摘要
为了提高去甲基金霉素的发酵效价,将金霉素链霉菌产生去甲基金霉素的变株的原生质体与金霉素高产变株热灭活的原生质体,用非交流电场法和交流电场法进行了电融合。在非交流电场法实验中。先用25％PEG将原生质体聚集,再以BAEKON 2000和SDⅡ型二种基因转移仪,采用几种不同的实验参数(脉冲强度、宽度和个数)进行融合,皆未能提高融合频率。交流电场法融合实验用SD3000细胞融合仪,当原生质体在交流电场(频率0.5MHz,强度800v/cm,作用时间60s)中形成串珠状后,施加直流脉冲(脉冲强度7kv/cm,宽度20μs,间隔0.5s,个数3),融合频率为2×10^(-4)左右,比50％PEG诱导的融合频率(2.5×10^(-5)高约一个数量级。对225株融合子进行了初筛发酵和效价测定。结果表明,其中效价高菌株所占百分数高于对照组(去甲基金霉素产生菌不同营养缺陷型的融合子),而且85％以上的融合子只产生去甲基组分。尽管融合子的发酵效价皆不稳定,但经过几次自然分离后仍获得了产量比出发菌株提高一倍左右并只产生去甲基组分较为稳定的融合子菌株。
In the strain-improvement program for a demethylch-lorotetracycline(DMCTC) producing mutant of Str. aureofaciens, it was crossed with a high-yield chlortetracycline (CTC) producer by protoplast electrofusion. The protoplasts of the second parent strain, the CTC producer, were inactivated by heat before fusion to attempt to reduce the proportion of fusants which synthesized both CTC and the demethylated derivatives. Two different types of gene transfer systems, BAEKON 2000 and SD Ⅱ, were employed for non-alternating current field electrofusion with various parameters, but no fusion frequencies were higher than that observed in control experiments. The alternating current field electrofusion was conducted with SD 3000 cell fusion apparatus. The optimum parameters (AC field:stength 800 V/cm, frequency 0.5MHz; DC pulse: strength 7 kv/cm,duration 20μs, number 3) resulted in fusant formation at frequency of 2.0×10^(-4), which was about one order of magnitude higher comparing with the PEG induced fusion.
The shake flask fermentation of the fusants showed that the percentage of high-titre strains isolated by crossing the DMCTC producer with the heat inactivated protoplasts of CTC producer was higher than that of strains obtained by fusion between the divergent auxotrophs of DMCTC producer. In addition, it was found that more than 85% fusants from DMCTC producer× CTC producer(inactivated)only produced DMCTC DMTC, and the others synthesized CTC and TC besides the demethylated components. These results implied that for improvement of productivity of one antibiotic producer, it might be useful to cross it by fusion with inactivated protoplasts of another strain which produces a closely related antibiotic of higher titre.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
1993年第3期163-168,共6页
Chinese Journal of Antibiotics
关键词
原生质体
电融合
金霉素链霉菌
Protoplast electrofusion
Streptomyces aureofaciens
Demethylchlorotetracycline
