论文总字数：25968字

摘 要

多不饱和脂肪酸具有预防心血管疾病、提高免疫力的功能，利用产油微生物获得多不饱和脂肪酸是微生物研究的热点课题。裂殖壶菌是应用到工业化发酵生产二十二碳六烯酸油脂的主要生产菌之一，具有生长周期短、油脂产量大以及培养条件温和等优点。裂殖壶菌主要通过迭代型脂肪酸聚酮合酶途径合成多不饱和脂肪酸，其中脱水酶DH功能域在这一途径中控制双键的合成。

本文利用课题组前期裂殖壶菌基因组，获得裂殖壶菌DH功能域的氨基酸序列信息，进一步利用Prot Param、SignalP 5.0 Server、Clustal X、MOGA-X等在线软件和数据库对裂殖壶菌DH功能域进行生物信息学分析。然后以大肠杆菌E.coli BL21为表达宿主，对来源于裂殖壶菌和希瓦氏菌的两个脱水酶功能域DH_1,2和DH₃进行体外表达，并通过检测脂肪酸的变化来研究DH功能域的催化效果。

生物信息学分析得知，裂殖壶菌两个DH功能域理论pI值在6到7之间，具有较弱的疏水性，不稳定指数小于40属于稳定蛋白，在大肠杆菌中半衰期大于10小时；没有信号肽，不属于分泌蛋白；翻译后修饰率在0.8%到0.9%之间，其中丝氨酸的修饰位点占25%以上；DH_1，2和FabA蛋白结构最为相近，DH₃和希瓦氏菌的DH功能域结构最为相近，二者说明两个DH功能域都具有脱水的功能，且DH_1，2很可能具有异构的功能；DH_1，2和DH₃蛋白与Schizochytrium sp. ATCC 20888的两个DH功能域有最近的亲缘关系。异源表达结果显示，裂殖壶菌中两个DH蛋白在合成脂肪酸时具有不同的偏好，DH_1,2促进多不饱和脂肪酸的合成，其中C17:1和C18:1提高分别提高了88.7%和21.0%，表达DH_1，2的大肠杆菌出现了大肠杆菌原本不产生的C18:2；而DH₃偏好于积累短链脂肪酸，18碳以下脂肪酸提高了8.4%。

关键词：裂殖壶菌 脂肪酸聚酮合酶 脱水酶 生物信息学 不饱和脂肪酸

Identification Dehydratase Domains in Polyunsaturated Fatty Acid Synthase

ABSTRACT

Polyunsaturated fatty acids have the functions of preventing cardiovascular diseases and improving immunity. The use of oleaginous microorganisms to obtain polyunsaturated fatty acids is a hot topic in microbial research. Schizochytrium is one of the most widely used oleaginous microorganisms used in industrial fermentation to produce polyunsaturated fatty acids. It has the advantages of short growth cycle, high lipid yield and mild culture conditions. Schizochytrium synthesizes polyunsaturated fatty acids mainly through an iterative polyketide synthase pathway(PKS), where the dehydratase(DH) domain controls the synthesis of double bonds in this pathway.

This thesis used the amino acid sequence information of the Schizochytrium DH functional domain obtained from the genome information of Schizochytrium. Bioinformatics analysis of the Schizochytrium DH domain was studied through the Prot Param, SignalP 5.0 Server, Clustal X, MOGA-X and other online software and databases. Then the prokaryotic organism E.coli was used as the expression host to express the two dehydratase functional domains DH_1,2 and DH₃ derived from Schizochytrium and Shewanella in vitro, and the catalytic effect of the DH functional domain was studied by detecting the profile changes of the fatty acids.

Online software analysis showed that the theoretical pI value of the two DH functional domains of Schizochytrium was between 6 and 7, and they had weake hydrophobicity. The instability index is less than 40 which means they are stable proteins. The half-life in E. coli is longer than 10 hours. There are no signal peptide, so they are not secreted protein. The post-translational modification rate is between 0.8% and 0.9%, of which serine modification sites account for more than 25%. DH_{1, 2} and FabA protein have the most similar structure, DH₃ and Shewanella DH function domain structure is the most similar. Results indicated that both DH domains have dehydration functions, and DH_{1, 2} are likely to have heterogeneous functions. DH_{1, 2} and DH₃ proteins are similar to the two DH domains of Schizochytrium sp. ATCC 20888. The results of heterologous expression showed that the two DH proteins in Schizochytrium had different preferences in the synthesis of fatty acids. DH_1,2 could promote the synthesis of polyunsaturated fatty acids. In particular, the percentage of C17:1 and C18:1increased by 88.7% and 21.0%. Moreover, E. coli expressed DH_{1, 2} synthesized C18:2which could not be originally produced by E. coli. DH3 preferred to accumulate short-chain fatty acids, and the percentage of fatty acids below C18 increased by 8.4%.

Keywords: Schizochytrium sp; Dehydratase; Enoyl reductase; Bioinformatics

目 录

摘 要 I

ABSTRACT II

前言 1

第一章 文献综述 2

1.1 多不饱和脂肪酸简介 2

1.2 多不饱和脂肪酸合成酶途径 2

1.2.1 整体酶催化过程 2

1.2.2 DH的催化 3

1.3 DH的研究方法 4

1.3.1 生物信息学方法 4

1.3.2 体外表达 4

1.4 本研究的意义 5

第二章 脱水酶DH功能域的初步鉴定及氨基酸序列分析 6

2.1 引言 6

2.2 材料和方法 6

2.2.1 实验所用生物信息库和软件 6

2.2.2 实验菌株和蛋白序列 7

2.2.3 蛋白序列分析 7

2.3 结果与讨论 8

2.3.1 DH_1，2和DH₃的基本性质分析 8

2.3.2 DH功能域的序列比对，二级结构预测以及进化树分析 10

2.3.3 三级结构预测 12

2.4 本章小结 13

第三章 脱水酶功能域体外表达与功能验证 14

3.1 引言 14

3.2 实验器材和方法 14

3.2.1 实验试剂 14

3.2.2 实验仪器 16

3.2.3 实验菌株、质粒、引物 16

3.2.4 DH表达载体的构建 16

3.2.5 培养基以及发酵参数 17

3.2.6 E. coli诱导表达 17

3.2.7 添加脂肪酸前体 18

3.2.8 检测方法 18

3.3 结果与讨论 18

3.3.1 表达不同来源DH功能域对大肠杆菌脂肪酸的影响 18

3.3.2 外源添加棕榈酸对重组大肠杆菌生物量的影响 19

3.3.3 外源添加棕榈酸对重组大肠杆菌脂肪酸比例的影响 20

3.4 本章小结 22

第四章 结论与展望 23

4.1 结论 23

4.2 展望 23

参考文献 25

致谢 28

前言

多不饱和脂肪酸（PUFA）是指含有不止一个双键且碳链长度大于18个碳原子的直链脂肪酸。多不饱和脂肪酸分为ω-3和ω-6两种，其中距羧基最远端的双键在倒数第3个碳原子上称之为ω-3，同理，距羧基最远的双键在倒数第六个碳原子上，则称为ω-6^[9]。近年来越来越多的研究表明，ω-3多不饱和脂肪酸如二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）具有预防心血管疾病、提高身体免疫力的功能^[1]。市场上的多不饱和脂肪酸通常来源于海洋鱼油，但随着海洋环境的恶化鱼油的质量大幅下降，多不饱和脂肪酸的产量和质量受到严重影响^[22]。使用产油微生物（Oleaginous microorganisms）高效获取高质量的多不饱和脂肪酸成为微生物研究的热点。

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