Screening And Metabolic Regulation Of Pseudomonas In Marine Habitat That Produced Phloroglucinol

Phloroglucinol has broad market prospect because of the good antibacterial and antiviral activities, and the applications as drugs intermediates of flavones or isoflavones which can cure cancer and cardiovascular disease. For the production of it, more and more attention has been paid to biosynthesis method due to the advantage of environment friendly compared with the chemical synthesis, especially with microorganisms. Previous studies have shown that Pseudomonads were the only natural microbial resources of phloroglucinol and its derivatives, however, there was no reports of phloroglucinol production strains from marine habitats. In this study, two strains which could produce phloroglucinol were selected from about 200 pseudomonads strains isolated from lower animals in the South China Sea, marine algae and sediments soils of the Bohai tidal zone respectively by chemistry and molecular screening. Then ultraviolet and microwave mutagenesis breeding study were carried out on the wild type strain. Finally, High Phloroglucinol-producing Mutant was selected and improved by optimization of medium and metabolic regulation. The results were as follows:1. Three different kinds of selective media were used for the isolation of Pseudomonads from samples of lower animals in the South China Sea, marine algae and sediments soils of the Bohai tidal zone.Data showed that the best one for selection was S1 medium which separation efficiency was 92.7%, the second one was KB+ medium with the separation efficiency of 79.2%, the worst one is KB medium which separation efficiency was 34.9%.2. Two phloroglucinol producing strains designated as LBF-93 and LBF-96 had been gained by gene probe method. By the sequence alignment of 16 S r RNA and the validated of Physiological and biochemical characteristics, LBF-93 and LBF-96 were idetified as Pseudomonas fluorescens and Pseudomonas brassicacearum respectively.3. A mutant strain named YLM96-01 was screened by complex mutagenesis of ultraviolet and microwave, and the phloroglucinol production of it reached the peak of 0.599g/L after 29 hours fermentation, which increased by 169% compared with that of the wild strain(0.223g/L).4. In order to improve the yield of phloroglucinol, Culture medium of mutant YLM96-01 was optimized through orthogonal design. Results showed that glycerol had the biggest effect on the expression of phloroglucinol, followed by peptone, glucose minimal impacted. The expression of phloroglucinol produced by strain YLM96-01 under the optimum cultivation reached 0.782g/L which increased by 30%.5. Results of metabolism regulation on the mutant strain YLM96-01 showed that the addition of metabolic inducer citric acid had no obvious effects on the phloroglucinol production. By contrast, the addition of 1% ethanol could significantly affected the expression of phloroglucinol. Production was reduced with 1% ethanol added in culture medium after incubation of 12 or 24 h, and increased 29% with ethanol added after incubation of 36 h than that in the ethanol unfed culture.