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• W3013258889 abstract "The introduction of the mevalonate pathway (MVA pathway) in recombinant Escherichia coli can improve the synthesis of terpenoids. But the imbalance expression of MVA pathway genes and accumulation of intermediates inhibit cell growth and terpenoids production. In this study, each gene of MVA pathway and key genes of lycopene synthesis pathway were cloned in plasmid to express in the recombinant E. coli LYC103 with optimizing the expression of the key genes of the 2-methyl-D-erythritol-4-phosphate pathway (MEP pathway), chromosome recombinant MVA pathway and the lycopene synthesis pathway. The results showed that the overexpression of ispA, crtE, mvaK1, idi and mvaD genes did not affect the cell growth, while lycopene production increased by 13.5%, 16.5%, 17.95%, 33.7% and 61.1% respectively, indicating that these genes may be the rate-limiting steps for the synthesis of lycopene. mvaK1, mvaK2, mvaD of MVA pathway were the rate-limiting steps and were in an operon. The mvaK1, mvaK2, mvaD operon was regulated by mRS (mRNA stabilizing region) library in front of mvaK1, obtaining strain LYC104. Lycopene yield of LYC104 was doubled and cell growth was increased by 32% compared with the control strain LYC103. CRISPR-cas9 technology was used to integrate idi into chromosome at lacZ site to obtain LYC105 strain. Cell growth of LYC105 was increased by 147% and lycopene yield was increased by 2.28 times compared with that of LYC103. In this study, each gene of lycopene synthesis pathway was expressed in plasmid to certify the rate-limiting gene based on the complete MVA pathway on the chromosome. Then the rate-limiting gene was integrated in chromosome with homologous recombination to release the rate-limiting, which providing a new strategy for the construction of high-yield strains for metabolic engineering.甲羟戊酸途径 (MVA 途径) 被引入重组大肠杆菌中，能够提高重组大肠杆菌中萜类化合物的合成能力。但因重组大肠杆菌中萜类化合物合成途径中间产物积累，导致细胞生长和萜类化合物合成受到限制。本研究在稳定表达MVA 途径以及优化2-甲基-D-赤藻糖醇-4-磷酸途径 (MEP 途径)、番茄红素合成途径关键基因表达的重组大肠杆菌LYC103 中，用质粒高表达MVA 途径和番茄红素合成途径关键基因，挖掘该途径的限速步骤。结果表明，ispA、crtE、mvaK1、idi 和mvaD 基因过表达后，细胞生长没有明显变化，番茄红素产量依次提高了13.5%、16.5%、17.95%、33.7%和61.1%，说明这几个基因可能是合成番茄红素的限速步骤。mvaK1、mvaK2、mvaD 三个基因在同一操纵子上，用mRNA 稳定区 (RNA stabilizing region) 进行启动子文库 (mRSL) 调控mvaK1，相当于对3 个基因同时调控。用高效基因组编辑技术 (CAGO) 对mvaK1 基因的mRNA 稳定区进行启动子文库的调控，得到菌株LYC104。番茄红素产量与对照菌株LYC103 相比增加了2 倍，细胞生长提高了32%。然后，利用CRISPR-Cas9 技术在染色体lacZ 位点整合idi 基因，得到LYC105 菌株。与出发菌株LYC103 相比，细胞生长提高了147%，番茄红素产量增加了2.28 倍。本研究在染色体上具有完整MVA 途径的基础上，利用质粒高表达单个基因挖掘限速步骤，用同源重组方法整合限速基因、解除限速，为代谢工程构建高产菌株提供新策略。." @default.
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• W3013258889 date "2020-01-25" @default.
• W3013258889 modified "2023-10-16" @default.
• W3013258889 title "[Role of rate-limiting step of mevalonate pathway in improving lycopene production in Escherichia coli]." @default.
• W3013258889 doi "https://doi.org/10.13345/j.cjb.190189" @default.
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