An Artificial Yeast Genetic Circuit Enables Deep Mutational Scanning

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An artificial yeast genetic circuit enables deep mutational scanning of an antimicrobial resistance protein Louis H. Scott, James C. Mathews, Gavin R. Flematti, Aleksandra Filipovska, and Oliver Rackham ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00121 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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ACS Synthetic Biology

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An artificial yeast genetic circuit enables deep mutational

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scanning of an antimicrobial resistance protein

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Louis H. Scotta, James C. Mathewsa, Gavin R. Flemattib, Aleksandra Filipovskaa,b, Oliver

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Rackhama,b,1

6 7 8

a

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University of Western Australia, Nedlands 6009, Australia.

Harry Perkins Institute of Medical Research and Centre for Medical Research, The

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b

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Australia.

School of Molecular Sciences, The University of Western Australia, Crawley 6009,

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To whom correspondence may be addressed. Email: [email protected].

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Key words: synthetic biology; tetracycline; structure-function relationships; TetX; antibiotic

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resistance; genetic selection; deep mutational scanning.

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ABSTRACT

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Understanding the molecular mechanisms underlying antibiotic resistance requires

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concerted efforts in enzymology and medicinal chemistry. Here we describe a new

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synthetic biology approach to antibiotic development, where the presence of tetracycline

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antibiotics is linked to a life-death selection in Saccharomyces cerevisiae. This artificial

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genetic circuit allowed the deep mutational scanning of the tetracycline inactivating

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enzyme TetX, revealing key functional residues. We used both positive and negative

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selections to confirm the importance of different residues for TetX activity, and profiled

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activity hotspots for different tetracyclines to reveal substrate-specific activity

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determinants. We found that precise positioning of FAD and hydrophobic shielding of

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the tetracycline are critical for enzymatic inactivation of doxycycline. However,

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positioning of FAD is suboptimal in the case of anhydrotetracycline, potentially

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explaining its comparatively poor degradation and potential as an inhibitor for this

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family of enzymes. By combining artificial genetic circuits whose function can be

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modulated by antimicrobial resistance determinants, we establish a framework to select

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for the next generation of antibiotics.


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Artificial genetic selections and screens provide invaluable biological insight for engineering

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new functions in organisms and biomolecules. Selections and screens in Saccharomyces

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cerevisiae

have

proven

exceptionally

1,2

important

for

elucidating

macromolecular

3

4

interactions , evolving novel functions , and resolving protein and pathway engineering

5

problems4,5. However, the power of yeast genetics has not yet been fully harnessed to tackle

6

the pressing issue facing human health: our failing arsenal of drugs against antibiotic

7

resistance. Resistance affects all antibiotic classes, including the broad-spectrum tetracyclines

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whose members are used extensively against a wide range of both Gram-positive and

9

negative bacteria, chlamydiae, mycoplasmas, rickettsiae and protozoa6. While antibiotic

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resistance against most classes typically occurs through enzymatic degradation, e.g. β-

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lactamase-mediated inactivation of penicillins7, this once underrepresented mode of

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resustance8 is now becoming recognized as a serious threat against tetracyclines, with the

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detection of inactivating enzymes in environmental8–10 and clinical samples11–16. The

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emergence of noncanonical tetracycline resistance provides a fresh impetus to develop new

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genetic selection tools to fight this threat.

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TetX was the first tetracycline inactivating enzyme to be characterized17. It is a flavin

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adenine dinucleotide (FAD)-dependent monooxygenase that acts through the regioselective

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monohydroxylation of tetracyclines, causing their spontaneous, non-enzymatic breakdown17–

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19

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bound to its FAD cofactor, and in complex with 7-iodtetracycline and 7-chlortetracycline19,

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and with minocycline and tigecycline22 have provided clues to the molecular mechanism by

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which TetX inactivates all known tetracyclines, including the third-generation ‘glycylcycline’

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tigecycline23, and eravacycline24. More recently, structures of distantly related Tet(50)

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tetracycline inactivating enzyme revealed two distinct conformations of the protein: one

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where FAD is poised to react with tetracyclines and another where FAD is in an unproductive

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conformation away from the substrate-binding site25. Interestingly a co-crystal structure with

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anhydrotetracycline (ATC), that has poor antibiotic activity, showed that it locked FAD in the

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unproductive state. This has led to the proposal that ATC could be used as an inhibitor of

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tetracycline inactivating enzymes, if co-administered with other tetracycline antibiotics.

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However, while Tet(50) and a number of related tetracycline inactivating enzymes isolated

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from soil samples cannot degrade ATC, TetX can, albeit slowly. Therefore, an outstanding

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question is which residues in TetX are responsible for differentiating between different

and loss of Mg2+ coordination that is crucial for ribosome binding20,21. Structures of TetX



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tetracyclines, and how can this information guide either the re-design of ATC as an enzyme

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inhibitor, or the re-design of other tetracycline members to evade inactivation?

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To answer this question, we used a synthetic biology approach in yeast to investigate

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the molecular mechanism behind the enzymatic inactivation of tetracyclines by TetX. We

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developed a series of artificial genetic circuits that permit the life-death selection for

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tetracyclines in yeast, enabling deep mutational scanning of the tetracycline resistance

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enzyme TetX. By performing multiple selections with different tetracyclines we identified

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key residues that are important for activity, thereby guiding the future rational design of new

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drugs and inhibitors. Our system provides a highly versatile and adaptable platform for drug-

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discovery and the investigation of diverse proteins and biomolecules.

