Methods of production of products of metabolic pathways

Abstract

A plurality of isolated polynucleotide sequences encoding enzymes of the astaxanthin pathway is disclosed. The polynucleotides include: (i) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and a first transcriptional regulatory sequence; (ii) a polynucleotide which encodes Beta-lycopene cyclase (lcy-B) and a second transcriptional regulatory sequence; (iii) a polynucleotide which encodes Beta-carotene ketolase (crtW) and a third transcriptional regulatory sequence; and wherein the first, second and third regulatory sequence are selected such that the expression of the Icy-B and the crtW is greater than a level of expression of the crtI. Methods of generating astaxanthin using the plurality of polynucleotide are also disclosed as well as bacterial cells comprising high levels of astaxanthin.

What is claimed is: 
     
         1 . A method of generating astaxanthin comprising expressing polynucleotides encoding enzymes of an astaxanthin pathway, the polynucleotides comprising:
 (i) a polynucleotide which encodes Phytoene dehydrogenase (crtI) and a first transcriptional regulatory sequence;   (ii) a polynucleotide which encodes Beta-lycopene cyclase (lcy-B) and a second transcriptional regulatory sequence;   (iii) a polynucleotide which encodes Beta-carotene ketolase (crtW) and a third transcriptional regulatory sequence; and   wherein said first, second and third regulatory sequence are selected such that the expression of said lcy-B and said crtW is greater than a level of expression of said crtI.   
     
     
         2 . The method of  claim 1 , wherein each of said first regulatory sequence, said second regulatory sequence and said third regulatory sequence is a ribosome binding site (RBS). 
     
     
         3 . The method of  claim 1 , wherein said regulatory sequences are selected such that the expression of said lcy-B and said crtW is at least five times greater than a level of expression of said crtI. 
     
     
         4 . The method of  claim 1 , wherein said regulatory sequences are selected such that the expression of said lcy-B and said crtW is at least ten times greater than a level of expression of said crtI. 
     
     
         5 . The method of  claim 1 , further comprising introducing into the cell a polynucleotide encoding a deoxyxylulose-5-phosphate synthase (DXS). 
     
     
         6 . The method of  claim 1 , wherein said expressing is effected in a bacterial cell. 
     
     
         7 . The method of  claim 2 , wherein each of said RBS is flanked by a spacer sequence. 
     
     
         8 . The method of  claim 1 , wherein each of said polynucleotides are comprised on a single expression vector. 
     
     
         9 . The method of  claim 1 , further comprising isolating the astaxanthin following said expressing. 
     
     
         10 . An isolated polynucleotide comprising:
 (i) a first RBS operatively linked to a first enzyme coding sequence;   (ii) a second RBS operatively linked to a second enzyme coding sequence; and   (iii) a third RBS operatively linked to a third enzyme coding sequence;   wherein said second RBS is selected such that the level of expression of said second enzyme coding sequence is greater than the level of expression of said first enzyme coding sequence;   wherein said third RBS is selected such that the level of expression of said third enzyme coding sequence is greater than the level of expression of said second enzyme coding sequence;   wherein said first enzyme, said second enzyme and said third enzyme are non-identical enzymes and each part of a biosynthesis pathway of an identical product of interest.   
     
     
         11 . The isolated polynucleotide of  claim 10 , wherein each of said RBS is flanked by a spacer sequence. 
     
     
         12 . The isolated polynucleotide of  claim 10 , wherein said product of interest is a protein. 
     
     
         13 . The isolated polynucleotide of  claim 10 , wherein said product of interest is selected from the group consisting of a food product, a pharmaceutical and a fuel.