为进一步提高生物法合成间苯三酚的效率,探究生物合成间苯三酚的分子机理以及更加高效的发酵条件和培养基成分.对来自荧光假单胞菌(Pseudomonas fluorescens)的phlD基因进行了克隆,在BL21(DE3)中重建间苯三酚的胞内合成途径,超表达多重抗药基因正向调节因子(marA)和乙酰辅酶A羧化酶(ACCase),并分析不同碳源及培养基离子环境对间苯三酚积累量的影响.结果显示,成功构建间苯三酚胞内合成途径,产量达到450 mg/L,marA基因能够提高工程菌对间苯三酚抵抗力,使间苯三酚产量提高到1060 mg/L;ACCase能够提高胞内丙二酰辅酶A的含量,使间苯三酚产量提高到2120 mg/L;葡萄糖、甘油和丙酮酸钠三种碳源的比较中,葡萄糖对合成间苯三酚最为有利,分别是甘油和丙酮酸钠的1.8倍和2.4倍;培养基中钙镁离子对间苯三酚的合成没有影响.marA和ACCase基因的超表达能大大提高间苯三酚生物合成的效率,以葡萄糖为碳源的无钙镁培养基为间苯三酚发酵的最适培养基.%This work is to further improve the efficiency of phloroglucinol biosynthesis,and to investigate the molecular mechanism of phloroglucinol biosynthesis and more efficient fermentation conditions and media components. We cloned the phlD gene from Pseudomonas fluorescens and reconstructed the intracellular synthesis pathway of phloroglucinol in the BL21(DE3). At the same time the multi-drug resistant regulator(marA)and acetyl-coA carboxylase(ACCase)were also super-expressed in the BL21(DE3),and the effects of different carbon sources and medium ion environment on the phloroglucinol accumulation were analyzed. The results showed that intracellular synthesis pathway of phloroglucinol was successfully established,and the yield reached 450 mg/L. The marA gene increased the resistance of engineering strain to phloroglucinol and thus phloroglucinol yield increased to 1060 mg/L. The ACCase improved the content of the intracellular malonyl-CoA,and increased the phloroglucinol yield up to 2120 mg/L. Compared to glycerin and sodium pyruvate,glucose was the most suitable for the phloroglucinol biosynthesis,and the phloroglucinol yield with glucose was 1.8 times and 2.4 times of that with glycerol and pyruvate, respectively. Calcium and magnesium ions in medium had no impact on the phloroglucinol synthesis. Conclusively,the superexpression of the marA and ACCase genes can greatly increase the efficiency of phloroglucinol biosynthesis,and the medium using glucose as carbon source and without no calcium and magnesium is the optimal for phloroglucinol biosynthesis.
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