Identification of Genes Essential for Fluorination and Sulfamylation within the Nucleocidin Gene Clusters of Streptomyces calvus and Streptomyces virens

Abstract

The gene cluster in Streptomyces calvus associated with the biosynthesis of the fluoro- and sulfamyl-metabolite nucleocidin was interrogated by systematic gene knockouts. Out of the 26 gene deletions, most did not affect fluorometabolite production, nine abolished sulfamylation but not fluorination, and three precluded fluorination, but had no effect on sulfamylation. In addition to nucI, nucG, nucJ, nucK, nucL, nucN, nucO, nucQ and nucP, we identified two genes (nucW_, nucA), belonging to a phosphoadenosine phosphosulfate (PAPS) gene cluster, as required for sulfamyl assembly. Three genes (orf(-3), orf2 and orf3) were found to be essential for fluorination, although the activities of their protein products are unknown. These genes as well as nucK, nucN, nucO and nucPNP, whose knockouts produced results differing from those described in a recent report, were also deleted in Streptomyces virens - with confirmatory outcomes. This genetic profile should inform biochemistry aimed at uncovering the enzymology behind nucleocidin biosynthesis.

Keywords: Streptomyces; fluorometabolite biosynthesis; gene knockouts; natural products; nucleocidin.

Figure 1

Structures of the known 4’‐fluoroadenosine metabolites and sulfamyl containing natural products.

Figure 2

Description of the nucleocidin BGC in nucleocidin producers.[ ¹⁴ , ¹⁵ ] The proposed BGCs, include a flanking region at the 5’‐end coding for enzymes with related activities (orf−8 to orf−5). The numbering is relative to oxido‐reductase gene, orf1, which was previously implicated as the start of the BGC. Genes encoding enzymes involved in sulfamylation are shown in red, with the non‐essential ones contained in dashed lines. Proteins of unknown function involved in fluorometabolite production are encoded by genes shown in black; the genes that are not essential for nucleocidin biosynthesis are in grey. Transcriptional regulator genes are in purple, membrane transporter genes in brown, genes coding for proteins involved in glucose transfer/removal are in yellow. Empty arrows represent a gene that is absent in some of the other producers (orf10). An asterisk denotes function annotations that were based on AlphaFold2 structure predictions and comparison to known protein folds. For the knockout phenotypes, No F‐Mets indicates abolishment of fluorometabolite production, F‐Met‐1 indicates production of F‐Met‐I 2 only, symbol (−) indicates that the deletion had no effect and the fluorometabolites were produced.

Figure 3

¹⁹F(¹H) NMR spectra of fluorometabolite profiles from cultures of 14 gene knockouts in and around the proposed nucleocidin 1 BGC. The resultant ¹⁹F{¹H} NMR signals represent either F‐Met‐II 3 (ca. −118 ppm), nucleocidin 1 (ca. −119.7 ppm) or F‐Met‐I 2(ca. −120.3 ppm).

Figure 4

PAPS gene clusters and their involvement in nucleocidin biosynthesis. A) The PAPS pathway highlighting the steps likely to involve NucA, NucB and NucW, the enzymes were found to play a role in nucleocidin biosynthesis. B) Schematic representation of PAPS clusters in S. calvus; the genes indicated with letters are homologues of acmA, acmB and acmW present in the (dealanyl)ascamycin BGC. C) ¹⁹F{¹H} NMR spectra of media extracts from cultures of nucA or nucW knockout strains; deletion of nucA was successfully complemented with a “native” expression plasmid. Signal assignments: F‐Met‐II 3 (ca. −118. ppm), nucleocidin 1 (ca. −119.7 ppm) and F‐Met‐I 2 (ca. −120.3 ppm).

Figure 5

Representative ¹⁹F{¹H} NMR spectra for media extracts from cultures of A) nine S. calvus knockout strains (black lines) along with gene complementation in trans (grey) and B) S. virens ΔnucN, ΔnucK and ΔnucO (magenta) together with gene complementation (brown). Sulfamylation is dependent on at least nine genes of the nucleocidin BGC. Signal assignments: F‐Met‐II 3 (ca. −118 ppm), nucleocidin 1 (ca. −119.7 ppm) and F‐Met‐I 2 (ca. −120.3 ppm).

Figure 6

Representative ¹⁹F{¹H} NMR spectra for media extracts from cultures of knockouts and complementation experiments with orf(−3), orf2 and orf3. A) Δorf(−3), Δorf2Δorf3, Δorf2 and Δorf3 (black lines) along with gene complementation in trans (grey) and B) S. virens Δorf(−3) and Δorf2Δorf3 (magenta) together with gene complementation (brown). Signal assignments: F‐Met‐II 3 (ca. −118 ppm), nucleocidin 1 (ca. −119.7 ppm) and F‐Met‐I 2 (ca. −120.3 ppm).

Figure 7

AlphaFold2 structure predictions for Orf2 and Orf(−3). A) Putative structure of Orf2 revealing a similarity to the triphosphate tunnel metalloenzyme family. B) Putative structure of Orf(−3), which shares fold similarity with cysteine/thiol dioxygenases. The framed inset shows a close up of the putative active site, modelled on the closest homologues, including three histidines (His115, His117 and His200), a tyrosine (Tyr73) and a tryptophan (Trp100).