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RESULTS

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An artificial genetic circuit for the life-death selection of tetracyclines in yeast. To

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develop a yeast strain whose growth on selective media depends on the presence of

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tetracyclines, we used a fusion between a variant of the tetracycline repressor (TetR)

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transcription factor and the transcription activating domain of the VP16 protein. When a

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tetracycline associates with this fusion protein, known as rtTA, it binds to a specific DNA

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sequence known as tetO and activates the transcription of nearby genes26. We constructed

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plasmids containing the GAL4 gene under control of tetO, such that the Gal4 transcription

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factor is produced when tetracyclines are present. To provide different levels of Gal4

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production responsiveness to the presence of tetracyclines, plasmids contained either two or

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seven copies of the tetracycline operator (tetO) DNA sequence upstream26 of GAL4. A series

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of GAL4 variants were used that combined strong or weak Kozak consensus sequences and

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start codons to further modulate the amount of Gal4 produced (Figure 1a). We coupled these

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tetracycline-responsive Gal4 expression plasmids with a yeast strain harbouring HIS3 and

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URA3 biosynthetic genes under control of Gal4-inducible upstream activating sequence

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(UASGAL) elements, to enable growth in media lacking histidine and uracil, respectively, if

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Gal4 is present (Figure 1b)27,28. Using URA3 as a reporter gene also provided a means for

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negative selection, as the enzyme encoded by URA3 will decarboxylate 5-fluoroorotic acid

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(5-FOA) to yield the toxic metabolite 5-fluorouracil (5-FU) (Figure 1c). Furthermore, the

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competitive inhibitor of the protein product of HIS3, 3-amino-1,2,4-triazole (3-AT), can be

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used to eliminate false positives that arise from uninduced leaky HIS3 expression in the

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absence of ATC (Figure 1c).

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Figure 1. An artificial genetic circuit for tetracycline selection in yeast. (a) Constitutively expressed rtTA (a rTetR and VP16 fusion) binds tetO when in complex with tetracyclines. Binding of tetO by rtTA induces the transcription of GAL4, and a greater number of tetO sites allows stronger GAL4 transcription. Plasmids with mutated GAL4 offer the following descending levels of translation: GAL4 with a strong Kozak consensus sequence; GAL4 with a weak Kozak; GAL4 with a weak start codon; and GAL4 with both a weak Kozak and a weak start codon. Nucleotide bases in bold represent the GAL4 start codon, bases in red represent mutants for altered translation. Lower case letters represent the most common bases that can nevertheless vary, whereas upper case letters indicate highly functionally important bases. The genetic circuit expression cascade is impeded if the inducer is degraded by a resistance factor (e.g. TetX). (b) Gal4 production by the genetic circuit will dictate the survival of yeast: too little Gal4 and the auxotrophic markers will not be expressed and yeast will not survive on media lacking histidine and uracil; Gal4 levels that activate the auxotrophic markers without toxicity enable yeast to survive on media lacking histidine and uracil; or pleiotropic effects of excess Gal4 will cause toxicity and the yeast will not survive. (c) Dependent on auxotrophic marker expression, activation of URA3 can be selected against using 5-fluoroorotic acid (5-FOA) that is metabolized into the cytotoxic compound fluorouracil (5-FU). Effects of leaky HIS3 expression in the absence of ATC can be countered using the competitive inhibitor 3-amino-1,2,4-triazole (3-AT).



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We performed a yeast survival assay to assess if any of the genetic circuits

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constructed could induce the expression of Gal4, in the presence of a tetracycline, to a

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sufficient level to activate the HIS3 and URA3 auxotrophic markers and allow yeast to

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survive on media lacking histidine and uracil (Figure 2a and Figure S1). We used ATC as an

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inducer as TetR is promiscuous in its tetracycline recognition, with the strongest affinity

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towards ATC [ATC.Mg]+ (Ka ~ 1011 M-1)29. Initially we confirmed that Gal4 is required for

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auxotrophic marker activation in our system as yeast containing the empty pCM251 plasmid

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(that does not express Gal4) did not survive on media lacking histidine and uracil (SC-L-T-U-

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H), whereas pGBK-Gal4 (that constitutively expresses Gal4) did (Figure 2a and Figure S1).

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Furthermore, we demonstrated that possible toxicity caused by rtTA association with

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ATC30,31 did not occur as yeast harboring empty pCM251 grew similarly on plasmid

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maintenance media (SC-L-T) irrespective of ATC concentration (Figure 2a and Figure S1).

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Next we analyzed growth rates for yeast containing the genetic circuits that expressed

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Gal4 through tetracycline induction. This revealed that they were responsive to ATC but with

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shifting dynamic ranges, with growth differences on media lacking histidine and uracil for

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circuits containing two tetO sites (tetO2) more pronounced than for those containing seven

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(tetO7) (Figure 2a and Figure S1). Interestingly, genetic circuits expressing GAL4 with a

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strong Kozak consensus sequence and strong start codon (ptetO2-Gal4 and ptetO7-Gal4), did

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not survive on media containing ATC (Figure 2a and Figure S1). We reasoned that this

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might be due to toxicity from ATC induced overexpression of the potent Gal4 transcription

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factor. Conversely, genetic circuits designed to attenuate Gal4 expression via Kozak (ptetO2-

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or ptetO7-Gal4-CCCC), start codon (ptetO2- or ptetO7-Gal4-TTG), or combined Kozak and

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start codon mutations for weak translation (ptetO2- or ptetO7-Gal4-CCCC-TTG) were not

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affected by ATC induced toxicity caused by Gal4 overexpression, as they grew similarly on

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plasmid maintenance media (SC-L-T) with or without ATC (Figure 2a and Figure S1). Of

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the circuits with attenuated Gal4 expression, yeast containing ptetO2-Gal4-TTG did not

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survive in the absence of ATC, yet showed strong growth in the presence of ATC on media

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lacking histidine and uracil (Figure 2a). This suggests Gal4 levels are only high enough to

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activate the HIS3 and URA3 reporter genes after ATC induced expression for this genetic

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circuit, thus providing a mechanism to select for the presence of tetracyclines in yeast. Use of

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the competitive inhibitor (3-AT) was not required for circuits containing ptetO2-Gal4-TTG,

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as it could robustly differentiate between media with or without tetracyclines. This system

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provides a useful genetic tool to link yeast growth to the presence of tetracyclines.


