An Artificial Yeast Genetic Circuit Enables Deep Mutational Scanning of an Antimicrobial Resistance Protein

Abstract

Understanding the molecular mechanisms underlying antibiotic resistance requires concerted efforts in enzymology and medicinal chemistry. Here we describe a new synthetic biology approach to antibiotic development, where the presence of tetracycline antibiotics is linked to a life-death selection in Saccharomyces cerevisiae. This artificial genetic circuit allowed the deep mutational scanning of the tetracycline inactivating enzyme TetX, revealing key functional residues. We used both positive and negative selections to confirm the importance of different residues for TetX activity, and profiled activity hotspots for different tetracyclines to reveal substrate-specific activity determinants. We found that precise positioning of FAD and hydrophobic shielding of the tetracycline are critical for enzymatic inactivation of doxycycline. However, positioning of FAD is suboptimal in the case of anhydrotetracycline, potentially explaining its comparatively poor degradation and potential as an inhibitor for this family of enzymes. By combining artificial genetic circuits whose function can be modulated by antimicrobial resistance determinants, we establish a framework to select for the next generation of antibiotics.

Building artificial genetic circuits to understand protein function

“…We recently used deep mutational scanning in yeast to investigate the structure and function of the antimicrobial resistance enzyme TetX (Scott et al, 2018). TetX is a flavindependent monooxygenase that can degrade all known tetracycline antibiotics (Yang et al, 2004).…”

“…Furthermore, codon usage optimization is recommended for all genes not originating from yeast -most gene synthesis companies offer this service. In our recent work, we found expression of the yeast codon usage optimized gene for TetX from the ADH1 promoter on a 2-micron plasmid was sufficient to destroy the tetracycline anhydrotetracycline (ATC) in yeast cultures, as detected by high performance liquid chromatography-mass spectrometry (HPLC-MS) (Scott et al, 2018). While a similar expression configuration can be an initial foundation for circuit design, expression may need to be adjusted during the final library selection to ensure enough sensitivity to detect mutations that cause subtle changes in function.…”

“…As TetX functions by degrading tetracyclines, a tetracycline inducible gene expression system was used to express the master regulator Gal4 in MaV204K (Scott et al, 2018). We built a genetic circuit where the reverse tetracycline-controlled transactivator (rtTA) can sense tetracyclines and bind to promoter elements upstream of the GAL4 gene, encoding the Gal4 transcription factor.…”

“…Our initial exploratory experiments demonstrated that induction of Gal4 expression by ATC caused toxicity to yeast (Scott et al, 2018). We hypothesized this may be caused by pleiotropic effects of Gal4 overexpression (Keren et al, 2016).…”

“…We will explain how variants in random mutant libraries can be selected for the gain, loss, or retention of protein activity, and how deep mutational scanning can reveal what and how prevalent these mutations are. We will illustrate this process by probing the structure and function relationship of residues within TetX, an emerging antimicrobial resistance threat that causes the enzymatic inactivation of all known tetracycline antibiotics (Scott, Mathews, Flematti, Filipovska, & Rackham, 2018).…”