Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1996-09-19
1998-07-28
Grimes, Eric
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
4352542, 43525421, 4353201, 536 232, C12N 119, C12N 1552, C12N 1563, C12P 706
Patent
active
057861862
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to recombinant DNA and to genetically-modified yeast for use in alcoholic fermentation.
BACKGROUND OF THE INVENTION
The familiar glycolytic pathway in Saccharomyces cerevisiae, in which sugars are converted into alcohol (the Embden-Meyerhof pathway; see Sequence A, below) has been studied intensively for many years because of its importance to the brewing, distilling, wine-making and baking industries, as well as for its inherent academic fascination. In the past, much effort has been devoted towards finding ways to get yeast to make more alcohol. This has taken two main directions: (1) the development of process monitoring and control procedures for product optimisation; and (2) strain selection and improvement programmes. Too often such ventures, having concentrated solely on the glycolytic pathway, have failed to take account of the physiological significance of the pathway to yeast.
Cuskey et al, J. Bacteriol. (June 1985) 162(3): 865-871, disclose the cloning of various genes specifying carbohydrate catabolism into strains of Pseudomonas. P. aeruginosa metabolises carbohydrates through the Entner-Doudoroff pathway (see Sequence B, below), and includes genes for glucose-6-phosphate dehydrogenase (ZWF), 6-phosphogluconate dehydratase (PGD) and 2-keto-3-deoxy-6-phosphogluconate aldolase (KGA); Cuskey et al produced a recombinant plasmid containing the genes for PGD and also glucokinase (GLK), but report that the structural genes for ZWF and KGA were not present on the cloned fragment.
Kawasaki et al, Biochem. Biophys. Res. Comm. (1982) 108(3):1107-1112, describe the cloning of various yeast glycolysis genes by complementation.
Banerjee et al, J. Gen. Microbiol. (1987) 133: 1099-1107, describe gluconeogenic mutations in P. aeruginosa, and recombinant plasmids carrying genes for fructose-bisphosphate aldolase (FBA), 3-phosphoglycerate kinase (PGK), NADP-linked glyceraldehyde-3-phosphate dehydrogenase (GAP) or KGA.
WO-A-8703006 describes yeasts having modified glycolysis rates, in order to increase the production of CO.sub.2, ethanol and other fermentation products, and to decrease biomass production.
The sequences of the eda and edd genes (encoding PGD and KGA) in E. coli are reported by Egan et al, J. Bacteriol. 174:4638-4646 (1992). Corrections to these sequences are disclosed by Carter et al, Gene 130:155-6 (1993).
The production of fuel alcohol by bacterial systems, e.g. Xymomonas, is known. Such systems have a narrow substrate range and low alcohol tolerance.
An object behind the present invention is to modify yeast to enable more efficient conversion of sugars to ethanol, and consequently reduced energy benefit to the yeast cell.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, yeast is genetically modified such that it is capable of expressing PGD (Enzyme Commission No. 4.2.1.12) and KGA (Enzyme Commission No. 4.1.2.14).
In addition to being able to express at least the enzymes given in Sequence A, and thereby convert a sugar to ethanol, unmodified yeast can express ZWF and phosphogluconate lactonase (PGL) which are the first two enzymes in Sequence B which are different from Sequence A. Accordingly, yeast of the invention provides a means whereby a sugar which is a conventional substrate for yeast-catalysed fermentation can be converted to pyruvate via Sequence B, i.e. the Entner-Doudoroff pathway.
According to a second aspect, novel recombinant DNA encodes PGD and KGA, and includes one or more sequences which promote transcription and translation of the enzymes in yeast.
DESCRIPTION OF THE INVENTION
The present invention is based on the observation that the production of ethanol is not the raison d'etre of a yeast cell. Ethanol is the waste product of an energy-yielding process that operates under conditions of low oxygen tension or high sugar concentration, enabling the yeast to grow and divide.
Energy yielded by yeast cultivation in the form of purine nucleoside triphosphates (such as ATP) is intended for cellular biosy
REFERENCES:
Temple et al., Bacteriol. 172:6396-6402, 1990.
Fraenkel, in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Amer. Soc. Microbiol., Washington, DC, pp. 142-150, 1987.
J. Bacteriol. (1992) 174 (14), 4638-46.
Mol. Microbiol. (1991), 5(12), 2901-11.
Dickinson John Richard
Lancashire William Edward
Malloch Richard Anthony
Grimes Eric
University College Cardiff Consultants Ltd.
Whitbread PLC
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