Microorganism strain and method for antibiotic-free, fermentative preparation of low molecular weight substances and proteins

Patent metadata

Field	Value
Publication number	US-11046732-B2
Application number	US-201515779773-A
Country	US
Kind code	B2
Filing date	Dec 11, 2015
Priority date	Dec 11, 2015
Publication date	Jun 29, 2021
Grant date	Jun 29, 2021

Abstract

The invention relates to a microorganism strain and a method for antibiotic-free fermentative preparation of low molecular weight substances and proteins. The microorganism strain for producing low molecular weight substances or proteins contains in its genome a mutation in a gene which brings about an auxotrophy in the strain. It further contains a production plasmid coding an enzyme for production of a low molecular weight substance or a recombinant protein and a functional copy of the gene whose chromosomal inactivation brings about the auxotrophy, wherein the auxotrophy is a non-feedable auxotrophy.

First claim

The invention claimed is:

1. A microorganism strain for producing low-molecular-weight substances or proteins, said microorganism strain comprising: (a) a genome containing a mutation in a gene, which mutation brings about an auxotrophy in the microorganism strain, and (b) a production plasmid encoding at least one enzyme for producing a low-molecular-weight substance or at least one recombinant protein and also a functional copy of the gene, the chromosomal inactivation of which brings about the auxotrophy, wherein the auxotrophy is a nonfeedable auxotrophy and the gene is a pyrH gene or a plsC gene or homologous genes thereof.

2. The microorganism strain as claimed in claim 1 , wherein the mutation of the gene brings about the nonfeedable auxotrophy in the strain, and leads to inactivation of said gene.

3. The microorganism strain as claimed in claim 1 , wherein the mutation of the gene brings about the nonfeedable auxotrophy in the strain, and leads to inactivation of an activity of a gene product coded by the gene.

4. A method for producing a strain as claimed in claim 1 , comprising: (i) introducing into said strain a temperature-sensitive plasmid comprising a functional copy of a pyrH gene or a functional copy of a plsC gene or homologous genes thereof; (ii) mutating or deleting in the genome of the strain the pyrH gene or the plsC gene or homologous genes thereof which leads to a nonfeedable auxotrophy in the strain; (iii) transforming said strain with a production plasmid comprising a gene encoding an enzyme for the production of a low-molecular weight substance or a recombinant protein and also a functional copy of the pyrH gene or a functional copy of the plsC gene or homologous genes thereof; and (iv) exchanging the temperature-sensitive plasmid for the production plasmid at a non-permissive temperature.

5. The method as claimed in claim 4 , wherein the temperature-sensitive plasmid has a temperature-sensitive origin of replication.

6. The method as claimed in claim 5 , wherein cells are exposed, immediately after the transformation with the production plasmid, to a temperature shock at 47-55° C. for 30-90 min and then further incubation is then carried out at the nonpermissive temperature of 37-45° C.

7. A method for producing low-molecular-weight substances or proteins comprising producing the low-molecular-weight substances or proteins with a microorganism strain as claimed in claim 1 by culturing said strain in an antibiotic-free fermentation medium.

8. A method for producing low-molecular-weight substances or proteins comprising producing the to substances or proteins with a microorganism strain as claimed in claim 1 by culturing said strain in an antibiotic-free fermentation medium.

Assignees

• Wacker Chemie Ag

Inventors

• Bornhoevd Carsten
• Thoen Marcel

Classifications

• C12N9/1074

  Cyclomaltodextrin glucanotransferase (2.4.1.19) · CPC title

• C12P13/12

  Methionine; Cysteine; Cystine · CPC title

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title