Li Zhenxia, Chen Qianqian, Tang Jinlei, Li Qingyan, Zhang Xueli
School of Horticulture and Garden, Henan Institute of Science and Technology, Xinxiang 453000, Henan, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Jan 25;36(1):77-89. doi: 10.13345/j.cjb.190189.
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.
在重组大肠杆菌中引入甲羟戊酸途径（MVA途径）可提高萜类化合物的合成。但MVA途径基因的不平衡表达和中间产物的积累会抑制细胞生长和萜类化合物的产生。在本研究中，将MVA途径的每个基因和番茄红素合成途径的关键基因克隆到质粒中，在重组大肠杆菌LYC103中表达，同时优化2-甲基-D-赤藓糖醇-4-磷酸途径（MEP途径）、染色体重组MVA途径和番茄红素合成途径关键基因的表达。结果表明，ispA、crtE、mvaK1、idi和mvaD基因的过表达不影响细胞生长，而番茄红素产量分别提高了13.5%、16.5%、17.95%、33.7%和61.1%，表明这些基因可能是番茄红素合成的限速步骤。MVA途径的mvaK1、mvaK2、mvaD是限速步骤且位于一个操纵子中。在mvaK1前通过mRS（mRNA稳定区）文库调控mvaK1、mvaK2、mvaD操纵子，获得菌株LYC104。与对照菌株LYC103相比，LYC104的番茄红素产量翻倍，细胞生长增加了32%。利用CRISPR-cas9技术将idi整合到染色体的lacZ位点，获得LYC105菌株。与LYC103相比，LYC105的细胞生长增加了147%，番茄红素产量提高了2.28倍。在本研究中，将番茄红素合成途径的每个基因在质粒中表达，基于染色体上完整的MVA途径鉴定限速基因。然后通过同源重组将限速基因整合到染色体中以解除限速，这为代谢工程高产菌株的构建提供了一种新策略。
