US 11,655,488 B1
Method and strains for reducing byproduct succinic acid in fermentation process of L-malic acid and use thereof
Hao Liu, Nanjing (CN); and Qing Xu, Nanjing (CN)
Assigned to Nanjing Haohe Biotechnology Co., Ltd., Nanjing (CN)
Filed by Nanjing Haohe Biotechnology Co., Ltd., Nanjing (CN)
Filed on May 16, 2022, as Appl. No. 17/744,778.
Claims priority of application No. 202111445669.8 (CN), filed on Dec. 1, 2021.
Int. Cl. C12P 7/46 (2006.01); C12N 15/80 (2006.01); C12N 9/00 (2006.01); C07K 9/00 (2006.01); C07K 14/38 (2006.01); C12N 9/02 (2006.01)
CPC C12P 7/46 (2013.01) [C07K 14/38 (2013.01); C12N 9/001 (2013.01); C12N 15/80 (2013.01); C12Y 103/00 (2013.01)] 3 Claims
 
1. A method of constructing an Aspergillus niger engineered strain, wherein the Aspergillus niger engineered strain is capable of reducing the byproduct succinic acid in a fermentation process for making L-malic acid, wherein fumaric acid reductase frdA and fumaric acid reductase flavoprotein subunit frdB are simultaneously knocked out from the Aspergillus niger engineered strain;
wherein the method comprises the following steps:
(1) respectively amplifying upstream and downstream sequence fragments of a gene frdA through PCR with a wild type Aspergillus niger ATCC1015 genome as a template, and recovering PCR products to respectively obtain target fragments; and cloning the upstream and downstream sequence fragments of the gene frdA into a vector pLH594, so as to construct a fumaric acid reductase frdA knockout vector pLH1067;
wherein the downstream sequence of the gene frdA is SEQ NO:3, and the upstream sequence of the gene frdA is SEQ NO: 4; and
transferring said vector pLH1067 into Aspergillus niger S489 under the mediation of Agrobacterium, and conducting transformant screening and hygromycin resistance gene recombination to obtain a frdA gene knockout strain K1; and
(2) respectively amplifying upstream and downstream sequence fragments of gene frdB through PCR with a wild type Aspergillus niger ATCC1015 strain genome as a template, and recovering PCR products to obtain target sequence fragments; and cloning the upstream and downstream target sequence fragments of the gene frdB into vector pLH594, so as to construct a fumaric acid reductase flavoprotein subunit frdB knockout vector pLH1162;
wherein the downstream sequence of the gene frdB is SEQ NO:7, and the upstream sequence of the gene frdB is SEQ NO: 8; and
transferring vector pLH1162 into the frdA gene knockout strain K1 under the mediation of Agrobacterium, and conducting transformant screening and hygromycin resistance gene recombination to obtain a frdA gene and frdB gene double-knockout strain K2, that is the Aspergillus niger engineered strain for reducing the byproduct succinic acid accumulation in the fermentation process for making L-malic acid.