基于代谢控制育种技术选育深黄被孢霉高产γ-亚麻酸菌株.docVIP

基于代谢控制育种技术选育深黄被孢霉γ-亚麻酸高产菌株 郝冉，胡申才* （武汉轻工大学生物与制药工程学院，武汉，430023） 摘要： 为了获得深黄被孢霉合成γ-亚麻酸的高产菌株，本研究采用了紫外照射和氮离子注入技术2种诱变方法来建立突变体库。然后，根据γ-亚麻酸代谢合成途径中的部分关键因子，设计并建立起了抗丙二酸和红四氮唑(TTC)筛选模型，该2种筛选模型分别旨在提高菌体生物量和不饱和脂肪酸含量。结果显示，采用丙二酸浓度为2 g/L的平板可筛选得到高产油脂菌株；在发酵培养的第54 h时收集菌体，用0.9 % TTC染色4 h，可作为TTC法的最佳染色条件。经过两轮不同方法的诱变筛选后，最终得到了一株具良好遗传稳定性的突变株I-0281，其GLA产量为889.80 mg/L，比原始菌株提高了60.27%。 关键词: γ-亚麻酸；菌种选育；筛选模型；抗丙二酸法；红四氮唑法 Screening of Mortierella isabellina mutants pertain to produce high-yield γ-linolenic acid by metabolic control breeding technique Hao Ran, Hu Shencai* (School of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, 430023) In order to obtain Mortierella isabellina mutants with the capability of high-yielding γ-linolenic acid, the mutant library was constructed by UV and N+ ions implantation technique, respectively. Then, according to some key enzymes involved in the metabolic synthetic pathway of γ-linolenic acid, the two screening models, anti-molonic acid and triphenyltetrazolium chloride (TTC), were designed and established, which are aimed to increase biomass or contents of unsaturated fatty acid. The results showed that the high-yield strains can be screened by resistant plate containing 2 g/L molonic acid; The TTC screening model was ascertained as collecting samples after fermenting for 54 h, stained 0.9 % by TTC for 4 h. Through 2 rounds screening via the two kinds of screening model, the I-0281 mutant strain was obtained, which has good genetic stability, and also the production of γ-linolenic acid was up to 889.80 mg/L, 60.27% higher than the wild strain. Key words: γ-linolenic acid; strain breeding; screening model; anti-molonic acid method; TTC method γ-亚麻酸（GLA）及其系列代谢物在人体的免疫系统、心血管系统、生殖系统、内分泌系统和神经系统中都具有重要而广泛的生理作用[1,2]。GLA在食品、保健品、医药品、化妆品和饲料等领域的应用越来越广泛，GLA的需求量越来越大。 1959年，Qsbond首次用化学合成法制备γ-亚麻酸，但从其原料、所用试剂以及操作条件来看，这种方法有一定难度而推广。目前，商业化生产GLA的资源主要为植物自然界中富含GLA的资源较少，受植物来源、地域、生长季节气候、加工生产成本等多因素影响，通过植物来生产GLA有一定的局限性[,4]。利用微生物发酵生产GLA未来