Wang Liang, Xiao Wen, Qiu Ting, Zhang Hongjian, Zhang Jianhua, Chen Xusheng
Key Laboratory of Industrial Biotechnology, School of Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
Microb Cell Fact. 2025 Apr 2;24(1):79. doi: 10.1186/s12934-025-02696-y.
Natamycin is a natural antibiotic with broad-spectrum antifungal activity, widely used in food preservation, medicine, and biological control. However, the relatively low biosynthetic capacity of producing strains limits further industrialization and broader applications of natamycin. Due to the complexity of cellular metabolism, evolutionary engineering is required for developing strains with enhanced natamycin biosynthetic capacity.
Here, protoplast fusion combined with phosphate tolerance screening was employed for the first time to enhance natamycin production of Streptomyces gilvosporeus. A high-yielding strain, GR-2, was obtained, with natamycin production twice that of the original strain. Transcriptomic analysis revealed that the natamycin biosynthetic gene cluster and several primary metabolic pathways were significantly upregulated in GR-2, likely contributing to its high production performance. Further experiments, including amino acid addition and reverse engineering, confirmed that branched-chain amino acid, nitrogen, and phosphate metabolism play crucial roles in promoting natamycin production. Silencing of the phosphate metabolism transcriptional regulators PhoP and PhoR led to a decreased expression of natamycin biosynthetic genes and significantly reduced natamycin production, highlighting the key role of these regulators in S. gilvosporeus. Based on omics data, co-expression of phoP and phoR in GR-2 resulted in the engineered strain GR2-P3, which exhibited a 25% increase in natamycin production in shake flasks. In a 5 L fermenter, GR2-P3 achieved a natamycin production of 12.2 ± 0.6 g·L⁻¹, the highest yield reported for S. gilvosporeus to date.
Our findings suggest that the high production performance of GR-2 is primarily due to the upregulation of the natamycin biosynthetic gene cluster and genes related to precursor supply. Increasing the intracellular supply of valine and glutamate significantly enhanced natamycin production. Additionally, the natamycin biosynthetic gene cluster is likely positively regulated by PhoP and PhoR. Our work presents a novel strategy for strain screening and evolution to improve natamycin production and identifies novel molecular targets for metabolic engineering.
纳他霉素是一种具有广谱抗真菌活性的天然抗生素，广泛应用于食品保鲜、医药和生物防治领域。然而，生产菌株相对较低的生物合成能力限制了纳他霉素的进一步工业化和更广泛应用。由于细胞代谢的复杂性，需要通过进化工程来培育具有增强纳他霉素生物合成能力的菌株。
在此，首次采用原生质体融合结合磷酸盐耐受性筛选来提高褐黄孢链霉菌的纳他霉素产量。获得了高产菌株GR-2，其纳他霉素产量是原始菌株的两倍。转录组分析表明，GR-2中纳他霉素生物合成基因簇和几条初级代谢途径显著上调，这可能是其高产性能的原因。包括添加氨基酸和逆向工程在内的进一步实验证实，支链氨基酸、氮和磷酸盐代谢在促进纳他霉素生产中起关键作用。沉默磷酸盐代谢转录调节因子PhoP和PhoR导致纳他霉素生物合成基因表达降低，纳他霉素产量显著减少，突出了这些调节因子在褐黄孢链霉菌中的关键作用。基于组学数据，在GR-2中共表达phoP和phoR得到工程菌株GR2-P3，该菌株在摇瓶中纳他霉素产量提高了25%。在5 L发酵罐中，GR2-P3的纳他霉素产量达到12.2±0.6 g·L⁻¹，是迄今为止报道的褐黄孢链霉菌的最高产量。
我们的研究结果表明，GR-2的高产性能主要归因于纳他霉素生物合成基因簇和与前体供应相关基因的上调。增加细胞内缬氨酸和谷氨酸的供应显著提高了纳他霉素产量。此外，纳他霉素生物合成基因簇可能受到PhoP和PhoR的正向调节。我们的工作提出了一种用于菌株筛选和进化以提高纳他霉素产量的新策略，并确定了代谢工程的新分子靶点。
