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Mutant cells having reduced expression of metallopeptidase, suitable for recombinant polypeptide production

A technology of cells and heterologous polypeptides, applied in the direction of recombinant DNA technology, enzymes, microorganisms, etc., can solve problems such as undisclosed metalloproteinase activity

Inactive Publication Date: 2012-04-11
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To our knowledge, no studies on this predicted ORF or putative encoded metalloprotease activity have been published since

Method used

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  • Mutant cells having reduced expression of metallopeptidase, suitable for recombinant polypeptide production
  • Mutant cells having reduced expression of metallopeptidase, suitable for recombinant polypeptide production
  • Mutant cells having reduced expression of metallopeptidase, suitable for recombinant polypeptide production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] Example 1. Construction of Mutant Bacillus licheniformis α-amylase Host

[0161] This example describes the construction of a B. licheniformis strain containing two copies of the gene encoding a secreted alpha-amylase. Mutants of this strain were then constructed by introducing a deletion in the gene encoding the putative metalloprotease BL00829.

[0162] The alpha-amylase used in this example was the JE1 alpha-amylase polypeptide originally produced by the alkaliphilic Bacillus sp. JP170. This is the gene contained in plasmid pTVB115, described in WO99 / 23211. This specific gene will be referred to as "je1" hereinafter.

[0163] MOL2650: Two-copy je1α-amylase strain

[0164] As described in Example 6 of WO2005 / 123915, the je1 gene was transferred to an integrating vector designed to allow integration of the alpha-amylase expression cassette into the chromosome of the Bacillus licheniformis strain, which already contained the amyL locus and xylA integrated at the A...

Embodiment 2

[0172] Construction of the Bacillus licheniformis β-amylase host of the mutation of embodiment 2

[0173] This example describes two Bacillus licheniformis strains containing the gene encoding a secreted β-amylase; the two strains are isogenic except for one that is mutated by deletion in the gene encoding the putative metalloprotease BL00829 of. The β-amylase used in this example was a β-amylase originally produced by the thermophilic bacterium Clostridium thermosulfurogenes, which replaced the native signal sequence with a heterologous secretion signal sequence derived from Bacillus licheniformis amyL . This particular hybrid gene will be referred to as "bmy1" hereinafter, and its nucleotide sequence is shown in SEQ ID NO:15.

[0174] AN411: Two-copy bmy1 beta-amylase strain

[0175] As described in Example 6 of WO2005 / 123915, the bmy1 gene (SEQ ID NO: 15) was transferred to an integrating vector designed to allow integration of the β-amylase expression cassette into th...

Embodiment 3

[0183] Example 3. Beta-amylase production in the Bacillus licheniformis strain from Example 2

[0184] A fed-batch fermentation process of the Bacillus licheniformis strain from Example 2 was performed as follows. All media were sterilized by methods known in the art. Unless otherwise described, tap water was used. The ingredient concentrations mentioned in the formulations below are the concentrations prior to any inoculation.

[0185] culture medium

[0186] LB agar: 10 g / l peptone from casein; 5 g / l yeast extract; 10 g / l sodium chloride; 12 g / l bacterial agar, adjusted to pH 7.0 + / - 0.2. A premix from Merck (LB-agar (Miller) 110283) was used.

[0187] M-9 Buffer: Disodium Hydrogen Phosphate, 2H 2 O 8.8g / l; Potassium dihydrogen phosphate 3g / l; Sodium chloride 4g / l; Magnesium sulfate, 7H 2 O 0.2 g / l (use deionized water in this buffer).

[0188] PRK-50: 110g / l soybean grits (soy grits); disodium hydrogen phosphate, 2H 2 O 5 g / l; defoamer (Struktol SB2121; Schill & S...

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Abstract

A mutated bacterial cell producing at least one heterologous polypeptide of interest, wherein said cell has a reduced expression-level of a polypeptide comprising an amino acid sequence at least 70% identical to the sequence shown in SEQ ID NO: 2, when compared with an otherwise isogenic but non-mutated cell; methods for producing said mutated cell; and methods for producing a polypeptide of interest using said mutated cell. SEQ ID NO: 2 represents a putative metalloprotease.

Description

[0001] sequence listing [0002] The present invention includes a Sequence Listing. technical field [0003] The present invention relates to mutant bacterial cells producing at least one heterologous polypeptide of interest, wherein said cells have a reduced composition comprising at least the sequence shown in SEQ ID NO: 2 compared to other isogenic but non-mutated cells. expression levels of polypeptides having 70% identical amino acid sequences; methods for producing said mutant cells; and methods for using said mutant cells to produce a polypeptide of interest. Background technique [0004] The recombinant industrial production of polypeptides, and in particular enzymes, is a highly competitive field in which the level of technology is very high. The industrial production of polypeptides in Bacillus is especially well-studied, and at present even steady improvements in polypeptide yield or productivity are required in this field. [0005] It has been known for a long ...

Claims

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Application Information

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IPC IPC(8): C12N9/56C12N15/75C12R1/07
CPCC12N15/75C12P21/02
Inventor A.K.尼尔森M.D.拉斯马森N.班克
Owner NOVOZYMES AS
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