|Alternative Title||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|
|Place of Conferral||北京|
|Keyword||Rubisco 大肠杆菌 Co2固定 乳酸 发酵|
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%。
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%。
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.
|MOST Discipline Catalogue||工学::生物工程|
|Table of Contents|
|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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