Biological methods for preparing a fatty dicarboxylic acid

Abstract

The technology relates in part to biological methods for producing a fatty dicarboxylic acid and engineered microorganisms capable of such production. Provided are engineered microorganisms capable of producing fatty dicarboxylic acids and products expressed by such microorganisms. Also provided are biological methods for producing fatty dicarboxylic acids.

1 - 75 . (canceled) 
     
     
         76 . A genetically modified yeast, comprising a heterologous acyl-CoA oxidase polypeptide or a heterologous acyl-CoA dehydrogenase polypeptide, which yeast is capable of producing a diacid from a feedstock comprising one or more components from a vegetable oil. 
     
     
         77 . The genetically modified yeast of  claim 76 , wherein the heterologous acyl-CoA oxidase polypeptide is a native polypeptide. 
     
     
         78 . The genetically modified yeast of  claim 76 , wherein the heterologous acyl-CoA oxidase polypeptide is an active, modified polypeptide. 
     
     
         79 . The genetically modified yeast of  claim 76 , wherein the heterologous acyl-CoA dehydrogenase polypeptide is a native polypeptide. 
     
     
         80 . The genetically modified yeast of  claim 76 , wherein the heterologous acyl-CoA dehydrogenase polypeptide is an active, modified polypeptide. 
     
     
         81 . The genetically modified yeast of  claim 76 , wherein the heterologous acyl-CoA oxidase polypeptide is selected from the group consisting of: a polypeptide having an amino acid sequence set forth in SEQ ID NO: 51 to SEQ ID NO: 3673. 
     
     
         82 . The genetically modified yeast of  claim 76 , wherein the heterologous acyl-CoA dehydrogenase polypeptide is selected from the group consisting of: SEQ ID NOs: 3679 to 3683, 3686, 3689, 3691, 3693, 3695, 3697, 3699, 3701 and 3703. 
     
     
         83 . The genetically modified yeast of  claim 76 , which is selected from the group consisting of: a  Candida  spp. yeast,  Yarrowia  spp. yeast,  Pichia  spp. yeast,  Saccharomyces  spp. yeast and  Kluyveromyces  spp. yeast. 
     
     
         84 . The genetically modified yeast of  claim 83 , which is selected from the group consisting of:  C. tropicalis, C. viswanathii, Y. lipolytica, P. pastoris, P. membranifaciens, P. kluyveri, P. guilliermondii, P. heedii, P. subpelliculosa, S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis, K. lactis  and  K. marxianus.    
     
     
         85 . The genetically modified yeast of  claim 76 , which comprises a genetic modification that reduces the activity of an enoyl CoA isomerase polypeptide, wherein the genetic modification disrupts a polynucleotide that encodes the enoyl CoA isomerase polypeptide. 
     
     
         86 . (canceled) 
     
     
         87 . The genetically modified yeast of  claim 85 , wherein the enoyl CoA isomerase polypeptide is a polypeptide native to the yeast. 
     
     
         88 . The genetically modified yeast of  claim 87 , wherein the yeast is a  Candida  spp. yeast. 
     
     
         89 . The genetically modified yeast of  claim 88 , wherein the enoyl CoA isomerase polypeptide comprises the amino acid sequence of SEQ ID NO: 3675 or 3677. 
     
     
         90 . (canceled) 
     
     
         91 . The genetically modified yeast of  claim 76 , which comprises a genetic modification that reduces the cytoplasmic or peroxisomal activity of an acyl-CoA synthetase (ACS) polypeptide. 
     
     
         92 - 99 . (canceled) 
     
     
         100 . The genetically modified yeast of  claim 76 , which comprises a genetic modification that reduces the activity of a PXA polypeptide. 
     
     
         101 - 108 . (canceled) 
     
     
         109 . The genetically modified yeast of  claim 76 , comprising one or more genetic modifications that reduce the activity of one or more native endogenous acyl-CoA oxidase polypeptides, wherein the one or more genetic modifications partially block beta oxidation activity. 
     
     
         110 - 134 . (canceled) 
     
     
         135 . An isolated nucleic acid, comprising a polynucleotide that encodes a modified acyl-CoA oxidase polypeptide from a yeast. 
     
     
         136 - 202 . (canceled) 
     
     
         203 . The genetically modified yeast of  claim 91 , wherein the genetic modification reduces the cytoplasmic or peroxisomal activity by disrupting a polynucleotide that encodes the ACS polypeptide, wherein the ACS polypeptide is an ACS1 polypeptide or ACS2 polypeptide. 
     
     
         204 . The genetically modified yeast of  claim 100 , wherein the genetic modification reduces the PXA activity by disrupting a polynucleotide that encodes the PXA polypeptide, wherein the PXA polypeptide is a PXA1 polypeptide or a PXA2 polypeptide, or a PXA1 polypeptide and a PXA2 polypeptide. 
     
     
         205 . A method for producing a diacid, comprising: a) contacting the genetically modified yeast of  claim 76  with a feedstock comprising a vegetable oil, wherein the yeast is capable of producing a diacid from a feedstock comprising one or more components from a vegetable oil; and b) culturing the yeast under conditions in which diacid is produced from the feedstock.