Methods for producing polypeptides in aspergillus mutant cells

A technology of Aspergillus and cells, applied in the direction of microorganism-based methods, methods using fungi, biochemical equipment and methods, etc., can solve problems that affect the cost of products and the time to put products on the market

Inactive Publication Date: 2009-08-19
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In many cases, these extensive analyzes are required for each batch production, affecting product cost and time to market

Method used

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  • Methods for producing polypeptides in aspergillus mutant cells
  • Methods for producing polypeptides in aspergillus mutant cells

Examples

Experimental program
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Effect test

Embodiment 1

[0236] A. Construction of a CPA-negative strain of Aspergillus oryzae Bz 14

[0237] Freeze-dried spores of Aspergillus oryzae Bz 14 strain were irradiated with an optimal dose of γ-rays, ranging from 1000Gy to 1250Gy, and these spores were plated on the plates of screening medium 1 at a density of 25-50 colonies / 9cm plate superior. Cyclopiazonic acid producing colonies formed a red counter (bottom of the colony) on selection medium 1 due to the presence of red insoluble CPA-Fe complexes.

[0238] Approximately 50,000 colonies were screened from irradiated spores and 154 CPA-deficient colonies were isolated, which were characterized by a cream / white appearance. After re-isolation, 64 strains still maintained a non-red appearance on the selection medium 1 plates. CPA was not detected in 52 strains using TLC-plug (plate) analysis. These strains were then cultured in MDU-1B medium under toxin-induced (34° C., 250 rpm, cultured for 5 days) shaking flask fermentation culture con...

Embodiment 2

[0255] Expression of lipase in Aspergillus oryzae strains JAL228 and BECh1

[0256] a. Construction of plasmid pCaHj493

[0257] The lipase plasmid pAHL (WO 97 / 07202) was digested with BamHI and SalI, and a 916bp lipase-encoding fragment was isolated.

[0258] pCaHj 483 was digested with BamHI and XhoI and the 6757 bp vector fragment was ligated to the lipase fragment as described in WO 98 / 00529. The ligation mixture was used to transform E. coli DH5α cells, and transformants containing the desired plasmid were isolated. This plasmid was named pCaHj493.

[0259] b. Transformation of pCaHj 493 into JaL228 and BECh-1 strains

[0260] pCaHj 493 was transformed into Aspergillus oryzae strains JaL228 and BECh1 by selection in acetamide as described in EP-A-0531372. Transformants were re-spored twice. Second reisolated spores of each transformant were tested for lipase production in shake flask and microtiter plate cultures.

Embodiment 3

[0262] A. Production of lipase in CPA-negative and CPA-positive Aspergillus oryzae strains

[0263] The lipase production of 18 JaL228 transformants and 30 BECh 1 transformants prepared as described in Example 2 in shake flask cultures was examined.

[0264] Cove N slant cultures of transformants were harvested with 10 ml of 0.1% Tween solution, and the spore suspension was used as inoculum in 100 ml of Gl-Gly medium and placed in 500 ml double baffle shake flasks. The medium was incubated on a rotary shaker at 250 rpm at 34°C for 24 hours. Then 10ml of the Gl-Gly culture was transferred to 100ml of 1 / 5MDU-2BP in a 500ml shake flask and continued at 34°C at 250rpm.

[0265] Samples were removed after 50 hours, filtered through Miracloth and centrifuged at 4000 xg. With a simple radioimmunodiffusion method (see Scand.J.Immuno.Vol.17, suppl.10, pages 41-56, (1983) "Technical Handbook of Immunoprecipitation Gel Technology", edited by N.H.Axelsen, Blackwell Scientific Publicatio...

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Abstract

A method is provided for producing a polypeptide of interest by (a) cultivating a mutant of a parent Aspergillus cell, wherein (i) the mutant comprises a first nucleic acid sequence encoding the polypeptide and a second nucleic acid sequence comprising a modification of at least one of the genes responsible for the biosynthesis or secretion of at least one toxin, and (ii) the mutant produces less of the toxin than the parent Aspergillus cell when cultured under the same conditions; and (b) isolating the polypeptide from the culture medium. Also, mutants of Aspergillus cells are provided, as well as methods for obtaining the mutant cells.

Description

field of invention [0001] The present invention relates to a method for producing a polypeptide of interest in toxin-deficient Aspergillus mutant cells. The present invention also relates to mutant strains of Aspergillus cells and methods for obtaining such mutant cells. Background of the invention [0002] In recent years, the use of recombinant host cells to express heterologous polypeptides has greatly simplified the mass production of some economically valuable polypeptides, such as key enzymes and secondary metabolites used in industry, while using other methods would face many difficulties, such as the production of polypeptides. Available in low yield or only purified from its natural source. A variety of expression systems are now available from which to choose for the production of any given polypeptide, including eubacterial and eukaryotic hosts. Selection of an appropriate expression system often depends not only on the ability of the host cell to produce suffic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C12R1/66C12N1/15C12N9/10C12N15/09C12N9/00C12N9/20C12N9/24C12N9/42C12N15/01C12N15/80C12P21/02C12R1/69
CPCC12N9/20C12N9/2482C12N9/1085C12N9/10C12N9/00C12P21/02C12N15/80C12R1/69C12Y302/01008C12R2001/69C12N1/145
Inventor 比约恩·E·克里斯坦森亨里克·莫尔加尔德斯文德·卡斯加尔德简·莱姆贝克
Owner NOVOZYMES AS
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