US 11,919,928 B2
Method for producing dihydrotentoxin
Takashi Kubo, Sapporo (JP); Masayuki Machida, Sapporo (JP); Tomonori Fujioka, Tokyo (JP); Shigenari Yamaguchi, Tokyo (JP); and Kiyoshi Kawai, Tokyo (JP)
Assigned to KUMIAI CHEMICAL INDUSTRY CO., LTD., Tokyo (JP)
Filed by KUMIAI CHEMICAL INDUSTRY CO., LTD., Tokyo (JP)
Filed on Oct. 12, 2021, as Appl. No. 17/499,599.
Application 17/499,599 is a division of application No. 16/085,502, granted, now 11,174,297, previously published as PCT/JP2017/010697, filed on Mar. 16, 2017.
Claims priority of application No. 2016-052806 (JP), filed on Mar. 16, 2016.
Prior Publication US 2022/0024996 A1, Jan. 27, 2022
Int. Cl. C07K 14/37 (2006.01); C07K 7/64 (2006.01); C12N 9/02 (2006.01); C12N 9/10 (2006.01); C12N 15/70 (2006.01); C12N 15/75 (2006.01); C12N 15/77 (2006.01); C12N 15/78 (2006.01); C12N 15/81 (2006.01); C12P 21/02 (2006.01); C12N 1/14 (2006.01)
CPC C07K 14/37 (2013.01) [C07K 7/64 (2013.01); C12N 9/0077 (2013.01); C12N 9/10 (2013.01); C12N 9/1007 (2013.01); C12N 15/70 (2013.01); C12N 15/75 (2013.01); C12N 15/77 (2013.01); C12N 15/78 (2013.01); C12N 15/81 (2013.01); C12P 21/02 (2013.01); C12N 1/14 (2013.01)] 8 Claims
 
1. A method for producing dihydrotentoxin, comprising the steps of:
culturing a transformant into which a tentoxin synthesis-related gene has been introduced, wherein the tentoxin synthesis-related gene encodes a protein having activity of nonribosomal peptide synthesis of dihydrotentoxin and containing the following modules in the order below from the N-terminal side:
a first module having a first adenylation domain comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 with adenylation activity, a first peptidyl carrier protein domain comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2 with peptidyl carrier protein activity, and a first condensation domain comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3 with condensation activity in that order from the N-terminal side;
a second module having a second adenylation domain comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4 with adenylation activity, a first N-methyltransferase domain comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 5 with N-methyltransferase activity, a second peptidyl carrier protein domain comprising the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6 with peptidyl carrier protein activity, and a second condensation domain comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 7 with condensation activity in that order from the N-terminal side;
a third module having a third adenylation domain comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 8 with adenylation activity, a third peptidyl carrier protein domain comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 9 with peptidyl carrier protein activity, and a third condensation domain comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 10 with condensation activity in that order from the N-terminal side; and
a fourth module having a fourth adenylation domain comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 11 with adenylation activity, a second N-methyltransferase domain comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 12 with N-methyltransferase activity, a fourth peptidyl carrier protein domain comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 13 with peptidyl carrier protein activity, and a fourth condensation domain comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14 with condensation activity in that order from the N-terminal side; and
collecting dihydrotentoxin from the culture supernatant.