Expression of recombinant tetracycline efflux pumps for the production of lysine or lysine-derived products, and methods and applications thereof

Patent metadata

Field	Value
Publication number	US-10400257-B2
Application number	US-201415517633-A
Country	US
Kind code	B2
Filing date	Oct 9, 2014
Priority date	Oct 9, 2014
Publication date	Sep 3, 2019
Grant date	Sep 3, 2019

Abstract

One aspect of the invention relates to a mutant polypeptide comprising the amino acid sequence of Escherichia coli tetracycline efflux pump A (TetA). Another aspect of the invention relates to a polynucleotide encoding a polypeptide of TetA, a fragment thereof, or a mutant thereof. Another aspect of the invention relates to a first expression plasmid vector comprising one or more first polynucleotides encoding a first polypeptide comprising a tetracycline efflux pump polypeptide, a fragment thereof or a mutant thereof, one or more second polynucleotides independently selected from the group consisting of a third polynucleotide encoding a third polypeptide comprising a lysine decarboxylase polypeptide, a fragment thereof or a mutant thereof, and a fourth polynucleotide encoding a fourth polypeptide comprising a lysine biosynthesis polypeptide, a fragment thereof or a mutant thereof. Another aspect of the invention relates to a transformant comprising one or more expression plasmid vectors as described herein in a host cell. Another aspect of the invention relates to a mutant host cell comprising one or more first, third or fourth polynucleotides as described herein integrated into a chromosome of a host cell. Another aspect of the invention relates to a method for producing a lysine comprising obtaining a transformant and/or mutant host cell as disclosed herein, culturing the transformant and/or mutant host cell under conditions effective for the expression of the lysine; and harvesting the lysine. Other aspects of the invention relate to methods for producing biobased cadaverine using the transformants disclosed herein, and biobased cadaverine prepared by the method disclosed herein, and polyamides formed using the biobased cadaverine as disclosed herein and compositions thereof.

First claim

What is claimed is:

1. A product, which is one of the following products I) through V):
I) a first polypeptide consisting of a mutant of the amino acid sequence SEQ ID NO: 2 (TetA) and fragments thereof, wherein the mutant of TetA or the fragment thereof is selected from the group consisting of amino acid sequences of SEQ ID NO: 32 (TetA aa1-96), TetA aa1-98, TetA aa1-99, TetA aa1-100, TetA aa1-101, TetA aa1-102, TetA aa1-103, TetA aa1-104, TetA aa1-124, TetA aa1-125, TetA aa1-126, TetA aa1-127, TetA aa1-128, TetA aa1-129, TetA aa1-130, TetA aa1-131, TetA aa1-132, TetA aa1-133, TetA aa1-155, TetA aa1-156, TetA aa1-157, TetA aa1-158, TetA aa1-159, TetA aa1-160, TetA aa1-161, TetA aa1-162, TetA aa1-182, TetA aa1-183, TetA aa1-184, SEQ ID NO: 30 (TetA (aa1-185)), TetA aa1-186, TetA aa1-187, TetA aa1-188, TetA aa1-189, TetA aa1-190, TetA aa1-191, TetA aa1-192, TetA aa1-193, TetA aa1-194, and TetA aa1-195;
II) a non-naturally occurring first polynucleotide encoding the first polypeptide of the product I);
III) a first expression plasmid vector comprising:
one or more first polynucleotides encoding the first polypeptide of the product I), and a backbone plasmid capable of autonomous replication in a host cell;
IV) a transformant comprising one or more the first expression plasmid vectors of the product III) in a host cell;
V) a mutant host cell comprising one or more first polynucleotides integrated into a chromosome of a host cell, wherein the first polynucleotide encodes the first polypeptide of the product I);
wherein the host cell in III), IV) and V) is selected from the group consisting of Pseudomonas, Escherichia, Corynebacterium , Bacilli, Hafnia, Brevibacterium, Lactobacillus, Lactococcus and Streptococcus.

2. A product of claim 1 , which is IV) the transformant, which further comprises one or more second expression plasmid vectors comprising one or more polynucleotides independently selected from the group consisting of a polynucleotide encoding a polypeptide comprising a tetracycline efflux pump polypeptide, a fragment thereof or a mutant thereof, a polynucleotide encoding a polypeptide comprising a lysine decarboxylase polypeptide, a fragment thereof or a mutant thereof, and a polynucleotide encoding a polypeptide comprising a lysine biosynthesis polypeptide, a fragment thereof or a mutant thereof; and
a backbone plasmid capable of autonomous replication in a host cell.

