Patent US10233468

High molecular weight PHA-producing microbe and method of producing high molecular weight PHA using same
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US10233468

1. A method for producing a polyhydroxyalkanoate (PHA) copolymer having an increased weight average molecular weight, comprising: (10) (9)

(1) culturing a PHA producing microorganism,

wherein at least a portion of a gene encoding a PHA degrading enzyme of the microorganism has been altered by at least one of substitution, deletion, insertion, and addition, to reduce or eliminate an activity of the PHA degrading enzyme,

wherein the PHA degrading enzyme has

(a) the amino acid sequence of SEQ ID NO:2, or

(b) an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2 and has a PHA degrading enzyme activity,

wherein the microorganism belongs to the genus Cupriavidus, and

(2) producing the PHA copolymer,

wherein the weight average molecular weight of the produced PHA copolymer is increased compared to the weight average molecular weight of the produced PHA copolymer in a microorganism wherein the gene encoding the PHA degrading enzyme has not been altered, and

wherein the produced PHA copolymer comprises at least two different type of units selected from the group consisting of 3-hydroxyhexanoic acid (3HH), 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), 3-hydroxyoctanoic acid (3HO), 4-hydroxybutyric acid (4HB), and a combination thereof.

2. The method according to claim 1, wherein at least a portion of a gene encoding an additional PHA degrading enzyme of the microorganism has been altered by at least one of substitution, deletion, insertion, and addition. (2) (0)

3. The method according to claim 2, wherein the additional PHA degrading enzyme has (1) (2)

(d) an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:17 and has a PHA degrading enzyme activity.

14. The method according to claim 3, wherein at least a portion of a gene encoding a PHA degrading enzyme of the microorganism has been altered by at least one of a substitution, deletion, insertion, and addition, wherein the PHA degrading enzyme has (0) (2)

13. The method according to claim 2, wherein the additional PHA degrading enzyme has (0) (2)

4. The method according to claim 1, wherein the microorganism further comprises: (0) (2)

5. The method according to claim 1, wherein the microorganism is Cupriavidus necator. (0)

6. The method according to claim 1, wherein the produced PHA copolymer comprises units derived from 3-hydroxyhexanoic acid. (0)

7. The method according to claim 1, wherein the culturing comprises culturing the microorganism in the presence of a carbon source which comprises a free fatty acid at a content of at least 50% based on the total content of fat and oil. (1) (0)

9. The method according to claim 1, wherein the produced PHA copolymer has a weight average molecular weight of at least 340×10⁴. (0)

10. The method according to claim 1, wherein the produced PHA copolymer is purified. (0)

11. The method according to claim 1, wherein a degradation activity of the PHA degrading enzyme in the microorganism is reduced to 10% or lower. (0)

12. The method according to claim 1, wherein the produced PHA copolymer is a P(3HB-co-3HH) copolymer, a P(3HB-co-3HV) copolymer, or a P(3HB-co-4HB) copolymer. (0)

15. The method according to claim 1, wherein the produced PHA copolymer comprises units derived from 3-hydroxybutyric acid. (0)

16. A method for producing a polyhydroxyalkanoate (PHA) copolymer having an increased weight average molecular weight, the method comprising: (1) (8)

(1) culturing a PHA producing microorganism, wherein the microorganism belongs to the genus Cupriavidus and in which a PHA synthase comprising the amino acid sequence of SEQ ID NO:15 has been introduced,

wherein

(a) a phaZ6 gene and a phaZ1 gene on the chromosome of Cupriavidus have been deleted;

(b) a phaZ6 gene and a phaZ2 gene on the chromosome of Cupriavidus have been deleted; or

(2) producing the PHA copolymer,

wherein the weight average molecular weight of the produced PHA copolymer is increased compared to the weight average molecular weight of the produced PHA copolymer in a Cupriavidus microorganism wherein the gene encoding the PHA degrading enzyme has not been altered.

17. The method according to claim 16, wherein the produced PHA copolymer is a P(3HB-co-3HH) copolymer, a P(3HB-co-3HV) copolymer, or a P(3HB-co-4HB) copolymer. (0)

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Patent US10233468

High molecular weight PHA-producing microbe and method of producing high molecular weight PHA using same

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