基因组改组选育蔗糖利用型L-乳酸高产菌株

Application of genome shuffling to screening of mutant from Lactobacillus for improving utilization of sucrose

对干酪乳杆菌进行紫外诱变和NTG诱变,得到蔗糖耐受性高的突变菌株,以此作为基因组改组的出发菌株,制得原生质体,将原生质体分别于紫外线和热灭活,致死率分别为89%和91.6%,在再生平板中培育,将存活的原生质体进行融合,获得的融合子通过蔗糖YE平板筛选,获得F1代,然后以F1代为出发菌,经过上述步骤得到了能够高效利用蔗糖发酵的F2代菌株。与野生型菌株比较发现,在15.0%蔗糖浓度条件下菌体旺盛生长,OD600达到3.11,较原始菌提高了0.70,发酵产酸量提高了61.0%,而且蔗糖酶活性比野生型有很大的提高,从0.54 U/mg cells提高到1.93 U/mg cells,提高了近4倍。

The original strain Lactobacillus was used as the starting strain for genome shuffling, and protoplasts were gained from it. The lethal protoplasts which were obtained from both ultraviolet irradiation and heat treatments fused together and then were subjected for protoplast regeneration. The fusants grew in the regenerative plates as the lethal damage cured by complementary repairing, repeated above steps, and sucrose-fermenting F2 stain was screened from the fusants via sucrose gradient plate. By comparing wild type, F2 strain was more efficient in sucrose utilizing and much more productive in lactic acid. The cell density （OD600） of F2 strain under the condition of 15.0% sucrose was 3.11, 0.70 more than that of the wild type. The production of lactic acid was 61.0% more than that of the wild type strain. The activity of invertase was also increased by 4 times, from 0.54U/rag cell to 1.93U/mg cell.