Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reissue Patent
1999-10-27
2001-09-25
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S140000, C435S145000, C435S252800, C435S849000
Reissue Patent
active
RE037393
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method to produce succinic acid, malic acid or fumaric acid, and more particularly this invention relates to a bacteria that produces high quantities of succinic acid, malic acid and fumaric acid.
2. Background of the Invention
Carboxylic acids hold promise as potential precursors for numerous chemicals. For example, succinic acid can serve as a feedstock for such plastic precursors as 1,4-butanediol (BDO), tetrahydrofuran, and gamma-butyrolactone. New products derived from succinic acid are under constant development, with the most notable of these being polyester which is made by linking succinic acid and BDO. Generally, esters of succinic acids have the potential of being new, “green” solvents that can supplant more harmful solvents and serve as precursors for millions of pounds of chemicals annually at a total market value of over $1 billion. Along with succinic acid, other 4-carbon dicarboxylic acids, such as malic acid, and fumaric acid also have feedstock potential.
The production of these carboxylic acids from renewable feedstocks (in this case through fermentation processes) is an avenue to supplant the more energy intensive methods of deriving such acids from nonrenewable sources. Succinate is an intermediate for anaerobic fermentations by propionate-producing bacteria but those processes result in low yields and concentrations.
Anaerobic rumen bacteria, such as Bacteroides ruminicola and Bacteroides amylophilus also produce succinate. However, rumen organisms are characteristically unstable in fermentation processes.
It has long been known that a mixture of acids are produced from E.coli fermentation, as elaborated in Stokes, J. L. 1949 “Fermentation of glucose by suspensions of Escherichia coli” J. Bacteriol. 5 7:147-158. However, for each mole of glucose fermented, only 1.2 moles of formic acid, 0.1-0.2 moles of lactic acid, and 0.3-0.4 moles of succinic acid are produced. As such, efforts to produce carboxylic acids fermentatively have resulted in relatively large amounts of growth substrates, such as glucose, not being converted to desired product.
Some bacteria, such as A. succiniciproducens, utilized in fermentation processes as outlined in U.S. Pat. No. 5,143,834 to Glassner et al., naturally produce succinic acid in moderate yields. However, this host organism converts at most 1 mole of carbohydrate to 1.33 moles of succinate and 0.67 moles of acetate. Production of the acetate co-product illustrates that one-third of the expensive glucose is not converted to succinate. Furthermore, the A. succiniciproducens host strain has been shown to be not highly osmotolerant in that it does not tolerate high concentrations of salts and is further inhibited by moderate concentrations of product. Lastly, A. succiniciproducens presents handling problems in that as an obligate anaerobe, procedures using the organism must be done in the absence of oxygen. Also, medium preparation for the inoculum requires the addition of tryptophan and also requires the mixing of four different solutions, one of which contains corrosive and toxic H
2
S.
A need exists in the art for a fermentation process to economically produce high amounts of carboxylic acids, such as succinic acid, malic acid and fumaric acid. The process should utilize low cost nutrients and substrates yet provide for high fermentation rates. To effect such a process, an osmotolerant, well-characterized facultative bacterial host is required to yield desired product in up to a 2:1 molar ratio of product-to-growth substrate.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for producing 4-carbon dicarboxylic acids that overcome many of the disadvantages of the prior art.
Yet another object of the present invention is to provide a strain of a facultative organism which produces concentrations of malic acid in the range of 100 grams per liter. A feature of the invention is the combination of a bacterium, which does not metabolize pyruvate to malic acid, with a malic enzyme gene. An advantage of the invention is the exclusive production of malic acid, or the production of malic acid and succinic acid.
Still another object of the present invention is to provide a strain of a facultative organism which produces succinic acid in a ratio of approximately 2:1 succinic to carbohydrate food source. A feature of the invention is the emergence of the strain after selective culturing techniques. An advantage of the invention is the economical production of succinic acid-producing mutants without the need for time consuming genetic manipulations of parent strains.
Briefly, a method for isolating succinic acid producing bacteria is provided comprising isolating a facultative organism lacking the capacity to catabolize pyruvate; increasing the biomass of the organism in an aerobic process; subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow; and isolating the mutants.
The invention also provides for a mutant characterized in that it produces a mixture of succinic acid, acetic acid and ethanol as fermentation products, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E. coli Group of Bacteria. On Aug. 20, 1997, this E. coli mutant was placed with the American Type Culture Collection as deposit #202021 and is designated herein as AFP 111. The ATCC is located at 12301 Parklawn Drive, Rockville, Md. 20852.
REFERENCES:
patent: 5143834 (1992-09-01), Glassner et al.
patent: 5521075 (1996-05-01), Guettler et al.
patent: 5573931 (1996-11-01), Guettler et al.
Donnelly Mark
Millard Cynthia S.
Stols Lucy
Cherskov & Flaynik
Lilling Herbert J.
The University of Chicago
LandOfFree
Mutant E. coli strain with increased succinic acid production does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Mutant E. coli strain with increased succinic acid production, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutant E. coli strain with increased succinic acid production will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2517987