Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1987-06-02
1990-07-31
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 681, 435 691, 435 712, 4351721, 4352523, 435320, 435189, 536 27, 935 60, 935 61, C12N 1500, C12P 2100, C07H 1512
Patent
active
049450522
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD OF THE INVENTION
This invention relates to DNA sequences, recombinant DNA molecules, organisms containing such sequences and molecules, the expression of certain enzymes by such organisms, and the production, by fermentation, of a Vitamin C precursor using such organisms and enzymes. More specifically, this invention relates to an expression vehicle and genetically modified organisms, transformed by that vehicle, that express enzymes used to convert glucose, or another carbon source, by fermentation to 2-keto-L-gluconic acid (2-KLG), a chemical precursor to Vitamin C (ascorbic acid).
BACKGROUND ART
There are several processes for producing Vitamin C. One process involves a number of chemical synthesis steps and one fermentation step. Briefly, the steps are hydrogenation of glucose to sorbitol, fermentation of sorbitol to sorbose using Acetobacter suboxydans, sorbose acetonization, diacetone sorbose oxidation to 2-KLG, esterification of 2-KLG, and conversion of the ester to ascorbic acid. This process is complex and requires a relatively high capital investment for an operating plant.
Another process involves two fermentation steps. The process starts with fermentation of glucose to 2,5-diketo-D-gluconate (2,5-DKG) by Erwinia sp.; fermentation of 2,5-DKG to 2-KLG by Corynebacterium sp.; esterification of 2-KLG; and conversion of the ester to ascorbic acid. One study has shown that D-gluconate and 2-keto-D-gluconate (2-KDG) are produced sequentially from glucose by Erwinia sp. before 2,5-DKG is produced in the first fermentation step. See T. Sonoyama et al., "Production of 2-keto-L-gulonic acid from D-glucose by Two-Stage Fermentation," App. and Envir. Microbiol., 43, 1064-69 (1982). This two-step fermentation process, although having a somewhat lower capital cost than the Acetobacter process, is still complex and expensive to operate.
Still another process for converting glucose to 2-KLG is referred to in European patent application No. 132,308. That application refers to the conversion of glucose to 2-KLG in a single step fermentation process. It first refers to Corynebacterium sp. ATCC 31090 as a source of a DNA sequence coding for a particular 2,5-DKG reductase (an enzyme that is said to catalyze the fermentation of 2,5-DKG to 2-KLG). This DNA sequence, with its own or a synthetic ribosome binding site, is then said to be inserted "downstream" of an E. coli trp or tac promoter or the pACYC184 CAT promoter in an expression vector. The vector is also said to contain a gene coding for tetracycline resistance or other selectable marker, and an origin of replication derived from plasmids ColE1, 115A, or RSF 1010. A host cell, Erwinia herbicola (ATCC 21998), is then said to be transformed with the vector. On fermentation this transformed cell is said to produce 2-KLG from glucose in one step. The conversion of glucose to 2-KLG in that process, however, is not fully satisfactory because the yield of 2-KLG is very low and the time of fermentation to obtain even that low yield is too long.
Accordingly, a single organism capable of converting a carbon source, such as glucose, to 2-KLG at acceptable rates and in a single fermentation step is still a goal that has not been attained.
SUMMARY OF THE INVENTION
The present invention solves the problem of finding a single organism capable of converting glucose, or other carbon source, into 2-KLG quickly and in high yield. In one embodiment, this invention provides an expression vehicle capable of transforming a host so that it performs all of the fermentation steps required for converting glucose, or other carbon source, to 2-KLG in a single fermentation at acceptable conversion rates, without intermediate product recovery or intermediate purification steps. The 2-KLG resulting from practice of the present invention may then be esterified and converted to ascorbic acid (Vitamin C), as in the conventional processes described above.
In contrast to the process of the present invention, the known commercial fermentation processes for converting glucose
REFERENCES:
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patent: 3959076 (1976-05-01), Sonoyama et al.
patent: 3963574 (1976-06-01), Sonoyama et al.
patent: 4731328 (1988-03-01), Maxwell
patent: 4757012 (1988-07-01), Estell et al.
patent: 4758514 (1988-07-01), Light et al.
Sonoyama et al., (1982), Apl. Env. Microbiol., 43:1064-1069.
T. Sonoyama et al., "Production of 2-Keto-L-Gulonic Acid from D-Glucose by Two-Stage Fermentation," Appl. Environ. Microbiol., 43, pp. 1064-1069, (1982).
Anderson et al., (1985), Science, 230:145-149.
Chemical Abstract, CA 105(3):23127, (1986), (JP 6163278, 4/1/86).
Grindley June
Hardy Kimber
Payton Mark A.
van de Pol Hendrick
Biogen Inc.
Brown Anne R.
Haley, Jr. James F.
Schwartz Richard A.
Storella John R.
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