A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening

Abstract

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His₆-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ~5-fold more effective than the canonical nitroreductase NfsB.

Keywords: Bacterial start codons; Environmental DNA screening; FatI restriction cloning; Functional metagenome screening; Metronidazole; Niclosamide; Nitroreductase; Synthetic biology; Targeted cell ablation.

Figure 1:. Key features of FatI expression vector pUCXMG.

Highlighted are the IPTG-inducible tac promoter; the lacO operator (repressor-binding) region; an XbaI site used in vector assembly; a strong ribosome binding sequence (RBS; derived from the T7 phage major capsid protein RBS); the start codon; the hexahistidine (His₆) tag; a thrombin cleavage sequence for His₆ tag removal; and the NcoI restriction site for insertion of FatI partially-digested eDNA fragments. Figure drawn using Geneious Prime version 2022.2. The full plasmid map and sequence are available as Supplementary Figure S2.

Figure 2:. Percentage of genes from sequenced genomes that initiate with ATG, (C)ATG or (G)ATG start codons, relative to genomic GC content.

The percentage of genes predicted to initiate with ATG (orange), (C)ATG (dark blue), or (G)ATG (light blue) start codons were sourced from 21,675 annotated bacterial genomes, derived from the National Centre for Biotechnology Information Assembly Database on June 7, 2021, and plotted relative to the total GC content of that genome. Data were analysed with Python 3.8.1, using Script 1 (Supplementary Files).

Figure 3:. Counter-screening of niclosamide-resistant E. coli 7TL eDNA variants to identify metronidazole sensitive strains.

910 niclosamide-resistant colonies were recovered from plating of E. coli 7TL cells transformed with the FatI eDNA library on LB agar amended with 0.5 μM niclosamide. Replicate LB cultures were established from each colony and grown for 4 h in either unamended media as a control or else media amended with 0.5 μM niclosamide or 1.5 mM metronidazole. The percentage growth of niclosamide-challenged cultures (A) or percentage growth inhibition of metronidazole-challenged cultures (B) were calculated relative to the unchallenged control. Panels A and B present data from a single set of representative 96-well plates (each of which contained a media-only blank well as well as one empty pUCX (black bar) and three pUCXMG:azoR_Ec controls (grey bars), the latter of which were expected to be niclosamide-resistant but not metronidazole-sensitive as per Supplementary Figure S1). Data were derived from two biological repeats and error bars represent 1 S.D., while the black dashed lines indicate the cut-off that was imposed to define niclosamide resistance (A) or high-level sensitivity to metronidazole (B). The full screening dataset is available in Supplementary File S3; overall, 78% of niclosamide-challenged cultures achieved at least 50% culture turbidity (OD₆₀₀) relative to control and 14% of metronidazole-challenged cultures were at least 80% growth-inhibited relative to control.

Figure 4:. SDS-PAGE analysis of E. coli 7TL cells expressing captured nitroreductases.

Enzymes were expressed from pUCXMG, without (top) or with (bottom) co-transformation by pRARE. Protein expression was induced and cultures incubated for 4.5 h, then cell densities normalised and loaded in the same order on each gel (except that there was no TdsD4 sample on the “+pRARE” gel as growth of the corresponding strain could not be achieved in liquid media). Control cultures of cells ± pRARE and expressing NfsA or NfsB from pUCX, or transformed by empty pUCX (V/O; vector only) were treated in identical fashion and analysed on a separate gel (rightmost panels).

Figure 5:. SDS-PAGE of recombinant His₆-tagged nitroreductases.

Each nitroreductase was purified by standard Ni/NTA chromatography post-expression from each respective pUCXMG cloning plasmid. Five micrograms of purified protein were loaded per lane.

Figure 6:. Cell ablation efficacy in transgenic zebrafish for neuronal cells expressing lead nitroreductase candidates.

A-E) Transgenic zebrafish larvae co-expressing the indicated nitroreductase and either yellow fluorescent protein (A-C,E) or mCherry (D) in cells of the central nervous system were exposed to a range of metronidazole concentrations to assess relative cell ablation efficacy. In initial tests, the MhqN2 line (E) showed >50% ablation at 1 mM metronidazole and was exposed to lower concentrations to enable measurement of an absolute EC₅₀. Bonferroni-corrected p′-values relative to the control condition (0 mM metronidazole) are indicated by asterisks: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (NS = not significant). F) Micrographs of MhqN2 expressing zebrafish larvae after 48 hours of exposure to control media (above) or media containing 10 mM metronidazole (below).