Biological methods for preparing adipic acid

Abstract

The technology relates in part to biological methods for producing adipic acid and engineered microorganisms capable of such production.

1 - 213 . (canceled) 
     
     
         214 . An engineered yeast comprising modifications that add or increase a hexanoate synthase activity, a monooxygenase activity, and a monooxygenase reductase activity, which yeast is capable of producing a diacid from a sugar. 
     
     
         215 . The engineered yeast of  claim 214 , which comprises increased copies of a polynucleotide encoding a polypeptide having the hexanoate synthase activity, the monooxygenase activity, or the monooxygenase reductase activity. 
     
     
         216 . The engineered yeast of  claim 214 , which comprises a heterologous promoter and/or 5′ UTR in genomic DNA in functional connection with a polynucleotide encoding a polypeptide having the hexanoate synthase activity, the monooxygenase activity, or the monooxygenase reductase activity. 
     
     
         217 . The engineered yeast of  claim 214 , wherein the hexanoate synthase activity is provided by a heterologous polypeptide. 
     
     
         218 . The engineered yeast of  claim 217 , wherein the heterologous polypeptide is encoded by a hexonate synthase subunit alpha (HEXA) gene, hexanoate synthase subunit beta (HEXB) gene, or a hexonate synthase subunit alpha (HEXA) gene and hexanoate synthase subunit beta (HEXB) gene. 
     
     
         219 . The engineered yeast of  claim 218 , wherein the HEXA gene, HEXB gene, or HEXA gene and HEXB gene are from an  Aspergillus  spp. fungus. 
     
     
         220 . The engineered yeast of  claim 219 , wherein the  Aspergillus  spp. fungus is  Aspergillus parasiticus.    
     
     
         221 . The engineered yeast of  claim 214 , which includes an increased number of copies of an endogenous polynucleotide that encodes a polypeptide having the monooxygenase activity. 
     
     
         222 . The engineered yeast of  claim 221 , wherein the endogenous polynucleotide encodes a CYP52A15 polypeptide, CYP52A16 polypeptide or CYP52A19 polypeptide. 
     
     
         223 . The engineered yeast of  claim 214 , which comprises a heterologous promoter in genomic DNA in functional connection with an endogenous polynucleotide that encodes a polypeptide having the monooxygenase activity. 
     
     
         224 . The engineered yeast of  claim 223 , wherein the endogenous polynucleotide encodes a CYP52A15 polypeptide, CYP52A16 polypeptide or CYP52A19 polypeptide. 
     
     
         225 . The engineered yeast of  claim 214 , which includes an increased number of copies of an endogenous polynucleotide that encodes a polypeptide having the monooxygenase reductase activity. 
     
     
         226 . The engineered yeast of  claim 225 , wherein the endogenous polynucleotide encodes a CPRA polypeptide or CPRB polypeptide. 
     
     
         227 . The engineered yeast of  claim 214 , which comprises a heterologous promoter in genomic DNA in functional connection with an endogenous polynucleotide that encodes a polypeptide having the monooxygenase reductase activity. 
     
     
         228 . The engineered yeast of  claim 227 , wherein the endogenous polynucleotide encodes a CPRA polypeptide or CPRB polypeptide. 
     
     
         229 . The engineered yeast of  claim 214 , which is a  Candida  spp. yeast. 
     
     
         230 . The engineered yeast of  claim 214 , which is a  Yarrowia  spp. yeast. 
     
     
         231 . The engineered yeast of  claim 230 , wherein the  Yarrowia  spp. yeast is  Yarrowia lipolytica.    
     
     
         232 . The engineered yeast of  claim 214 , wherein the diacid is adipic acid.