Biological methods for preparing adipic acid
The technology relates in part to biological methods for producing adipic acid and engineered microorganisms capable of such production.
1. A method for producing adipic acid, comprising:
(a) contacting a Candida yeast with a feedstock comprising a mixture of fatty acids from a vegetable oil, wherein the Candida yeast comprises genetic alterations that:
(i) reduce or eliminate the amount of a POX4 polypeptide, thereby partially blocking beta oxidation activity,
(ii) add or increase a CYP52A15, CYP52A16, or CYP52A19 monooxygenase activity, and
(iii) add or increase a monooxygenase reductase activity; and
(b) culturing the Candida yeast under conditions in which adipic acid is produced.
2. The method of claim 1 , wherein the vegetable oil is a palm oil.
3. The method of claim 1 , wherein the vegetable oil is a soybean oil.
4. The method of claim 1 , wherein the vegetable oil is a fatty acid soap stock.
5. The method of claim 1 , wherein the Candida yeast is a genetically modified ATCC20336 yeast.
6. The method of claim 1 , wherein the genetic alteration that adds or increases the monooxygenase reductase activity comprises (i) increasing the copy number of a CPRA coding sequence, or (ii) modifying or adding a promoter in operable connection with a CPRA coding sequence.
7. The method of claim 1 , wherein the genetic alteration that adds or increases the monooxygenase reductase activity comprises (i) increasing the copy number of a CPRB coding sequence, or (ii) modifying or adding a promoter in operable connection with a CPRB coding sequence.
8. The method of claim 1 , wherein the genetic alteration that adds or increases the monooxygenase reductase activity comprises (i) increasing the copy number of a CPRA coding sequence, or (ii) modifying or adding a promoter in operable connection with a CPRA coding sequence; and (iii) increasing the copy number of a CPRB coding sequence, or (iv) modifying or adding a promoter in operable connection with a CPRB coding sequence.
9. The method of claim 1 , wherein the Candida yeast comprises a genetic alteration that adds or increases a CYP52A15 monooxygenase activity.
10. The method of claim 9 , wherein the genetic alteration increases the copy number of a polynucleotide that encodes a polypeptide having the CYP52A15 monooxygenase activity.
11. The method of claim 9 , wherein the genetic alteration comprises modifying or adding a promoter in operable connection with a polynucleotide that encodes a polypeptide having the CYP52A15 monooxygenase activity.
12. The method of claim 1 , wherein the Candida yeast comprises a genetic alteration that adds or increases a CYP52A16 monooxygenase activity.
13. The method of claim 12 , wherein the genetic alteration increases the copy number of a polynucleotide that encodes a polypeptide having the CYP52A16 monooxygenase activity.
14. The method of claim 12 , wherein the genetic alteration comprises modifying or adding a promoter in operable connection with a polynucleotide that encodes a polypeptide having the CYP52A16 monooxygenase activity.
15. The method of claim 1 , wherein the Candida yeast comprises a genetic alteration that adds or increases a CYP52A19 monooxygenase activity.
16. The method of claim 15 , wherein the genetic alteration increases the copy number of a polynucleotide that encodes a polypeptide having the CYP52A19 monooxygenase activity.
17. The method of claim 15 , wherein the genetic alteration comprises modifying or adding a promoter in operable connection with a polynucleotide that encodes a polypeptide having the CYP52A19 monooxygenase activity.
18. The method of claim 1 , wherein the adipic acid is produced at a level of at least 0.4 grams per liter of culture medium.