US2015087035A1PendingUtilityA1

Biological methods for preparing adipic acid

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Assignee: VERDEZYNE INCPriority: May 3, 2011Filed: Sep 21, 2012Published: Mar 26, 2015
Est. expiryMay 3, 2031(~4.8 yrs left)· nominal 20-yr term from priority
Inventors:Stephen PicataggioTom Beardslee
C12N 9/0042C12N 9/0073C12P 7/44C12N 9/001C12Y 103/03006C12Y 602/01001C12Y 602/01003
52
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Claims

Abstract

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

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method for producing adipic acid, comprising:
 (a) contacting a  Candida  yeast with a feedstock comprising a vegetable oil, wherein the  Candida  yeast comprises:
 (1) a genetic alteration that reduces or eliminates POX4 activity but not POX5 activity, 
 (2) a genetic alteration that reduces or eliminates an ACS1 acyl-CoA synthetase activity, and 
 (3) a genetic alteration that reduces or eliminates a FAT1 long chain acyl-CoA synthetase activity; and 
   (b) culturing the  Candida  yeast under conditions in which adipic acid is produced at a yield of 2.20 grams per liter of culture medium or greater.   
     
     
         2 . The method of  claim 1 , wherein the adipic acid is produced at a yield of 2.53 grams per liter of culture medium or greater. 
     
     
         3 . The method of  claim 1 , wherein the adipic acid is produced at a yield of 3.73 grams per liter of culture medium or greater. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the vegetable oil is a palm oil, soybean oil or coconut oil. 
     
     
         5 . The method of any one of  claims 1  to  3 , wherein the vegetable oil comprises oleic acid. 
     
     
         6 . The method of any one of  claims 1  to  3 , wherein the vegetable oil is a soap stock. 
     
     
         7 . The method of any one of  claims 1  to  6 , wherein the POX4 activity is eliminated. 
     
     
         8 . The method of  claim 7 , wherein the  Candida  yeast comprises a disruption, deletion or knockout of (i) a polynucleotide that encodes a POX4 polypeptide or (ii) a promoter operably linked to a polynucleotide that encodes a POX4 polypeptide. 
     
     
         9 . The method of  claim 8 , wherein the POX4 polypeptide comprises the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 39. 
     
     
         10 . The method of any one of  claims 1  to  9 , wherein the  Candida  yeast comprises a genetic alteration that increases the POX5 activity. 
     
     
         11 . The method of  claim 10 , wherein the POX5 activity is increased by an increased amount of an enzyme comprising (i) the amino acid sequence of SEQ ID NO: 40, (ii) an amino acid sequence 90% or more identical to (i), or (iii) an amino acid sequence that includes 1 to 10 amino acid substitutions, insertions or deletions with respect to (i). 
     
     
         12 . The method of any one of  claims 1  to  11 , wherein the genetic alteration of (3) eliminates the FAT1 long chain acyl-CoA synthetase activity. 
     
     
         13 . The method of  claim 12 , wherein the  Candida  yeast comprises a disruption, deletion or knockout of (i) a polynucleotide that encodes a FAT1 polypeptide or (ii) a promoter operably linked to a polynucleotide that encodes a FAT1 polypeptide. 
     
     
         14 . The method of  claim 13 , wherein the FAT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 51. 
     
     
         15 . The method of  claim 13 , wherein the FAT1 polypeptide is encoded by a polynucleotide comprising a nucleotide sequence that is 81% or more identical to SEQ ID NO: 50. 
     
     
         16 . The method of any one of  claims 1  to  15 , wherein the genetic alteration of (2) eliminates the ACS1 acyl-CoA synthetase activity. 
     
     
         17 . The method of  claim 16 , wherein the  Candida  yeast comprises a disruption, deletion or knockout of (i) a polynucleotide that encodes a ACS1 polypeptide or (ii) a promoter operably linked to a polynucleotide that encodes a ACS1 polypeptide. 
     
     
         18 . The method of  claim 17 , wherein the ACS1 polypeptide comprises the amino acid sequence of SEQ ID NO: 49. 
     
     
         19 . The method of  claim 17 , wherein the ACS1 polypeptide is encoded by a polynucleotide comprising a nucleotide acid sequence that is 84% or more identical to SEQ ID NO: 48. 
     
     
         20 . The method of any one of  claims 1  to  19 , wherein  Candida  yeast comprises a genetic modification that increases an acyl-CoA hydrolase activity. 
     
     
         21 . The method of  claim 20 , wherein the genetic modification that increases the acyl-CoA hydrolase activity increases an ACH activity. 
     
