Method for producing fibroin-like protein

Patent metadata

Field	Value
Publication number	US-11117937-B2
Application number	US-201815989400-A
Country	US
Kind code	B2
Filing date	May 25, 2018
Priority date	Nov 25, 2015
Publication date	Sep 14, 2021
Grant date	Sep 14, 2021

Abstract

A method for producing a fibroin-like protein is provided. A fibroin-like protein is produced by a method of culturing Escherichia coli having a gene encoding the fibroin-like protein in a medium, inducing expression of the gene encoding the fibroin-like protein, and collecting the fibroin-like protein, wherein accumulation of an organic acid at the time of inducing the expression is reduced, and wherein the gene is expressed under control of a tryptophan promoter.

First claim

The invention claimed is:

1. A method for producing a fibroin-like protein, the method comprising:
(A) culturing Escherichia coli having a gene encoding the fibroin-like protein in a medium;
(B) inducing expression of the gene encoding the fibroin-like protein during said culturing; and
(C) collecting the fibroin-like protein,
wherein when said inducing expression occurs, accumulation of an organic acid is reduced, and
wherein the gene is expressed under the control of a tryptophan promoters;
wherein said inducing expression occurs by depleting tryptophan in the medium, adding 3-beta-indoleacrylic acid to the medium, or a combination thereof;
wherein the fibroin-like protein is selected from the group consisting of:
(X) fibroin,
(Y) a fibrous protein having a sequence similar to a repetitive sequence of fibroin, and
(Z) combinations thereof;

wherein the sequence similar to a repetitive sequence of fibroin is represented by formula (I),
REP1-REP2 . . . (I);
wherein the fibrous protein having a sequence similar to a repetitive sequence of a fibroin comprises 2 or more repetitions of the sequence represented by formula (I);
wherein, independently for each repetition, said REP 1 is a continuous amino acid sequence of one or more of alanine and glycine, and the length of said REP1 is 2 to 20 residues; and
wherein, independently for each repetition, said REP2 is a continuous amino acid sequence comprising amino acids selected from the group consisting of glycine, serine, glutamine, and alanine; wherein the total number of glycine, serine, glutamine, and alanine residues is 40% or more of the total number of amino acid residues of the REP2, and the length of the REP2 is 2 to 200 residues.

2. The method according to claim 1 , wherein said inducing expression occurs either by depleting tryptophan in the medium, or by depleting tryptophan in the medium and adding 3-beta-indoleacrylic acid to the medium.

3. The method according to claim 1 , wherein the concentration of tryptophan in the medium is lowerthan 50 mg/L during said depleting.

4. The method according to claim 1 ,
wherein the step (A) comprises a culture period A1 and a culture period A2,
wherein the culture period A2 occurs after the culture period A1,
wherein the concentration of tryptophan in the medium during the culture period A1 is 50 mg/L or higher, and
wherein the concentration tryptophan in the medium during the culture period A2 is lower than 50 mg/L.

5. The method according to claim 4 , wherein said inducing expression occurs at the timepoint of switching from the culture period A1 to the culture period A2.

6. The method according to claim 1 , wherein at the beginning of said culturing, the medium contains tryptophan at a concentration of 50 mg/L or higher.

7. The method according to claim 1 , wherein 3-beta-indoleacrylic acid is added to the medium before said culturing but said inducing expression occurs by depleting tryptophan in the medium during said culturing.

8. The method according to claim 1 , wherein said inducing expression occurs by adding 3-beta-indoleacrylic acid to the medium during said culturing.

9. The method according to claim 1 , wherein the amount of organic acid that has accumulated in the medium at the time of said inducing the expression is 3.3 g/L or lower.

10. The method according to claim 1 , wherein OD620 at the time of said inducing the expression is 50 or more.

11. The method according to claim 1 , wherein said accumulation of the organic acid is reduced by limiting the amount of a carbon source during a period before said inducing the expression.

12. The method according to claim 11 , wherein the carbon source in the medium is limited to 1.0 g/L or lower during the period before said inducing the expression.

13. The method according to claim 11 , wherein the carbon source is glucose.

14. The method according to claim 1 , wherein the accumulation of the organic acid is reduced by modifying the Escherichia coli so that an ability to produce the organic acid is reduced.

15. The method according to claim 1 , wherein the Escherichia coli is auxotrophic for tryptophan.

Assignees

• Ajinomoto Kk
• Spiber Inc

Inventors

• Nakase Kentaro
• Takashima Yoshinori
• Nagahiko Takeshi

Classifications

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/71 Primary

  Expression systems using regulatory sequences derived from the trp-operon · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• C07K2319/02

  containing a signal sequence · CPC title

• C07K2319/035

  containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins · CPC title