Long chain dibasic acid with low content of long chain dibasic acid impurity of shorter carbon-chain and preparation method thereof

Patent metadata

Field	Value
Publication number	US-12018311-B2
Application number	US-202217814972-A
Country	US
Kind code	B2
Filing date	Jul 26, 2022
Priority date	Jul 6, 2018
Publication date	Jun 25, 2024
Grant date	Jun 25, 2024

Abstract

The present invention relates to a long-chain dibasic acid with low content of long-chain dibasic acid impurity of shorter carbon chain, to the preparation of a long-chain dibasic acid producing strain by directed evolution of POX gene and homologous recombination, and to the production of a long-chain dibasic acid with low content of long-chain dibasic acid impurity of shorter carbon chain by using the strain. The present invention also relates to a strain containing a mutated promoter, wherein, when a long-chain dibasic acid is produced by fermentation of this strain, the content of the acid impurity of shorter carbon chain in the fermentation product is significantly reduced.

First claim

The invention claimed is:

1. A method of producing a long chain dibasic acid or a fermentation broth by fermentation using a microorganism, comprising culturing the microorganism, and optionally isolating and/or purifying the long chain dibasic acid from the culture product,
wherein the microorganism comprising an isolated mutated POX gene or a variant thereof,
wherein said isolated mutated POX gene or a variant thereof comprises: [i] a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:24, and [ii] a deletion of any one nucleotide base in the promoter of said POX gene from nucleic acid residues 266-275 of SEQ ID NO:24.

2. The method of claim 1 , wherein the isolated mutated POX gene or a variant thereof comprises
[a] a nucleic acid sequence as set forth in SEQ ID NOs:27 or 29, or
[b] a nucleic acid sequence having at least 85%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95% or 99.96% sequence identity to SEQ ID NO:24.

3. The method of claim 1 , wherein the microorganism is:
(i) selected from the group consisting of Corynebacterium, Geotrichum candidum, Candida, Pichia, Rhodotroula, Saccharomyces and Yarrowia;
(ii) yeast; or
(iii) Candida tropicalis or Candida sake.

4. The method of claim 1 , wherein the long-chain dibasic acid is:
(i) selected from the group consisting of C9 to C22 long-chain dibasic acids;
(ii) selected from the group consisting of C9 to C18 long-chain dibasic acids;
(iii) one or more selected from the group consisting of C10 dibasic acid, C11 dibasic acid, C12 dibasic acid, C13 dibasic acid, C14 dibasic acid, C15 dibasic acid and C16 dibasic acid;
(iv) at least one or more of C10 to C16 dibasic acids, or at least one or more of n-C10 to C16 dibasic acids; or
(v) at least one or more selected from the group consisting of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

5. The method of claim 1 , further comprising extraction and purification of the long-chain dibasic acid, or, wherein the fermentation broth obtained by fermentation is subject to extraction and purification treatment to obtain a final long-chain dibasic acid product.

6. The method of claim 5 , wherein the extraction and purification include sterilization and acidification of the fermentation broth, and following the sterilization and acidification of the fermentation broth, said method further comprises a solid-liquid separation of a long-chain dibasic acid precipitate obtained after the acidification, and/or solvent crystallization of long-chain dibasic acid precipitate.

7. The method of claim 1 , wherein the fermentation broth includes a fermentation broth containing a salt of a long-chain dibasic acid produced during a process for producing the long-chain dibasic acid by biological fermentation.

8. The method of claim 6 , wherein:
(i) the sterilization is carried out by membrane filtration;
(ii) the acidification of the fermentation broth refers to an acidification treatment of a clear liquid containing a salt of a long-chain dibasic acid obtained after membrane filtration, and the salt of the long chain dibasic acid is converted to a precipitate of the long chain dibasic acid by adding an acid;
(iii) the solid-liquid separation refers to separation of the obtained long-chain dibasic acid precipitate from a mother liquor obtained after the acidification treatment of the clear liquid, which includes separation by filtration or/and centrifugation;
(iv) the acidification is carried out using an inorganic acid;
(v) the end point pH of the acidification is less than 5;
(vi) the solvent crystallization refers to dissolving the long-chain dibasic acid precipitate in an organic solvent, and crystallizing the long-chain dibasic acid by cooling\evaporation\solvating-out, and then separating the crystals to obtain a more purified long-chain dibasic acid; and/or
(vii) the extraction and purification further includes decoloration of the fermentation broth containing a salt of a long-chain dibasic acid.

9. The method of claim 8 , wherein:
(i) the sterilization is carried out by a ceramic membrane filtration process;
(ii) when a ceramic membrane is used for membrane filtration, the pre-membrane pressure is 0.2 to 0.4 MPa, and/or the pore diameter of the filter membrane is 0.05 to 0.2 μm;
(iii) the inorganic acid is sulfuric acid, hydrochloric acid, nitric acid, or an acid mixture thereof;
(iv) the end point pH of acidification is less than 4.0; and/or
(v) the decoloration comprises adding activated carbon to the fermentation broth or the clear liquid containing a salt of a long-chain dibasic acid for decoloration treatment, and then removing the activated carbon by filtration after the decoloration treatment.

10. The method of claim 9 , wherein:
(i) the activated carbon is added in an amount of 0.1 to 5 wt % or 1 to 3 wt %, relative to the amount of the long-chain dibasic acid contained in the fermentation broth or the clear liquid; and/or
(ii) where activated carbon is used for decoloration, the decoloration temperature is 85 to 100° C., and the decoloration time is 15 to 165 min.

11. The method of claim 8 , wherein the organic solvent includes one or more of alcohol, acid, ketone and ester.

12. The method of claim 11 , wherein:
(i) the alcohol includes one or more of methanol, ethanol, isopropanol, n-propanol, and n-butanol;
(ii) the acid includes acetic acid;
(iii) the ketone includes acetone; and/or
(iv) the ester includes ethyl acetate and/or butyl acetate.

13. The method of claim 8 , wherein the precipitate of a long-chain dibasic acid is dissolved in an organic solvent and then decolorized, and then is separated to obtain a clear liquid and a more purified long-chain dibasic acid.

14. The method of claim 13 , wherein after being separated, the clear liquid is subjected to cooling crystallization.

15. The method of claim 14 , wherein the cooling crystallization includes cooling to 65 to 80° C., incubating for 1 to 2 hours, then cooling to 25 to 35° C., and crystallizing.

Assignees

• Cathay Biotech Inc
• Cibt America Inc

Inventors

• Liu Wenbo
• Xu Min
• Yang Chen
• Chou Howard
• Liu Xiucai

Classifications

• C12P7/44 Primary

  Polycarboxylic acids · CPC title

• C12R2001/74

  Candida tropicalis · CPC title

• C12Y103/03006

  Acyl-CoA oxidase (1.3.3.6) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N15/01

  Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor · CPC title