     
         22 . The method of  claim 21 , wherein the ACH activity is an ACHA activity, an ACHB activity or an ACHA and an ACHB activity. 
     
     
         23 . The method of  claim 22 , wherein the ACHA activity is provided by an increased amount of an enzyme comprising (i) the amino acid sequence of SEQ ID NO: 43, (ii) an amino acid sequence 90% or more identical to (i), or (iii) an amino acid sequence that includes 1 to 10 amino acid substitutions, insertions or deletions with respect to (i), and the ACHB activity is provided by an increased amount of an enzyme comprising (iv) the amino acid sequence of SEQ ID NO: 45, (v) an amino acid sequence 90% or more identical to (iv), or (vi) an amino acid sequence that includes 1 to 10 amino acid substitutions, insertions or deletions with respect to (iv). 
     
     
         24 . The method of any one of  claims 1  to  23 , wherein the  Candida  yeast comprises a genetic modification that increases one or more monooxygenase activities. 
     
     
         25 . The method of embodiment  24 , wherein the one or more monooxygenase activities are chosen from: a CYP52A13 activity, a CYP52A14 activity, a CYP52A15 activity, a CYP52A16 activity, and a CYP52A19 activity. 
     
     
         26 . The method of  claim 25 , wherein the CYP52A13 activity is provided by an increased amount of an enzyme comprising (i) the amino acid sequence of SEQ ID NO: 63, (ii) an amino acid sequence 90% or more identical to (i), or (iii) an amino acid sequence that includes 1 to 10 amino acid substitutions, insertions or deletions with respect to (i). 
     
     
         27 . The method of  claim 25 , wherein the CYP52A14 activity is provided by an increased amount of an enzyme comprising (i) the amino acid sequence of SEQ ID NO: 65, (ii) an amino acid sequence 90% or more identical to (i), or (iii) an amino acid sequence that includes 1 to 10 amino acid substitutions, insertions or deletions with respect to (i). 
     
     
         28 . The method of  claim 25 , wherein the CYP52A15 activity is provided by an increased amount of an enzyme comprising (i) the amino acid sequence of SEQ ID NO: 67, (ii) an amino acid sequence 90% or more identical to (i), or (iii) an amino acid sequence that includes 1 to 10 amino acid substitutions, insertions or deletions with respect to (i). 
     
     
         29 . The method of  claim 25 , wherein the CYP52A16 activity is provided by an increased amount of an enzyme comprising (i) the amino acid sequence of SEQ ID NO: 69, (ii) an amino acid sequence 90% or more identical to (i), or (iii) an amino acid sequence that includes 1 to 10 amino acid substitutions, insertions or deletions with respect to (i). 
     
     
         30 . The method of  claim 25 , wherein the CYP52A19 activity is provided by an increased amount of an enzyme comprising (i) the amino acid sequence of SEQ ID NO: 75, (ii) an amino acid sequence 90% or more identical to (i), or (iii) an amino acid sequence that includes 1 to 10 amino acid substitutions, insertions or deletions with respect to (i). 
     
     
         31 . The method of any one of  claims 1  to  30 , wherein the  Candida  yeast comprises a genetic modification that increases a monooxygenase reductase activity. 
     
     
         32 . The method of  claim 31 , wherein the genetic modification that increases the monooxygenase reductase activity increases a CPR activity. 
     
     
         33 . The method of  claim 31 , wherein the CPR activity is a CPRB activity. 
     
     
         34 . The method of  claim 33 , wherein the CPRB activity is provided by an increased amount of an enzyme comprising (i) the amino acid sequence of SEQ ID NO: 81, (ii) an amino acid sequence 90% or more identical to (i), or (iii) an amino acid sequence that includes 1 to 10 amino acid substitutions, insertions or deletions with respect to (i). 
     
     
         35 . The method of any one of  claims 1  to  34 , wherein the  Candida  yeast is a genetically modified  Candida  strain ATCC20692. 
     
     
         36 . A method for producing adipic acid, comprising:
 (a) contacting a  Yarrowia  yeast with a feedstock comprising a vegetable oil, wherein the  Yarrowia  yeast comprises:
 (1) a genetic alteration that reduces or eliminates POX4 activity but not POX5 activity, 
 (2) a genetic alteration that reduces or eliminates an ACS1 acyl-CoA synthetase activity, and 
 (3) a genetic alteration that reduces or eliminates a FAT1 long chain acyl-CoA synthetase activity; and 
   (b) culturing the yeast under conditions in which adipic acid is produced at a yield of 2.20 grams per liter of culture medium or greater.   
     
     
         37 . The method of  claim 36 , wherein the  Yarrowia  yeast is a  Yarrowia lipolytica  yeast.

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