Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1998-04-22
2001-04-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S232000, C435S320100, C435S252300
Reexamination Certificate
active
06210937
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to synthesis of aromatic compounds by the conversion of biomass-derived carbon sources. More particularly, the invention relates to cloned genes, transformed hosts carrying such cloned genes, and methods of use thereof for producing selected aromatic compounds by the biocatalytic conversion of glucose and other sugars capable of being used in the biosynthesis of such aromatic compounds. Selected genes of the
E. coli
common aromatic pathway have been cloned and expressed in bacterial hosts. A host carrying vectors for over-expression of the selected genes of the common aromatic pathway plus an additional gene or genes for converting chorismate, the final product of the common aromatic pathway, to a selected aromatic compound results in production of substantial amounts of such selected aromatic compound.
Chorismate is an intermediate in biosynthetic pathways that lead to the production of many aromatic compounds. Because of the large number of aromatic pathways that branch from chorismate, the biosynthetic pathway used by organisms to produce chorismate is often known as the “common aromatic pathway.” This pathway is also known as the shikimate pathway because shikimate was the first identified intermediate in the pathway.
Efficient and cost-effective biosynthetic production of chorismate and its biosynthetic derivatives require that carbon sources such as glucose, lactose, galactose, and other sugars be converted to the selected product in high percentage yields. Accordingly, it is valuable from the standpoint of industrial biosynthetic production of aromatic compounds or other biosynthetic derivatives of chorismate to increase the flux of carbon sources into and through the common aromatic pathway, thereby enhancing biosynthesis of chorismate and its derivatives.
The present invention provides for enhanced commitment of cellular carbon sources to enter and flow through the common aromatic pathway by transferring into host cells genetic elements encoding enzymes that catalyze synthesis of the initial carbon compounds of the common aromatic pathway, genetic elements encoding selected enzymes of the common aromatic pathway, and genetic elements encoding enzymes that catalyze conversion of chorismate to a selected aromatic compound. The genetic elements can be in the form of extrachromosomal plasmids, cosmids, phages, or other replicable elements configured for carrying these genetic elements for expression in a host cell.
U.S. Pat. No. 5,168,056 to Frost discloses cloning and expression of transketolase and optionally the aroF gene and/or aroB gene for enhancing diversion of carbon resources into the common aromatic pathway. U.S. Pat. No. 5,272,073 to Frost & Draths describes a method for synthesizing catechol from a carbon source, such as glucose, by creating a pathway that diverges from the common aromatic pathway for conversion of dehydroshikimate to protocatechuate and then to catechol. This divergent pathway is induced by transforming a host with recombinant DNA carrying the transketolase, DAHP synthase, and 3-dehydroquinate synthase genes. U.S. Pat. No. 5,008,190 and U.S. Pat. No. 5,030,567 to Lee et al. describe cloning of the aroF gene and the pheA gene for increasing the biosynthesis of phenylalanine. EP 77196 discloses cloning of a gene that specifies biosynthesis of a DAHP synthase that is resistant to feedback inhibition by aromatic amino acids. R. Meuller et al., 43 Appl. Microbiol. Biotech. 985-88 (1995); M. Seibert et al., 140 Microbiol. 897-904 (1994); G. Wu et al., 139 J. Gen. Microbiol. 17995-1805 (1993); B. P. Nichols et al., 174 J. Bacteriol. 5309-16 (1992); M. Siebert et al., 307 FEBS Lett. 347-50 (1992); L. Heide et al., 175 J. Bacteriol. 5728-29 (1993); H. Matsude et al., JP 96107789, disclose cloning of the chorismate pyruvate lyase gene that encodes the enzyme for converting chorismate to 4-hydroxybenzoic acid. U.S. Pat. No. 5,487,987 to J. Frost et al. discloses synthesis of adipic acid from biomass-derived carbon sources by expression of 3-dehydroshikimate dehydratase and other enzymes for conversion of 3-dehydroshikimate to adipic acid. WO 94/08015 by Frost et al. teaches the synthesis of quinic acid from glucose by cloning and expressing enzymes in the early stages of the common aromatic pathway for synthesis of dehydroquinate and subsequent conversion to quinic acid. WO 95/33843 by Frost et al. describes enhanced efficiency of production of aromatic compounds by cloning and expressing 3-dehydroquinate synthase, shikimate kinase, 5-enolpyruvoyl-shikimate-3-phosphate synthase, and chorismate synthase and optionally with transketolase and DAHP synthase. All of these processes are inadequate for the production of commercially acceptable levels of selected aromatic compounds for which chorismate is a precursor.
In view of the foregoing, it will be appreciated that cloned genes of the common aromatic pathway and additional genes for converting chorismate to a selected aromatic compound, transformed hosts carrying such cloned genes, and methods of using such cloned genes and transformed hosts for producing the selected aromatic compound would be a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to molecularly clone selected genes of the
E. coli
common aromatic pathway and additional genes for converting chorismate, the final product of the common aromatic pathway, to a selected aromatic compound.
It is another object of the invention to provide transformed hosts carrying the selected cloned genes for over-expression of the selected enzymes of the common aromatic pathway and such additional enzymes necessary for converting chorismate to a selected aromatic compound.
It is also an object of the invention to provide methods for synthesizing a selected aromatic compound by cloning and expressing selected genes of the common aromatic pathway and such additional genes necessary for converting chorismate to the selected aromatic compound.
These and other objects can be achieved by providing a method for producing a compound for which chorismate is an anabolic precursor comprising the steps of:
(a) transforming a microorganism with at least one recombinant plasmid, the at least one recombinant plasmid comprising at least one plasmid vector into which DNA segments that code for DAHP synthase, transketolase, PEP synthase, chorismate synthase, shikimate kinase, EPSP synthase, DHQ synthase, and one or more enzymes for converting chorismate into the compound have been inserted;
(b) culturing the transformed microorganism under conditions that promote the synthesis of the compound.
In preferred embodiment of the invention, the compound is p-hydroxybenzoic acid and the one or more enzymes for converting chorismate comprises chorismate pyruvate lyase.
REFERENCES:
patent: 4621153 (1986-11-01), Hatch
patent: 4681852 (1987-07-01), Tribe
patent: 5008190 (1991-04-01), Lee et al.
patent: 5030567 (1991-07-01), Lee et al.
patent: 5168056 (1992-12-01), Frost
patent: 5169768 (1992-12-01), Backman
patent: 5272073 (1993-12-01), Frost et al.
patent: 5409830 (1995-04-01), Lim et al.
patent: 5487987 (1996-01-01), Frost et al.
patent: 0077196 A2 (1983-04-01), None
patent: 8-107789 (1996-04-01), None
patent: 87/00202 (1987-01-01), None
patent: WO 94/08015 (1994-04-01), None
patent: WO 95/33843 (1995-12-01), None
patent: 96/08567 (1996-03-01), None
M. Siebert et al., “Formation of 4-Hydroxybenzoate inEscherichia coli:Characterization of the ubiC Gene and Its Encoded Enzyme Chorismate-Pyruvate Lyase”, Microbiology 140: 897-904, 1994.*
G. Gosset et al., “A Direct Comparison of Approches For Increasing Carbon Flow to Aromatic Biosynthesis inEscherichia coli” J. Indust. Microbiol. 17(1):47-52, Jul. 1996.
Amaratunga Mohan
Bulmer Deborah K.
Johnson Bruce F.
Ward Thomas E.
Watkins Carolyn S.
Bechtel BWXT Idaho LLC
Clayton Howarth & Cannon
Prouty Rebecca E.
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