米多霉素产生菌原生质体融合研究

Study on protoplasts fusion of mildiomycin-producing strains

从土壤中筛选出的链轮丝菌Streptoverticillium rimofaciensZD615发酵生产米多霉素的产量较低,但可以通过添加米多霉素的前体提高米多霉素产量;经紫外诱变获得的链轮丝菌Streptoverticillium rimofaciensZD519产米多霉素的产量相对较高,但丧失了前体利用能力,菌体生长还受前体的抑制.对链轮丝菌ZD615和ZD519原生质体制备最优条件的研究结果表明,甘氨酸质量浓度为0.01 g/mL的菌丝培养基和2 g/L的溶菌酶质量浓度最适于实验菌株原生质体的制备和再生;通过研究紫外灭活ZD615和热灭活ZD519的原生质体的双亲本融合条件,发现当化学融合时间为5 min时,可得到最大的双亲融合率(5.2%).从多种融合子中选育出一株高产链轮丝菌51-19,该菌株能够利用米多霉素的前体物质高产米多霉素,米多霉数产量比出发菌株ZD615提高了170%,达到了1 015 mg/L.

The productivity of Streptoverticillium rimofaciens ZD615 could be improved efficiently by the addition of several mildiomycin＇s precursor into the fermentation medium, but the final yield was still low. One mutant （Streptoverticilliurn rimofaciens ZD519） could achieve a relatively high productivity of mildiomycin, although the addition of different precursors could not improve the production of mildiomycin and even inhibited mycelia growth. The conditions of preparing protoplasts from Streptoverticilliurn rimo- faciens ZD615 and ZD519 were optimized. The results showed that hypha medium containing 0.01 g/mL glycine was most suitable for the preparation of the protoplast, and that lysozyme concentration of 2 g/L was optimal for preparation and regeneration of the protoplast. The protoplasts of Streptoverticillium rirnofaciens ZD615 and ZD519 were inactivated by UV radiation and heating, respectively, and the fusion between biparental inactivated protoplasts was used to obtain the high-yielding mildiomycin strain. The highest rate of biparental fusion （5.2%） was obtained when the fusion time was set at 5 min. A high-yield strain 51-19 was obtained from different fusion mutants which could utilize mildiomycin＇s precursor to produce mildiomycin effectively, with the productivity being as high as 1015 mg/L, about 170% higher than that of the parental strain （ZD615）.