Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-02-07
2001-10-16
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S155000, C435S252330, C435S320100
Reexamination Certificate
active
06303352
ABSTRACT:
FIELD OF THE INVENTION
The invention is drawn to microorganisms and their use in the production of 1,2-propanediol via microbial fermentation of common sugars. More specifically, the present invention is drawn to recombinant microorganisms having reductive enzyme activity or activities which enable the recombinant microorganism to ferment common sugars to 1,2-propanediol.
BIBLIOGRAPHY
Complete bibliographic citations to the references mentioned below are included in the Bibliography section, immediately preceding the claims. Each of the references mentioned below is incorporated herein by reference in its entirety.
DESCRIPTION OF THE PRIOR ART
1,2-Propanediol (1,2-PD; also known as propylene glycol) is a major commodity chemical with an annual production greater than one billion pounds in the United States. The major utilization of 1,2-PD is in unsaturated polyester resins, liquid laundry detergents, pharmaceuticals, cosmetics, antifreeze and de-icing formulations.
1,2-PD is conventionally produced from petrochemicals. Unfortunately, several toxic chemicals, such as chlorine, propylene oxide, and propylene chlorohydrin are either required or are produced as by-products in the conventional synthesis. In the conventional route, 1,2-PD is produced by the hydration of propylene oxide, which is obtained from propylene. The synthetic process produces racemic 1,2-PD, an equimolar mixture of the two enantiomers. This chemical process has a number of disadvantages, including the use of large quantities of water to minimize the production of polyglycols. The major problem, however, with the conventional synthetic route to 1,2-PD arises in the production of its intermediate, propylene oxide.
Propylene oxide is manufactured by one of two standard commercial processes: the chlorohydrin process or the hydroperoxide process. The chlorohydrin process involves toxic chlorinated intermediates and the use of caustic or lime. Additionally, this process may result in air emissions of propylene chlorohydrin and chlorine. (Franklin Associates, Ltd. (1994).) The hydroperoxide process involves oxidation of propylene by an organic hydroperoxide and results in the stoichiometric co-production of either tert-butanol or 1-phenyl ethanol. This make the economics of the production of propylene oxide via the hydroperoxide route directly related to the market for the co-produced byproducts. (Gait (1973).)
It is known that 1,2-PD is produced by several organisms when grown on exotic sugars. As early as 1937, the fermentation of L-rhamnose to 1,2-PD (later shown to be the S enantiomer) was described by Kluyver and Schnellen (1937). In
E. coli
and a variety of other microorganisms, L-rhamnose and L-fucose are metabolized to L-lactaldehyde and dihydroxyacetone phosphate. (Sawada and Takagi (1964) and Ghalambor and Heath (1962), respectively.) Under aerobic conditions, L-lactaldehyde is oxidized in two steps to pyruvate (Sridhara and Wu (1969)). Under anaerobic conditions, however, L-lactaldehyde is reduced to S-1,2-PD by a nicotinamide adenine nucleotide (NAD)-linked 1,2-propanediol oxidoreductase (EC 1.1.1.77). The S-1,2-PD produced diffuses into the extra-cellular medium.
Although a variety of microorganisms, including
E. coli,
produce S-1,2-PD from 6-deoxyhexose sugars, Obradors et al. (1988), this route is not commercially feasible because these sugars are extremely expensive. The least expensive of these 6-deoxyhexose sugars, L-rhamnose, currently sells for approximately $325 per kilogram (Pfanstiehl Laboratories, Chicago, Ill.).
In the mid-1980's, organisms capable of fermenting common sugars, such as glucose and xylose, to R-1,2-PD were discovered. See, for instance, Tran-Din and Gottschalk (1985).
