Daptomycin production enhancement by ARTP mutagenesis and fermentation optimization in Streptomyces roseosporus.

AIMS: We evaluated whether the randomness of mutation breeding can be regulated through a double-reporter system. We hope that by establishing a new precursor feeding strategy, the production capacity of industrial microorganisms after pilot scale-up can be further improved. METHODS AND RESULTS: In this study, the industrial strain Streptomyces roseosporus L2796 was used as the starter strain for daptomycin production, and a double-reporter system with the kanamycin resistance gene Neo and the chromogenic gene gusA was constructed to screen for high-yield strain L2201 through atmospheric and room temperature plasma (ARTP). Furthermore, the composition of the culture medium and the parameters of precursor replenishment were optimized, resulting in a significant enhancement of the daptomycin yield of the mutant strain L2201(752.67 mg/l). CONCLUSIONS: This study successfully screened a high-yield strain of daptomycin through a double-reporter system combined with ARTP mutation. The expression level of two reporter genes can evaluate the strength of dptEp promoter, which can stimulate the expression level of dptE in the biosynthesis of daptomycin, thus producing more daptomycin. The developed multi-stage feeding rate strategy provides a novel way to increase daptomycin in industrial fermentation.

Identification and Characterization of a New Regulator, TagR, for Environmental Stress Resistance Based on the DNA Methylome of Streptomyces roseosporus.

DNA methylation is a defense that microorganisms use against extreme environmental stress, and improving resistance against environmental stress is essential for industrial actinomycetes. However, research on strain optimization utilizing DNA methylation for breakthroughs is rare. Based on DNA methylome analysis and KEGG pathway assignment in Streptomyces roseosporus, we discovered an environmental stress resistance regulator, TagR. A series of in vivo and in vitro experiments identified TagR as a negative regulator, and it is the first reported regulator of the wall teichoic acid (WTA) ABC transport system. Further study showed that TagR had a positive self-regulatory loop and m4C methylation in the promoter improved its expression. The ΔtagR mutant exhibited better hyperosmotic resistance and higher decanoic acid tolerance than the wild type, which led to a 100% increase in the yield of daptomycin. Moreover, enhancing the expression of the WTA transporter resulted in better osmotic stress resistance in Streptomyces lividans TK24, indicating the potential for wide application of the TagR-WTA transporter regulatory pathway. This research confirmed the feasibility and effectiveness of mining regulators of environmental stress resistance based on the DNA methylome, characterized the mechanism of TagR, and improved the resistance and daptomycin yield of strains. Furthermore, this research provides a new perspective on the optimization of industrial actinomycetes. IMPORTANCE This study established a novel strategy for screening regulators of environmental stress resistance based on the DNA methylome and discovered a new regulator, TagR. The TagR-WTA transporter regulatory pathway improved the resistance and antibiotic yield of strains and has the potential for wide application. Our research provides a new perspective on the optimization and reconstruction of industrial actinomycetes.

m4C DNA methylation regulates biosynthesis of daptomycin in Streptomyces roseosporus L30.

Despite numerous studies on transcriptional level regulation by single genes in drug producing Actinomyces, the global regulation based on epigenetic modification is not well explored. N4-methylcytosine (m4C), an abundant epigenetic marker in Actinomycetes' genome, but its regulatory mechanism remains unclear. In this study, we identify a m4C methyltransferase (SroLm3) in Streptomyces roseosporus L30 and multi-omics studies were performed and revealed SroLm3 as a global regulator of secondary metabolism. Notably, three BGCs in ΔsroLm3 strain exhibited decreased expression compared to wild type. In-frame deletion of sroLm3 in S.roseosporus L30 further revealed its role in enhancing daptomycin production. In summary, we characterized a m4C methyltransferase, revealed the function of m4C in secondary metabolism regulation and biosynthesis of red pigment, and mapped a series of novel regulators for daptomycin biosynthesis dominated by m4C methylation. Our research further indicated that m4C DNA methylation may contribute to a metabolic switch from primary to secondary metabolism in Actinomyces.

Improving the Yield and Quality of Daptomycin in Streptomyces roseosporus by Multilevel Metabolic Engineering.

Daptomycin is a cyclic lipopeptide antibiotic with a significant antibacterial action against antibiotic-resistant Gram-positive bacteria. Despite numerous attempts to enhance daptomycin yield throughout the years, the production remains unsatisfactory. This study reports the application of multilevel metabolic engineering strategies in Streptomyces roseosporus to reconstruct high-quality daptomycin overproducing strain L2797-VHb, including precursor engineering (i.e., refactoring kynurenine pathway), regulatory pathway reconstruction (i.e., knocking out negative regulatory genes arpA and phaR), byproduct engineering (i.e., removing pigment), multicopy biosynthetic gene cluster (BGC), and fermentation process engineering (i.e., enhancing O2 supply). The daptomycin titer of L2797-VHb arrived at 113 mg/l with 565% higher comparing the starting strain L2790 (17 mg/l) in shake flasks and was further increased to 786 mg/l in 15 L fermenter. This multilevel metabolic engineering method not only effectively increases daptomycin production, but can also be applied to enhance antibiotic production in other industrial strains.

A novel strategy of gene screen based on multi-omics in Streptomyces roseosporus.

Daptomycin is a new lipopeptide antibiotic for treatment of severe infection caused by multi-drug-resistant bacteria, but its production cost remains high currently. Thus, it is very important to improve the fermentation ability of the daptomycin producer Streptomyces roseosporus. Here, we found that the deletion of proteasome in S. roseosporus would result in the loss of ability to produce daptomycin. Therefore, transcriptome and 4D label-free proteome analyses of the proteasome mutant (Δprc) and wild type were carried out, showing 457 differential genes. Further, five genes were screened by integrated crotonylation omics analysis. Among them, two genes (orf04750/orf05959) could significantly promote the daptomycin synthesis by overexpression, and the fermentation yield in shake flask increased by 54% and 76.7%, respectively. By enhancing the crotonylation modification via lysine site mutation (K-Q), the daptomycin production in shake flask was finally increased by 98.8% and 206.3%, respectively. This result proved that the crotonylation modification of appropriate proteins could effectively modulate daptomycin biosynthesis. In summary, we established a novel strategy of gene screen for antibiotic biosynthesis process, which is more convenient than the previous screening method based on pathway-specific regulators. KEY POINTS: • Δprc strain has lost the ability of daptomycin production • Five genes were screened by multi-omics analysis • Two genes (orf04750/orf05959) could promote the daptomycin synthesis by overexpression.