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乙酰辅酶A衍生产品生物平台细胞的构建与研究
Alternative Title
刘敏
Thesis Advisor赵广
2016-05
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline生物化学与分子生物学
Keyword大肠杆菌 平台细胞 代谢工程 乙酰辅酶a 间苯三酚 3-羟基丙酸
Abstract自上世纪以来,化石燃料资源枯竭以及全球气候变暖等问题日益加重,能源与环境的问题已经成为现代社会亟需解决的重大问题。生物法合成因其原料可再生,环境友好等优势,逐渐受到各国政府的高度重视。乙酰辅酶A是生物体内的核心代谢中间产物,同时也可作为多种生物基产品的合成前体。本文基于乙酰辅酶A的代谢途径,通过全局性、多水平、多途径的代谢改造组合调控,构建通用的大肠杆菌平台细胞,适用于乙酰辅酶A衍生的一类生物基产品的生产。该平台细胞具有通用性、高效性等优势,具有较大的市场应用价值。 本文选取全局调控因子基因arcA和csrB、局部调控因子基因iclR、乙酸代谢相关功能基因ackA和acs进行组合调控。首先针对arcA和iclR的基因改造对乙酰辅酶A衍生产品生物合成的影响进行深入研究。选择两种市场应用广泛的乙酰辅酶A衍生产品间苯三酚(PG)和3-羟基丙酸(3HP)进行检测,研究发现arcA和iclR敲除均能够提高葡萄糖的利用率和细胞产率,同时显著控制乙酸副产物积累。敲除菌株的PG和3HP产量均提高2倍以上。由此表明,arcA、iclR的单基因改造能够促进乙酰辅酶A衍生产品的生物合成。 利用PG的生物合成对arcA敲除、iclR敲除、csrB过表达、ackA敲除、acs过表达5种单基因改造的效果进行比较分析。5种单基因改造均能不同程度提高PG的产量,其中arcA敲除效果最为显著,能够将PG产量提高2.5倍,进而将arcA与其他四种基因改造进行组合调控,发现arcA的组合调控均比arcA单基因改造效果差。因此,arcA不参与后续平台细胞的构建。将iclR敲除、csrB过表达、ackA敲除、acs过表达4种基因改造进行不同的组合调控后,利用PG发酵检测发现,不同分子改造进行组合后,对PG的生物合成产生了不同程度的调控作用。其中多组合突变菌株Q2637、Q2628、Q2140和Q2627的PG产量比对照菌株提高3.5倍以上。csrB、ackA和acs三组合突变的PG产量最高,达到2.12±0.09g/l,比对照菌株的产量0.41±0.02 g/l提高5倍。与PG发酵结果类似,csrB、ackA和acs三组合突变菌株的3HP产量最高,达到3.49±0.33 g/l,比对照1.75±0.11 g/l提高约2倍。根据PG和3HP的发酵检测结果得出:csrB、ackA和acs的组合调控可以用于构建一株通用的大肠杆菌平台细胞,以促进乙酰辅酶A衍生产品的生物合成。 在PG和3HP的发酵过程中对平台细胞的生长状况、耗糖状态及发酵产物进行评估分析。PG发酵过程中,组合突变菌株与对照菌株的细胞生长及耗糖速度无明显差异, 3HP发酵过程中,组合突变菌株的细胞生长及耗糖速度明显低于对照菌株。然而,PG和3HP重组菌株的细胞产率高于对照菌株,由此表明,csrB、ackA和acs的组合调控能够提高葡萄糖的利用率。该平台细胞不仅能够显著提高PG和3HP的生物合成,还能有效控制乙酸副产物积累,乙酸副产物的浓度均降低90%以上。 综上所述,由csrB、ackA和acs组合调控构建的平台细胞,能够为乙酰辅酶A衍生产品的生物合成提供良好的表达宿主,同时有效提高葡萄糖利用率,控制乙酸副产物积累,降低微生物工业生产的成本,具有重要的工业应用价值。
Other AbstractThe energy crisis and environmental pollution have become the serious problems that need to be addressed urgently since the twentieth century. Biological method is attracted highly attention due to the renewable materials and few pollution. In this study, we constructed a platform cell factory of Escherichia coli for the production of acetyl-CoA-derived chemicals through metabolic engineering of global regulation, multi-level and multi-path. This platform cell factory is versatile, high efficiency and has a great market value. We chose five genetic modifications of arcA knockout, iclR knockout, csrB overexpression, ackA knockout and acs overexpression in this study. Firstly, The two chemicals, phloroglucinol (PG) and 3-hydroxypropionate (3HP) both derived from acetyl-CoA, were used to evaluate the effects of arcA and iclR konckout on acetyl-CoA-derived chemicals production. The mutants significantly increased the glucose utilization and cell yield than the corresponding controls. The production of PG and 3HP was increased by more than 2 times. In additon, the mutants showed surprising efficiency to overcome acetate excretion. The effects of arcA knockout, iclR knockout, csrB overexpression, ackA knockout and acs overexpression on PG produciton were compared and discussed. The five single genetic modificaion improved PG production in different degrees. The arcA mutant showed the best result for improving PG production by 2.5 folds. Then, the combined regulation strains were constructed between arcA and other genetic modifications. However, the PG produciton of combined regulation strains was lower than that of the arcA single mutant. So the platform cell factory was constructed without containing arcA konckout. The different recombinant strains were constructed with iclR knockout, csrB overexpression, ackA knockout and acs overexpression. The recombinant strains of Q2637,Q2628,Q2140 and Q2627 increased PG production by more than 3.5 folds. The combined regulation of csrB, ackA and acs improved PG production by 5 folds, from 0.41±0.02g/l to 2.12±0.09g/l. and 3HP production was improved by 2 folds, from1.75± 0.11 g/l to 3.49± 0.33 g/l. Considering all these, the combined regulation of csrB, ackA and acs can be used to constructed a platform cell factory to improve the acetyl-CoA-derived chemicals prodution. The cell growth, glucose utilizaiton and fermentation products of the platform cell factory were also discussed during the production of PG and 3HP. The platform cell factory showd similar cell growth and glucose consumption curve to the control strains. However, the cell yield of recombinant strains was higher than the control strains. The platform cell factory can not only increase the production of PG and 3HP,but also overcome acetate excretion. The acetate concentration was decreased by more than 90%. In conlusion, the platform cell factory that was engineered of csrB, ackA and acs would be a good host for acetyl-CoA-derived chemicals production. It would be widely used in industry because of the higher glucose utilization, lower acetate excretion and lower production cost.
Department材料生物技术中心
Subject Area生物学
Date Available2019-09-01
Subtype博士 ; 学位论文
Language中文
Document Type学位论文
Identifierhttp://ir.qibebt.ac.cn/handle/337004/9752
Collection生物基材料组群
Affiliation中国科学院青岛生物能源与过程研究所
Recommended Citation
GB/T 7714
刘敏. 乙酰辅酶A衍生产品生物平台细胞的构建与研究[D]. 北京. 中国科学院研究生院,2016.
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