Health & Environmental Research Online (HERO)


Print Feedback Export to File
5032830 
Technical Report 
Manufacture of TATB and TNT from Biosynthesized Phloroglucinols 
Frost, JW 
2010 
GRA and I 
e 2 
In route to the microbial synthesis of mono-O-methylphloroglucinols, phloroglucinol O-methyl transferase (POMT) from Rosa chinensis var. spontanea has been successfully de novo synthesized in codon-optimized form for expression in E. coli, which is the host currently used for microbial synthesis of phloroglucinol (PG) from glucose. The specific activity of heterologously- expressed, codon-optimized POMT is at 0.02 U/mg, which is 5-fold higher than the specific activity of POMT purified to homogeneity from Rosa chinensis var. spontanea petals. Similarly, orcinol O-methyl transferase (OOMT) protein sequence was identified from GenBank, codon optimized for E. coli expression using the DNA2.0 algorithm, and synthesized. Expressing this gene in E. coli gave a specific activity at 0.006 U/mg using crude lysate. Efforts had been made to create the first E. coli mono-O-methylphloroglucinol synthesizing construct. E. coli PG1/pKIT1.008 synthesized 0.7 g/L methoxyresorcinol in the medium under fed-batch fermentor-controlled conditions. A methionine supplementation strategy was developed to give a 40% increase in mono-O-methylphloroglucinol production under fed-batch fermentor-controlled conditions. It is believed that mono-O-methylphloroglucinol is toxic to E. coli cells and difficulties were experienced in maintaining a healthy culture in the fermentor. To circumvent this issue, resin-based extractive fermentation was carried out as an external loop throughout the run. By using a strong anion- exchange resin under optimized conditions, E. coli PG1/pKIT1.008 synthesized 2 g/L mono-O-methylphloroglucinol in 70 h after inoculation. Simultaneously, efforts had been made in both strain development and optimizing fermentation conditions for microbial phloroglucinol synthesis. Under optimized resin-based extractive fermentation, E. coli PG1/pKIT10.080 synthesized 25 g/L of phloroglucinol.