基于ARTP诱变的安丝菌素P-3高产菌株高通量筛选

High-throughput Screening of Ansamitocin P-3 High-yield Strains Based on ARTP Mutagenesis

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。

The bacterial maytansinoid,ansamitocin P-3 belongs to macrolactam antibiotic and exhibits potent antitumor activities.Due to the important medicinal value and broad market prospect,improving its production has become an important issue worldwide.In this study,two high-yield ansamitocin mutants of Actinosynnema pretiosum,M13 and M144,were selected by atmospheric and room temperature plasma(ARTP)mutagenesis,combined with 24-well microtiter plate fermentation and high-throughput screening using yeast Filobasidium uniguttulatum as indicator.Through the optimization of the high-throughput screening process,the best parameters were obtained.First of all,the indicator yeast reached mid-logarithmic growth when cultured in liquid culture in shaking flask for 15 hours.Moreover,when the inoculation volume of seed culture from shake flask to the microtiter plate was 10%,and the incubation time of mixed indicator yeast and ansamitocin was 30 h,a linear growth of indicator yeast was detected between 0.2~0.7μg/mL of AP-3.Under these conditions,the standard curve between the OD_(600)value and the concentrations of AP-3 was determined with y=0.0228x+1.8377,R^(2)=0.9906.With a 15%yield increase as the selection threshold,two stable high-yield mutants,M13 and M144,were finally selected after 10rounds of consecutive cultivation.Furthermore,next-generation genome sequencing was performed for these two mutants,and mutant loci were identified in each strain.This study provids theoretical basis for studying the high-yield mechanisms of AP-3 and establishing metabolic network for Actinosynnema pretiosum ATCC 31280.