Methods of monitoring metabolic pathways
View Patent ↗The present invention provides methods and compositions for monitoring cofactors and metabolites of a metabolic pathway of interest. The subject compositions and methods are particularly suited for monitoring the mevalonate pathway in a variety of cells. The invention also provides fermentation methods for the production of isoprenoids.
1. A method of quantitating a cofactor and a mevalonate pathway metabolite, comprising:
(a) providing an extract of a cell comprising a cofactor and a mevalonate pathway metabolite, wherein the extract exhibits a pH value of 8 or greater; and
(b) quantitating said cofactor and said metabolite from said extract.
2. A method of quantitating a cofactor and a mevalonate pathway metabolite, comprising:
(a) quenching cell metabolism in a plurality of cells having at least one cofactor and metabolite by contacting the cells with a quenching solvent resulting in a first mixture, wherein the first mixture has a temperature that is equal to or less than about −25° C.;
(b) adding an extraction solvent to the first mixture resulting in a second mixture wherein the pH of the second mixture has a pH value of 8 or greater; and
(c) quantitating said cofactor and said metabolite from the second mixture.
3. The method in claim 2 wherein the quenching solvent is a liquid at −80° C.
4. The method in claim 2 wherein the quenching solvent comprises methanol and glycerol and remains a liquid at −80° C.
5. The method of claim 2 wherein the metabolite includes at least acetoacetyl CoA and the method further comprises dividing the second mixture into at least two aliquots, wherein one of the aliquot is acidified and used to quantify acetoacetyl CoA and the other aliquot is used to quantitate a cofactor or metabolite that is not acetoacetyl CoA.
6. The method of claim 1 or 2 , wherein the cofactor is selected from the group consisting of NAD + , NADP + , NADH, NADPH, AMP, ADP, and ATP, and wherein the metabolite is selected from the group consisting of farnesyl pyrophosphate, geranyl pyrophosphate, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, mevalonate, mevalonate phosphate, and mevalonate pyrophosphate.
7. The method of claim 1 or 2 , wherein the step of quantitating is effected by mass spectrometry.
8. The method of claim 1 or 2 , wherein the step of quantitating is effected by liquid chromatography in conjunction with mass spectrometry.
9. The method of claim 1 or 2 , wherein the cell is prokaryotic.
10. The method of claim 1 or 2 , wherein the cell is eukaryotic.
11. The method of claim 1 or 2 , wherein the cell is a bacterial cell.
12. The method of claim 1 or 2 , wherein the cell is a yeast cell.
13. A method comprising:
(a) quenching cell metabolism in a plurality of cells having at least one cofactor and metabolite by contacting the cells with a quenching solvent resulting in a first mixture, wherein the first mixture has a temperature that is equal to or less than about −25° C.;
(b) separating the cells from the supernatant in the first mixture resulting in a cell pellet;
(c) isolating the cell pellet;
(d) adding an extraction solvent to the cell pellet resulting in a second mixture wherein the pH of the second mixture has a pH value of 8 or greater;
(e) breaking open the cells in the second mixture;
(f) separating the cell debris from the supernatant in the second mixture;
(g) dividing the supernatant in the second mixture into at least two aliquots;
(h) acidifying one of the at least two aliquots; and,
(i) quantitating the at least one cofactor and metabolite in the at least two aliquots.
14. The method of claim 13 , wherein the acidified aliquot is used to quantitate a CoA or ATP and the non-acidified aliquot is used to quantitate a cofactor or metabolite that is not a CoA or ATP.
15. The method of claim 13 , wherein the acidified aliquot is used to quantitate acetoacetyl CoA and the non-acidified aliquot is used to quantitate a cofactor or metabolite that is not acetoacetyl CoA.
16. The method of claim 13 , wherein the method quantitates each cofactor from the group consisting of NAD+, NADP+, NADH, NADPH, AMP, ADP, and ATP and each metabolite of the mevalonate pathway from the group consisting of farnesyl pyrophosphate, geranyl pyrophosphate, isopentyl pyrophosphate, dimethylallyl pyrophosphate, mevalonate, mevalonate phosphate, and mevalonate pyrophosphate, and wherein the step of quantifying is effected by liquid chromotagraphy in conjunction with mass spectrometry.
17. The method of claim 13 , wherein the cofactor and the metabolite are quantitated with a relative standard deviation (RSD) of less than 13%.