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 .mu.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.