Expression of polypeptides involved in lysine decarboxylation, and methods and applications thereof

Patent metadata

Field	Value
Publication number	US-10647976-B2
Application number	US-201415321800-A
Country	US
Kind code	B2
Filing date	Jun 26, 2014
Priority date	Jun 26, 2014
Publication date	May 12, 2020
Grant date	May 12, 2020

Abstract

The expression plasmid vectors comprise a polynucleotide sequence encoding Ldc2 polypeptide, a fragment, and/or a mutant. A backbone plasmid is capable of autonomous replication in a host cell. The host cell is not a P. aeruginosa cell. Transformants are transformed with expression plasmid vector. The transformants are not P. aeruginosa. Mutant host cells comprise a polynucleotide sequence encoding Ldc2 polypeptide, a fragment and/or a mutant that has been integrated into the host cell chromosome. A polypeptide, a fragment and/or a mutant comprise Ldc2. A non-naturally occurring polynucleotide, and/or a mutant encodes polypeptide comprising Ldc2. Biobased cadaverine is produced using the transformants and the biobased cadaverine is prepared by the method. Polyamides are formed using the biobased cadaverine and compositions.

First claim

What is claimed is:

1. A product, selected from the group consisting of:
I) a transformant comprising an expression plasmid vector in a host cell that is not a Pseudomonas aeruginosa ( P. aeruginosa ) cell, the expression plasmid vector comprising: a polynucleotide encoding one or more polypeptides, each polypeptide consisting of the amino acid sequence of a mutant of SEQ ID NO: 4 (lysine decarboxylase (Ldc2)); and a backbone plasmid capable of autonomous replication in the host cell,
wherein the mutant of SEQ ID NO:4 (Ldc2) is selected from the group consisting of the amino acid sequences of SEQ ID NO: 6 (Ldc2 S111C), SEQ ID NO: 11 (Ldc2 N262T), SEQ ID NO: 12 (Ldc2 K265N), SEQ ID NO: 13 (Ldc2 S111C/N262T), SEQ ID NO: 14 (Ldc2 S111C/K265N), SEQ ID NO: 15 (Ldc2 N262T/K265N), and SEQ ID NO: 16 (Ldc2 S111C/N262T/K265N);
II) an expression plasmid vector comprising: a polynucleotide encoding one or more polypeptides, each polypeptide consisting of the amino acid sequence of a mutant of SEQ ID NO: 4 (Ldc2); and a backbone plasmid capable of autonomous replication in a host cell, wherein the host cell is not a P. aeruginosa cell,
wherein the mutant of SEQ ID NO:4 (Ldc2) is selected from the group consisting of the amino acid sequences of SEQ ID NO: 6 (Ldc2 S111C), SEQ ID NO: 11 (Ldc2 N262T), SEQ ID NO: 12 (Ldc2 K265N), SEQ ID NO: 13 (Ldc2 S111C/N262T), SEQ ID NO: 14 (Ldc2 S111C/K265N), SEQ ID NO: 15 (Ldc2 N262T/K265N), and SEQ ID NO: 16 (Ldc2 S111C/N262T/K265N);
III) a first mutant host cell comprising a polynucleotide integrated into a chromosome of the host cell, wherein the polynucleotide encodes one or more polypeptides, each polypeptide consisting of the amino acid sequence of a mutant of SEQ ID NO: 4 (Ldc2),
wherein the mutant of SEQ ID NO:4 (Ldc2) is selected from the group consisting of the amino acid sequences of SEQ ID NO: 6 (Ldc2 S111C), SEQ ID NO: 11 (Ldc2 N262T), SEQ ID NO: 12 (Ldc2 K265N), SEQ ID NO: 13 (Ldc2 S111C/N262T), SEQ ID NO: 14 (Ldc2 S111C/K265N), SEQ ID NO: 15 (Ldc2 N262T/K265N), and SEQ ID NO: 16 (Ldc2 S111C/N262T/K265N);
IV) a non-naturally occurring polynucleotide encoding a polypeptide consisting of the amino acid sequence of a mutant of SEQ ID NO: 4, wherein the mutant of SEQ ID NO: 4 is selected from the group consisting of the amino acid sequences of SEQ ID NO: 6 (Ldc2 S111C), SEQ ID NO: 11 (Ldc2 N262T), SEQ ID NO: 12 (Ldc2 K265N), SEQ ID NO: 13 (Ldc2 S111C/N262T), SEQ ID NO: 14 (Ldc2 S111C/K265N), SEQ ID NO: 15 (Ldc2 N262T/K265N), and SEQ ID NO: 16 (Ldc2 S111C/N262T/K265N); and
V) a non-naturally occurring polynucleotide comprising the polynucleotide sequence of a mutant of SEQ ID NO: 17.

