Fig 1.
Production of CA by the recombinant strains.
S. clavuligerus/pIB139 (purple), and S. clavuligerus/pIBLD (blue).
Fig 2.
Aerial mycelium formation of S. clavuligerus on GYM medium with apramycin 40 µg mL-1 (1) S. clavuligerus/ pIB139.
(2) S. clavuligerus/ pIBLD. (a) After 5 days of growth. (b) After 10 days of growth.
Fig 3.
Volcano plot showing differentially expressed genes.
Cyan dots indicate the significantly upregulated and downregulated genes. Pink dots indicate genes that were not significantly different in the two groups. The X-axis depicts the log2 fold-change in expression, while the Y-axis represents the statistical significance of each gene. A) 72 hours, B) 24 hours.
Fig 4.
Predicted STRING networks for the DEGs at 72 h of cultivation, visualized in Cytoscape.
The network in red corresponds to the up-regulated genes with some representative highlighted clusters. Cluster 1A: Bacteriophage T4, Gp19, tail tube. 1B: Fatty acid and lipid metabolic process. 1C: Sesquiterpenoid and triterpenoid biosynthesis. 1D: Organic acid metabolic process. 1F: Cysteine and methionine metabolism and 1F: Lanthionine synthetase C-like. In blue, the network for the down-regulated genes with some representative highlighted clusters. 2A: Penicillin and cephalosporin biosynthesis. Cluster 2B: Sulfur metabolism. 2C: aminopeptidase activity and whiB. 2D: Glycine betaine transport; 2E: Beta-lactamase and acetyltransferase domain.
Fig 5.
Ontology enrichment analysis (FDR <= 0.05) generated in ShinyGO v0.741 for S. clavuligerus/pIBLD at 72 h.
A) Up-regulated genes. B) Down-regulated genes.
Fig 6.
Ontology enrichment analysis (FDR<=0.05) generated in ShinyGO v0.741150 for DEGs at 24 hours of growth.
A) Up-regulated genes. B) Down-regulated genes.
Fig 7.
Up-regulated genes related to the Phage-like protein in S. clavuligerus.
A) Phage-related genes and their potential organization in S. clavuligerus ATCC27064 chromosome. B) Potential interaction network for phage-related genes predicted using STRING DB (version 12.0). 1.Terpene synthase (CRV15_RS00255). 2 BTAD putative transcriptional regulator (CRV15_RS27960). C) Phage-related genes and their potential organization in S. clavuligerus ATCC 27064 plasmid. Genes that were not overexpressed are highlighted in grey.
Fig 8.
Cross-species ortholog fold-change comparison under opposite BldD perturbations.
Hexbin plot of RBH orthologs (n = 3,653) comparing S. coelicolor bldD null mutant transcriptome (KO) logFC (x-axis) vs sign-inverted S. clavuligerus bldD overexpression (OE) fold-change (y-axis; − logFC). Red points highlight GlcNAc/amino-sugar orthologs (n = 74); Spearman ρ and p-values are shown for the global set and this subset.
Fig 9.
Genes from the CA and cephamycin C gene clusters that were downregulated in S. clavuligerus/pIBLD at 72 h.
In purple, CA genes. In yellow, cephamycin C genes.
Fig 10.
Schematic diagram of the enzymes that were overexpressed in a potential peptidoglycan biosynthesis pathway based on those in other bacteria [29,31].
In red the enzymes that were overexpressed in this work. The enzymes and substrate are described in the text.