Study on the regulatory mechanism of NsdAsr on rimocidin biosynthesis in Streptomyces rimosus M527

Abstract

Background: We previously identified a regulator NsdA_sr, which negatively regulated rimocidin biosynthesis in Streptomyces rimosus M527. However, the exact regulatory mechanism of NsdA_sr on rimocidin production remains unknown.

Results: In this study, firstly, transcriptomic data demonstrated that the differentially expressed genes resulting from the over-expression of nsdA_sr were primarily associated with several key metabolic pathways, including glycolysis, oxidative phosphorylation, and ribosome-related genes, all of which were downregulated. This directly impacted the concentrations of CoA and NADH, as confirmed by concentration measurement assays. Subsequently, the results of the ChIP-seq experiments revealed that NsdA_sr directly binds to 49 target genes. Notably, these include RS18275 and RS18290 (both involved in fatty acid degradation) as well as rpoB (related to DNA transcription). The validity of the ChIP-seq assay for these three genes was further supported by in vitro electrophoretic mobility shift assays. Regarding RS18275 and RS18290, the results revealed that the binding of NsdA_sr to these elements led to the downregulation of gene expression. This, in turn, resulted in a decrease in the levels of butyryl-CoA and malonyl-CoA, which are known precursors for rimocidin biosynthesis. Consequently, this negatively impacted on the biosynthesis of rimocidin. In the case of rpoB, the results indicated that NsdA_sr binding led to a downregulation of overall protein levels. This was determined by enzymatic activity of report gene GUS and Western blot assay. Consequently, this resulted in a decrease in rimocidin yield.

Conclusion: This study reveals NsdA_sr's dual role in limiting rimocidin production by suppressing metabolic precursors and modulating protein expression. Integrated transcriptomic and ChIP-seq analyses provide critical insights into its regulatory mechanisms.

Keywords: Streptomyces rimosus; ChIP-Seq; GUS; NsdAsr; Precursor supply.

Fig. 1

Volcano-plots of differentially expressed genes. Each dot in the graph represents a gene, and the red and blue dots correspond to genes that are significantly upregulated and downregulated, respectively, while the gray dots are not significant

Fig. 2

Gene organization of rim gene cluster in the genome of Streptomyces rimosus M527 and rimocidin biosynthetic pathway, where acetyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, butanoyl-CoA and malonyl-CoA are involved in. Proposed model for rimocidin and CE-108 biosynthesis in S. diastaticus var. 108 (Seco et al., 2004), R: -CH₃ (CE-108, acetyl-CoA as the starter unit); R: -CH₂CH₂CH₃ (rimocidin, butanoyl-CoA as the starter unit). The functional annotation of genes in rim gene cluster is consistent with the previous report [40]

Fig. 3

The KEGG analysis of selected DEGs and the resulting schematic overview of primary metabolic changes in S. rimosus M527-NA_sr: the number behind DEG was the log₂ (fold change) of transcription level, the dashed arrow indicated multi-step reactions, the downregulated pathway was highlighted in blue (fabG: 3-oxoacyl-ACP reductase FabG; RS20365: enoyl-CoA hydratase; RS20375: enoyl-CoA hydratase/isomerase family protein)

Fig. 4

Determination of intracellular NADH and NADPH concentrations in S. rimosus M527/M527-NA_sr. ^**Indicates highly statistically significant results (P value < 0.01)

Fig. 5

HPLC analysis of rimocidin production from fermentation extracts of the WT strain S. rimosus M527, M527-NA_sr, and M527-NA_his

Fig. 6

Analysis and comparison of dry cell weight (DCW) in WT strain S. rimosus M527, recombinant strain M527-NA_sr, and M527‐NA_his in shake‐flask culture experiment. Error bars indicate SD of samples performed in triplicate

Fig. 7

Verification of selected target genes of M527-NA_his by ChIP-qPCR. Statistical significance: *0.01 <P < 0.05, **P < 0.01

Fig. 8

Butanoate, propanoate metabolism and fatty acid degradation pathway in S. rimosus M527-NA_his

Fig. 9

In vitro EMSA of NsdA_sr binding to the putative promoter regions of genes rpoB(a), RS18275(b) and RS18290(c). His₆-NsdA_sr protein was incubated with biotinylated promoter probes, with a 100× molar excess of unlabeled competitor DNA added in competition assays. NsdA_sr protein binding putative promoter region of genes rpoB(a), RS18275(b) and RS18290(c). The symbols “+” or “−” in the top row indicate the presence or absence of probes and competitors. Lane 1: biotin-labeled DNA probe; lane 2: biotin-labeled DNA probe plus NsdA_sr protein; lane 3: a 100-fold excess of unlabeled specific competitor plus NsdA_sr protein. DNA-protein binding conditions: 0.04 pmol/μL biotinylated probe with 10 μg NsdA_sr

Fig. 10

Transcript levels of rpoB, RS18275, and RS18290 were compared between WT S. rimosus M527 and recombinant strain M527-NA_his using quantitative RT-PCR, with error bars representing standard deviation from three biological replicates (**P<0.01)

Fig. 11

Determination of intracellular butanoyl-CoA and malonyl-CoA in S. rimosus M527 and M527-NA_sr. Data are shown as mean ± SD from three biological replicates. (**P<0.01)

Fig. 12

Determination of the GUS activity and total protein content of S. rimosus M527-GA_his and M527-NGA_his. a: Western blot detection of M527-GA_his and M527-NGA_his. M: 150 kDa protein Marker. Lane 1, negative control M527-ES; Lane 2-3, M527-GA_his-1 48 h/72 h protein; Lane 4-5, M527-GA_his-2 48 h/72 h; Lane 6-7, M527-NGA_his-1 48 h/72 h; Lane 8-9, M527-NGA_his-2 48 h/72 h. b: Western blot detection of M527-GA_his and M527-NGA_his. M: 150 kDa protein Marker. Lane 1, negative control M527-ES; Lane 2-3, M527-GA_his-3 48 h/72 h protein; Lane 4-5, M527-GA_his-4 48 h/72 h; Lane 6-7, M527-NGA_his-3 48 h/72 h; Lane 8-9, M527-NGA_his-4 48 h/72 h. c: GUS activity phenotype detection. 1: M527-ES-48 h/72 h; 2: M527-GA_his-48 h/72 h; 3: M527-NGA_his-48 h/72 h