Recombinant microorganism having ability to produce poly(lactate-coglycolate) or copolymer thereof from xylose and method for preparing poly(lactate-coglycolate) or copolymer thereof by using same

• US 10,640,774 B2
• Filed: 12/13/2016
• Issued: 05/05/2020
• Est. Priority Date: 01/28/2016
• Status: Active Grant

• Current Assignee: Korea Advanced Institute of Science and Technology
• Original Assignee: Korea Advanced Institute of Science and Technology
• Inventors: Lee, Sang Yup, Choi, So Young, Park, Si Jae
• Primary Examiner(s): Raghu, Ganapathirama
• Application Number:
  US16/071,680
• Publication Number:
  US 20190024093A1
• Time in Patent Office: 1,239 Days
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/10 Transferases (2.) ribonucleases C12N9/22
• CPC Class Codes: C12N 9/104 Aminoacyltransferases (2.3.2)
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12Y 101/01002 Alcohol dehydrogenase (NADP+) (1.1.1.2), i.e. aldehyde reductase
• CPC Class Codes: C12Y 101/01028 D-Lactate dehydrogenase (1.1.1.28)
• CPC Class Codes: C12Y 101/01113 L-Xylose 1-dehydrogenase (1.1.1.113)
• CPC Class Codes: C12Y 102/03003 Pyruvate oxidase (1.2.3.3)
• CPC Class Codes: C12Y 103/01006 Fumarate reductase (NADH) (1.3.1.6)
• CPC Class Codes: C12Y 197/01004 [Formate-C-acetyltransferase]-activating enzyme (1.97.1.4)
• CPC Class Codes: C12Y 203/01176 Propanoyl-CoA C-acyltransferase (2.3.1.176)
• CPC Class Codes: C12Y 301/01068 Xylono-1,4-lactonase (3.1.1.68)

Abstract

The present invention relates to a recombinant microorganism having the ability to produce poly(lactate-co-glycolate) and its copolymers from xylose, and more particularly to a recombinant microorganism having the ability to produce poly(lactate-co-glycolate) and its copolymers without having to supply a glycolate precursor from an external source, and a method of producing a poly(lactate-co-glycolate) copolymers using the same.

Claims

1. A recombinant microorganism having the ability to produce poly(lactate-co-glycolate), wherein a polyhydroxyalkanoate synthase-encoding gene, a propionyl-CoA transferase-encoding gene, a xylose dehydrogenase-encoding gene, and a xylonolactonase-encoding gene are introduced in a microorganism having the ability to produce lactate from pyruvic acid, wherein the recombinant microorganism has an ability to produce poly(lactate-co-glycolate) without supplying glycolate.

2. The recombinant microorganism of claim 1, wherein the polyhydroxyalkanoate synthase is PHA synthase derived from Pseudomonas sp. 6-19 or a mutant enzyme of PHA synthase, which has an amino acid sequence selected from the following amino acid sequences:

an amino acid sequence comprising at least one mutation selected from the group consisting of E130D, S325T, S477G, S477F, S477Y, S477G and Q481K in the amino acid sequence of SEQ ID NO;

1;

an amino acid sequence (PhaC1202) comprising mutations of E130D and Q481K in the amino acid sequence of SEQ ID NO;

1;

an amino acid sequence (PhaC1301) comprising mutations of E130D, S325T and Q481K in the amino acid sequence of SEQ ID NO;

1;

an amino acid sequence (PhaC1310) comprising mutations of E130D, S477F and Q481K in the amino acid sequence of SEQ ID NO;

1;

an amino acid sequence (PhaC1437) comprising mutations of E130D, S325T, S477G and Q481K in the amino acid sequence of SEQ ID NO;

1; and

an amino acid sequence (PhaC1439) comprising mutations of E130D, S325T, S477F and Q481K in the amino acid sequence of SEQ ID NO;

1.

3. The recombinant microorganism of claim 1, wherein the propionyl-CoA transferase is a Pct540 enzyme having the amino acid sequence of SEQ ID NO:

2.