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Finally, we tested if addition of 5-FOA to the media could enable negative selection

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with the same genetic circuits. Using a yeast survival assay, our findings indicate that URA3

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expression, with associated 5-FOA mediated toxicity, only occurs in the presence of

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tetracyclines when yeast harbors ptetO2-Gal4-TTG (Figure 2a). Therefore, using ptetO2-

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Gal4-TTG we have developed a positive-negative selection system for tetracyclines that can

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be used to select for or against growth of an engineered yeast.

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Tetracycline selection in yeast can detect antimicrobial resistance. We investigated how

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our genetic circuits behaved in the presence of an antimicrobial resistance determinant, and if

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TetX could inactivate ATC before inducing significant GAL4 expression (Figure 1a and

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Figure 2b). A synthetic and yeast expression-optimized gene for tetX from Bacteroides

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fragilis was cloned into a constitutive expression plasmid and transformed into yeast. High

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performance liquid chromatography-mass spectrometry (HPLC-MS) confirmed that yeast

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heterologously expressing TetX can destroy ATC in liquid culture (Figure 2c). Next, we

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confirmed that TetX can inactivate ATC before induction of GAL4 expression in our genetic

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circuits, as yeast expressing TetX grew similarly in the presence or absence of ATC (Figure

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2a and Figure S1). Importantly, TetX impeded ATC-dependent survival on media lacking

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histidine and uracil; and conversely, alleviated 5-FOA mediated toxicity in the presence of

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ATC for yeast harboring the genetic circuit ptetO2-Gal4-TTG (Figure 2a). Taken together,

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our results show that engineered yeast containing the artificial genetic circuit ptetO2-Gal4-

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TTG can effectively select for, and against, the activity of antimicrobial resistance systems.

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Next, we evaluated this selection for library screening by plating single colonies of yeast

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harboring ptetO2-Gal4-TTG on media lacking histidine and uracil but with varying ATC

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concentrations (Figure 2d). Single colony counts revealed ATC concentration dependent

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survival, and that co-expression of TetX did not allow survival (Figure 2d). Therefore we

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present the first comprehensive strategy to select for and against tetracyclines and tetracycline

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resistance in yeast, and that this selection is suitable for use in library screens.

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Figure 2. An artificial genetic selection for tetracyclines in yeast. (a) Survival assay for yeast harboring genetic circuits when grown in the presence of 2 µg/ml ATC or ATC and 0.1 % 5FOA on auxotrophic media. Yeast plates contained SC media as indicated in the figure: SCL-T was used as plasmid maintenance media, and SC-L-T-U-H as media to test for histidine and uracil biosynthesis. pCM251 is a negative control that does not express GAL4, whereas pGBK-Gal4 is a positive control that expresses GAL4 constitutively. The genetic circuits were plated in order of decreasing Gal4 translation. Each genetic circuit was grown in the presence or absence of a plasmid constitutively expressing TetX. For each selective condition, yeast were inoculated as neat and as 10-fold serial dilutions. (b) TetX converts (1) ATC into (2) 11a-hydroxy-anhydrotetracycline, which then undergoes spontaneous degradation. (c) HPLC-MS analysis for the extracted ion chromatogram for ATC [M+H = 427.1]: i) trace for the ATC standard at 100 µg/ml produces a peak (% relative intensity) that elutes ~17 min; ii) trace for the organic extract of yeast pellet harboring empty pACT2 after culture in the presence of 20 µg/ml ATC for 72 hours also produces a peak corresponding to ATC with the addition of a peak of lower intensity corresponding to the epimer of ATC (EATC); iii) trace for the organic extract of yeast pellet harboring pACT2-TetX after culture in the presence of 20 µg/ml ATC for 72 hours produces peaks of lower intensity for ATC and EATC than seen in (ii), indicating their degradation by TetX; iv) trace for the organic extract of yeast pellet harboring empty pACT2 does not produce a peak for ATC as it does not occur naturally in yeast culture. (d) Quantitative growth assay for ATC induced sensitivity in yeast. Negative control contains empty pCM251 and pACT2 and does not express GAL4, positive control contains pGBK-Gal4 and pACT2 and constitutively expresses GAL4, yeast harboring the selection plasmid ptetO2-Gal4-TTG were grown without (pACT2) or with (pACT2-TetX) a plasmid constitutively expressing TetX. Yeast were grown on SC-L-T-U-H media supplemented with ATC as indicated on the figure to test for histidine and uracil biosynthesis resulting from the induction of Gal4 by ATC. Bars represent mean colony forming unit (CFU) counts (n=6). Error bars indicate standard error of the mean (SEM). 8 ACS Paragon Plus Environment

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Investigation of TetX structure and function using an artificial genetic circuit. Next, we

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coupled our artificial genetic selection to random mutagenesis and deep sequencing to

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investigate the relationship between protein structure and function of the antimicrobial

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resistance factor TetX. We rationalized that yeast harboring ptetO2-Gal4-TTG could select

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for loss of function mutants of TetX, as these mutants would not degrade a tetracycline-

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family inducer required for survival on media lacking histidine and uracil. Conversely, we

7

reasoned that media containing inducer and 5-FOA would select for mutants that retained

8

their function, as TetX would degrade tetracyclines before induction of GAL4 expression and

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downstream URA3. ATC and doxycycline (DOX) were used as inducers. Not only does ATC

10

strongly activate the genetic circuit, it also acts as an inhibitor of other tetracycline

11

inactivating enzymes, including Tet(50, 51, 55 and 56)25. A deeper understanding of the

12

interaction between ATC and TetX could guide its rational re-design as a broader tetracycline

13

inactivating enzyme inhibitor for use in combination therapies. Similar to ATC, DOX

14

displays remarkable affinity to TetR [DOX.Mg]+ (Ka ~ 1010 M-1)29, but better represents

15

conventional tetracycline antibiotics through its conserved ribosomal binding lower periphery

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(Figure S2). Understanding the intricacies of DOX destruction may help direct the design of

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next generation tetracyclines that can overcome enzymatic inactivation.

