Improved propionibacterium strains for the production of propionic acid

Patent metadata

Field	Value
Publication number	US-2019071697-A1
Application number	US-201615762332-A
Country	US
Kind code	A1
Filing date	Sep 29, 2016
Priority date	Sep 30, 2015
Publication date	Mar 7, 2019
Grant date	—

Abstract

Disclosed herein are new strains of Propionibacterium and methods for the biosynthetic production of propionic acid.

First claim

1 . An isolated Propionibacterium strain wherein said strain produces a higher yield of propionic acid relative to P. acidipropionici strain ATCC 55737, wherein said isolated strain comprises a modified gene, relative to strain P. acidipropionici strain ATCC 55737, wherein
A. said modified gene is
a) the ABC polar amino acid transporter gene;
b) the Cytochrome C biogenesis gene;
c) the ABC multiple sugar transporter gene,
d) a combination of modified genes for each of the ABC polar amino acid transporter and the Cytochrome C biogenesis protein; or
e) a combination of modified genes for each of the ABC polar amino acid transporter gene, the Cytochrome C biogenesis gene and the ABC multiple sugar transporter gene; or
B. wherein said modified gene is
a) the large subunit ribosome gene;
b) the long chain acyl-CoA synthetase
c) the cation diffusion facilitator or
d) a combination of modified genes for each of the large subunit ribosome, the long chain acyl-CoA synthetase, and the cation diffusion facilitator.

2 . The isolated strain of claim 1 wherein said modified gene is
a) the ABC polar amino acid transporter gene;
b), the Cytochrome C biogenesis gene;
c) the ABC multiple sugar transporter gene;
d) a combination of modified genes for each of the ABC polar amino acid transporter and the Cytochrome C biogenesis protein; or
e) a combination of modified genes for each of the ABC polar amino acid transporter gene, the Cytochrome C biogenesis gene and the ABC multiple sugar transporter gene encodes an altered gene product for the ABC polar amino acid transporter and/or the Cytochrome C biogenesis protein.

3 . (canceled)

4 . The isolated Propionibacterium strain of claim 2 wherein said strain comprises one or more of the following gene modifications:
i) SNP G1917729A located in the promoter of the putative ABC multiple sugar transporter gene (XLOC_000692);
ii) SNP C2293187T located in the putative ABC transporter of polar amino acids gene (XLOC_000834); and
iii) SNP C1487806T in the cytochrome c biogenesis gene (XLOC_001967).

5 . The isolated strain of claim 4 wherein said strain comprises i), ii) and iii).

6 . The isolated strain of claim 2 wherein said strain is P. acidipropionici strain WGS 7, deposited with the American Type Culture Collection (ATCC), on Sep. 2, 2016 under ATCC Accession No. PTA 123476.

7 . A method of producing a novel Propionibacterium strain having enhanced growth rates or propionic acid production relative to P. acidipropionici ATCC 4875 or P. acidipropionici ATCC 55737, said method comprising
exchanging genetic material through genomic shuffling, between a first set of Propionibacterium strains selected from the group consisting of P. acidipropionici ATCC 4875, P. acidipropionici ATCC 55737 or a strain generated from genomic shuffling between P. acidipropionici ATCC 4875 and P. acidipropionici ATCC 55737, P. acidipropionici ATCC 4965, P. intermedium ATCC 14072, or P. jensenii ATCC 9617, and a second set of Propionibacterium strains comprising the P. acidipropionici strain F3E8, deposited with the American Type Culture Collection (ATCC), on Jun. 25, 2015 under ATCC Accession No. PTA-122267, culturing the strains generated by the genomic shuffling between the first and second set of Propionibacterium strains to identify a novel Propionibacterium strain having enhanced growth rates or propionic acid production relative to that produced by P. acidipropionici ATCC 55737 when the strains are cultured under identical conditions.

