Improving the production of the micafungin precursor FR901379 in an industrial production strain.
Improving the production of the micafungin precursor FR901379 in an industrial production strain.
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DOI：
10.1186/s12934-023-02050-0
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发表时间：
2023-03-06
影响因子：
6.4
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中科院分区：
文献类型：
--
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米卡芬净是一种棘白菌素型抗真菌药，用于临床治疗侵袭性真菌感染。它是由磺化脂六肽FR 901379（一种由丝状真菌Coleophoma empetri产生的非核糖体肽）半合成的。然而，FR 901379的低发酵效率增加了米卡芬净生产的成本，并阻碍了其广泛的临床应用。本研究通过系统代谢工程在C. empetri MEFC 09.首先，通过过表达限速酶细胞色素P450 McfF和McfH来优化FR 901379的生物合成途径，这成功地消除了不需要的副产物的积累并增加了FR 901379的产量。在此基础上，对编码β-1，3-葡聚糖合成酶的抗病基因进行了体内功能研究。CEfks 1的缺失影响生长并导致更多的球形细胞。此外，还鉴定了调控FR 901379生物合成的转录激活因子McfJ，并将其应用于代谢工程。过量表达mcfJ使FR 901379的产量从0.3 g/L显著增加到1.3 g/L。最后，构建了共表达mcfJ、mcfF和mcfH的工程菌，在5L的生物反应器中进行分批补料培养，效价达到4.0g/L。该研究代表了FR 901379生产的显著改进，并为建立其他棘白菌素的高效真菌细胞工厂提供指导。在线版本包含补充材料，可通过10.1186/s12934-023-02050-0获得。
Micafungin is an echinocandin-type antifungal agent used for the clinical treatment of invasive fungal infections. It is semisynthesized from the sulfonated lipohexapeptide FR901379, a nonribosomal peptide produced by the filamentous fungus Coleophoma empetri. However, the low fermentation efficiency of FR901379 increases the cost of micafungin production and hinders its widespread clinical application. Here, a highly efficient FR901379-producing strain was constructed via systems metabolic engineering in C. empetri MEFC09. First, the biosynthesis pathway of FR901379 was optimized by overexpressing the rate-limiting enzymes cytochrome P450 McfF and McfH, which successfully eliminated the accumulation of unwanted byproducts and increased the production of FR901379. Then, the functions of putative self-resistance genes encoding β-1,3-glucan synthase were evaluated in vivo. The deletion of CEfks1 affected growth and resulted in more spherical cells. Additionally, the transcriptional activator McfJ for the regulation of FR901379 biosynthesis was identified and applied in metabolic engineering. Overexpressing mcfJ markedly increased the production of FR901379 from 0.3 g/L to 1.3 g/L. Finally, the engineered strain coexpressing mcfJ, mcfF, and mcfH was constructed for additive effects, and the FR901379 titer reached 4.0 g/L under fed-batch conditions in a 5 L bioreactor. This study represents a significant improvement for the production of FR901379 and provides guidance for the establishment of efficient fungal cell factories for other echinocandins. The online version contains supplementary material available at 10.1186/s12934-023-02050-0.
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