4. The recombinant microorganism of claim 1, wherein the chromosomal promoter of a lactate dehydrogenase-encoding gene is replaced with a strong promoter selected from the group consisting of trc, tac, pBAD, trp, lacUV5, and T7.

5. The recombinant microorganism of claim 1, wherein one or more genes selected from the group consisting of a glucose PTS enzyme IIBC component-encoding gene, an aldehyde-alcohol dehydrogenase-encoding gene, a pyruvate-formate lyase-encoding gene, a fumarate reductase-encoding gene, a pyruvate oxidase-encoding gene, a did gene that is a lactate dehydrogenase-encoding gene, a malate synthase-encoding gene, and a glycolate oxidase-encoding gene is deleted from the recombinant microorganism.

6. The recombinant microorganism of claim 1, wherein a beta-ketothiolase-encoding gene and an acetoacetyl-CoA reductase-encoding gene are further introduced in the microorganism, and the recombinant microorganism has the ability to produce poly(lactate-co-glycolate-co-3-hydroxybutyrate).

7. The recombinant microorganism of claim 6, wherein one or more genes selected from the group consisting of a glucose PTS enzyme IIBC component-encoding gene, an aldehyde-alcohol dehydrogenase-encoding gene, a pyruvate-formate lyase-encoding gene, a fumarate reductase-encoding gene, a pyruvate oxidase-encoding gene, a did gene that is a lactate dehydrogenase-encoding gene, a malate synthase-encoding gene, and a glycolate oxidase-encoding gene is further deleted from the recombinant microorganism.

8. The recombinant microorganism of claim 1, wherein a CoA-dependent succinate semialdehyde dehydrogenase-encoding gene, and a 4-hydroxybutyrate dehydrogenase-encoding gene are further introduced in the microorganism, and the recombinant microorganism has the ability to produce poly(lactate-co-glycolate-co-4-hydroxybutyrate).

9. The recombinant microorganism of claim 8, wherein a glucose PTS enzyme IIBC component-encoding gene, an aldehyde-alcohol dehydrogenase-encoding gene, a pyruvate-formate lyase-encoding gene, a fumarate reductase-encoding gene, a pyruvate oxidase-encoding gene, a did gene that is a lactate dehydrogenase-encoding gene, a malate synthase-encoding gene, and a glycolate oxidase-encoding gene are further deleted from the recombinant microorganism.

10. A method for producing poly(lactate-co-glycolate), comprising the steps of:

(a) producing poly(lactate-co-glycolate) by culturing the recombinant microorganism having the ability to produce poly(lactate-co-glycolate) of claim 1 without supplying glycolate; and

(b) recovering the produced poly(lactate-co-glycolate).

11. A method for producing poly(lactate-co-glycolate-co-3-hydroxybutyrate) comprising the steps of:

(a) producing poly(lactate-co-glycolate-co-3-hydroxybutyrate) by culturing the recombinant microorganism having the ability to produce poly(lactate-co-glycolate-co-3-hydroxybutyrate) of claim 6 without supplying glycolate; and

(b) recovering the produced poly(lactate-co-glycolate-co-3-hydroxybutyrate).

12. A method for producing poly(lactate-co-glycolate-co-4-hydroxybutyrate) comprising the steps of:

(a) producing poly(lactate-co-glycolate-co-4-hydroxybutyrate) by culturing the recombinant microorganism having the ability to produce poly(lactate-co-glycolate-co-4-hydroxybutyrate) of claim 8 without supplying glycolate; and

(b) recovering the produced poly(lactate-co-glycolate-co-4-hydroxybutyrate).

13. A method for producing poly(lactate-co-glycolate-co-hydroxycarboxylic acid) comprising the steps of:

(a) producing poly(lactate-co-glycolate-co-hydroxycarboxylic acid) by culturing the recombinant microorganism of claim 1 without supplying glycolate in the presence of hydroxycarboxylic acid; and

(b) recovering the produced poly(lactate-co-glycolate-co-hydroxycarboxylic acid).

14. The method of claim 13, wherein the hydroxycarboxylic acid is selected from the group consisting of 2-hydroxyisovalerate, 5-hydroxyvalerate, and 6-hydroxyhexanoate.