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Library construction and differential substrate deep mutational scanning. We

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constructed a mutant TetX library that was analyzed by deep mutational scanning, and also

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by genotyping individual mutants prior to phenotypic assays (Figure 3). We achieved a

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mutation rate for tetX

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of 0.14% with error-prone PCR (EP-PCR). Library transformation generated 1.8x107 primary

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transformants, that were further expanded for four generations before plating on media for

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plasmid maintenance, for a loss of ability to degrade ATC or DOX, or for the retention of

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ATC degradation. The mutant populations were analyzed by targeted deep sequencing of the

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tetX open reading frame (Figure S3). Non-synonymous mutation counts at each amino acid

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position of TetX on plasmid maintenance media were compared to those recorded on media

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that selected for a loss or retention of TetX function. This allowed the calculation of either

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the enrichment or depletion, as a log2 fold change, of loss of function or neutral mutations at

31

each amino acid position of TetX (Figure 4a). On average, non-synonymous mutations were

32

enriched after selection with ATC or DOX but were depleted after selection with ATC in

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combination with 5-FOA (Figure 4b). These results reflect that loss of function mutations are



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more common, however neutral mutations also occur, and validate the system’s ability to

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select for both deleterious and neutral mutations.

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Figure 3. An artificial selection to elucidate TetX structure and function relationships. Using error-prone PCR (EP-PCR), we added mutations to those already formed during gBlock synthesis to produce a library of tetX genes. We harnessed yeast’s innate ability for homologous recombination to rapidly and efficiently assemble a mutant library in MaV204K containing ptetO2-Gal4-TTG. tetX mutants were selected for loss or retention of function. The resulting colonies were further analyzed by yeast survival assays and Sanger sequencing, or by deep mutational scanning using next generation sequencing.



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FAD positioning is not favorable for ATC destruction. After selection on either ATC,

2

DOX, or ATC and 5-FOA, non-synonymous mutation enrichment or depletion ratios for

3

amino acid residues known to contribute to FAD binding19 were projected onto the structure

4

of TetX (Figure 4c and Table S1). Mutation ratios at these sites followed two trends: largely

5

enriched when selected for on ATC or DOX; or conversely, depleted when selected for on

6

ATC together with 5-FOA, which is consistent with the requirement of FAD for TetX

7

function17. However, there was a large difference in mutation enrichment or depletion seen at

8

P318, where it was required for DOX destruction but inconsequential for ATC destruction.

9

Normally, stacking interactions between the dimethylbenzene ring of flavin and P318

10

contribute to FAD binding, helping to place the isoalloxazine at a short (~5.9 Å) distance

11

from the C11a hydroxylation site19. As loss of this interaction is not deleterious for ATC

12

destruction, this implies precise positioning of the isoalloxazine in TetX to be less than ideal

13

for ATC degradation, and that it may even be deleterious for its hydroxylation.

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Figure 4. Deep mutational scanning of TetX. (a) Log2 fold change in non-synonymous mutation frequency following selection for a loss of function (ATC and DOX selections), or for retention of function (ATC with 5-FOA selection) plotted against all residues for TetX. A positive log2 fold change represents the enrichment of a mutation following selection, whereas a negative log2 fold change represents a depletion. Enriched mutations when selected for in the presence of ATC or DOX cause a loss of TetX activity. Conversely depleted mutations have a neutral effect on this selection. Enriched mutations when selected for in the presence of ATC and 5-FOA are neutral for TetX activity. Conversely depleted mutations cause a loss of TetX activity. (b) Average TetX amino acid mutation (log2 fold change unselected versus selected) after selection. Error bars indicate standard error of the mean (SEM). (c) Surface representation of TetX based on PDB: 4A9922. Enrichment or depletion of mutations after each selection, for residues involved in FAD binding, are represented as a color spectrum. Colors indicate the log2 fold change in non-synonymous mutational frequency at the indicated position. FAD is represented as sticks.



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TetX tolerates mutation differently depending on the tetracycline substrate. We next

2

searched for additional differences in amino acid mutations that were dependent on the

3

tetracycline used for selection. To do this, the relative fold-change in enrichment or depletion

4

of mutations between ATC and DOX selections was calculated, revealing key differences in a

5

number of functional sites required for either ATC or DOX destruction (Figure 5a). To better

6

represent these relationships, preferential mutation ratios were projected onto the structure of

7

TetX (Figure 5b). We observed clustering of differential residue mutation preferences along

8

the FAD binding channel and around the substrate binding tunnel. These preferences were

9

less significant distal from these sites. We predicted that these sensitivities stem mostly from

10

differences in substrate shape; ATC’s ring system is less prone to torsion than other

11

tetracyclines and is thus conformationally distinct within the binding pocket32. In light of this,

12

we examined the enrichment or depletion of mutations at residues involved in positioning the

13

ATC or DOX substrate relative to FAD.

14 15

Positioning of ATC and DOX in TetX is similar. Active site positioning of tetracyclines

16

has been shown to occur through hydrogen bonding interactions with the substrate A-ring and

17

residues Q192, R213, H234 and G236 of TetX19. Also, a solvent bridge required by Q192 for

18

hydrogen bonding is reported to be stabilized by S23822. Mutation enrichment was seen for

19

all these residues after ATC selection, except for R213, which was found to be 1.3-fold more

20

enriched in DOX selection than ATC (Figure 5c and Table S1). This suggests that A-ring

21

positioning is similar for both ATC and DOX, and that binding of ATC by TetX is unlikely to

22

be drastically different from other tetracyclines. ATC is slightly more planar in the C-D ring

23

region than DOX32, and this difference likely explains enrichment of R213. Loss of binding

24

at R213 may contribute to better ATC destruction by permitting it to swing, placing C11a

25

more proximal to FAD for hydroxylation. Furthermore, R213 is likely a steric hindrance only

26

to ATC and not DOX due to ATC’s greater rigidity.

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DOX destruction is more sensitive to a loss of hydrophobic shielding than for ATC.

