Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2002-05-07
2004-06-15
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S252300, C435S252330, C435S252400, C435S190000, C435S233000
Reexamination Certificate
active
06750049
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to the production of 1,2,3,4-tetrahydroxybenzene and more specifically, to methods of producing 1,2,3,4-tetrahydroxybenzene from the bioconversion of a carbon source.
BACKGROUND OF THE INVENTION
Polyhydroxy benzenes and quinones possessing the oxygenation pattern of 1,2,3,4-tetrahydroxybenzene 1 (
FIG. 1
) often display biological activity. Aurantiogliocladin 2 and fumigatin 3 (
FIG. 1
) are antibiotics. Vischer, E. B.,
J. Chem. Soc
. 815 (1953): Baker, W. et al.,
J. Chem. Soc
. 820 (1953); Baker, W. et al.,
J. Chem. Soc
. 670 (1941). Coenzyme Q
n=10
4 (
FIG. 1
) is an essential antioxidant in humans protecting low density lipoproteins from atherosclerosis-related oxidative modification. Ingold, K. U. et al.,
PNAS
(USA) 90:45 (1993); Stocker, R. et al.,
PNAS
(USA) 88:1646 (1991) Steinberg, D.,
Circulation
84:1420 (1991). Dillapiole 5 (
FIG. 1
) is a pyrethrin synergist and is responsible for the sedative effect of
Perilla frutescens
leaves. Honda, G. et al.,
Chem. Pharm. Bull
. 36:3153 (1988): Tomar, S. S. et al.,
Agric. Biol. Chem
. 50:2115 (1986).
The current method of preparing 1,2,3,4-tetrahydroxybenzene uses pyrogallol as the synthetic starting material. Pyrogallol is converted to aminopyrogallol using a four-step synthesis. Aminopyrogallol is then hydrolyzed to give 1,2,3,4-tetrahydroxybenzene. Conversion of pyrogallol to 1,2,3,4-tetrahydroxybenzene requires the use of such reagents as phosgene, solvents such as pyridine and xylene, and has a nitroaromatic as a synthetic intermediate.
It would also be desirable to provide an improved method for producing derivatives of 1,2,3,4-tetrahydroxybenzene. Particularly, it would be desirable to provide a method for producing 1,2,3-trihydroxybenzene (pyrogallol). It would also be desirable if such a method were cost efficient and employed readily available materials. Currently, 1,2,3-trihydroxybenzene is obtained by thermal decarboxylation of gallic acid. However, gallic acid is isolated from natural sources such as gall nuts and tara powder and therefore is in limited supply.
It would thus be desirable to provide an improved method for producing 1,2,3,4-tetrahydroxybenzene. It would also be desirable if such a method was cost-efficient, using inexpensive starting materials. It would be further desirable if the method employed non-toxic compounds and was environmentally benign.
SUMMARY OF THE INVENTION
A bioengineered synthesis scheme for the production of 1,2,3,4-tetrahydroxybenzene from a carbon source is provided. In one embodiment, the bioconversion methods of the present invention comprise the steps of microbe-catalyzed conversion of a carbon source to myo-2-inosose followed by acid-catalyzed dehydration of myo-2-inosose to produce 1,2,3,4-tetrahydroxybenzene. As shown in the synthesis scheme of
FIG. 2
, the microbe-catalyzed conversion step of the present invention requires four enzymes. In one embodiment, the microbe-catalyzed conversion comprises the conversion of a carbon source to myo-inositol by a recombinant microbe and the subsequent conversion of myo-inositol to myo-2-inosose catalyzed by a second microbe. In another embodiment, the recombinant microbe is
Escherichia coli
designed to cause the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. In yet another embodiment, the conversion of myo-inositol to myo-2-inosose is catalyzed by the microbe
Gluconobacter oxydans
. Acid-catalyzed dehydration of the resulting myo-2-inosose yields 1,2,3,4-tetrahydroxybenzene.
The biocatalytic synthesis of 1,2,3,4-tetrahydroxybenzene provided herein is environmentally benign, economically attractive, and utilizes abundant renewable sources as a starting material.
Methods are also provided for the production of derivatives of 1,2,3,4-tetrahydroxybenzene, particularly Coenzyme Q 1,2,3-trihydroxybenzene (pyrogallol). In one embodiment 1,2,3-trihydroxybenzene is produced by reduction of 1,2,3,4-tetrahydroxybenzene. In a preferred embodiment, the reduction is achieved by catalytic hydrogenation followed by hydrolysis.
Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.
REFERENCES:
Posternak , T. The Cyclitols, p. 164, Holden Day, San Francisco, CA 1965.*
Majumder et al. (1997) Biochem Biophys Acta 1348:245-256.*
Nelson et al. (1998) Plant Cell 10:753-764.
Frost John W.
Hansen Chad A.
Board of Trustees operating Michigan State University
Harness & Dickey & Pierce P.L.C.
Prouty Rebecca E.
Steadman David J
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