Metabolic perturbation of Streptomyces albulus by introducing NADP-dependent glyceraldehyde 3-phosphate dehydrogenase

Abstract

The available resources of Streptomyces represent a valuable repository of bioactive natural products that warrant exploration. Streptomyces albulus is primarily utilized in the industrial synthesis of ε-poly-L-lysine (ε-PL). In this study, the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans was heterologously expressed in S. albulus CICC11022, leading to elevated intracellular NADPH levels and reduced NADH and ATP concentrations. The resulting perturbation of S. albulus metabolism was comprehensively analyzed using transcriptomic and metabolomic methodologies. A decrease in production of ε-PL was observed. The expression of gapN significantly impacted on 23 gene clusters responsible for the biosynthesis of secondary metabolites. A comprehensive analysis revealed a total of 21 metabolites exhibiting elevated levels both intracellularly and extracellularly in the gapN expressing strain compared to those in the control strain. These findings underscore the potential of S. albulus to generate diverse bioactive natural products, thus offering valuable insights for the utilization of known Streptomyces resources through genetic manipulation.

Keywords: NADP-dependent glyceraldehyde 3-phosphate dehydrogenase; Streptomyces albulus; bioactive natural products; metabolic perturbation; transcriptome and metabolome; ε-poly-L-lysine.

FIGURE 1

Effect of gapN expression on intracellular NADPH, NADH and ATP concentrations. **P < 0.01; ***P < 0.001.

FIGURE 2

Effects of heterologous gapN expression on cell growth, glucose consumption, and ε-PL production.

FIGURE 3

KEGG annotation analysis of the DEGs. (A) Classification of DEGs with KEGG annotation results. (B) Noteworthy DEGs within specific KEGG pathways.

FIGURE 4

Intracellular and extracellular metabolomic comparisons of the gapN-expressing strain and the control strain. (A) Venn plot of the differential intracellular and extracellular metabolites. (B) Volcano plot of the differential intracellular metabolites. (C) Volcano plot of the differential extracellular metabolites. (D) KEGG enrichment analysis based on intracellular metabolites. (E) KEGG enrichment analysis based on extracellular metabolites. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 5

Effects of gapN expression on the ε-PL biosynthesis pathway. The green lines, arrows, and gene names represent genes whose expression was significantly upregulated at each step, whereas the red lines, arrows and gene names represent genes whose expression was significantly downregulated. The numbers that follow the gene names are the FCs in gene expression. The green and red metabolite names correspond to the upregulated and downregulated metabolites detected by metabolomics, respectively. zwf, glucose-6-phosphate dehydrogenase; pgl, 6-phosphogluconolactonase; pfk, 6-phosphofructokinase; ppc, phosphoenolpyruvate carboxylase; pckA, phosphoenolpyruvate carboxykinase; poxB, pyruvate oxidase; acdAB, acetate-CoA ligase (ADP-forming); gltA, citrate synthase; aceA, isocitrate lyase; aceB, malate synthase A; lysC, aspartate kinase; asd, aspartate-semialdehyde dehydrogenase; ectA, L-2,4-diaminobutyrate acetyltransferase; ectB, diaminobutyrate-2-oxoglutarate transaminase; ectC, L-ectoine synthase; ectD, ectoine hydroxylase; dapA, 4-hydroxytetrahydrodipicolinate synthase; dapD, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase; argD, acetylornithine aminotransferase; lysA, diaminopimelate decarboxylase; pls, polylysine synthetase; and pld, ε-PL degrading enzyme.

FIGURE 6

Effects of gapN expression on SM-BGC expression and SM biosynthesis in S. albulus CICC11022. (A) Regulation of the 36 SM-BGCs by gapN expression. The numbers on the x-axis correspond to the SM-BGC numbers. (B) The predicted secondary metabolites of the SM-BGCs that were identified in the metabolomic data. The numbers in parentheses after the metabolite names are the SM-BGC numbers. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

FIGURE 7

Expression profiles and VIP analysis of the metabolites that were upregulated both intracellularly and extracellularly.