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Expression system used for the antibiotic-free production of polypeptides

An expression system and DNA sequence technology, applied in the field of expression systems that produce polypeptides in the absence of antibiotics, can solve the problems of poverty, complexity, and methods for increasing the formation rate of products, and achieve the effect of high yield

Inactive Publication Date: 2011-01-19
AB多酶两合公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For prokaryotes, the disadvantage of this approach is that after transformation, only one copy of the gene is present in the host, and the method of integrating other copies of the same gene in order to increase the rate of product formation via gene dosage effects is rather complicated
[0016] In conclusion, it must be stated that, despite several years of experience in the production of polypeptides by biotechnological methods, so far, no possibility has been provided to produce them using multiple copies of the relevant genes, without resorting to expensive or ecologically affecting substances such as antibiotics. Selected utility system
Due to the complexity of the media used industrially (WO2004 / 078953), or because the system still contains antibiotic resistance genes, different methods of selection by auxotrophic markers have also so far given rather poor results

Method used

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  • Expression system used for the antibiotic-free production of polypeptides
  • Expression system used for the antibiotic-free production of polypeptides
  • Expression system used for the antibiotic-free production of polypeptides

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[0083] Molecular biology work was performed according to standard methods, eg as described in guidelines by Sambrook and Russell (2001, Molecular cloning. Cold Spring Harbor Laboratory Press). All kits and enzymes used were applied according to the respective manufacturer's instructions.

example 1

[0084] Example 1: Isolation of the gpsA gene in Bacillus amyloliquefaciens

[0085] To isolate the gpsA gene, chromosomal DNA of Bacillus amyloliquefaciens (AB Enzymes GmbH strain collection) was prepared by means of the QIAGEN DNeasy Tissue kit (Qiagen, Hilden), and the gpsA gene was amplified thereon by PCR. Primers were thus used that hybridized upstream and downstream of the gpsA gene, so that the gene was fully amplified. The primers are sequences derived from the flanking regions of gpsA as determined by the sequencing in Example 2a). Amplification was performed using the following primers:

[0086] SEQ ID NO. 3: AL 1_1198 gctgttaagccgccgagcttcgttg,

[0087] SEQ ID NO. 4: AR_180C taatcccatagcaccaagcgcaaaccac.

[0088] A DNA fragment containing 1591 bp was completely sequenced using the method of Sanger et al. (1977, Proc. Natl. Acad. Sci. USA 74, 5463-5467). The sequencing primers used are listed in Table 1.

[0089] Table 1: Primers used for complete sequencing of ...

example 2

[0092] Example 2: Deletion of the gpsA gene in Bacillus amyloliquefaciens

[0093] Elimination of the gene gpsA on the chromosome of B. amyloliquefaciens was performed by deletion of the vector. The procedure is based on Vehmaanper Description by et al. (1991). Plasmid PE194 (described in the same article) was chosen as vector for gpsA deletion. The advantage of this vector is that it does not have a temperature-dependent origin of replication. At 28°C, pE194 can replicate in cells so that at this temperature, pE194 can be selected first for successful transformation. Subsequently, cells containing the vector were incubated at 46°C. At this temperature, the vector no longer replicates and selection pressure is exerted when the plasmid integrates into the chromosome via one of the two regions of homology (upstream and downstream of gpsA). Subsequently, further homologous recombination via another (second) region of homology results in deletion of gpsA. Further recombinat...

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Abstract

The invention relates to an expression system for the production of one or several target polypeptide(s), comprising a host cell, in whose genome the DNA sequence coding the glycerine-3-phosphate dehydrogenase is inactivated or partially or fully deleted and which is transformed with an extrachromosomal element that comprises a DNA Sequence coding the target polypeptide(s) and the glycerine-3-phosphate dehydrogenase. According to the invention, both the host cell genome and the extrachromosomal element do not carry any antibiotic-resistant gene.

Description

technical field [0001] The present invention relates to a microbial expression system that produces polypeptides based on the use of extrachromosomal DNA, thereby making it unnecessary to use antibiotic marker genes for host cell selection (proteins derived from said genes confer antibiotic resistance on cells, so These genes are also known as antibiotic resistance genes, resistance genes, antibiotic markers, or antibiotic selection markers), but instead use the enzyme encoding glycerol-3-phosphate dehydrogenase (also known as NAD(P)H-dependent dihydroxyacetone phosphate reductase , NAD(P)H-dependent 3-phosphate glycerol dehydrogenase, 3-phosphate glycerol dehydrogenase (NADP), 3-phosphate glycerol synthase, biosynthetic 3-phosphate glycerol dehydrogenase, L-3-phosphate Glycerol: DNA sequence of NAD (P) oxidoreductase), so desired polypeptides (eg xylanase) are produced without the addition of antibiotics. Antibiotic resistance genes are absent in the expression system. The ...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N9/04
CPCC12N15/75C12N9/0006
Inventor 斯爱琳·卡多特啼娜·普劳斯璐斯·史维尔德福哲布如诺·文因特
Owner AB多酶两合公司
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