Compositions and methods of biosynthesizing carotenoids and their derivatives

Abstract

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

1 - 25 . (canceled) 
     
     
         26 . A recombinant microorganism comprising at least one nucleic acid construct comprising:
 a) a nucleic acid sequence encoding a lycopene ϵ-cyclase enzyme encoded by LCYe from  Lactuca sativa ; 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 sequences, and   wherein the microorganism is selected from  Yarrowia lipolytica  and  Saccharomyces cerevisiae , and the microorganism further comprises at least one nucleic acid construct comprising a nucleic acid sequence encoding a geranylgeranyl diphosphate synthase enzyme fused in frame with a farnesyl diphosphate synthase enzyme, wherein the nucleic acid sequence is operably linked to one or more expression control sequences.   
     
     
         27 . The recombinant microorganism of  claim 26 , wherein the nucleic acid construct comprises a nucleic acid sequence encoding a lycopene ϵ-cyclase enzyme with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 87. 
     
     
         28 . The recombinant microorganism of  claim 26 , wherein the microorganism further comprises at least one nucleic acid construct comprising a nucleic acid sequence encoding a phytoene dehydrogenase enzyme from  Mucor circinelloides , wherein the nucleic acid sequence is operably linked to one or more expression control sequences. 
     
     
         29 . The recombinant microorganism of  claim 26 , wherein the microorganism further comprises at least one nucleic acid construct comprising a nucleic acid sequence encoding a phytoene synthase enzyme which is a bifunctional lycopene cyclase/phytoene synthase from  Mucor circinelloides , wherein the nucleic acid sequence is operably linked to one or more expression control sequences. 
     
     
         30 . The recombinant microorganism of  claim 28 , wherein the microorganism further comprises at least one nucleic acid construct comprising a nucleic acid sequence encoding a phytoene synthase enzyme which is a bifunctional lycopene cyclase/phytoene synthase from  Mucor circinelloides , wherein the nucleic acid sequence is operably linked to one or more expression control sequences. 
     
     
         31 . The recombinant microorganism of  claim 26 , wherein the at least one nucleic acid construct further comprises a nucleic acid sequence encoding a phytoene dehydrogenase enzyme from  Mucor circinelloides , wherein the nucleic acid sequence is operably linked to one or more expression control sequences. 
     
     
         32 . The recombinant microorganism of  claim 26 , wherein the at least one nucleic acid construct further comprises a nucleic acid sequence encoding a phytoene synthase enzyme which is a bifunctional lycopene cyclase/phytoene synthase from  Mucor circinelloides , wherein the nucleic acid sequence is operably linked to one or more expression control sequences. 
     
     
         33 . The recombinant microorganism of  claim 31 , wherein the at least one nucleic acid construct further comprises a nucleic acid sequence encoding a phytoene synthase enzyme which is a bifunctional lycopene cyclase/phytoene synthase from  Mucor circinelloides , wherein the nucleic acid sequence is operably linked to one or more expression control sequences. 
     
     
         34 . The recombinant microorganism of  claim 26 , wherein one or more of the nucleic acid sequences of the at least one nucleic acid construct is operably linked to an intron-containing transcriptional elongation factor TEF promoter (TEFIN). 
     
     
         35 . The recombinant microorganism of  claim 26 , wherein one or more of the nucleic acid sequences of the at least one nucleic acid construct is operably linked to an export protein promoter (EXP1). 
     
     
         36 . The microorganism of  claim 26 , wherein the microorganism is selected from a microorganism genetically engineered to inhibit the expression of lycopene β-cyclase and a microorganism naturally not capable of expressing lycopene β-cyclase. 
     
     
         37 . The recombinant microorganism of  claim 26 , wherein the nucleic acid construct is codon optimized for expression in the microorganism. 
     
     
         38 . The recombinant microorganism of  claim 26 , wherein said microorganism comprises lycopene. 
     
     
         39 . The recombinant microorganism of  claim 26 , wherein said microorganism produces α-ionone. 
     
     
         40 . The recombinant microorganism of  claim 26 , wherein said microorganism produces pure R(+)-α-ionone. 
     
     
         41 . A recombinant microorganism comprising at least one nucleic acid construct comprising:
 a) a nucleic acid sequence encoding a lycopene ϵ-cyclase enzyme encoded by LCYe from  Lactuca sativa ; 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 sequences, and   wherein the microorganism is selected from  Yarrowia lipolytica  and  Saccharomyces cerevisiae , and wherein one or more of the nucleic acid sequences of the at least one nucleic acid construct is operably linked to an intron-containing transcriptional elongation factor TEF promoter (TEFIN).   
     
     
         42 . The microorganism of  claim 41 , wherein the microorganism is selected from a microorganism genetically engineered to inhibit the expression of lycopene β-cyclase and a microorganism naturally not capable of expressing lycopene β-cyclase. 
     
     
         43 . The recombinant microorganism of  claim 41 , wherein said microorganism comprises lycopene. 
     
     
         44 . The recombinant microorganism of  claim 41 , wherein said microorganism produces α-ionone. 
     
     
         45 . The recombinant microorganism of  claim 41 , wherein said microorganism produces pure R(+)-α-ionone.