Production of alpha-(R)-(E)-(+)-ionone in recombinant Saccharomyces cerevisiae

Abstract

This invention provides improved biological synthesis of the apocarotenoid α-ionone in Saccharomyces cerevisiae. The final native step involved in the natural apocarotenoid pathway depends on an endogenous farnesyl pyrophosphate synthase (FPPs). From there, heterologous geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene desaturase (crtl), lycopene ε-cyclase (LycE) and a Carotenoid Cleavage Dioxygenase (CCD1) are required to complete the synthesis of α-ionone. Lycopene ε-cyclase from lettuce (Lactuca sativa) or modified cyclase from Arabidopsis thaliana was used to overproduce lycopene which was then cleaved by the carotenoid cleavage dioxygenase from Petunia hybrida (Ph-CCD1).

What is claimed is: 
     
         1 . A method of producing enantiomerically pure (R)-(E)-(+)-alpha-ionone in  Saccharomyces cervisiae  comprising the steps of:
 (a) constructing recombinant yeast cells that overexpress native nucleic acids, or modified versions thereof, encoding at least one enzyme of the mevalonate pathway for synthesizing farnesyl pyrophosphate, wherein expression of the one or more enzymes is under control of constitutive or inducible promoters;   (b) modifying the recombinant yeast cells from step (a) to further comprise heterologous nucleic acids that encode enzymes of an apocarotenoid pathway for synthesizing alpha-ionone, wherein said apocarotenoid pathway includes (i) an enzyme that condenses farnesyl pyrophosphate and isopentenyl pyrophosphate to form geranylgeranyl pyrophosphate, (ii) an enzyme that condenses two molecules of geranylgeranyl pyrophosphate to form phytoene, (iii) an enzyme that converts phytoene to lycopene, (iv) an enzyme that cyclizes lycopene to form δ-carotene and/or ε-carotene, and (v) an enzyme that cleaves δ-carotene and/or ε-carotene to produce alpha-ionone.   
     
     
         2 . The method of  claim 1 , wherein the yeast cells are selected from the group consisting of  Pichia pastoris, Yarrowia lipolytica , and  Saccharomyces cerevisiae.    
     
     
         3 . The method of  claim 1 , wherein the enzyme that condenses farnesyl pyrophosphate with isopentenyl pyrophosphate is a geranylgeranyl pyrophosphate synthase (CrtE). 
     
     
         4 . The method of  claim 3 , wherein the geranylgeranyl pyrophosphate synthase (CrtE) is from  Xanthophyllomyces dendrorhous.    
     
     
         5 . The method of  claim 1 , wherein the enzyme that cyclizes lycopene is lycopene ε-cyclase (LcyE) from one or more of the following:  Zea mays, Chromochloris zofingiensis, Marchantia polymorpha  and  Lactuca sativa.    
     
     
         6 . The method of  claim 1 , wherein the enzyme that cleaves δ-carotene and/or ε-carotene is an oxidoreductase. 
     
     
         7 . The method of  claim 6 , wherein the oxioreductase is selected from the group consisting of monooxygenase, dioxygenase, and peroxidase. 
     
     
         8 . The method of  claim 7 , wherein the monooxygenase and dioxygenase are CCDs. 
     
     
         9 . The method of  claim 6 , wherein the oxioreductase is a CCD1 from  Petunia hybrida, Osmanthus fragrans, Arabidopsis thaliana , or  Vitis vinifera.    
     
     
         10 . The method of  claim 1 , wherein the nucleic acids are expressed using yeast centromeric plasmids, high copy number plasmids or integration plasmids, and integrated into specific and stable yeast genomic sites. 
     
     
         11 . The method of  claim 1 , wherein the alpha-ionone is produced in an amount greater than 1 mg per gram of dry cell weight. 
     
     
         12 . The method of  claim 11 , wherein the alpha-ionone is produced in less than 100 hours. 
     
     
         13 . The method of  claim 1 , wherein the recombinant yeast cells are transformed to include nucleic acids having the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:10. 
     
     
         14 . The method of  claim 1 , wherein the recombinant yeast cells are transformed to include nucleic acids having the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:10.