3. A product of claim 1 , which is IV) the transformant, wherein the backbone plasmid is an E. coli expression plasmid vector.

4. A product of claim 3 , wherein the backbone plasmid is selected from the group consisting of pUC18, pUC19, pBR322, pACYC, pET, pSC101 and any derived plasmids thereof.

5. A product of claim 1 , which is IV) the transformant, wherein the expression plasmid vector further comprises one or more polynucleotides encoding a polypeptide comprising an antibiotic resistance protein, a fragment thereof, or mutant thereof.

6. A product of claim 5 , wherein the antibiotic resistance protein comprises a tetracycline resistance protein or an oxytetracycline-resistance protein (Otr).

7. A product of claim 6 , wherein the antibiotic resistance protein is selected from the group consisting of OtrA, OtrB, OtrC, and Tcr3.

8. A product of claim 1 , which is IV) the transformant, wherein the first polynucleotide is selected from the group consisting of SEQ ID NO: 29 (tetA (nt 1-558)) and SEQ ID NO: 31 (tetA (nt 1-291)).

9. A product of claim 8 , wherein the first polypeptide is selected from the group consisting of SEQ ID NO: 30 (TetA (aa1-185)) and SEQ ID NO: 32 (TetA (aa1-96)).

10. A product of claim 2 , wherein
the polynucleotide encoding a polypeptide comprising a lysine decarboxylase polypeptide is SEQ ID NO: 41 (cadA), fragments thereof, and mutants thereof, and
the polynucleotide encoding a polypeptide comprising a lysine biosynthesis polypeptide is selected from the group consisting of sucA, ppc, aspC, lysC, asd, dapA (dihydrodipicolinate synthase), dapB, dapD, argD, dapE, dapF, lysA, ddh, pntAB, cyoABE, gadAB, ybjE, gdhA, gltA, sucC, gadC, acnB, pflB, thrA, aceA, aceB, gltB, aceE, sdhA, murE, speE, speG, puuA, puuP, ygjG, fragments thereof, and mutants thereof.

11. A product of claim 2 , wherein
the polypeptide comprising a lysine decarboxylase polypeptide is SEQ ID NO: 6 (CadA), fragments thereof, and mutants thereof, and
the polypeptide comprising a lysine biosynthesis polypeptide is selected from the group consisting of SucA, Ppc, AspC, LysC, Asd, DapA (dihydrodipicolinate synthase), DapB, DapD, ArgD, DapE, DapF, LysA, Ddh, PntAB, CyoABE, GadAB, YbjE, GdhA, GltA, SucC, GadC, AcnB, PflB, ThrA, AceA, AceB, GltB, AceE, SdhA, MurE, SpeE, SpeG, PuuA, PuuP, and YgjG, fragments thereof, and mutants thereof.

12. A product of claim 2 , wherein the polypeptide comprising a lysine biosynthesis polypeptide is selected from the group consisting of SEQ ID NO: 3 (LysC), SEQ ID NO: 4 (DapA), SEQ ID NO: 5 (LysA), SEQ ID NO: 22 (Asd), SEQ ID NO: 23 (DapB), SEQ ID NO: 24 (DapD), SEQ ID NO: 25 (AspC), SEQ ID NO: 26 (LysC-1), SEQ ID NO: 27 (LysC-2), and SEQ ID NO: 28 (S-LysC).

13. A product of claim 1 , which is IV) the transformant, wherein the host cell is from the genus Hafnia, Escherichia , or Corynebacterium.

14. A product of claim 13 , wherein the host cell is from the species Hafnia alvei, Escherichia coli , or Corynebacterium glutamicum.

15. A method for producing a lysine comprising:
obtaining the transformant of the product IV) and/or the mutant host cell of the product V) of claim 1 ;
culturing the transformant and/or mutant host cell under conditions effective for the expression of the lysine; and
harvesting the lysine.

16. A method for producing cadaverine (1,5-pentanediamine) comprising:
1a) cultivating the transformant of the product IV) and/or the mutant host cell of the product V) of claim 1 ;
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
• Cibt America Inc

Inventors

• Chou Howard
• Liu Xiucai

Classifications

• C07K14/36

  from Actinomyces; from Streptomyces (G) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12N15/77

  for Corynebacterium; for Brevibacterium · CPC title

• C12P13/001

  Amines; Imines · CPC title