Abstract: 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.

Key words: phloroglucinol, MarA, ACCase, PhlD