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产丙烷基因工程大肠杆菌的构建及脂肪醛去甲酰加氧酶的蛋白质工程改造
张磊
导师吕雪峰
2016-05
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物化学与分子生物化学
关键词大肠杆菌 丙烷 脂肪醛去甲酰加氧酶 缬氨酸途径 理性设计 定向进化 烃传感器
摘要脂肪烃类生物燃料的理化性质接近传统的化石燃料,具有能量密度高、吸湿性低的特征,因而与现有的能源基础设施相兼容。利用微生物生产脂肪烃类生物燃料受到了学术和工业界的广泛关注。其中,丙烷作为一种短链脂肪烃,更以其燃烧清洁、低温室气体排放量等优点,在交通运输、房屋加热和食物烹调等方面得到了广泛应用。虽然有文章报道了基于大肠杆菌的脂肪酸系统或梭菌的丁醇代谢构建的丙烷合成途径,但上述途径提供的正丁醛底物有限,制约了丙烷产量的继续提升。针对上述问题,本文通过改造大肠杆菌缬氨酸途径,过表达蓝细菌的脂肪醛去甲酰加氧酶(aldehyde-deformylating oxygenase, ADO)基因,构建了一条新的丙烷合成途径,解决了底物供给不足这一制约性因素,并结合蛋白质理性设计,进一步提高了丙烷产量。此外,本文利用自然界微生物中的烃诱导信号途径,构建了一种可感应大肠杆菌胞内脂肪烃含量的生物传感器,并利用该传感器对ADO开展了定向进化研究。主要研究内容及结果如下: (1)采用两步无痕法敲除了大肠杆菌BW25113中的yqhD、adhE、adhP、eutG、yiaY、yjgB、fucO、yahK和DkgA九个醛还原酶基因,并同时敲除了frdABCD、ldhA、pflB和转录因子fnr四个基因,构建了一株醛还原酶活性缺失的大肠杆菌BW25113(DE3) Δ13,生成并累积了大量的异丁醛(1.1 g/L)。通过在该菌株中过表达基于缬氨酸途径构建的丙烷合成途径,实现了丙烷的生物合成(91 μg/L)。 (2)为提高ADO(Prochlorococcus marinus MIT 9313_PMT1231)对异丁醛的催化效率,利用蛋白质理性设计,将ADO活性中心的部分氨基酸残基突变为侧链位阻效应更小的残基,拓宽了底物进入通道,消除了异丁醛进入催化反应中心时的位阻效应。理性设计得到的ADO突变体(I127G、I127G/A48G)在体外加醛实验及酶活实验中的效率都有显著改善,并将丙烷从头合成的产量提高了三倍(267 μg/L)。 (3)针对脂肪烃高通量检测手段缺乏的问题,通过整合来自不动杆菌(Acinetobacter baylyi)和假单胞菌(Pseudomonas oleovorans)烃诱导信号途径中的基因元件,再以绿色荧光蛋白(green fluorescent protein, GFP)作为报告基因,在大肠杆菌中构建了一种能够感应脂肪烃并产生荧光响应的生物传感器。该传感器的荧光强度能够定性反映胞内脂肪烃的含量,为脂肪烃生物燃料研究领域内相关基因的定向进化和高通量筛选创造了条件。 (4)利用构建的脂肪烃传感器,结合易错PCR技术和流式细胞分选技术(fluorescence activated cell sorting, FACS),对ADO(Synechococcus elongates PCC7942_orf1593)实施了定向进化研究,经过两轮筛选,最终获得了脂肪烃产量提高了三倍的突变体。这一工作首次尝试将定向进化技术应用于ADO的改造,取得了较好的进展,为通过基因工程进一步提高微生物脂肪烃产量提供了有益的启示。
其他摘要Given the concerns about fossil fuel depletion and climatic change, exploiting renewable and environment-friendly energy has become attractive. Short-chain alkane propane has widespread applications in vehicles, cooking, and ambient heating owning to its excellent features such as higher energy density, cleaner combustion, less greenhouse gas emission, and fine compatibility. Hence, producing renewable propane from microbial platforms attracts increasing concerns from academia and industrial community. Previous propane biosynthetic pathways based on fatty acid biosynthesis (FASII) of E. coli or CoA-dependent butanol pathway from Clostridium sp. were restricted by the low availability of precursor. To address this obstacle, we constructed a new biosynthetic pathway for propane production by assembling the engineered valine pathway of E. coli and cyanobacterial aldehyde-deformylating oxygenase (ADO). The new propane pathway can produce abundant isobutyraldehyde and overcomes the low availability of precursor in propane production. After improving the activity of ADO (Prochlorococcus marinus MIT 9313_PMT1231) via rational design aiming at substrate channel, we increased the propane productivity by three times. We also developed a fluorescent biosensor for rapid and in situ monitoring of alkane synthesis in E. coli and carried out several rounds of directed evolution on ADO (Synechococcus elongates PCC7942_orf1593). The main results and conclusions are summarized as follows: (1) A two-step markerless recombination method was applied to delete 13 genes of BW25113, namely yqhD, adhE, adhP, eutG, yiaY, yjgB, fucO, yahK, DkgA, frdABCD, ldhA, pflB, and fnr, resulting an aldehyde reductases (ALR)-deprived strain BW25113(DE3) Δ13. The strain produced sufficient isobutyraldehyde precursor (1.1 g/L) and finally achieved de novo synthesis of propane (91 μg/L) by assembling the engineered valine pathway and cyanobacterial ADO. (2) To overcome the poor activity of ADO on isobutyraldehyde, rational design was applied to engineer the ADO active center to accommodate branched-chain isobutyraldehyde. After extensive screening of ADO mutants via whole-cell assay method, we identified two ADO mutants (I127G, I127G/A48G) which exhibited higher catalytic activity for isobutyraldehyde and improved propane productivity by three times (267 μg/L). (3) Given the lack of fast and high-throughput intracellular alkane detection methods in biofuels research field, we reassembled the two natural alkane-responsive plugins AlkR-PalkM from Acinetobacter baylyi and AlkS-PalkB from Pseudomonas oleovorans to develop a synthetic chimera alkane response biosensor in E. coli with green fluorescent protein (GFP) as reporter. The alkane biosensor provides a visualized and high-throughput way of monitoring the alkane synthesis in E. coli. (4) Combining the alkane biosensor, error-prone PCR, and fluorescence activated cell sorting (FACS) together, several rounds of directed evolution were carried out on ADO, resulting in about three-fold increase on fatty alkane productivity.
作者部门微生物代谢工程
学科领域微生物代谢工程
公开日期2017
学位类型博士 ; 学位论文
语种中文
文献类型学位论文
条目标识符http://ir.qibebt.ac.cn/handle/337004/9767
专题微生物代谢工程研究组
作者单位中国科学院青岛生物能源与过程研究所
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张磊. 产丙烷基因工程大肠杆菌的构建及脂肪醛去甲酰加氧酶的蛋白质工程改造[D]. 北京. 中国科学院大学,2016.
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