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<p>Fungal biosynthetic gene clusters (BGCs) are often co-expressed, creating competition for shared precursors and limiting desired metabolite accumulation. The marine-derived fungus <i>Penicillium</i> sp. KWF31 produces paraherquamide A (PHQA) alongside penicillic acid derivatives and pigments. Using a phenotype-guided strategy and comparative BGCs analysis, we disrupted the penicillic acid backbone gene <i>g322</i> and pigment-associated polyketide synthase (PKS) genes to relieve pathway competition. While Δ<i>g322</i> abolished dihydropenicillic acid but intensified pigmentation, deleting the pigment PKS gene <i>g4411</i> eliminated colony pigmentation yet; neither single mutant increased PHQA. In contrast, the double mutant Δ<i>g322</i>Δ<i>g4411</i> removed both major byproduct branches and increased PHQA production by ~ 42.3% relative to the parental Δ<i>ku70</i> strain without compromising growth. To elucidate the underlying mechanisms, we integrated comparative transcriptomics with RT-qPCR validation, establishing a “Push-Pull” metabolic reprogramming model. This model reveals that the disruption of competitive pathways triggers a systemic regulatory cascade: comparative transcriptomics with RT-qPCR validation the “Push” effect is driven by the preservation of precursor pools (e.g., downregulation of acetyl-CoA hydrolase), while the “Pull” effect is realized through systemic energy reallocation from ribosome biogenesis toward the transcriptional activation of the PHQA BGC. Notably, deletion of <i>g591</i>, a <i>wA</i> homolog involved in spore pigmentation, reduced PHQA to ~ 30.9% of the control, and this defect persisted in Δ<i>g591</i>Δ<i>g4411</i>, indicating a <i>g4411</i>-independent connection between <i>wA</i>-associated genetics and PHQA output. These results show that dual disruption of competing polyketide pathways, integrated with a systems-level understanding of metabolic flux, is required to effectively enhance PHQA accumulation in a native <i>Penicillium</i> producer.</p>

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Dual disruption of competitive polyketide pathways enhances paraherquamide A biosynthesis

  • 期刊论文

  • 发表日期：

    2026年7月22日

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  • Hongling Xiang,
  • Yike Wang,
  • Yuangui Tang,
  • Yunlu Cui,
  • Lei Yan,
  • Pinmei Wang,
  • Jinzhong Xu

摘要

Fungal biosynthetic gene clusters (BGCs) are often co-expressed, creating competition for shared precursors and limiting desired metabolite accumulation. The marine-derived fungus Penicillium sp. KWF31 produces paraherquamide A (PHQA) alongside penicillic acid derivatives and pigments. Using a phenotype-guided strategy and comparative BGCs analysis, we disrupted the penicillic acid backbone gene g322 and pigment-associated polyketide synthase (PKS) genes to relieve pathway competition. While Δg322 abolished dihydropenicillic acid but intensified pigmentation, deleting the pigment PKS gene g4411 eliminated colony pigmentation yet; neither single mutant increased PHQA. In contrast, the double mutant Δg322Δg4411 removed both major byproduct branches and increased PHQA production by ~ 42.3% relative to the parental Δku70 strain without compromising growth. To elucidate the underlying mechanisms, we integrated comparative transcriptomics with RT-qPCR validation, establishing a “Push-Pull” metabolic reprogramming model. This model reveals that the disruption of competitive pathways triggers a systemic regulatory cascade: comparative transcriptomics with RT-qPCR validation the “Push” effect is driven by the preservation of precursor pools (e.g., downregulation of acetyl-CoA hydrolase), while the “Pull” effect is realized through systemic energy reallocation from ribosome biogenesis toward the transcriptional activation of the PHQA BGC. Notably, deletion of g591, a wA homolog involved in spore pigmentation, reduced PHQA to ~ 30.9% of the control, and this defect persisted in Δg591Δg4411, indicating a g4411-independent connection between wA-associated genetics and PHQA output. These results show that dual disruption of competing polyketide pathways, integrated with a systems-level understanding of metabolic flux, is required to effectively enhance PHQA accumulation in a native Penicillium producer.