Transformed microorganism for producing PHA copolymer comprising 3HH monomer unit at high composition rate and method for producing PHA using same
The present application provides: a transformed microorganism for producing a PHA copolymer containing 3HH monomer unit at a higher composition ratio, specifically, a transformed microorganism comprising a PHA synthase gene capable of synthesizing a PHA copolymer containing 3HH monomer unit and a gene encoding a protein having (R)-specific enoyl-CoA hydratase activity, characterized in that, in the transformed microorganism, the expression of a gene encoding at least one β-ketothiolase enzyme having thiolysis activity for β-keto-(C6) acyl-CoA (i.e., β-ketohexanoyl-CoA) is inhibited, thereby losing or reducing the enzyme activity; and a method for producing a PHA copolymer containing 3HH monomer unit, comprising a step of culturing the transformed microorganism.
1. A transformed microorganism that is a bacterium belonging to the genus Cupriavidus , comprising:
a PHA synthase gene capable of synthesizing a polyhydroxyalkanoate (PHA) copolymer comprising 3-hydroxyhexanoate (3HH) monomer unit and 3-hydroxybutanoate (3HB) monomer unit; and
a gene encoding a protein having (R)-specific enoyl-CoA hydratase activity,
wherein in the transformed microorganism, at least two genes encoding a β-ketothiolase enzyme are disrupted such that the activity of the β-ketothiolase enzyme having thiolysis activity for β-ketohexanoyl-CoA is reduced or lost as compared to a corresponding non-transformed microorganism, resulting in increase in a 3HH ratio in a PHA copolymer produced in the transformed microorganism compared to that in a non-transformed microorganism,
wherein a phaA gene is not disrupted in the transformed microorganism, and
wherein the at least two genes encoding the β-ketothiolase enzyme is a combination of the gene (i) and gene (ii):
(i) a bktB gene comprising the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having 85% or higher sequence identity to the nucleotide sequence of SEQ ID NO: 7; and
(ii) an A1528 gene having comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having 85% or higher sequence identity to the nucleotide sequence of SEQ ID NO: 8.
2. The transformed microorganism according to claim 1 , wherein in the transformed microorganism, expression of the gene encoding a protein having (R)-specific enoyl-CoA hydratase activity is enhanced as compared to a corresponding non-transformed microorganism.
3. The transformed microorganism according to claim 1 , wherein expression of the PHA synthase gene is enhanced as compared to a corresponding non-transformed microorganism.
4. The transformed microorganism according to claim 1 , wherein the bacterium belonging to the genus Cupriavidus is Cupriavidus necator.
5. The transformed microorganism according to claim 4 , wherein the Cupriavidus necator is Cupriavidus necator H16 strain.
6. A method for producing a PHA copolymer comprising 3HH monomer unit and 3HB monomer unit, the method comprising:
culturing the transformed microorganism of claim 1 using a carbon source comprising an oil or a fat or a fatty acid; and
recovering a PHA copolymer comprising 3HH monomer unit and 3HB monomer unit.
7. The method according to claim 6 , wherein the PHA copolymer is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HH)).
8. The transformed microorganism according to claim 1 , wherein in the transformed microorganism, the at least two genes encoding the at last one β-ketothiolase enzyme having thiolysis activity for β-ketohexanoyl-CoA is disrupted such that the thiolysis activity of the β-ketothiolase enzyme is lost.
9. The transformed microorganism according to claim 1 , wherein in the transformed microorganism, the at least two genes encoding the at last one β-ketothiolase enzyme having thiolysis activity for β-ketohexanoyl-CoA is disrupted such that the thiolysis activity of the β-ketothiolase enzyme is reduced as compared to a corresponding non-transformed microorganism.
10. The transformed microorganism according to claim 1 , wherein the bktB gene comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 7.
11. The transformed microorganism according to claim 1 , wherein the A1528 gene comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
12. The transformed microorganism according to claim 1 , wherein the reduction of the activity of the β-ketothiolase enzyme in the microorganism is caused by genome editing technique, CRISPR/Cas system, homologous recombination technique, transposon technique, modification of a promoter region for the gene, modification of a ribosome binding sequence for the gene, or a combination thereof.
13. The transformed microorganism according to claim 1 , wherein a value of produced PHA amount-reducing rate×3HH composition ratio-increasing rate is at least 0.95.