Methods of monitoring metabolic pathways

Abstract

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 - 17 . (canceled) 
     
     
         18 . An extract from a cell comprising one or more cofactors and one or more mevalonate pathway metabolites, wherein the extract exhibits a pH value of 8 or greater and comprises an alcohol, and wherein the cofactor and the metabolite are stable in said extract for at least about 8 hours. 
     
     
         19 . The extract of  claim 18  wherein the one or more cofactors and one or more mevalonate pathway metabolites are stable in said extract for at least about 12 hours. 
     
     
         20 . The extract of  claim 18  wherein the alcohol is methanol. 
     
     
         21 . The extract of  claim 18  that is quenched at a temperature below −23° C. with a quenching solvent that is a liquid at −80° C. 
     
     
         22 - 29 . (canceled) 
     
     
         30 . The extract of  claim 18 , wherein the one or more cofactors are selected from the group consisting of NAD + , NADP + , NADH, NADPH, AMP, ADP and ATP. 
     
     
         31 . The extract of  claim 18 , wherein the one or mevalonate pathway metabolites are selected from the group consisting of acetyl CoA, acetoacetyl-CoA, 3-hydroxy-3-methylglutaryl-CoA, farnesyl pyrophosphate, geranyl pyrophosphate, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, mevalonate, mevalonate phosphate, and mevalonate pyrophosphate. 
     
     
         32 . The extract of  claim 18 , wherein the cell is prokaryotic. 
     
     
         33 . The extract of  claim 18 , wherein the cell is eukaryotic. 
     
     
         34 . The extract of  claim 18 , wherein the cell is a bacterial cell. 
     
     
         35 . The extract of  claim 18 , wherein the cell is a yeast cell. 
     
     
         36 . The extract of  claim 34 , wherein the yeast is  Saccharomyces cerevisiae.    
     
     
         37 . The extract of  claim 18 , wherein the alcohol is ethanol. 
     
     
         38 . The extract of  claim 18 , wherein the extract exhibits a pH value of 9 or greater. 
     
     
         39 . The extract of  claim 18 , wherein the extract further comprises a basic buffer. 
     
     
         40 . The extract of  claim 39 , wherein the basic buffer is an ammonium salt. 
     
     
         41 . The extract of  claim 40 , wherein the ammonium salt is ammonium chloride or ammonium acetate. 
     
     
         42 . The extract of  claim 39 , wherein the ratio of alcohol to basic buffer in the extract is 1:1. 
     
     
         43 . The extract of  claim 39 , wherein the ratio of alcohol to basic buffer in the extract is greater than 2. 
     
     
         44 . The extract of  claim 18 , wherein the cell comprises an enzyme that catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-coenzyme A. 
     
     
         45 . The extract of  claim 44 , wherein the enzyme is acetyl-CoA thiolase. 
     
     
         46 . The extract of  claim 18 , wherein the cell comprises an enzyme that catalyzes the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) from acetyl-coenzyme A and acetoacetyl-CoA. 
     
     
         47 . The extract of  claim 46 , wherein the enzyme is HMG-CoA synthase. 
     
     
         48 . The extract of  claim 18 , wherein the cell comprises an enzyme that catalyzes the formation of mevalonate from HMG-CoA. 
     
     
         49 . The extract of  claim 48 , wherein the enzyme is HMG-CoA reductase. 
     
     
         50 . The extract of  claim 18 , wherein the cell comprises an enzyme that catalyzes the formation of mevalonate 5-phosphate from mevalonate. 
     
     
         51 . The extract of  claim 50 , wherein the enzyme is mevalonate kinase. 
     
     
         52 . The extract of  claim 18 , wherein the cell comprises an enzyme that catalyzes the formation of mevalonate 5-pyrophosphate from mevalonate 5-phosphate. 
     
     
         53 . The extract of  claim 52 , wherein the enzyme is phosphomevalonate kinase. 
     
     
         54 . The extract of  claim 18 , wherein the cell comprises an enzyme that catalyzes the formation of isopentenyl pyrophosphate from mevalonate 5-pyrophosphate. 
     
     
         55 . The extract of  claim 54 , wherein the enzyme is mevalonate pyrophosphate decarboxylase. 
     
     
         56 . The extract of  claim 18 , wherein the cell comprises an enzyme that catalyzes the conversion of isopentenyl pyrophosphate to dimethylallyl pyrophosphate. 
     
     
         57 . The extract of  claim 56 , wherein the enzyme is isopentenyl pyrophosphate isomerase. 
     
     
         58 . The extract of  claim 18 , wherein the cell comprises an enzyme that catalyzes the sequential condensation reactions of dimethylallyl pyrophosphate with two units of 3-isopentenyl pyrophosphate to form farnesyl pyrophosphate. 
     
     
         59 . The extract of  claim 58 , wherein the enzyme is farnesyl pyrophosphate synthase.