29

Hydrophobic shielding in the binding site is offered by F224, M215, G321 and M37519, or

30

F224, F319 and P31822 dependent on the substrate bound. Mutation enrichment across all

31

these residues was seen after selection on DOX, but only at M215, G321 and F319 after ATC

32

selection (Table S1). Residue F224, required for both minocycline and 7-iodtetracycline

33

hydrophobic shielding19,22, was 7.3-fold more enriched for DOX than ATC inactivation

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(Figure 5c). 14 ACS Paragon Plus Environment

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Figure 5. Differences in functionally important residues for degradation of ATC versus DOX. (a) Mutability was calculated as a fold change in mutation preference between ATC and DOX and plotted against the length of TetX. Values of 0.1 to -0.1 were omitted prior to calculation. (b) Mutability scores from (a) were projected on a surface representation of TetX bound to minocycline based on PDB: 4A9922. Purple represents residues important for activity when selected on DOX, and orange represents those important for activity when selected on ATC. FAD and minocycline are represented as sticks in green or yellow, respectively. (c) Residues for substrate and FAD positioning within the binding pocket are shown. A curved arrow in the last image indicates putative ATC positioning within the binding pocket caused by increased planarity through its C-D ring system. This positioning could lead to steric hindrance with R213, and evasion of hydrophobic shielding offered by F224. FAD and minocycline are represented as sticks in green or yellow, respectively.


Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

This result indicates hydrophobic shielding is more important for DOX destruction than for

2

ATC, nevertheless it is still required for both. Again, this is likely a result of differing

3

planarity between the two molecules. Possibly, the rigidity of ATC positions its hydrophobic

4

edge towards the hydrophilic portion of the substrate binding pocket, where mutation to

5

residues involved in hydrophobic shielding have less effect this substrate’s already

6

unfavorable binding.

7

The findings from our deep mutation scanning experiments suggest that the rigidity

8

and planarity of ATC position it in a configuration distinct from DOX within the binding

9

pocket. This distinct conformation leaves the hydrophilic edge of ATC more distal from the

10

FAD isoalloxazine than the edge of DOX during destruction. This difference rationalizes why

11

ATC is poorly degraded, and although its positioning in TetX is suboptimal, it is not

12

sufficiently distinct to act as a strong inhibitor like it does for Tet(50)9,25.

13 14

Deep mutational scanning results are consistent with individually isolated mutants. We

15

examined a selection of individual mutants isolated from ATC selection plates prior to deep

16

mutational scanning to test if they also showed variation at the FAD and substrate binding

17

sites. 324 individual mutant colonies that survived selection were isolated and their

18

phenotype confirmed (Figure S4). Mutants with desired phenotypes were further examined

19

by yeast survival assays and Sanger sequencing. Of those with single non-synonymous amino

20

acid changes, mutations were predicted to disrupt either FAD or substrate binding. For

21

example, mutant Y45F is predicted to disrupt FAD binding. Y45 ordinarily precedes the

22

FAD-binding residues G46 and R47, itself anchoring the underside of the cofactor-binding

23

pocket to other regions of the protein. Substitution with a hydrophobic phenylalanine residue

24

removes an important hydrogen bond donor, partially negating the anchoring effect of residue

25

45 (Figure 6b). This substitution was more highly enriched for ATC than DOX selections

26

(Table S1), and this sensitivity is shown by an increased ability to impede ATC inactivation

27

(Figure 6a). The mutations M316I and M316V also modify the substrate specificity of TetX

28

by altering FAD binding. Both mutations result in reduced steric force exerted on the

29

isoalloxazine moiety of FAD, allowing repositioning of FAD relative to the substrate (Figure

30

S5). These mutations were enriched for both ATC and DOX selections (Table S1). Yeast

31

survival assays show these mutations impede TetX function, although with differing

32

severities (Figure 6a). In terms of substrate binding, the M215T mutant displayed poor

33

specificity for ATC and DOX with a



Page 18 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Figure 6. TetX mutants at FAD and substrate binding sites cannot inactivate tetracyclines. (a) Qualitative growth assay for TetX mutant altered specificity. Yeast survival assay for TetX mutants grown in the presence of 2 µg/ml ATC, 2 µg/ml DOX, and 2 µg/ml ATC with 0.1 % 5-FOA. Yeast plates contained SC media as indicated on the figure. For each selective condition, yeast was inoculated as neat and as 10-fold serial dilutions. (b) Residue Y45 stabilizes the underside of TetX FAD cofactor-binding site by hydrogen bonding with other residues, acting as an anchor. The mutation Y45F interferes with this stabilization. Electron density map is depicted as mesh for selected residues. Mutations are simulated with PyMOL based on PDB: 4A9922. Minocycline and FAD are represented sticks in yellow or green respectively. (c) In WT TetX, the hydrophobic edge of tetracycline substrates is shielded from solvent exposure by non-polar amino acid residues and makes van der Waals contacts with same. The activity of TetX is sensitive to mutations that introduce polarity into this hydrophobic pocket. Electron density maps are depicted as mesh. Mutations are simulated with PyMOL based on PDB: 4A9922. Minocycline is represented as sticks in yellow.


Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

single subtle amino-acid change in the substrate binding pocket. M215 is ordinarily involved

2

in hydrophobic shielding19, and electron density mapping suggests this dissipates with

3

polarization (Figure 6c). M215 is enriched in both ATC and DOX selections (Table S1). A

4

yeast survival assay shows this mutation to impede TetX function, yet some activity still

5

remains as growth does not reach the same level as for the control lacking TetX (Figure 6a).

6

These results confirm TetX requires precise placement of both FAD and substrate for

7

enzymatic activity, and that variation of either can reduce its activity.

8 9

DISCUSSION

10

Drug discovery has typically revolved around, or expanded on, established methodologies

11

developed through incremental improvement. Although rare, breakthrough technologies have

12

disrupted classical antibiotic research33,34, providing a means to sift through the vast and

13

historically unattainable chemical diversity offered in nature. While recent advances are

14

significant, and synthetic gene circuits have shed light on fungal drug resistance30,35,

15

antibiotic research will benefit tremendously from additional innovative technologies. Here

16

we present a novel synthetic biology approach to drug discovery, with an artificial selection

17

that aims to help guide the creation of the next generation of antimicrobials.

