Production and separation of 3-hydroxypropionic acid
The disclosure provides methods and apparatus for producing 3-hydroxypropionic acid or a salt thereof, for removing 3-hydroxypropionic acid from aqueous solution (e.g., aqueous broth), and for using it to make various chemicals.
1. A nucleic acid, comprising a first and a second promoter, wherein either the first is an inducible promoter and is inducible by a small molecule; the first and second promoters are operably linked to a first gene; and the first gene encodes a protein involved in the synthesis of 3-hydroxypropionic acid (3-HP), a salt of 3-HP, or coenzyme B12, wherein a second 10 codons at the 5′-terminus of the first gene are optimized for translation in Pseudomonas denitrificans and a codon frequency for each amino acid in the optimized second 10 codons is more than 0.
2. The nucleic acid of claim 1 , wherein codon optimization comprises:
measuring a codon frequency of each amino acid in a gene encoding a native Pseudomonas denitrificans protein; and
using the codon frequency of the native proteins to replace the second 10 codons at the 5′-terminus of the first gene with an optimized 10 codons, wherein the codon for each amino acid of the optimized 10 codons is present at the same frequency as the codon frequency of the native proteins.
3. The nucleic acid of claim 2 , wherein the nucleic acid comprises a sequence that is 95% identical to a sequence selected from the group consisting of SEQ ID Nos: 29, 30, and 31.
4. The nucleic acid of claim 1 , wherein the first gene comprises a gene encoding first gene is involved in the synthesis of 3-hydroxypropionic acid (3-HP) a salt of 3-HP or coenzyme B 12 .
5. The nucleic acid of claim 4 , wherein the first gene comprises a gene encoding kgsA and comprises a sequence selected from the group consisting of SEQ ID NO: 29-31.
6. The nucleic acid of claim 5 , wherein codon optimization comprises:
measuring a codon frequency of each amino acid in a gene encoding a native Pseudomonas denitrificans protein; and
using the codon frequency of the native proteins to replace the second 10 codons at the 5′-terminus of the first gene with an optimized 10 codons, wherein the codon for each amino acid of the optimized 10 codons is present at the same frequency as the codon frequency of the native proteins.
7. The nucleic acid of claim 3 , wherein the first gene comprises a gene encoding kgsA and comprises a sequence selected from the group consisting of SEQ ID NO: 29-31.
8. The nucleic acid of claim 7 , wherein codon optimization comprises:
measuring a codon frequency of each amino acid in a gene encoding a native Pseudomonas denitrificans protein; and
using the codon frequency of the native proteins to replace the second 10 codons at the 5′-terminus of the first gene with an optimized 10 codons, wherein the codon for each amino acid of the optimized 10 codons is present at the same frequency as the codon frequency of the native proteins.
9. The nucleic acid of claim 2 , wherein the first gene comprises a gene encoding kgsA and comprises a sequence selected from the group consisting of SEQ ID NO: 29-31.
10. The nucleic acid of claim 9 , wherein codon optimization comprises:
measuring a codon frequency of each amino acid in a gene encoding a native Pseudomonas dennrificans protein; and
using the codon frequency of the native proteins to replace the second 10 codons at the 5′-terminus of the first gene with an optimized 10 codons, wherein the codon for each amino acid of the optimized 10 codons is present at the same frequency as the codon frequency of the native proteins.
11. The nucleic acid of claim 1 , wherein the first gene comprises a gene encoding kgsA and comprises a sequence selected from the group consisting of SEQ ID NO: 29-31.
12. The nucleic acid of claim 1 , wherein the nucleic acid comprises a sequence that is 95% identical to a sequence selected from the group consisting of SEQ ID Nos: 29, 30, and 31.
13. The nucleic acid of claim 12 , wherein the first gene comprises a gene encoding first gene is involved in the synthesis of 3-hydroxypropionic acid (3-HP) a salt of 3-HP or coenzyme B 12 .
14. The nucleic acid of claim 1 , wherein the nucleic acid comprises a sequence that is 95% identical to SEQ ID No: 30.
15. The nucleic acid of claim 14 , wherein codon optimization comprises:
measuring a codon frequency of each amino acid in a gene encoding a native Pseudomonas denitrificans protein; and
using the codon frequency of the native proteins to replace the second 10 codons at the 5′-terminus of the first gene with an optimized 10 codons, wherein the codon for each amino acid of the optimized 10 codons is present at the same frequency as the codon frequency of the native proteins.
16. The nucleic acid of claim 1 , wherein the first gene comprises a gene encoding kgsA and comprises a sequence consisting of SEQ ID NO: 30.
17. The nucleic acid of claim 16 , wherein codon optimization comprises:
measuring a codon frequency of each amino acid in a gene encoding a native Pseudomonas denitrificans protein; and
using the codon frequency of the native proteins to replace the second 10 codons at the 5′-terminus of the first gene with an optimized 10 codons, wherein the codon for each amino acid of the optimized 10 codons is present at the same frequency as the codon frequency of the native proteins.
18. The nucleic acid of claim 17 , wherein the first gene comprises a gene encoding first gene is involved in the synthesis of 3-hydroxypropionic acid (3-HP) a salt of 3-HP or coenzyme B 12 .
19. The nucleic acid of claim 16 , wherein the first gene comprises a gene encoding first gene is involved in the synthesis of 3-hydroxypropionic acid (3-HP) a salt of 3-HP or coenzyme B 12 .
20. The nucleic acid of claim 2 , wherein the nucleic acid comprises a sequence that is 95% identical to a sequence selected from the group consisting of SEQ ID Nos: 29, 30, and 31, and wherein the first gene comprises a gene encoding first gene is involved in the synthesis of 3-hydroxypropionic acid (3-HP) a salt of 3-HP or coenzyme B 12 .