其他题名A study of construction of a metabolic pathway for lactic acid production by fixed CO2 in E. coli and screening of Rubisco with high carboxylation activity
学位论文
学位类型硕士
导师张晓黎
2019-11-25
学位授予单位中国科学院研究生院
学位授予地点北京
学位名称工程硕士
学位专业生物工程
关键词Rubisco大肠杆菌Co2固定乳酸发酵
摘要卡尔文循环(Calvin-Benson-Bassham, CBB）是从无机碳生产有机生物质的主要途径，核酮糖-1,5-二磷酸羧化/加氧酶（Rubisco）是CBB循环固定CO2第一步反应中的关键酶和限速酶，Rubisco的羧化活性直接影响自养生物通过CBB循环进行CO2固定的能力，在作物生产和全球碳循环中起着关键作用。
Rubisco对CO2的催化效率和选择性极低，而且其活性中心高度保守，利用现有的理性设计或定向进化手段对其进行改造收效甚微。因此，设计高效的高活性Rubisco筛选体系，探索未知的Rubisco，是目前的研究热点。此外，利用Rubisco的固碳活性合成生物基化学产品已成为微生物固碳研究的重要方向。目前已利用Rubisco在蓝藻、酿酒酵母中实现了产乙醇、异丁醛、L-丁醇和甘油等化学产品，但利用Rubisco在大肠杆菌Escherichia coli中固定CO2产乳酸的研究尚未见报道。因此，本研究尝试在大肠杆菌体内建立一条Rubisco固定CO2产乳酸的代谢途径。主要结果如下：
1）在大肠杆菌中构建了一条甘油-木糖-Rubisco-PRK产乳酸代谢途径，其乳酸的理论得率为107.8%，13C标记的乳酸理论得率为25%。通过发酵条件优化和发酵菌株比较，最终确定发酵条件为：厌氧条件下，发酵培养基为M9，缓冲液为HEPES/NaHCO3，诱导温度为22℃，发酵菌株为E. coli BW25113ΔfrdΔpflB::ldhA。在此发酵条件下，该菌株的乳酸产量为2.51 g/L，乳酸得率为92.04%，13C标记的净乳酸得率为19.66%。
2）通过细胞生长状态初筛和乳酸产量及13C标记的乳酸得率复筛，构建了适用于筛选高效Rubisco羧化活性的筛选体系。与已有筛选体系相比，该体系具有高通量、准确度高、更简便的优势。
3）利用本课题构建的高效Rubisco羧化活性筛选体系，从23个带有不同来源Rubisco的E. coli中筛选到BW-RBC9菌株，其乳酸产量为3.1 g/L；乳酸得率为93.46%，接近理论得率107.8%；13C标记的净乳酸得率21.83%，接近理论得率25%。
综上，本课题构建的甘油-木糖-Rubisco-PRK产乳酸代谢途径实现了Rubisco在大肠杆菌中固定CO2产乳酸的功能；建立了一套适用于高通量筛选Rubisco的筛选体系，并成功筛选到高羧化活性的Rubisco，为今后利用Rubisco在大肠杆菌中固定CO2产乳酸并实现产业化进行了有益的探索。
其他摘要Calvin-Benson-Bassham (CBB) is the main way to produce organic biomass from inorganic carbon. Ribulose-1,5-diphosphate carboxylation/oxygenase (Rubisco) is a key and rate-limiting enzyme in the first step of CBB to fix CO2. The carboxylation activity of Rubisco directly affects the ability of fixing CO2 through the CBB, and thus Rubisco plays a key role in crop production and the global carbon cycle.
Rubisco's catalytic efficiency and selectivity of CO2 are extremely low. Moreover, the active center of Rubisco is highly conservative, and it has little effect on transforming it by rational design or directed evolution. Therefore, designing an efficient screening system suitable for higher carboxylation activity and exploring and discovering unknown Rubisco2 are the current research hotspots. In addition, the use of Rubisco's carbon-fixing carboxylation activity to synthesize bio-based chemical products has become an important direction for microbial carbon fixation research. Although some chemical products, such as ethanol, isobutyraldehyde, L-butanol, and glycerol, have been produced in cyanobacteria, Saccharomyces cerevisiae and other microorganisms using Rubisco to fix CO2, studies on using Rubisco to fix CO2 for producing lactic acid in Escherichia coli have not been reported. Therefore, this study attempts to establish a Rubisco-fixed CO2 lactic acid production pathway in E. coli. The main results are following:
(1) The Glycerol-Xylose-Rubisco-PRK lactic acid metabolism pathway was constructed in E. coli. The theoretical yield of lactic acid was 107.8%, and the theoretical yield of 13C-labeled lactic acid was 25%. Through optimization of fermentation conditions and comparison of fermentation strains, the optimal fermentation conditions were determined as: under anaerobic conditions, M9 was the fermentation medium, the buffer was HEPES / NaHCO3, and the induction temperature was 22° C; the optimal fermentation strain was E. coli BW25113ΔfrdΔpflB::ldhA. The lactic acid yield of this strain was 2.51 g/L, the yield of lactic acid was 92.04%, and the net yield of lactic acid labeled with 13C was 19.66%.