18 19

Our artificial genetic circuits can select for or against the presence or absence of

20

tetracyclines in yeast. This selection is a vital step towards broadening our waning antibiotic

21

collection, with three examples of potential applications. First, this selection can aid the yet to

22

be realized biosynthesis of tetracyclines in eukaryotes; it can permit rapid prototyping by

23

providing an accessible readout on the success of pathway engineering experiments,

24

obviating the need for comparatively slower high throughput screening methodologies.

25

Second, vast libraries of compounds can be selected for that not only retain key antibiotic

26

motifs, but also evade resistance through evolved tailoring. This is conceivable as the

27

tetracycline repressor (TetR) is a promiscuous regulator that detects the lower conserved

28

periphery of tetracyclines essential for bacteriostatic activity29. Moreover, tetracycline

29

substitutions at positions C2 and C4-C9 have varied effects on TetR binding, both positive

30

and negative based on hydrogen bonding, hydrophilic interactions and steric hindrance29.

31

With the addition of an antimicrobial resistance determinant (such as TetX), contrasting

32

recognition by TetR could screen for desired tetracycline modifications made by massive

33

libraries of tailoring enzymes. Third, utilizing a transcriptional regulator as a sensor has

34

consequences for the discovery and engineering of other antibiotics and small molecules far 19 ACS Paragon Plus Environment


Page 20 of 32

1

beyond this current system. There are currently over 200,000 TetR family members in public

2

sequence databases. The small molecules recognized by these proteins are extremely diverse,

3

including many desirable natural products. Furthermore, the atomic structures are known for

4

about 200 of these, and experiments have shown that protein engineering approaches can be

5

used to redesign these to bind new ligands36. Therefore, the concept of linking recognition of

6

a natural product, via a TetR protein derivative, to life-death selections in yeast should be

7

generally applicable to most natural products of interest. In principle, variants constructed

8

with different eukaryotic activators could overcome the Gal4-mediated toxicity encountered

9

in the study. However, not only could this toxicity be harnessed as a selection in its own

10

right, but we also found that attenuating the level of Gal4 translation produced a series of

11

circuits that were all responsive to tetracycline but with shifting dynamic ranges. Our genetic

12

circuits therefore provide a significant addition to the synthetic biology toolbox37.

13 14

Drug resistance can evolve via non-coding or synonymous mutations, as well as copy

15

number alterations and epigenetic changes38. While our system could be adapted to study

16

these important mechanisms, we focused on coding mutations that offer structure-function

17

insights that can applied to drug design, as this has the best therapeutic potential. Since

18

recognition of tetracyclines by inactivating enzymes is mediated in large part by the same

19

hydrophilic binding pattern that enables bacteriostatic activity, it is unlikely that an effective

20

antibiotic could be engineered to escape inactivation19. Instead, a specialized inactivating

21

enzyme inhibitor provides an achievable alternative goal for rational drug design. Previous

22

studies suggest ATC represents an “evolutionarily-privileged lead compound” for the design

23

of an inhibitor, owing to its natural inhibitory effect on tetracycline inactivating enzymes9,25;

24

this strong inhibition is however not observed for TetX. Broadening the effective inhibitory

25

spectrum of ATC to include TetX and its homologues is thus an immediate goal. Our work

26

demonstrates that positioning of ATC relative to FAD in TetX is unfavorable for inactivation.

27

Furthermore, rare mutations distal from FAD and substrate binding sites can have detrimental

28

effects on activity. Three speculative approaches for inhibitor design exploiting these findings

29

are possible. First, we suggest positioning of the tetracycline substrate for hydroxylation by

30

the isoalloxazine of FAD is highly affected by its planarity. Furthermore, planar positioning

31

of ATC is crucial for its inhibitory effect against tetracycline inactivating enzymes25.

32

Flattening of the ATC A-ring in plane with the rest of the ring system is a recurrent theme,

33

and it stands to reason that inhibition of tetracycline-inactivating enzymes including TetX

34

could be maximized by forcing greater ATC planarity throughout the A-D ring system. A 20 ACS Paragon Plus Environment

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1

second approach is indicated by sensitive hydrophobic shielding interactions. ATC’s rigidity

2

positions its hydrophobic edge away from residues that would ordinarily shield

3

hydrophobicity. Introducing limited polarity to the hydrophobic edge of ATC may

4

consolidate this positioning, in effect locking it in a location that is unfavorable for enzymatic

5

inactivation. An added benefit of this polar substitution is potential improvement of ATC’s

6

safety profile, since hydrophobic interactions with mammalian serum proteins appear to

7

mediate much of ATC’s toxicity39. Third, our results also indicate approaches to inhibitor

8

design not relying on ATC modification. Although rarer, residues distal from the active site

9

were identified as important for TetX activity. These sites therefore represent potential targets

10

for novel small-molecule inhibitors. The structure-guided targeting of non-catalytic residues

11

has attracted attention as a strategy for the inhibition of tyrosine-kinase enzymes including

12

human epidermal growth factor receptor (EGFR)40. However, extensive modelling and

13

experimentation is still required to devise an approach to inhibition by these means.

14 15

CONCLUSION

16

We demonstrate how a synthetic biology approach can be a valuable tool in the fight against

17

antimicrobial resistance. We constructed artificial genetic circuits in yeast to select for

18

tetracyclines by linking their presence to the expression of the transcription factor Gal4.

