precursors,l-phenylglycine, inStreptomyces pristinaespiralis. To our knowledge, this is the first report describing the interplay between PAA catabolism and antibiotic biosynthesis inStreptomycesstrains. Considering that the PAA catabolic pathway and its regulation by PaaR are widespread in antibiotic-producing actinomycetes, it could be suggested that PaaR-dependent regulation of antibiotic biosynthesis might commonly exist.