Transformant for producing dodecanedioic acid and method for producing dodecanedioic acid

Patent metadata

Field	Value
Publication number	US-12286663-B2
Application number	US-202318189272-A
Country	US
Kind code	B2
Filing date	Mar 24, 2023
Priority date	Sep 29, 2022
Publication date	Apr 29, 2025
Grant date	Apr 29, 2025

Abstract

A gene editing system of Candida viswanathii includes a Candida viswanathii , a first gene editing fragment and a second gene editing fragment. The first gene editing fragment successively includes a first homology arm and a screening gene. The second gene editing fragment is connected to a C-terminus of the first gene editing fragment and includes a second homology arm, a Cas9 expression cassette and a sgRNA cassette. The Cas9 expression cassette successively includes a Cas9 promoter, a Cas9 gene and three nuclear localization sequences. The sgRNA cassette successively includes a sgRNA promoter, a first ribozyme, a targeting sequence, a scaffold and a second ribozyme. The first gene editing fragment and the second gene editing fragment are constructed as a linear fragment for gene editing of a chromosome of the Candida viswanathii.

First claim

What is claimed is:

1. A transformant for producing dodecanedioic acid, comprising:
a host cell, wherein the host cell is Candida viswanathii ; and
at least three exogenous genes, wherein the at least three exogenous genes comprise a CYP52A19 gene, a CPRb gene and a FAO2 gene, and the at least three exogenous genes are integrated into a chromosome of the host cell.

2. The transformant for producing dodecanedioic acid of claim 1 , wherein the at least three exogenous genes are integrated into a POX2 gene of the chromosome of the host cell.

3. The transformant for producing dodecanedioic acid of claim 2 , wherein the at least three exogenous genes comprise a POS5 gene.

4. A method for producing dodecanedioic acid, comprising:
providing a reaction substrate, wherein the reaction substrate comprises dodecane; and
performing a fermentation step, wherein the reaction substrate is inoculated with the transformant for producing dodecanedioic acid of claim 1 , and then is cultured at a fermentation condition for a fermentation time to obtain a fermented substance, and the fermented substance comprises dodecanedioic acid.

5. The transformant for producing dodecanedioic acid of claim 1 , wherein the at least three exogenous genes are integrated in the chromosome of the host cell using a gene editing system, and the gene editing system comprises:
a first gene editing fragment, which successively comprises a first homology arm and a screening gene; and
a second gene editing fragment connected to a C-terminus of the first gene editing fragment, which successively comprises a second homology arm, a Cas9 expression cassette and a sgRNA cassette, wherein the Cas9 expression cassette successively comprises a Cas9 promoter, a Cas9 gene and three nuclear localization sequences, the sgRNA cassette successively comprises a sgRNA promoter, a first ribozyme, a targeting sequence, a scaffold and a second ribozyme, and the first gene editing fragment and the second gene editing fragment are constructed as a linear fragment for gene editing of the chromosome of the host cell;
wherein the first homology arm and the second homology arm respectively correspond to a specific fragment of a gene on the chromosome of the host cell, and the targeting sequence corresponds to a specific sequence of the gene on the chromosome of the host cell;
wherein the first gene editing fragment and/or the second gene editing fragment comprise the at least three exogenous genes.

6. The transformant for producing dodecanedioic acid of claim 5 , wherein the Cas9 promoter is P PGK1 , and the sgRNA promoter is P TDH1 .

7. The transformant for producing dodecanedioic acid of claim 6 , wherein the first ribozyme is tRNA Ala having the sequence of SEQ ID NO: 12, and the second ribozyme is hepatitis delta virus (HDV) having the sequence of SEQ ID NO: 11.

8. The transformant for producing dodecanedioic acid of claim 5 , wherein the screening gene of the first gene editing fragment further comprises a first screening gene fragment, the second gene editing fragment further comprises a second screening gene fragment at an N-terminus of the second homology arm, the first screening gene fragment has a homologous fragment, the second screening gene fragment has the homologous fragment, and the first screening gene fragment and the second screening gene fragment are recombined into the linear fragment by the homologous fragment of the first screening gene fragment and the homologous fragment of the second screening gene fragment.

Assignees

• Univ Nat Tsing Hua
• Chang Chun Plastics Co Ltd
• Chang Chun Petrochemical Co Ltd
• Dairen Chemical Corp

Inventors

• Hu Yu-Chen
• Pham Nam Ngoc
• Chou June-Yen
• Wang Hsing-Yun
• Liu Vincent Jianan

Classifications

• C12Y114/14

  with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14) · CPC title

• C12Y101/0302 Primary

  Long-chain-alcohol oxidase (1.1.3.20) · CPC title

• C12Y101/01

  with NAD+ or NADP+ as acceptor (1.1.1) · CPC title

• C12Y207/01086

  NADH kinase (2.7.1.86) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title