Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1998-11-30
2001-08-28
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S252300, C435S252330, C435S320100
Reexamination Certificate
active
06280986
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to improved recombinant bacteria transformed with heterologous DNA coding for alcohol dehydrogenase and pyruvate decarboxylase, which are effective for use in the production of ethanol.
2. Description of the Prior Art
In the last decade, a major goal of biofuels research has been to metabolically engineer microorganisms to ferment multiple sugars from biomass or agricultural wastes to fuel ethanol. L. O. Ingram and his colleagues were first to report a metabolically engineered bacterium for high levels of alcohol production (U.S Pat. Nos. 5,000,000; 5,424,202; and 5,482,846). The cloned
Zymomonas mobilis
genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) were combined to form the PET operon which was transformed into
Escherichia coli
on pUC-based plasmid to form pLOI295 (Ingram et al., 1987, Appl Environ. Microbiol., 53:2420-2425). The resultant recombinant strain produced over 3.4% wt/v ethanol from glucose in media containing ampicillin (Amp) with positive selection pressure for the plasmid. PET operon plasmids and gene expression were improved by changes in promoters (Ingram and Conway, 1988, Appl. Environ. Microbiol., 54:397-404), insertion of a tetracycline (Tc) gene to form pLOI297 for plasmid selection and selection of hardier
E. coli
strains (Alterthum and Ingram, 1989, Appl. Environ. Microbiol., 55:1943-1948). Although the last workers showed
E. coli
B maintained pLOI297 for 25 generations in the absence of Tc, we have found rapid plasmid losses after this time and recently Lawford and Rousseau (1995, Biotechnol. Lett., 17:751-756; and 1995, Appl. Biochem. Biotechnol., 57/58:277-292) have shown even more rapid declines in levels of ethanol production. A considerably more stable strain was developed by Ohta et al. (1991, Appl. Environ. Microbiol., 57:893-900) by integrating the PET operon and chloramphenicol (Cm) resistance gene into the
E. coli
chromosome. The resultant
E. coli
strains did not require Cm in the growth media for retention of the PET operon, but ethanol production levels were much lower in the absence of Cm, presumably due to reduced PET gene copy number. When mutants were selected for resistance to high levels (600 &mgr;g/ml ) of Cm, high levels of ethanol production were restored as shown with strain KO11. However, Lawford and Rousseau (ibid) have found that these mutants also lose the ability to produce high levels of ethanol in the absence of Cm.
Although the bacteria genetically engineered to contain the PET operon produce high levels of ethanol, these recombinants require antibiotics in the growth media to maintain genetic stability and high ethanol productivity. In the absence of such antibiotics the bacterial population rapidly lose the ability to produce high levels of ethanol.
SUMMARY OF THE INVENTION
We have now discovered recombinant bacteria transformed with heterologous DNA coding for alcohol dehydrogenase (adh) and pyruvate decarboxylase (pdc), which are effective for use in the production of ethanol, but which do not require the presence of antibiotics in the culture medium to maintain genetic stability and high ethanol productivity. These recombinant bacteria are produced using mutant host strains which are substantially deficient in the ability to fermentatively reduce pyruvate. When grown in an anaerobic environment, the recombinant pyruvate mutants transformed with the adh and pdc genes are genetically stable, maintaining the inserted genes and ethanol productivity even in the absence of antibiotics.
In accordance with this discovery, it is an object of this invention to provide improved recombinant bacteria that are effective for use in the production of ethanol.
It is also an object of this invention to provide recombinant bacteria that are genetically stable and maintain high ethanol productivity in an anaerobic environment in the absence of antibiotics or other chemical selective agents.
REFERENCES:
patent: 5000000 (1991-03-01), Ingram et al.
patent: 5424202 (1995-06-01), Ingram et al.
patent: 5482846 (1996-01-01), Ingram et al.
Fairoz Mat-Jan et al. “Mutants ofEscherichia coliDeficient in the Fermentative Lactate Dehydrogenase”Journal of Bacteriology, vol. 171, No. 1 (Jan. 1989) pp. 342-348.
R.B. Hespell et al. “Stabilization of pet Operon Plasmids and Ethanol Production inEscherichia coliStrains Lacking Lactate Dehydrogenase and Pyruvate Formate-Lyase Activities”Applied and Environmental Microbiology, vol. 62, No. 12 (Dec. 1996) pp. 4594-4597.
Wu, G. et al., MutantsE. coliaffected in respiration...Journal of General Microbiology 139 (8), pp. 1795-1805, Apr. 1993.*
Ohta, K. et al. Genetic Improvement ofE. coli...Applied and Environmental Microbiology 57(4) pp. 893-900, Apr. 1991.
Bothast Rodney J.
Dien Bruce S.
Hespell Robert B.
Odelson David A
Wyckoff Herbert A.
Deck Randall E.
Fado John D.
Gansheroff Lisa
Odelson David A
Silverstein M. Howard
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