Enhancing rufomycin production by CRISPR/Cas9-based genome editing and promoter engineering in Streptomyces sp. MJM3502

Abstract

Streptomyces sp. MJM3502 is a promising producer of rufomycins, which are a class of potent anti-tuberculosis lead compounds. Although the structure, activity, and mechanism of the main rufomycin 4/6 and its analogs have been extensively studied, a significant gap remains in our understanding of the genome sequence and biosynthetic pathway of Streptomyces sp. MJM3502, and its metabolic engineering has not yet been reported. This study established the genetic manipulation platform for the strain. Using CRISPR/Cas9-based technology to in-frame insert the strong kasO∗p promoter upstream of the rufB and rufS genes of the rufomycin BGC, we increased rufomycin 4/6 production by 4.1-fold and 2.8-fold, respectively. Furthermore, designing recombinant strains by inserting the kasO∗p promoter upstream of the biosynthetic genes encoding cytochrome P450 enzymes led to new rufomycin derivatives. These findings provide the basis for enhancing the production of valuable natural compounds in Streptomyces and offer insights into the generation of novel active natural products via synthetic biology and metabolic engineering.

Keywords: Anti-Tuberculosis; Genome mining; Promoter engineering; Rufomycin; Streptomyces sp. MJM3502.

Fig. 1

Phylogenomic analysis of Streptomyces sp. MJM3502. (A) Phylogenetic analysis based on the 16s rRNA sequence. (B) A heatmap generated with OrthoANI values calculated using OAT software. (C) A collinearity diagram of Streptomyces sp. MJM3502 with Streptomyces sp. 3507 and Streptomyces sp. 8412. Each colored block (called locally collinear blocks) represents a region of a homologous sequence that aligns to part of another genome and is free from internal rearrangement. The colored lines indicate which blocks in each genome are homologous.

Fig. 2

Analysis of the biosynthetic potential and metabolites of Streptomyces sp. MJM3502. (A) Circular genome map of Streptomyces sp. MJM3502 (9,314,038 bp), highlighting various BGCs for secondary metabolites. The outermost circle denotes genomic DNA with color-coded BGCs, while the inner circles represent GC content and GC skew. (B) Comparative analysis of the rufomycin/ilamycin BGC in Streptomyces sp. MJM3502, S. atratus ATCC 14046, and S. atratus SCSIO ZH16. (C) The main chemical structures of rufomycin and ilamycin derivatives produced by Streptomyces sp. MJM3502 and S. atratus SCSIO ZH16. Structural differences, such as hydroxyl (OH), methyl (CH₃), and carboxyl (COOH) groups, are marked by colored circles.

Fig. 3

Intergeneric conjugal transfer using spore conjugation and CRISPR/Cas9-based genome editing and screening for selected single-colony mutation. (A) Pick single exconjugants and streak on plates with and without apramycin, respectively, and grow at 28 °C for 3 or 4 days. (B) Colonies with reduced growth on the plate with apramycin were transferred in liquid media for losing the plasmid. (C) Replicate the grown colonies to plates with and without apramycin to confirm plasmid loss. (D) Extract genomic DNA of apramycin-sensitive single colonies from mycelium on the nonselective plate, followed by PCR amplification to check mutation.

Fig. 4

The design of plasmids to introduce the powerful promoter kasO∗p into MJM3502. (A) A schematic representation of the design positions where the promoter was inserted in the BGC of rufomycin. (B) The strategy of promoter engineering in six positions using CRISPR/Cas9. (C) A schematic representation of the primer design to verify kasO∗p knock-in mutants. (D) PCR amplification products using templates of genomic DNA from the WT (W) and the engineered promoter knock-in strains at position PRO1-PRO6 (P1–P6). M (1 kb maker); UHA and DHA are both 2 kb; UHA + kasO∗p and DHA + kasO∗p bands are both 2.1 kb.

Fig. 5

CRISPR/Cas9-based knock-in of the kasO∗p promoter to enhance the production of rufomycin in Streptomyces sp. MJM3502. (A) UPLC analyses of fermentation broth in the strains of Streptomyces sp. PRO1, PRO3, and PRO4. (B) The increasing ratio of rufomycin in the engineered strains Streptomyces sp. PRO1, PRO3, and PRO4. (C) qRT-PCR analysis of key gene expression after normalization to the housekeeping hrdB gene for both the WT and the engineered strains of Streptomyces sp. PRO2, PRO3, and PRO4. The error bars represent the standard deviation of biological triplicates.

Fig. 6

The generation of potential novel rufomycin derivatives and a putative gene in the rufomycin BGC in different promoter engineering mutants. (A) The UPLC-MS/MS spectrum of rufomycin analogs in WT MJM3502 and PRO2. (B) The sequence alignments of ruf∗ with other transposases in the Streptomyces strain.

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