Mechanisms of Enhanced Natamycin Biosynthesis in Streptomyces gilvosporeus 717: An Integrated Genomic and Transcriptomic Analysis

Technical Summary: Mechanisms of Enhanced Natamycin Biosynthesis in Streptomyces gilvosporeus 717

Problem Statement
Natamycin, a polyene macrolide antifungal, is a critical agent for food preservation and medical therapy. While random mutagenesis (e.g., ARTP) is an established strategy for improving production yields, the specific genetic and transcriptional mechanisms driving high-yield phenotypes remain poorly understood. Previous studies often rely on descriptive transcriptomics that link yield changes to broad metabolic patterns without resolving which specific genetic variants trigger stage-specific responses or how these responses converge to enhance accumulation. There is a need to move beyond empirical strain optimization to a mechanism-informed understanding of how regulatory rewiring, rather than just structural gene modification, drives high productivity.

Methodology
The study employed an integrated multi-omics framework combining whole-genome resequencing (WGS) and time-resolved transcriptomics (RNA-seq) on a high-producing mutant, Streptomyces gilvosporeus 717, derived from the parental strain 712 via Atmospheric and Room-Temperature Plasma (ARTP) mutagenesis.

• Strain Development: Strain 717 was selected for stable high-yield production after ARTP treatment and rigorous screening.
• Fermentation: Production was validated in 15 L fed-batch bioreactors, with samples collected at 72 h (biosynthetic initiation) and 120 h (high-yield accumulation).
• Genomic Analysis: WGS was performed to identify high-confidence variants (SNPs and InDels) in strain 717 relative to 712. Variants were annotated using SnpEff to determine their location (coding vs. non-coding) and functional impact.
• Transcriptomic Analysis: RNA-seq was conducted at 72 h and 120 h to identify differentially expressed genes (DEGs).
• Integration: A tri-layer mechanistic framework was applied to correlate genetic variations with stage-specific transcriptional responses. Protein-protein interaction (PPI) networks were constructed to map candidate genes to the natamycin biosynthetic gene cluster (BGC).

Key Results

1. Phenotypic Improvement: Strain 717 achieved a natamycin titer of 21.15 g/L in 15 L fed-batch fermentation, a 74.2% increase over the parental strain 712. The mutant exhibited faster biomass accumulation and stable growth without compromising secondary metabolism.
2. Genomic Architecture: WGS identified 320 high-confidence variants in strain 717. Crucially, 93.1% (298/320) of these variants were located in non-coding, intergenic, or upstream regulatory regions. Only 6.9% (22 variants) affected coding sequences, suggesting that the high-yield phenotype is driven primarily by regulatory rewiring rather than structural gene alterations.
3. Stage-Dependent Transcriptional Reprogramming:
   • Early Stage (72 h): Characterized by the upregulation of ABC transporter components, including ATP-binding proteins and permeases, indicating enhanced energy-dependent membrane transport.
   • Late Stage (120 h): Shifted toward increased expression of substrate-binding proteins (SBPs) for nutrient acquisition (phosphate, nitrogen, carbon) and upregulation of propionate metabolism and methylmalonyl-CoA supply pathways.
   • BGC Activation: The natamycin biosynthetic gene cluster showed significant transcriptional activation in strain 717, particularly at 120 h, covering core PKS components, tailoring enzymes, and regulators.
4. Integrated Candidate Genes: By cross-referencing WGS variants with RNA-seq data, three core candidate genes were prioritized:
   • B1H19_RS30395: A HAD family hydrolase with a stop-gained SNP but high expression, potentially altering phosphate homeostasis and signal transduction.
   • B1H19_RS33295: An NAD(P)-binding protein with a disruptive in-frame insertion and upregulation, linked to redox balance and NADPH supply.
   • B1H19_RS08875: A nonribosomal peptide synthetase (NRPS) with a frameshift mutation but high expression, suggesting the disruption of a competing secondary metabolic pathway to redirect resources to natamycin.

Significance and Claims
The paper claims to establish a systematic multi-omics evidence chain that deciphers the molecular mechanisms governing high natamycin biosynthesis. The authors assert that the high productivity of strain 717 is associated with regulatory-region variation and phase-specific transcriptional rewiring rather than extensive coding-sequence changes.

The study proposes a "variant-transcription-phenotype" framework that links genetic variation to altered phosphate metabolism, redox balance, and competing metabolic pathways. The identified candidate genes and functional modules (involving membrane transport, nutrient acquisition, precursor supply, and BGC expression) are presented as potential targets for mechanism-guided improvement. The authors position this work as a step toward shifting natamycin biosynthesis from empirical strain optimization to rational, mechanism-informed engineering, offering a methodological template applicable to other polyketide antibiotic producers. The findings suggest that coordinated changes in transport, nutrient acquisition, and precursor supply collectively support enhanced production, providing a basis for future reverse genetic validation and combinatorial engineering strategies.