Compositions and methods of biosynthesizing carotenoids and their derivatives

Abstract

The present invention relates to compositions and methods of producing carotenoids and carotenoid derivatives.

What is claimed: 
     
         1 . A recombinant microorganism comprising at least one nucleic acid construct comprising:
 a) a nucleic acid sequence encoding a lycopene β-cyclase enzyme; and   b) a nucleic acid sequence encoding a carotenoid cleavage dioxygenase enzyme;   wherein the nucleic acid sequences are operably linked to one or more expression control sequence, and wherein the microorganism further comprises lycopene.   
     
     
         2 . The microorganism of  claim 1 , wherein the lycopene β-cyclase is selected from a lycopene cyclase enzyme of a bifunctional lycopene cyclase/phytoene synthase encoded by carRP of  Mucor circinelloides , lycopene cyclase/phytoene synthase encoded by carRA of  Phycomyces blakesleeanus , and lycopene cyclase encoded by crtY from  Erwinia herbicola.    
     
     
         3 . The microorganism of  claim 1 , wherein the carotenoid cleavage dioxygenase enzyme is encoded by CCD1 from  Daucus carota.    
     
     
         4 . The microorganism of  claim 1 , wherein the microorganism is selected from a microorganism genetically engineered to inhibit the expression of lycopene α-cyclase and a microorganism naturally not capable of expressing lycopene α-cyclase. 
     
     
         5 . The microorganism of  claim 1 , wherein the microorganism is selected from  Yarrowia lipolytica, Saccharomyces cerevisiae , and  E. coli.    
     
     
         6 . The microorganism of  claim 1 , comprising β-ionone. 
     
     
         7 . The recombinant microorganism of  claim 1 , wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a carotenoid cleavage dioxygenase enzyme with at least 80% identity to an amino acid sequence of SEQ ID NO: 88. 
     
     
         8 . The recombinant microorganism of  claim 1 , wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a lycopene β-cyclase enzyme with at least 80% identity to an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO: 64 and SEQ ID NO: 67. 
     
     
         9 . A recombinant microorganism comprising at least one nucleic acid construct comprising
 a) a nucleic acid comprising a sequence encoding a phytoene synthase enzyme; and   b) a nucleic acid comprising a sequence encoding a phytoene dehydrogenase enzyme;   wherein the nucleic acid sequences are operably linked to one or more expression control sequences.   
     
     
         10 . The microorganism of  claim 9 , wherein the microorganism is selected from  Yarrowia lipolytica  and  Saccharomyces cerevisiae.    
     
     
         11 . The microorganism of  claim 9 , wherein the phytoene synthase enzyme is selected from phytoene synthase of lycopene cyclase/phytoene synthase encoded by carRP of  Mucor circinelloides  and phytoene synthase of lycopene cyclase/phytoene synthase encoded by carRA of  Phycomyces blakesleeanus , wherein the lycopene cyclase/phytoene synthase enzyme is modified to decrease lycopene cyclase activity. 
     
     
         12 . The microorganism of  claim 9 , wherein the phytoene dehydrogenase enzyme is encoded by CarB of  Mucor circinelloides.    
     
     
         13 . The microorganism of  claim 9 , wherein the microorganism further comprises a nucleic acid comprising a sequence encoding a farnesyl diphosphate synthase enzyme and a geranylgeranyl diphosphate synthase enzyme, wherein the nucleic acid sequence is operably linked to one or more expression control sequences. 
     
     
         14 . The microorganism of  claim 13 , wherein the nucleic acid sequence encoding a farnesyl diphosphate synthase enzyme is fused in frame with a geranylgeranyl diphosphate synthase enzyme, and wherein the nucleic acid sequence is operably linked to one or more expression control sequences. 
     
     
         15 . The microorganism of  claim 13 , wherein the microorganism is  Yarrowia lipolytica , the geranylgeranyl diphosphate synthase enzyme is selected from geranylgeranyl diphosphate synthase from  Yarrowia lipolytica  and geranylgeranyl diphosphate synthase from  Xanthophyllomyces dendrorhous , and the farnesyl diphosphate synthase enzyme is from  Yarrowia lipolytica.    
     
     
         16 . The microorganism of  claim 13 , wherein the microorganism is  Saccharomyces cerevisiae , the geranylgeranyl diphosphate synthase enzyme and the farnesyl diphosphate synthase enzyme are from  Saccharomyces cerevisiae.    
     
     
         17 . The microorganism of  claim 13 , wherein the microorganism further comprises a nucleic acid sequence encoding a geranyl pyrophosphate synthase enzyme from  Saccharomyces cerevisiae , wherein the nucleic acid sequence is operably linked to one or more expression control sequences. 
     
     
         18 . The microorganism of  claim 17 , wherein the microorganism further comprises a nucleic acid sequence encoding a truncated 3-hydroxy-3-methyl-glutaryl-CoA reductase (tHMG1) from  Saccharomyces cerevisiae.    
     
     
         19 . The microorganism of  claim 9 , wherein the microorganism is  E. coli.    
     
     
         20 . The microorganism of  claim 19 , wherein the phytoene synthase enzyme and the phytoene dehydrogenase enzyme are from  Erwinia herbicola.    
     
     
         21 . The microorganism of  claim 19 , wherein the microorganism further comprises a nucleic acid comprising a sequence encoding a geranylgeranyl pyrophosphate synthase from  Erwinia herbicola.    
     
     
         22 . The microorganism of  claim 9 , comprising lycopene. 
     
     
         23 . The recombinant microorganism of  claim 9 , wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a phytoene synthase enzyme with at least 80% identity to an amino acid sequence encoded by SEQ ID NO: 65. 
     
     
         24 . The recombinant microorganism of  claim 9 , wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a phytoene dehydrogenase enzyme with at least 80% identity to an amino acid sequence of SEQ ID NO: 61. 
     
     
         25 . The recombinant microorganism of  claim 14 , wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a farnesyl diphosphate synthase enzyme fused in frame with a geranylgeranyl diphosphate synthase enzyme, with at least 80% identity to an amino acid sequence of SEQ ID NO: 74.