(2) Constructed a selection system suitable for screening efficient Rubisco carboxylation activity by preliminary screening of cell growth status and rescreening of the production of lactic acid and 13C-labeled lactic acid. Compared with the original screening system, this system has the advantages of high throughput, high accuracy, and simplicity.
?(3) BW-RBC9 strain was screened from 23 E. coli strains with different sources of Rubisco by using our constructed selection system for Rubisco with high carboxylation activity. The production of lactic acid was 3.1 g/L and the yield of lactic acid was 93.46%, which was close to the theoretical yield of 107.8%. The net yield of lactic acid labeled with 13C was 21.83%, which was close to the theoretical yield of 25%.
In summary, our project realized Rubisco's fixation of CO2 to produce lactic acid in E. coli by the constructed glycerol-xylose-Rubisco-PRK metabolism pathway. Also, we established a screening system suitable for high-throughput screening of Rubisco and found a Rubisco with high-carboxylase activity. The study is a useful exploration of using Rubisco to fix CO2 for producing lactic acid in E. coli and realizing industrial production in the future.
目录目? 录??????????????????????????????????????????
第1章 引 言... 1
1.1? 微生物固碳... 1
1.2? 核酮糖-1,5-二磷酸羧化/加氧酶（Rubisco）... 1
1.3? Rubisco筛选方法的研究现状... 5
1.4? 大肠杆菌中Rubisco固碳的代谢工程改造和应用... 7
1.5? 课题研究意义和目标... 9
第2章 材料与方法... 11
2.1? 实验材料... 11
2.1.1? 质粒与菌株... 11
2.1.2? 引物... 12
2.1.3? 试剂... 13
2.1.4? 培养基... 14
2.1.5? 主要仪器... 15
2.2? 分子生物学实验方法... 15
2.2.1? PCR扩增目标基因... 15
2.2.2? 限制性酶切与连接... 16
2.2.3? CRISPR基因敲除... 17
2.2.4? 大肠杆菌感受态细胞的制备与转化... 18
2.2.5? 菌落PCR鉴定... 20
2.2.6? DNA合成与测序... 20
2.3? 菌株培养方法... 21
2.3.1? 固体培养基筛选方法... 21
2.3.2? 改造大肠杆菌产乳酸发酵方法... 21
2.4? 检测方法... 22
2.4.1? 木糖、乳酸、甘油的检测... 22
2.4.2? Rubisco活性检测... 23
第3章? 大肠杆菌中利用Rubisco固定CO2产乳酸途径的构建... 25
3.1? 引言... 25
3.2? 甘油-Rubisco-PRK代谢途径的建立... 26
3.2.1 E. coli HX-030中甘油-Rubisco-PRK代谢途径的评价... 27
3.2.2 E. coli EB204中甘油-Rubisco-PRK代谢途径的评价... 29
3.3? 甘油-木糖-Rubisco-PRK代谢途径的建立... 33
3.3.1 E. coli EB204中甘油-木糖-Rubisco-PRK代谢途径产乳酸能力和固碳能力的评价??? 34
3.4? Rubisco固碳产乳酸发酵方式的优化... 37
3.4.1? 发酵培养基的选择和优化... 37
3.4.2? 发酵温度的选择... 41
3.4.3? 厌氧和微好氧发酵的选择... 43
3.5? Rubisco固碳产乳酸大肠杆菌的选择... 45
3.6? 本章小结... 50
第4章 高固碳活性Rubisco筛选办法的构建... 53
4.1? 引言... 53
4.2? 大肠杆菌活性定向选择系统对不同来源Rubisco固碳活性的评价... 53
4.2.1? 大肠杆菌活性定向选择系统的设计原理... 53
4.2.2? 大肠杆菌活性定向选择系统中不同羧化活性Rubisco的比较... 54
4.3? 新构建的高固碳活性Rubisco筛选办法的评估... 56
4.3.1? 高固碳活性Rubisco筛选办法的设计原理... 56
4.3.2? 高固碳活性筛选体系中不同羧化活性Rubisco的比较... 57
4.4? 本章小结... 59
第5章 高羧化活性Rubisco固碳元件的筛选... 61
5.1? 引言... 61
5.2? 筛选获得高固碳活性的Rubisco元件... 61
5.3? 产乳酸代谢途径的改造... 64
5.3.1? 代谢途径优化前后发酵结果比较... 67
5.4? 本章小结... 69
第6章? 结论与展望... 71
6.1? 结论... 71
6.2? 展望... 71
参考文献... 73
附? 录... 77
致? 谢... 79
作者简历及攻读学位期间发表的学术论文与研究成果... 81
页数96
语种中文
文献类型学位论文
条目标识符http://ir.yic.ac.cnhttp://ir.yic.ac.cn/handle/133337/24121
专题中科院烟台海岸带研究所知识产出_学位论文
推荐引用方式
GB/T 7714学位论文. 大肠杆菌中固定CO2产乳酸代谢途径的构建和高固碳活性核酮糖-1,5-二磷酸羧化酶的筛选研究[D]. 北京. 中国科学院研究生院,2019.
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