8 . The improved Propionibacterium strain method of claim 7 wherein said first set of strains is selected from the group consisting of P. acidipropionici ATCC 4875 and P. acidipropionici ATCC 55737 and said second set of strains comprises P. acidipropionici strain F3E8, deposited with the American Type Culture Collection (ATCC), on Jun. 25, 2015 under ATCC Accession No. PTA-122267 wherein said new strain has an increase in total yield (g/g) of propionic acid relative to that produced by P. acidipropionici ATCC 55737 when the two strains are cultured under identical conditions.

9 . The method of claim 7 wherein said first set of strains comprises a library of Propionibacterium strains formed by combining strains generated from 2, 3 or 4 of the following:
i) genomic shuffling between P. acidipropionici ATCC 4875 and P. acidipropionici ATCC 55737;
ii) genomic shuffling between P. acidipropionici ATCC 4875 and P. acidipropionici ATCC 4965;
iii) genomic shuffling between P. acidipropionici ATCC 4875 and P. acidipropionici ATCC 14072; and
iv) genomic shuffling between P. acidipropionici ATCC 4875 and P. jensenii ATCC 9617.

10 . The isolated Propionibacterium strain of claim 1 , wherein said modified gene is
a) the large subunit ribosome; b) the long chain acyl-CoA synthetase; c) the cation diffusion facilitator or d) a combination of modified genes for each of the large subunit ribosome, the long chain acyl-CoA synthetase, and the cation diffusion facilitator.

11 . (canceled)

12 . The isolated strain of claim 10 comprising
(i) a modified large ribosomal RNA gene comprising a nucleotide substitution at position 1441 relative to the same sequence in P. acidipropionici ATCC 55737 or positions according to Table 1 relative to same sequence in P. acidipropionici ATCC 4875;
(ii) a modified long chain acyl-CoA synthetase gene comprising a nonsense codon after nucleotide position 1500 and 2269 relative to the sequence of in P. acidipropionici ATCC 55737 and 264, 1650, and 2295 relative to P. acidipropionici ATCC 4875; or
(iii) a modified cation diffusion facilitator gene encoding a gene product comprising an amino acid substitution at amino acid position 318 relative to the sequence amino acid sequence in P. acidipropionici ATCC 55737 and positions according to Table 1 relative to P. acidipropionici ATCC 4875.

13 . (canceled)

14 . The isolated strain of claim 10 wherein the strain comprises an arginine deiminase regulon that is duplicated and said duplicated arginine deiminase regulon comprises a deletion at position 37 and said strain further comprises a duplication of the whole ribosomal RNA.

15 . (canceled)

16 . The isolated strain of claim 10 wherein said strain is a P. acidipropionici strain comprising a large subunit ribosome sequence of SEQ ID NO: 15, a long chain acyl-CoA synthetase sequence of SEQ ID NO: 16, and a cation diffusion facilitator sequence of SEQ ID NO: 13.

17 - 19 . (canceled)

20 . The P. acidipropionici strain F3E8, deposited with the American Type Culture Collection (ATCC), on Jun. 25, 2015 under ATCC Accession No. PTA-122267, or a variant of said deposited strain.

21 . A method for producing propionic acid, said method comprising the steps of
culturing a P. acidipropionici strain of claim 1 under conditions suitable for growth of the strain; recovering the propionic acid produced by said strain.

22 - 24 . (canceled)

25 . The method of claim 7 wherein the first set of strains is generated from genomic shuffling between P. acidipropionici ATCC 4875 and P. acidipropionici ATCC 55737.

26 . The method of claim 7 wherein the first set of strains is generated from genomic shuffling between P. acidipropionici ATCC 4875 and P. acidipropionici ATCC 14072.

27 . The method of claim 7 wherein the first set of strains is generated from genomic

Assignees

• Univ Queensland

Inventors

• Luna-Flores Carlos H
• Nielsen Lars K
• Marcellin Esteban
• Stowers Christopher C
• Martins Bianca F
• Cox Brad M

Classifications

• C07K14/195

  from bacteria · CPC title

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C12Y203/01086

  Fatty-acyl-CoA synthase (2.3.1.86) · CPC title

• C12N15/03

  Bacteria · CPC title

• C12P7/52 Primary

  Propionic acid; Butyric acids · CPC title