Screening a New Type of Schizochxtrium Strain Producing DHA Using Starch by Protoplast Fusion Technology

doi:10.13560/j.cnki.biotech.bull.1985.2015.02.012

Abstract

Abstract: Schizochxtrium B4D1 and Aspergillus niger CGMCC 3.316 as original strains, a new strain of Schizochxtrium was selected using starch for producing DHA. The protoplasts from two parents strains were obtained respectively with Lysing enzyme. The study was investigated on the effect of culture time, culture method, enzymolysis time on the protoplasts preparation. Finally, the protoplasts fusion was done under the PEG mediated condition. The optimum condition of protoplast fusion was as follows：PEG6000 concentration of 40%, fusion temperature of 30℃, fusion time of 10 min. Furthermore, the fusion rate reached 1.9% at the optimum condition. Through comparing its colony characteristics, color, morphology and purified spore culture, a new strain has been selected using starch as carbon source. RAPD verification showed that the fusant having integrated genome of B4D1 and CGMCC 3.316 expressed more genetic information from B4D1.

Key words: Schizochytrium, DHA, protoplast, fusion, starch

Wang Di, Lu Fuping, Wang Haijun, Li Demao, Chen Shulin, Zhang Ke. Screening a New Type of Schizochxtrium Strain Producing DHA Using Starch by Protoplast Fusion Technology[J]. Biotechnology Bulletin, 2015, 31(2): 84-90.

References

[1] 陈诚. 裂殖壶菌（Schizochytrium limacinum SR21）发酵制备二十二碳六烯酸（DHA）过程的初步研究[D]. 杭州: 浙江大学, 2007.
[2]Armenta RE, Valentine MC. Single-cell oils as a source of omega-3 fatty acids：an overview of recent advances[J]. Journal of the American Oil Chemists’ Society, 2013, 90（2）：167-182.
[3] 王申强. 裂殖壶菌产DHA的发酵工艺研究及高产菌株选育 [D]. 无锡: 江南大学, 2013.
[4]Wu ST, Yu ST, Lin LP. Effect of culture conditions on docosahexae-noic acid production by Schizochytrium sp. S31[J]. Process Bio-chemistry, 2005, 40（9）：3103-3108.
[5]常桂芳. 氧对裂壶藻利用甘油产DHA影响机制及其高密度发酵控制策略的研究[D]. 无锡: 江南大学, 2013.
[6]陈宁, 王艳萍, 王一芃, 张克旭. 利用淀粉直接发酵生产肌苷菌株的构建[J]. 微生物学报, 1997（3）：184-189.
[7]刘虹, 程秀芳, 张志焱, 谷巍. 利用原生质体融合技术选育高产虾青素酵母菌株[J]. 中国饲料, 2013（11）：5-9.
[8]Manger-Jacob F, Muller T, Janssen M, et al. Isolation and sequencing of a new glucoamylase gene from an Aspergillus niger aggregate strain（DSM 823） molecularly classified as Aspergillus tubingensis [J]. Antonie Van Leeuwenhoek, 2005, 88（3-4）：267-275.
[9]Yokochi T, Honda D, Higashihara T, Nakahara T. Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21[J]. Applied Microbiology and Biotechnology, 1998, 49（1）：72-76.
[10]Cheng R, Ma R, Li K, et al. Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium[J]. Microbiol Res, 2012, 167（3）：179-186.
[11] 何忠宝. 原生质体融合构建葡萄酒降酸酵母的研究[D]. 杨凌：西北农林科技大学, 2004.
[12] 姚婷婷, 王正祥. 黑曲霉原生质体的制备、再生及转化条件 [J]. 食品与生物技术学报, 2006（4）：116-120.
[13]骆健美, 李建姝, 王艳婷, 王敏. 褐黄孢链霉菌双亲灭活原生质体融合的研究[J]. 现代化工, 2008（S2）：349-351, 353.
[14]王春丽, 武改红, 陈畅, 陈树林. 黑曲霉原生质体诱变选育β-葡萄糖苷酶高产菌株[J]. 生物工程学报, 2009, 12：1921-1926.
[15]王岁楼, 王海翔, 杨志萍, 等. 酵母原生质体制备、融合及高产β-胡萝卜素融合子的筛选[J]. 食品科学, 2013（9）：99-103.
[16]何小妮, 蒋波, 王玉海, 等. 非对称灭活双亲原生质体融合法选育产果糖基转移酶的米曲霉新菌株的研究[J]. 现代食品科技, 2013（5）：993-997.
[17] 林义, 钟添华, 骆祝华, 等. 尼罗红染色法筛选产油酵母及定量检测胞内油脂含量的研究[J]. 微生物学通报, 2012（1）：125-137.
[18] 吕雪梅, 杨关福, 张细权. RAPD分析中遗传距离计算方法的比较[J]. 华南农业大学学报, 1997（S1）：93-100.
[19] 王福荣. 生物工程分析与检验[M]. 北京：中国轻工业出版社, 2010：141-142.
[20] 徐新丽. 原生质体融合技术选育纤维素发酵产油菌株[J]. 武夷学院学报, 2011（2）：27-31.
[21] 张淑君. 原生质体融合构建纤维素发酵产油脂菌株的研究[D]. 泰安: 山东农业大学, 2012.