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 genetically modified Candida yeast, comprising:
(a) a POX5 polypeptide;
(b) 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, whereby POX4 activity is reduced or removed;
(c) 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, whereby FAT1 activity is reduced or removed, which polynucleotide that encodes the FAT1 polypeptide comprises a nucleotide sequence that is 81% or more identical to SEQ ID NO: 50; and
(d) 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, whereby the ACS1 activity is reduced or removed, which polynucleotide that encodes the ACS1 polypeptide comprises a nucleotide acid sequence that is 84% or more identical to SEQ ID NO: 48.
2. The Candida yeast of claim 1 , wherein the POX4 activity is removed.
3. The Candida yeast of claim 1 , wherein the POX4 polypeptide comprises an amino acid sequence that is 90% or more identical to SEQ ID NO: 39.
4. The Candida yeast of claim 3 , wherein the POX4 polypeptide comprises the amino acid sequence of SEQ ID NO: 39.
5. The Candida yeast of claim 1 , wherein the FAT1 activity is removed.
6. The Candida yeast of claim 1 , wherein the FAT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 51.
7. The Candida yeast of claim 1 , wherein the ACS1 activity is removed.
8. The Candida yeast of claim 1 , wherein the ACS1 polypeptide comprises the amino acid sequence of SEQ ID NO: 49.
9. The Candida yeast of claim 1 , comprising (i) increased copy number of a polynucleotide that encodes the POX5 polypeptide or (ii) a promoter inserted and operably linked to a polynucleotide that encodes the POX5 polypeptide.
10. The Candida yeast of claim 9 , wherein the POX5 polypeptide comprises an amino acid sequence that is 90% or more identical to SEQ ID NO:40.
11. The Candida yeast of claim 10 , wherein the POX5 polypeptide comprises the amino acid sequence of SEQ ID NO: 40.
12. The Candida yeast of claim 1 , comprising (i) increased copy number of a polynucleotide that encodes a CYP52A14, CYP52A15, CYP52A16 or CYP52A19 polypeptide or (ii) a promoter inserted and operably linked to a polynucleotide that encodes a CYP52A14, CYP52A15, CYP52A16 or CYP52A19 polypeptide.
13. The Candida yeast of claim 12 , comprising (i) increased copy number of a polynucleotide that encodes a CYP52A19 polypeptide or (ii) a promoter inserted and operably linked to a polynucleotide that encodes a CYP52A19 polypeptide.
14. The Candida yeast of claim 12 , wherein the polynucleotide that encodes the CYP52A15 polypeptide comprises a nucleotide sequence of SEQ ID NO: 16.
15. The Candida yeast of claim 12 , wherein the polynucleotide that encodes the CYP52A16 polypeptide comprises a nucleotide sequence of SEQ ID NO: 17.
16. The Candida yeast of claim 12 , wherein the polynucleotide that encodes the CYP52A19 polypeptide comprises a nucleotide sequence of SEQ ID NO: 20.
17. The Candida yeast of claim 1 , wherein the yeast that is genetically modified is Candida strain ATCC20692.
18. The Candida yeast of claim 1 , wherein the yeast that is genetically modified is Candida strain ATCC20336.