2. A product of claim 1 , which is I) the transformant, wherein the polynucleotide sequence comprises the polynucleotide sequence of a mutant of SEQ ID NO: 3, wherein the mutant of SEQ ID NO:3 encodes one of the mutants of SEQ ID NO:4 (Ldc2).

3. A product of claim 1 , which is I) the transformant, wherein the host cell is an Escherichia coli ( E. coli ) cell.

4. A product of claim 3 , wherein the polynucleotide sequence has been codon optimized for optimal polypeptide expression in the E. coli cell.

5. A product of claim 3 , wherein the backbone plasmid is an E. coli expression plasmid vector.

6. A product of claim 5 , wherein the backbone plasmid is selected from the group consisting of pUC18, pUC19, pBR322, pACYC, pET, pSC101, and any derived plasmids thereof.

7. A product of claim 5 , wherein the expression plasmid vector further comprises a promoter polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 5, which is positioned upstream from the polynucleotide sequence.

8. A product of claim 1 , which is I) the transformant, wherein the host cell is a Hafnia alvei ( H. alvei ) cell.

9. A product of claim 1 , which is II) the expression plasmid vector, wherein the host cell is an E. coli cell.

10. A product of claim 9 , wherein the polynucleotide sequence is a codon optimized polynucleotide for optimal polypeptide expression in the E. coli cell.

11. A product of claim 9 , wherein the backbone plasmid is an E. coli expression plasmid vector.

12. A product of claim 11 , wherein the backbone plasmid is selected from the group consisting of pUC18, pUC19, pBR322, pACYC, pET, pSC101, and any derived plasmids thereof.

13. A product of claim 12 , wherein the expression plasmid vector further comprises a promoter polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 5, which is positioned upstream from the polynucleotide sequence.

14. A product of claim 1 , which is II) the expression plasmid vector, wherein the host cell is a H. alvei cell.

15. A product of claim 1 , which is III) the first mutant host cell, wherein the host cell is an E. coli cell.

16. A product of claim 1 , which is IV) the non-naturally occurring polynucleotide, comprising a polynucleotide sequence encoding Ldc2 further comprising one or more mutations selected from the group consisting of a mutation at nucleotide position 331, a mutation at nucleotide position 332, a mutation at nucleotide position 333, a mutation at nucleotide position 784, a mutation at nucleotide position 785, a mutation at nucleotide position 786, a mutation at nucleotide position 793, a mutation at nucleotide position 794, and a mutation at nucleotide position 795.

17. A product of claim 16 , wherein the mutation at the nucleotide position 332 is mutated to G, the mutation at the nucleotide position 785 is mutated to a C, and the mutation at the nucleotide position 795 is mutated to a T or C.

18. A product of claim 16 , wherein the polynucleotide sequence encoding Ldc2 is the polynucleotide sequence of SEQ ID NO: 3 or a codon-optimized polynucleotide thereof.

19. A product of claim 18 , wherein the codon-optimized polynucleotide is the polynucleotide sequence of SEQ ID NO: 17.

20. A product of claim 16 , selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.

21. A product of claim 1 , which is IV) the non-naturally occurring polynucleotide, wherein the polynucleotide sequence has been codon optimized for optimal polypeptide expression in an E. coli cell.

22. A product of claim 1 , which is VI) the non-naturally occurring polynucleotide, wherein the polynucleotide sequence has been codon optimized for optimal polypeptide expression in an E. coli cell.

23. A method for producing a polypeptide comprising the amino acid sequence of a mutant of SEQ ID NO: 4 (Ldc2), comprising:
obtaining the first mutant host cell of claim 1 III) and/or the transformant of claim 1 I);
culturing the first mutant host cell and/or the transformant under conditions effective for the expression of the polypeptide; and
harvesting the polypeptide.

24. A method for producing cadaverine (1,5-pentanediamine) comprising:
1a) cultivating the first mutant host cell of claim 1 III) and/or the transformant of claim 1 I);
1b) producing cadaverine using the culture obtained from step 1a) to decarboxylate lysine; and
1c) extracting and purifying cadaverine using the culture obtained from step 1b).

Assignees

• Cathay R&D Center Co Ltd
• Cathay Ind Biotech Ltd
• Cathay Biotech Inc
• Cibt America Inc

Inventors

• Chou Howard
• Li Naiqiang
• Liu Xiucai

Classifications

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12Y401/01018

  Lysine decarboxylase (4.1.1.18) · CPC title