Clostridiun sphenoides
produces R-1,2-PD via a methylglyoxal intermediate. In this pathway, dihydroxyacetone phosphate (DHAP) is converted to methylglyoxal (MG) by the action of methylglyoxal synthase. The MG is reduced stereospecifically to give D-lactaldehyde. The D-lactaldehyde is then further reduced to give R-1,2-PD. The commercial production of 1,2-PD by
C. sphenoides
is severely limited, however, by the fact it is only produced under phosphate limitation; it is both difficult and expensive to obtain commercial-grade medium components which are free of phosphate. Additionally, only low titers of 1,2-PD are achieved.
Thermoanaerobacterium thermosaccharolyticum HG-
8 (formerly
Clostridium thermosaccharolyticum,
ATCC 31960) also produces R-1,2-PD via methylglyoxal. Cameron and Cooney (1986). As with
C. sphenoides,
DHAP is converted to MG. The MG is then reduced at the aldehyde group to yield acetol. The acetol is then further reduced at the ketone group to give R-1,2-PD. For both
C. sphenoides
and
T. thermosaccharolyticum
HG-8, the enzymes responsible for the production of 1,2-PD have not been identified or cloned.
SUMMARY OF THE INVENTION
The invention is directed to a method of producing 1,2-propanediol by fermentation of sugars. The method comprises culturing a microorganism which expresses one or more enzymes which catalyze production of 1,2-propanediol from intracellular methylglyoxal in a medium containing a sugar carbon source other than a 6-deoxyhexose sugar, whereby the sugar carbon source is metabolized into 1,2-propanediol. Preferably, the method utilizes a recombinant organism containing one or more recombinant genes whose encoded gene products catalyze the reduction of methylglyoxal to 1,2-propanediol.
More specifically, the invention is directed to a method of producing 1,2-propanediol by fermentation with recombinant
E. coli
or yeast which comprises culturing a recombinant
E. coli
or yeast in a medium containing a sugar carbon source selected from the group consisting of arabinose, fructose, galactose, glucose, lactose, maltose, sucrose, xylose, and combinations thereof. The recombinant
E. coli
or yeast includes one or more recombinant genes which encode enzymes selected from the group consisting of aldose reductase, glycerol dehydrogenase, or combinations thereof.
The invention is also drawn to a synthetic operon which enables the production of 1,2-propandiol in a microorganism transformed to contain the operon. The operon includes one or more genes whose encoded gene products catalyze the reduction of methylglyoxal to 1,2-PD and a promoter sequence operationally linked to the one or more genes.
In a preferred embodiment, the synthetic operon includes at least one promoter sequence, a gene selected from the group consisting of an aldose reductase gene, a glycerol dehydrogenase gene, and combinations thereof; and a gene selected from the group consisting of a methylglyoxal synthase gene, a pyridine nucleotide transferase gene, and combinations thereof, wherein the genes are operationally linked to the promoter.
The invention is also drawn to
E. coli
transformed to contain the synthetic operon.
In short, the present invention is drawn to the use of microorganisms, preferably recombinant
E. coli
or
S. cerevisiae,
which express reductive enzyme activity which enables them to produce 1,2-PD, presumably via a reductive pathway leading from methylglyoxal to acetol (or lactaldehyde) to 1,2-PD.
If a recombinant microorganism is utilized, the gene sequences encoding the reductive enzyme activity may reside on plasmids within the microorganism, or the gene sequences may be integrated into the chromosome. It is preferred that the recombinant gene sequences be integrated into the genome of the microorganism.
The invention utilizes microorganisms which express enzymes which enable the production of 1,2-PD from the fermentation of common sugars. As used herein, the term “common sugars” refers to readily available sugars including, but not limited to, arabinose, fructose, galactose, glucose, lactose, maltose, sucrose, and xylose. Specifically excluded from the term “common sugars” are 6-deoxyhexose sugars such as rhamnose and fucose.
While not being limited to a particular cellular mode of action, it is thought that by properly manipulating enzyme activity, intracellular MG i
Altaras Nedim E.
Cameron Douglas C.
Shaw Anita J.
DeWitt Ross & Stevens S.C.
Leone, Esq. Joseph T.
Nashed Nashaat T.
Wisconsin Alumni Research Foundation
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