利用全基因组改组技术提高SubtilosinA的产量

The breeding for high-yield subtilosina strain with genome shuffling

以Bacillus subtilis nja A1B2-2为出发菌株,在最佳的原生质体形成、再生和融合条件的基础上,将前期经过理化诱变筛选的四株高产菌株进行两轮基因组改组。结果表明,当以0.6mol/L Na Cl为高渗洗涤液,0.1mg/m L溶菌酶酶解40min后,涂布在以SMM为高渗体系的NB再生培养基上,原生质体形成率达到95.49%,再生率为89.25%。优化原生质体的融合条件,融合率达到了1.39×10-3。在此基础上将四株高产Subtilosin A菌株进行两轮全基因组改组,结合双亲灭活的筛选方法,挑选出一株遗传性状稳定的高产菌株R2-264,产量达17.59mg/L,比出发菌株提高了3.58倍。

Bacillus subtilis nja A1B2-2 as the starting strain,based on the optimum conditions for protoplast formation,regeneration and protoplasts fusion,genome shuffling was carried out between four mutant strains screened from phys-chem mutagenesis. The results showed when the hypertonic washing solution 0.6mol/L Na Cl,0.1mg/m L lysozyme treating for 40 min,spreading on gregeneration medium NB with hypertonic SMM,protoplast formation rate and the regeneration rate respectively reached 95.49% and 89.25%. After optimizing protoplast fusion conditions,its rate was up to 1.39 ×10^-3. Then two round of genome shuffling was launched between four high-yield Subtilosin A strains. With the selection system of double inactivation of parental protoplasts,a stable and high-yield strain R2-264 was selected out with the maximum production of Subtilosin A at 17.59mg/L,which was 3.58 times higher than the starting strain.