美登素家族的安丝菌素P-3(ansamitocin,AP-3)属于大环内酰胺类抗生素,有极强的抗肿瘤活性。鉴于其重要的药用价值和广阔的市场前景,提高AP-3的产量成为国内外研究重点。本研究采用常压室温等离子体(atmospheric and room temperature pl...美登素家族的安丝菌素P-3(ansamitocin,AP-3)属于大环内酰胺类抗生素,有极强的抗肿瘤活性。鉴于其重要的药用价值和广阔的市场前景,提高AP-3的产量成为国内外研究重点。本研究采用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变技术,并结合24深孔板发酵及以线黑粉酵母(Filobasidium uniguttulatum)为指示菌的高通量生物测定筛选技术,最终筛选到两株AP-3产量稳定提高15%的珍贵束丝放线菌(Actinosynnema pretiosum)的突变株——M13和M144。并对高通量生测筛选技术进行了优化,最终确定实验过程的最佳参数:在液体摇瓶YPD中培养15 h时线黑粉酵母指示菌株可达到对数生长中期,且当摇瓶转接至孔板的接种量为10%时,指示菌与AP-3混合培养至30 h达到稳定期,可以明显观察到指示菌在0.2~0.7μg/mL AP-3浓度范围内的线性生长变化,酶标仪OD_(600)读数与AP-3浓度之间的标准曲线为y=0.0228x+1.8377,R^(2)=0.9906。以AP-3产量提高15%作为筛选指标,可筛选出高产突变株。对经高通量生测筛选出的突变株经10轮传代发酵后,最终筛选出两株AP-3产量稳定提高的突变株M13和M144。经二代重测序后,可定位突变株内部的突变位点,为今后从全基因组层面解析AP-3的高产原因,以及建立珍贵束丝放线菌ATCC31280的代谢网络提供理论基础。展开更多
The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and con...The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and contains genes for polyketide assembly, precursor synthesis, post-polyketide synthesis modification, and regulation. Within the biosynthetic gene cluster, asm8 encodes an 1117-amino-acid protein with a high degree of similarity to the large ATP-binding LuxR family-type regulators. In the current study, we determined that inactivation of asm8 by gene replacement in ATCC 31565 resulted in the complete loss of ansamitocin production, and that complementation with a cloned asm8 gene restored ansamitocin biosynthesis. Interestingly, the disruption of asm8 decreased the transcription of genes responsible for 3-amino-5-hydroxybenzoate (AHBA) formation, the starter unit required for ansamitocin biosynthesis. Subsequently, feeding of exogenous AHBA to the asm8 mutant restored ansamitocin biosynthesis, which showed that Asm8 is a specific positive regulator in AHBA biosynthesis. In addition, investigation of asm8 homologs identified two new ansamitocin producers, and inactivation of the asm8 homolog in A. pretiosum ATCC 31280 abolished ansamitocin production in this strain. Characterization of the positive regulator Asm8 and discovery of the two new ansamitocin producers paves the way for further improving production of this important antitumor agent.展开更多
文摘美登素家族的安丝菌素P-3(ansamitocin,AP-3)属于大环内酰胺类抗生素,有极强的抗肿瘤活性。鉴于其重要的药用价值和广阔的市场前景,提高AP-3的产量成为国内外研究重点。本研究采用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变技术,并结合24深孔板发酵及以线黑粉酵母(Filobasidium uniguttulatum)为指示菌的高通量生物测定筛选技术,最终筛选到两株AP-3产量稳定提高15%的珍贵束丝放线菌(Actinosynnema pretiosum)的突变株——M13和M144。并对高通量生测筛选技术进行了优化,最终确定实验过程的最佳参数:在液体摇瓶YPD中培养15 h时线黑粉酵母指示菌株可达到对数生长中期,且当摇瓶转接至孔板的接种量为10%时,指示菌与AP-3混合培养至30 h达到稳定期,可以明显观察到指示菌在0.2~0.7μg/mL AP-3浓度范围内的线性生长变化,酶标仪OD_(600)读数与AP-3浓度之间的标准曲线为y=0.0228x+1.8377,R^(2)=0.9906。以AP-3产量提高15%作为筛选指标,可筛选出高产突变株。对经高通量生测筛选出的突变株经10轮传代发酵后,最终筛选出两株AP-3产量稳定提高的突变株M13和M144。经二代重测序后,可定位突变株内部的突变位点,为今后从全基因组层面解析AP-3的高产原因,以及建立珍贵束丝放线菌ATCC31280的代谢网络提供理论基础。
基金supported by grants from the Ministry of Science and Technology of China (2012CB721005, 2012AA02A706, 2011ZX08009-001, 2012AA022107)the National Natural Science Foundation of China (31070070, 31121064)+1 种基金Ministry of Education of China (20110073110048)the SJTU-UM Collaborative Program
文摘The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and contains genes for polyketide assembly, precursor synthesis, post-polyketide synthesis modification, and regulation. Within the biosynthetic gene cluster, asm8 encodes an 1117-amino-acid protein with a high degree of similarity to the large ATP-binding LuxR family-type regulators. In the current study, we determined that inactivation of asm8 by gene replacement in ATCC 31565 resulted in the complete loss of ansamitocin production, and that complementation with a cloned asm8 gene restored ansamitocin biosynthesis. Interestingly, the disruption of asm8 decreased the transcription of genes responsible for 3-amino-5-hydroxybenzoate (AHBA) formation, the starter unit required for ansamitocin biosynthesis. Subsequently, feeding of exogenous AHBA to the asm8 mutant restored ansamitocin biosynthesis, which showed that Asm8 is a specific positive regulator in AHBA biosynthesis. In addition, investigation of asm8 homologs identified two new ansamitocin producers, and inactivation of the asm8 homolog in A. pretiosum ATCC 31280 abolished ansamitocin production in this strain. Characterization of the positive regulator Asm8 and discovery of the two new ansamitocin producers paves the way for further improving production of this important antitumor agent.
