Effect of Heterologous Expression of the vgb Gene Mediated by Different Promoters on Novonestmycin Production

ZHANG Yifan; LIU Xiaoyan; WAN Zhongyi; FANG Wei; ZHU Lei; CHEN Ling; CAI Jun; ZHOU Ronghua; WANG Changgao; MIN Yong

doi:10.15933/j.cnki.1004-3268.2025.04.009

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.