裂壶藻脂肪酸合酶途径合成多不饱和脂肪酸的研究 <sup>*</sup>

doi:10.13523/j.cb.20180904

中国生物工程杂志

2018, Vol. 38

Issue (9): 27-34 DOI: 10.13523/j.cb.20180904

研究报告

裂壶藻脂肪酸合酶途径合成多不饱和脂肪酸的研究 ^*

马淑霞^1,³,张玲²,闫晋飞^1,^**(

),游松³

¹ 国家海洋局天津海水淡化与综合利用研究所天津 300192
² 首都儿科研究所附属儿童医院北京 100020
³ 沈阳药科大学沈阳 110016

Study on the Synthesis of Polyunsaturated Fatty Acids by FattyAcid Synthase Pathway of Schizochytrium sp.

Shu-xia MA^1,³,Ling ZHANG²,Jin-fei YAN^1,^**(

),Song YOU³

¹ The Institute of Seawater Desalination and Comprehensive Utilization, Tianjin 300192, China
² Children’s Hospital, Capital Institute of Pediatrics,Beijing 100020,China;
³ Shenyang Pharmaceutical University, Shenyang 110016, China

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摘要：

裂壶藻作为一种生产DHA的重要菌种,其脂肪酸合成途径除了已经被广泛了解的聚酮合酶(PKS)途径外,还残存着作为真核微生物中常见的脂肪酸合酶(FAS)途径中的功能酶和中间产物。经过前期的研究,运用基因工程手段,将载有与裂壶藻近缘藻种中已知的Δ12-desaturase表达模块和相关筛选标记构建入载体,通过电击转化导入Schizochytrium sp.ATCC20888中, 以期望修复裂壶藻中FAS途径的合成能力。通过分子水平检测确定目的功能基因已稳定转化后,再由气相色谱分析转化株油脂中相关脂肪酸含量分布情况。经过长期筛选验证,最终证明获得了两株阳性转化株,其生物量初步测定分别较野生株提高11.14%和4.12%,其油脂中DHA含量分别较野生株提高了19.50%及14.65%。

关键词： 基因工程; 脂肪酸合酶途径; 裂壶藻; DHA

Abstract:

As an excellent species for the production of DHA, Schizochytrium sp. has two distinct pathways for producing DHA, fatty acid synthase pathway and polyketide synthase pathway, of which fatty acid synthase pathway played limited role in the DHA synthesis process due to its low activity of one or more of the enzymes, resulted in its only responsible for some short chain fatty acid synthesis. In order to enhance the ability of fatty acid synthase pathway for producing DHA in Schizochytrium sp. to improve the DHA content of its oil, the exogenous △12-desaturase expression plasmid with screening markers for Schizochytrium sp.ATCC20888 from the brown algae was constructed, then Schizochytrium sp. were transformed through electroporation. After long-term screening, two positive transformants were obtained by DNA and RNA identification. Their initial biomass measurements were 11.14% and 4.12% higher than the wild strains while the DHA content in oils and fats were 19.50% and 14.65% higher than the wild strains.

Key words: Genetic engineering Fatty acid synthase pathway Schizochytrium sp. DHA

收稿日期: 2018-04-09 出版日期: 2018-10-12

基金资助: * 2015年中央级公益性科研院所基本科研业务费专项资金(K-JBYWF-2015-T19)

通讯作者: 闫晋飞 E-mail: jinfei_yan@qq.com

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引用本文:

马淑霞,张玲,闫晋飞,游松. 裂壶藻脂肪酸合酶途径合成多不饱和脂肪酸的研究 ^*[J]. 中国生物工程杂志, 2018, 38(9): 27-34.

Shu-xia MA,Ling ZHANG,Jin-fei YAN,Song YOU. Study on the Synthesis of Polyunsaturated Fatty Acids by FattyAcid Synthase Pathway of Schizochytrium sp.. China Biotechnology, 2018, 38(9): 27-34.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180904 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I9/27

图1 表达载体pBlu2SKP-Pht△12

图2 质粒pBlu2SKP-Pht△12-1的构建

图3 质粒pBlu2SKP-Pht△12-2的构建

图4 质粒pBlu2SKP-Pht△12的构建

图5 抗性筛选实验结果

图6 DNA水平筛选转化株

图7 转化株的RT-PCR结果

表1 野生株及转化株的生物量比较

图8 转化株与野生株的气相色谱图

表2 转化株与野生株部分脂肪酸在其油脂中的百分含量

图9 合成多不饱和脂肪酸的FAS途径[5]

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