Abstract: To enhance the production of the novel agricultural antibiotic novonestmycin，Streptomyces sp.HBERC‐20821 was used as the host strain.Five engineered strains were constructed using different promoters：ermEp，kasOp，kasOp₃，kasOp*，and SP44‑SR12.The expression of the Vitreoscilla hemoglobin gene(vgb)and its differential expression under various promoters were validated by quantitative real‐time PCR(qRT‐PCR)，and the strain with the highest novonestmycin yield was identified through parallel shake flask fermentation experiments.The bioactivity of the functional VHb protein in the selected strain was confirmed using carbon monoxide difference spectroscopy.Furthermore，parallel bioreactor fermentations were conducted to investigate the impact of heterologous expression of the vgb gene，on novonestmycin production.The results showed that five engineered strains containing different promoters were successfully constructed.Under the mediation of the kasOp series promoters，vgb expression was significantly higher than that achieved by the traditional ermEp promoter，with increases ranging from 9‐to 35‐fold；notably，the kasOp₃ promoter mediated vgb expression was 35‐fold that of ermEp.In shake flask fermentations，the parental strain HBERC‐20821 produced 545.01 mg/L of novonestmycin，while the engineered strain 20821‐kasOp‐vgb(20821/PKV)achieved the highest novonestmycin yield with an increase of 46.30% over the parental strain，reaching 797.30 mg/L.Carbon monoxide difference spectroscopy confirmed the bioactivity of the VHb protein in 20821/PKV，as evidenced by the characteristic absorption peak at 420 nm of its CO‐bound complex.In bioreactor fermentations，the parental strain produced 495.67 mg/L of novonestmycin，whereas the yield from 20821/PKV was 803.18 mg/L，corresponding to a 62.04% increase.In summary，the kasOp promoter effectively enhanced vgb expression，thus significantly improving novonestmycin production in the engineered strain 20821/PKV.

Key words: Novonestmycin, Streptomyces, vgb gene, Promoter, Heterologous expression

摘要： 为提升新型农用抗生素诺沃霉素的产量，以链霉菌（Streptomyces sp.）HBERC-20821为材料，分别利用5 种启动子ermEp、kasOp、kasOp₃、kasOp*和SP44-SR12构建工程菌株，利用实时荧光定量PCR(qRT-PCR)验证透明颤菌血红蛋白基因(vgb)的表达及其在不同启动子介导下的表达量差异，并通过摇瓶平行发酵试验筛选出诺沃霉素产量最高的工程菌株。利用一氧化碳差示光谱法检测所筛选菌株中透明颤菌血红蛋白(VHb)的生物活性，并将所筛选的工程菌株与原始菌株进行发酵罐平行发酵培养，探究vgb基因异源表达对诺沃霉素产量的影响。结果表明，试验成功构建了含有不同启动子的5种工程菌株，在kasOp系列启动子介导下，vgb基因表达量显著高于传统ermEp启动子，提升幅度达9~35倍，其中kasOp₃启动子介导的vgb基因表达量最高，为ermEp启动子的35倍。在摇瓶平行发酵培养中，原始菌株HBERC-20821诺沃霉素产量为545.01 mg/L，kasOp启动子介导下的工程菌株20821-kasOp-vgb（20821/PKV）诺沃霉素产量最高，较原始菌株提高46.30%，产量达到797.30 mg/L；一氧化碳差示光谱试验证实工程菌株20821/PKV中VHb蛋白具有生物活性，其与一氧化碳形成的复合物在420 nm波长处展现出特征吸收峰。发酵罐平行发酵培养试验显示，原始菌株诺沃霉素产量为495.67 mg/L，工程菌株20821/PKV诺沃霉素产量较原始菌株提高62.04%，产量达到803.18 mg/L。综上，在kasOp启动子介导下，工程菌株20821/PKV诺沃霉素产量得到有效提升。

关键词: 诺沃霉素, 链霉菌, 透明颤菌血红蛋白基因, 启动子, 异源表达