Enhanced sensitivity of an Ah-receptor system in yeast through condition modification and use of mammalian modifiers

Abstract

Proteins, such as the Ah receptor (AHR), hold potential as sensors to detect ligands in environmental and biological samples, and may also serve as tools to regulate biosynthetic and industrial processes. The AHR is also a prototype system for the PAS superfamily that can sense and mediate adaptation to signals as diverse as light, voltage, oxygen and an array of small molecules. The yeast, S. cerevisiae, has proven to be an important model to study the signal transduction of sensors like the AHR because of its ease of use, numerous available strategies for genetic manipulation, and capacity for heterologous expression. To better understand the utility of sensor proteins as components of yeast detection systems, we characterized a chimeric AHR-LexA system that drives expression from a Lex operator (LexO) driven, beta-galactosidase (β-Gal) reporter. In this report, we demonstrate that improvements in assays sensitivity and pharmacology can arise from the careful optimization of yeast growth phase and the duration of ligand exposure. We also report that the coexpression of heterotypic modifiers from mammalian cells (e.g., the ARA9 and ARA3 proteins), can improve yeast assay performance. We propose that complementing these assay improvements with previously reported yeast mutations described by others will expand the utility of the AHR for biotechnology applications.

Keywords: Ah receptor; Assay; Ligands; Polycyclic aromatic hydrocarbons; Reporter; Yeast.

Graphical abstract

Fig. 1

Domain map of the AHR and LexA-AHR fused protein. Schematic diagram of the murine AHR^b1 structure domains and the LexA substitution made on the N- terminal side of the AHR. Specifically, the bHLH domain, which contains the DNA binding domain of AHR, was swapped for the DNA binding domain of LexA.

Fig. 2

Growth Curve of the yeast L40AHRNΔ166 SD-TRP media. A: Growth curve experiment in SD-Trp media with the yeast L40 strain harboring the plasmid pBTMAHRN∆166-B1. Cultures were grown for up to 32 h (30 °C; 220 rpm) taking OD₆₀₀ measurements every two hours. All data points are average of a replica of at least 3 experiments. B: Cultures were grown for 16 h (Early Log Phase), 20 h (Mid Log Phase), and 24 h (Late Log Phase). When the cultures reached their set time they were exposed to BNF for two hours at 30 °C; 220 rpm. β-Gal activity was measured to determine AHR activation. Data points reflect 3 replicate experiments with error bars representing standard deviation.

Fig. 3

Linearity of the response after a 1:2 cell dilution. An overnight culture of the L40Δ166 was divided and diluted to an OD₆₀₀ of 0.1 and 0.5. Each cell dilution was serially diluted even further to 1:2 dilutions. Each sample was exposed to 10 µM BNF for 2 h. All data points are average of a replica of 3 experiments with error bars representing standard deviation. The β-Gal units were determined on a ClarioStar plate reader.

Fig. 4

Time of exposure. Cultures of L40pBTMAHRN∆166-B1 were exposed to direct dilution (1:4) of BNF for 15 min, 30 min, 1 h, 2 h, 4 h and 16 h at 30 °C; 220 rpm. β-Gal activity was measured to determine AHR activation. β-Gal units are reported as relative light units (RLU). All data points are average of a replica of at least 3 experiments with error bars representing standard deviation.

Fig. 5

Structures of chemicals used in this study.

Fig. 6

Pharmacology of the yeast system. Ligand-induced activity measured using the yeast system and a mouse hepatoma cell line. Dose-response curves for L40pBTMAHRN∆166-B1 yeast reporter system and for the H1L6.1c mouse hepatoma cells. Cultures were exposed to ligands for two hours at 30 °C; 220 rpm. β-Gal or luciferase activities were measured to determine AHR activation. Units were converted to percent using the maximal and minimal response from all the ligands used in the experiment. All data points are average of a replica of 3 experiments with error bars representing standard deviation.

Fig. 7

Influence of modifiers on chimeric system in yeast. The LexA-AHR chimeric system’s response to BNF (PL703, blue triangles), compared to its expression with the coexpression of the full length ARA9 modifier (PL703 + PL810, brown squares), the full length ARA3 modifier (PL703 + PL1142, green triangles) and the ARA3 modifier with its BTB domain deleted (PL703 + PL1417, red circles).