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Manufacture of TATB and TNT from Biosynthesized Phloroglucinols
DRATHS CORP OKEMOS MI
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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 Umg, 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 Umg using crude lysate. Efforts had been made to create the first E. coli mono-O-methylphloroglucinol synthesizing construct. E. coli PG1pKIT1.008 synthesized 0.7 gL 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 PG1pKIT1.008 synthesized 2 gL 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 PG1pKIT10.080 synthesized 25 gL of phloroglucinol.
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