19

Pleiotropic effects of Gal4 overexpression provided the unexpected ability to select against

20

tetracyclines. With Gal4 translation optimized, we then showed the capacity to select for

21

tetracyclines. This selection was lost upon addition of the resistance factor TetX, providing

22

the framework to study its structure and function by deep mutational scanning. Information

23

gained from mutational studies of TetX provides valuable insights on how to potentially

24

overcome its resistance, with the rational re-design of ATC as an inhibitor the most

25

promising. Our findings show precise positioning of FAD and substrate to be critical for

26

inactivation, revealing leads for the modification of ATC based on modified planarity and

27

hydrophobicity. Future work towards the synthesis of tetracycline inactivating enzyme

28

inhibitors will benefit greatly from these insights. Furthermore, our artificial genetic selection

29

has many applications in the directed evolution of next generation antibiotics beyond those

30

presented here.



Page 22 of 32

1

METHODS

2

Microbial culture. Escherichia coli DH10B [F- endA1 recA1 galE15 galK16 nupG rpsL

3

∆lacX74 Φ80lacZ∆M15 araD139 ∆(ara,leu)7697 mcrA ∆(mrr-hsdRMS-mcrBC) λ-]

4

(Invitrogen) was grown and maintained on lysogeny broth (LB) or agar (1% w/v tryptone,

5

0.5% w/v yeast extract, 1% w/v NaCl and 1.5% agar). E. coli was grown at 37°C and liquid

6

cultures agitated with shaking at 180 rpm. E. coli was rendered chemically competent and

7

used for plasmid construction with traditional molecular biology techniques. Saccharomyces

8

cerevisiae MaV204K41 (MATα, trp1–901, leu2–3, 112, his3∆200, ade2–101∆::kanMX, gal4∆,

9

gal80∆, SPAL10::URA3, UASGAL1::HIS3, GAL1::lacZ) was grown and maintained on

10

synthetic complete (SC) media or agar (0.017% w/v yeast nitrogen base without amino acids

11

and ammonium sulphate, 0.05% ammonium sulphate, 0.005% amino acid drop out powder,

12

2% w/v glucose, 2% agar, pH 5.6) supplemented with amino acids. MaV204K was used as

13

parent strain for all yeast work and transformed as described by Gietz and Woods42.

14

Tetracyclines are light and heat sensitive43. Media was cooled to 50°C before addition of

15

anhydrotetracycline (ATC) and doxycycline (DOX) and then shielded from light during all

16

experiments.

17 18

Plasmids. The amino acid sequence for TetX from Bacteroides fragilis (GenBank:

19

AAA27471.1) was back-translated and codon-optimized for S. cerevisiae expression using

20

Gene Designer (ATUM)44. A robust Kozak consensus sequence comprising six adenine bases

21

5′ of the start codon was added to ensure strong translation initiation before synthesis by

22

GeneArt and ligated as a HindIII fragment to pACT2 (Clontech) digested with the same

23

enzyme to yield pACT2-TetX, placing TetX under control of the constitutive ADH1

24

promoter. pGBK-RC-No-BD was created by removing the region of pGBK-RC41 encoding

25

the Gal4 DNA binding domain but leaving the multiple cloning site (MCS) proceeded by an

26

ADH1 promoter intact. GAL4 was amplified by PCR from wild-type yeast genomic DNA and

27

ligated to pGBK-RC-No-BD digested with BamHI and NotI, placing GAL4 under control of

28

the ADH1 promoter and yielding pGBK-Gal4.

29 30

Multiple plasmids were constructed for the tetracycline conditional expression of Gal4 in

31

yeast. First, a robust Kozak consensus sequence comprising six adenine bases 5´ from the

32

start codon was introduced to GAL4 when amplified from wild type yeast genomic DNA by

33

PCR. GAL4 was then cloned into pCM251 and pCM25226 using NotI and BamHI, placing it

34

downstream of tetO2 or tetO7 in pCM251 and pCM252 respectively to yield ptetO2-Gal4 and 22 ACS Paragon Plus Environment

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1

ptetO7-Gal4. Second, the Kozak consensus sequence immediately 5´ of the GAL4 start codon

2

in ptetO2-Gal4 and ptetO7-Gal4 was altered by Quick Change site directed mutagenesis to

3

bases least commonly found in yeast, from AAAGATG to CCCCATG (start codon bold), to

4

yield ptetO2-Gal4-CCCC and ptetO7-Gal4-CCCC. Third, and also using Quick Change site

5

directed mutagenesis, the ATG start codon of GAL4 in both ptetO2-GAL4 and ptetO7-GAL4

6

was replaced with the less common TTG, producing ptetO2-Gal4-TTG and ptetO7-Gal4-TTG.

7

Finally, both previously mentioned mutagenesis strategies were combined to yield ptetO2-

8

Gal4-CCCC-TTG and ptetO7-Gal4-CCCC-TTG from ptetO2-GAL4 and ptetO7-GAL4

9

respectively, with both plasmids altered to contain the following DNA sequence: CCCCTTG

10

(GAL4 start codon in bold).

11 12

Survival assays. 1 OD600 units of yeast harvested from a fresh overnight culture was

13

suspended in 1 ml Tris-EDTA (TE) buffer (10 mM Tris, 1 mM EDTA, pH 8). Each yeast

14

suspension was used to make 1/10 serial dilutions in TE buffer. Five µl of each dilution was

15

inoculated as standing droplets onto agar plates supplemented as indicated in the figure

16

legends. The plates were imaged after 2-3 days growth at 30°C.

17 18

Quantitative growth assay. 1 OD600 units of yeast harvested from a fresh overnight culture

19

was suspended in 1 ml TE. Yeast suspensions were then diluted by a factor of 10-4 via serial

20

ten-fold dilution in TE buffer. 150 µl of final dilute yeast suspension was inoculated on

21

selective SC agar plates. The plates were imaged after 2 days growth at 30°C.

22 23

Detection of anhydrotetracycline in yeast by mass spectrometry. S. cerevisiae cultures

24

spiked with ATC were grown at 30°C with shaking at 200 rpm for 3 days. After incubation,

25

0.05 g of Na2EDTA was added once the cultures were acidified to pH 3 with 3 M H2SO4.

