【作者】 张燕；

【导师】 黄凤洪；

【作者基本信息】 华中农业大学 ， 食品科学， 2011， 硕士

【摘要】 脂肪酸是人类重要的营养来源,其中的多不饱和脂肪酸(Polyunsaturated Fatty Acids, PUFAs)是人体重要的生理活性物质。多不饱和脂肪酸中的亚油酸(Linoleic acid, LA)和α-亚麻酸(α-linoleic acid, ALA)是人体的必需脂肪酸,为人体合成下游长链PUFAs所必需。利用微生物发酵法生产PUFAs,不仅具有成本低廉、生产周期短以及受环境影响小等优点,而且更是生产长链的稀缺PUFAs的重要资源。本研究以橘林油脂酵母(Lipomyces kononenkoae)为实验材料,克隆与亚油酸和α-亚麻酸合成相关的Δ9-和△15-脂肪酸去饱和酶基因(lkfad9和lkfad15),并验证功能。主要得到以下结果：1.通过对培养基成分和培养条件的优化试验,找出橘林油脂酵母发酵生产PUFAs的主要影响因素,确定其高产PUFAs的最优化培养基及培养条件。2.利用脂肪酸去饱和酶(Fatty Acid Desaturase, FAD)的保守区域设计简并引物,克隆橘林油脂酵母Δ9-和△15-FAD的核心编码序列,并采用基因组步移及逆转录PCR等技术,克隆了两个基因完整结构基因序列。生物信息学分析,两个基因均属于脂肪酸脱氢酶超家族,与其他酵母中相应的FAD具有较高同源性。3.将基因lkfadl5分别转入毕赤酵母(Pichia pastoris) GS115和酿酒酵母(Saccharomyces cerevisiae) INVScI,发酵重组酵母,诱导外源基因表达后收集菌体,气相色谱分析重组菌株的脂肪酸成分。结果显示,基因lkfadl5能通过催化LA变为ALA而提高毕赤酵母GS115中ALA的含量(从4.68%增加到21.93%),并且能仅利用油酸作为底物,在酿酒酵母INVScI中产出LA和ALA,这个结果显示lkfad15同时具有△12-和△15-FAD的活性。本研究证实了橘林油脂酵母△15-脂肪酸去饱和酶基因lkfadl5同时具有△12-和△15-FAD脱氢作用的双功能,为利用双功能的脂肪酸去饱和酶LKFAD15在转基因生物中富集ω-3系列多不饱和脂肪酸提供了重要的应用基础。更多还原

【Abstract】 Fatty acids are important nutrient of human. Among them, polyunsaturated fatty acids (PUFAs) are particularly important for human physical activities. Linoleic acid (LA) andα- linoleic acid (ALA) are substrates of the PUFAs, which are essential fatty acids (EFA) for human. Microbial could proliferate rapidly by lot cost materials without much environmental impaction, produce PUFAs by oleaginous microbial fermentation could be lots of advantages. So that it could be good prospects to develop genetically modified (GM) oleaginous microbial strains for very long chain PUFAs production.In this study, the coding sequences ofΔ9- andΔ15-fatty acid desaturase gene (lkfad9 and lkfad15) were cloned from Lipomyces kononenkoae and heterologous expressed in yeast expression systems.The main results of this study are as follows:1. The fermentation condition of Lipomyces kononenkoae was optimized by medium composition and culture condition optimization to product high yield polyunsaturated fatty acids.2. The core DNA sequence of lkfad9 and lkfad15 were amplified using degenerate primers according to the conservative amino acid sequences. And then, the total coding sequences of these two genes were cloned using Genome-Walking and reverse transcript PCR techniques. Bioinformatic analysis results showed that these two genes are members of the fatty acid desaturase superfamily which reveal high homology to the corresponding desaturase genes of the other yeasts.3. The coding sequence of lkfad15 was transformed into Pichia pastoris GS115 and Saccharomyces cerevisiae INVScI respectively. The recombinant yeasts were cultured and induced to express lkfad15. Fatty acids components of the recombinant yeasts were analyzed by gas chromatography. The results shown that Ikfad15 could increase the ALA content (4.68% to 21.93%) in GS115 by transform LA to ALA, and it even could produce both LA and ALA in INVScI use only oleic acid as substrate. It reveals that LKFAD15 have the functions of bothΔ12- andΔ15 -desaturases.This work proved the LKFAD15 has the ability of bothΔ12- andΔ15 -desaturase, it is the basis work for productω-3 PUFAs in GM organisms using bi-functional desaturase LKFAD15.更多还原