Metabolically engineered organisms for the production of added value bio-products
The present invention relates to genetically engineered organisms, especially microorganisms such as bacteria and yeasts, for the production of added value bio-products such as specialty saccharide, activated saccharide, nucleoside, glycoside, glycolipid or glycoprotein. More specifically, the present invention relates to host cells that are metabolically engineered so that they can produce said valuable specialty products in large quantities and at a high rate by bypassing classical technical problems that occur in biocatalytical or fermentative production processes.
1. A metabolically engineered bacterium or yeast for production of fructose-6-phosphate or GDP-fucose, characterized in that said bacterium or yeast:
a) has been genetically modified by introducing a heterologous gene encoding a carbohydrate hydrolase capable of splitting sucrose into glucose and fructose;
b) comprises a fructokinase to catalyze conversion of fructose to fructose-6-phosphate; and
c) has been further genetically modified to prevent loss of fructose-6-phosphate via glycolysis due to genetic disruption of an endogenous gene encoding a phosphofructokinase, a phosphoglucose isomerase, or a combination thereof
and wherein said bacterium or yeast comprises:
(i) a mannose-6-phosphate isomerase to catalyze conversion of fructose-6-phosphate to mannose-6-phosphate,
(ii) a phosphomannomutase to catalyze conversion of mannose-6-phosphate to mannose-1-phosphate;
(iii) a mannose-1-phosphate guanylyltransferase to catalyze conversion of mannose-1-phosphate to GDP-mannose;
(iv) a GDP mannose dehydratase to catalyze conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-mannose; and
(v) a GDP fucose synthase to catalyze conversion of GDP-4-dehydro-6-deoxy-mannose to GDP-fucose.
2. The metabolically engineered bacterium or yeast according to claim 1 , wherein an endogenous gene encoding a phosphofructokinase has been disrupted.
3. The metabolically engineered bacterium or yeast according to claim 1 , wherein an endogenous gene encoding a phosphoglucose isomerase has been disrupted.
4. The metabolically engineered bacterium or yeast according to claim 1 , wherein an endogenous gene encoding a phosphofructokinase and an endogenous gene encoding a phosphoglucose isomerase have been disrupted.
5. The metabolically engineered bacterium or yeast according to claim 1 , wherein said bacterium or yeast has been further genetically modified to prevent loss of fructose-6-phosphate via conversion to fructose due to genetic disruption of an endogenous gene encoding a phosphatase.
6. The metabolically engineered bacterium or yeast according to claim 1 , wherein said bacterium or yeast comprises a hexokinase to catalyze conversion of glucose to glucose-6-phosphate.
7. The metabolically engineered bacterium or yeast according to claim 6 , wherein said hexokinase is a glucokinase.
8. The metabolically engineered bacterium or yeast according to claim 6 , wherein said bacterium or yeast has been further genetically modified to promote conversion of glucose-6-phosphate into biomass by genetic disruption of an endogenous gene encoding a phosphoglucomutase, an endogenous gene encoding a glucose-1-phosphate adenylyltransferase, or a combination thereof.
9. The metabolically engineered bacterium or yeast according to claim 1 , wherein said carbohydrate hydrolase is an invertase.
10. The metabolically engineered bacterium or yeast according to claim 1 , wherein said bacterium or yeast has been further genetically modified by introducing one or more heterologous gene selected from the group consisting of: a heterologous gene encoding a mannose-6-phosphate isomerase, a heterologous gene encoding a phosphomannomutase, a heterologous gene encoding a mannose-1-phosphate guanylyltransferase, a heterologous gene encoding a GDP mannose, and a heterologous gene encoding a GDP fucose synthase.
11. The metabolically engineered bacterium or yeast according to claim 1 , wherein said bacterium is Escherichia coli or wherein said yeast is Saccharomyces cerevisiae.
12. The metabolically engineered bacterium or yeast according to claim 1 , wherein the metabolically engineered bacterium or yeast is a metabolically engineered bacterium.
13. The metabolically engineered bacterium or yeast according to claim 12 , wherein the bacterium is Escherichia coli.
14. The metabolically engineered bacterium or yeast according to claim 1 , wherein the metabolically engineered bacterium or yeast is a metabolically engineered yeast.
15. The metabolically engineered bacterium or yeast according to claim 14 , wherein the yeast is Saccharomyces cerevisiae.
16. The metabolically engineered bacterium or yeast according to claim 1 , wherein said phosphofructokinase catalyzes conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, and said phosphoglucose isomerase catalyzes conversion of fructose-6-phosphate to glucose-6-phosphate.
17. A method for the production of fructose-6-phosphate or GDP-fucose, comprising the steps of:
i) cultivating the metabolically engineered bacterium or yeast according to claim 1 , and
ii) extracting and purifying the GDP-fucose or fructose-6-phosphate.