原生质体融合技术构建棕榈油酸高产酵母菌株

Construction of high palmitoleic acid-producing yeast by protoplast fusion

采用原生质体融合技术进行产棕榈油酸酵母Saccharomy cescerevisiaeNo.12.926和产脂酵母RhodotorulaNo.12.908的融合研究,获得了棕榈油酸高产酵母工程菌株。实验结果表明,原生质体形成的最佳条件为:对数期酵母No.12.926和No.12.908用2%蜗牛酶于30℃分别酶解1.5和2h。在最佳条件下,酵母No.12.926和No.12.908原生质体形成率分别为94%和80%,再生率分别为75%和60%。原生质体融合由聚乙二醇诱导。将得到的融合子进行多次传代培养优选,获得了遗传性状稳定的融合菌株。融合子的生物量为亲株的两倍多,其细胞形态和菌落颜色与亲株有差别。产脂和产棕榈油酸分析表明,融合子的产脂量为菌体干重的48.53%,其中棕榈油酸占油脂总量的47.29%,为菌体干重的22.95%。

High palmitoleic acid-producing strains were obtained by protoplast fusion between Saccharomyces cerevisiae No. 12.926 producing palmitoleic acid and Rhodotorula No. 12.908 producing oil and fat. The conditions of protoplast generation of two parental strains were optimized as： strain No.12.926 and No.12.908 at logarithmic phase of their growth were treated by 2% snailase at 30℃ for 1.5 and 2 hour, respectively. Under the optimized conditions, the rates of protoplast generation of strain No.12.926 and No.12.908 reached 94% and 80%, respectively and their regeneration were 75% and 60%, respectively. The fusants were stable in genetic by the analysis of genetic stability. The biomass of fusants was more than two times of their parental strains and cell morphology and colony color of fusant were different from those of their parental strains. The weight of oil and fat producing by fusants was 48.5% of dried cell. Palmitoleic acid was 47.29% of total oil and fat, which accounted for 22.95% of dried cell.