26

Cells were collected by centrifugation, washed three times with ultra-pure water and

27

resuspended in 1 ml 0.5% Tween-20 and 100 µl of glass beads. Cells were then lysed by

28

vigorous mixing at 4°C for 20 min. Lysed cells were extracted with 7 ml of ethyl

29

acetate:methanol:acetic acid (89:10:1) twice. Organic extracts were dried under N2 and then

30

re-constituted in 1 ml MeOH before analysis by high performance liquid chromatography –

31

mass spectrometry (HPLC-MS). Detection of anhydrotetracycline was undertaken using a

32

Waters Alliance e2695 HPLC connected to a Waters LCT Premier XE time-of-flight (TOF)

33

mass spectrometer with an electrospray ionization source (ESI). Separation was achieved

34

with an Alltima C18 column (150 x 2.1 mm id, 5 µm, Grace Discovery Sciences) using a 23 ACS Paragon Plus Environment


Page 24 of 32

1

flow rate of 0.3 ml/min. The column was eluted with 5% acetonitrile/95% H2O (+ 0.1%

2

formic acid) before increasing to 100% acetonitrile/H2O (+ 0.1% formic acid) over 50 min

3

and held for 5 min. 5 µl of sample was injected onto the column which was held at 25ºC. The

4

mass spectrometer was operated in both positive and negative mode. Cone and desolvation

5

gas flows were set to 20 and 650 l/hr, respectively. The capillary voltage was set at 3 kV, the

6

source temperature at 80°C, and the desolvation temperature at 350°C.

7 8

EP-PCR library generation. The synthetic tetX sequence obtained from pOA-RC-yTetX

9

was re-synthesized as a gBlock (IDT), with the silent mutations A438G and A441G to

10

remove a 9-bp-long adenine homopolymer. The gBlock template was designed to contain 193

11

bp (5´) and 108 bp (3´) overlap with the pACT2 vector to allow subsequent assembly in yeast

12

by homologous recombination. Low fidelity PCR with DreamTaq polymerase was used to

13

produce a diverse library of tetX sequences. For a 50 µl reaction, 32 ng of TetX gBlock

14

template was amplified using 2.5 U DreamTaq Polymerase, 200 µM dNTPs, 280 µM of the

15

primers

16

GCCGACAACCTTGATTGGAG-3´) in DreamTaq buffer with 3.2% v/v dimethyl sulfoxide

17

(DMSO). Reaction mixtures were subjected to 95°C denaturation for 2 min, 20 cycles of

18

95°C for 2 min, 68°C (decreasing by 0.65°C per cycle) for 30 seconds, and 68°C for 1:20

19

min. This was followed by 20 cycles of 95°C for 2 min, 55°C for 30 seconds, 68°C for 1:20

20

min. Reactions were held at 4°C after a final elongation step for 5 min at 72°C. 60 PCR

21

reactions were pooled and DNA isolated via standard ethanol precipitation techniques. Vector

22

was prepared by digesting pACT2 with HindIII and the 7371 bp fragment extracted from

23

agarose gel, yielding 210 µg.

S6fwd

(5´-AGTTTGCCGCTTTGCTATCA-3´)

and

S6rev

(5´-

24 25

TetX library screening. Mutant tetX genes generated by EP-PCR and linearized pACT2

26

were transformed into MaV204K harboring ptetO2-Gal4-TTG by the lithium acetate method.

27

The pACT-TetX library was assembled in vivo by yeast homologous recombination. The

28

primary yeast library was expanded for four additional doublings, and then inoculated on the

29

surface of agar plates containing either 2 µg/ml ATC (25% of total library), 2 µg/ml DOX

30

(25% of total library), or 2 µg/ml ATC and 0.1% 5-FOA (2 % of library). An aliquot (2 %) of

31

unselected library was also harvested. Selection plates were incubated for 48 hours at 30°C.

32

Individual colonies were isolated in TE buffer, taking care to leave behind most of the

33

colony, and then inoculated as non-standardized 5 µl volumes to selective SC agar plates

34

prior to Sanger sequencing (AGRF, Perth, Australia) and yeast survival assays. 24 ACS Paragon Plus Environment

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1 2

Next generation sequencing. Colonies surviving selection were harvested and their plasmid

3

DNA (from 3x106 cells) extracted by digestion with zymolyase prior to alkaline lysis. The

4

mutant tetX population from each selection was amplified by PCR as six separate overlapping

5

fragments of 20. Reads were also trimmed of sequences originating from

18

PCR primers using Geneious to purge mutation errors originating from oligonucleotide

19

synthesis. Mutant variant frequency in the unselected population was compared to those in

20

the selected populations and enrichment values were calculated using Enrich246 software.

21 22

ASSOCIATED CONTENT

23

Supporting Information includes Supporting Table S1 and Supporting Figures (Figures S1-

24

S5). This material is available free of charge via the internet at http://pubs.acs.org.

25 26

ACKNOWLEDGEMENTS

27

We thank Samuel A. Raven and Anima Poudyal for experimental assistance. This work was

28

supported by fellowships, scholarships and grants from the Australian Research Council

29

(DP140104111 to A.F. and O.R and FT110100304 to GRF), the National Health and Medical

30

Research Council (APP1058442, APP1045677 to A.F. and O.R.), and the Cancer Council

31

Western Australia (to O.R.). We thank T. Ito for the MaV204K yeast strain and

32

EUROSCARF for the pCM251 and pCM252 plasmids. The authors acknowledge the

33

facilities and technical assistance at the Centre for Microscopy, Characterisation & Analysis,



Page 26 of 32

1

The University of Western Australia, a facility funded by the University, State and

2

Commonwealth Governments.

3 4

Notes

5

The authors declare no conflict of interest.


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Page 32 of 32

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For Table of Contents Use Only

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for

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An artificial yeast genetic circuit enables deep mutational scanning of an antimicrobial resistance protein

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Louis H. Scott, James C. Mathews, Gavin R. Flematti, Aleksandra Filipovska,

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Oliver Rackham

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An Artificial Yeast Genetic Circuit Enables Deep Mutational Scanning

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