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Stable Genomic Integration of Multiple Polynucleotide Copies

a technology of polynucleotide and genomic integration, which is applied in the field of stable genomic integration of multiple polynucleotide copies, can solve the problems of difficult to achieve proper chromosomal integration, and achieve the effect of increasing the number of polynucleotide copies and increasing the production of gene products

Inactive Publication Date: 2008-04-10
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for simultaneously integrating multiple copies of a gene encoding a polypeptide of interest into the host cell chromosome, while also providing the means for initiating transcription of the gene after the proper integration. This method involves introducing a polynucleotide construct containing a recognition sequence of a site specific recombinase and the gene of interest into the host cell, followed by the recombinase-mediated integration of the gene into the chromosome. The simultaneous integration of multiple copies of the gene can be done using a low-copy number construct, which is more efficient than separate integration events. The invention also provides a host cell designed for this purpose and a method for producing the polypeptide of interest.

Problems solved by technology

It may often be difficult to achieve proper chromosomal integration in a host cell of a gene encoding a polypeptide of interest, when the gene is introduced into the cell on a DNA construct, even a low-copy number construct, while being actively transcribed from a promoter on the construct.
This is particularly true for polypeptides that are inhibitory or perhaps even toxic to the host cell above a certain concentration.

Method used

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  • Stable Genomic Integration of Multiple Polynucleotide Copies
  • Stable Genomic Integration of Multiple Polynucleotide Copies
  • Stable Genomic Integration of Multiple Polynucleotide Copies

Examples

Experimental program
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Effect test

example 1

Construction of a B. subtilis Strain with Two TP901-1 attB Sites in the Chromosome

[0162]As the first step, an 161 bp attB site (attB161, SEQ ID NO: 21) was integrated in the xyl locus in B. subtilis strain PL1801, resulting in the strain AEB43. Integration was obtained by double cross-over of a DNA fragment which contains attB161 adjacent to the cat gene, surrounded by an upstream and a downstream region of the xyl locus. The upstream and downstream xyl fragments were obtained with PCR on chromosomal DNA from B. subtilis using primers that are suitable for amplifying regions of sufficient size for an efficient integration by homologous recombination (0.5 kb or more). By PCR, these fragments were joined with the attB161 fragment (obtained from Lactococcus lactis subsp. cremoris 3-107) and with the cat gene, yielding chloramphenicol (Cm) resistance.

[0163]This xylup-attB-cat-xyldown fragment was introduced into PL1801 by transformation and the transformants were plated on Cm containing...

example 2

Construction of an Integrase-Donor Plasmid

[0166]The TP901-1 integrase is needed to perform the recombination between the attB and attP sites. The expression of the integrase can be placed under the control of a constitutive or an inducible promoter. In plasmid pAEB146 expression of the integrase is under the control of the Pxyl-promoter, which is induced from a low to a high level of activity upon the addition of xylose. pAEB146 has a temperature sensitive replicon functional in Bacillus and the oriT region from plasmid pUB 110 which enables conjugation, and thus can be used in both B. subtilis and B. lichenformis. Alternatively, the integrase can be expressed from a plasmid which has a different kind of replicon, or it could be integrated into the chromosome.

example 3

Construction of an attP Plasmid

[0167]pAEB153 contains the minimal attP site of TP901-1 (SEQ ID NO: 23) determined in Brøndsted and Hammer (1999) App. Environ. Microbiol. 65 752-758, but a larger attP region can also be used, or a smaller, if it is still active in recombination. Replication of pAEB153 is dependent on donation of replication protein from another plasmid with the pE194 replicon such as pAEB146. Alternatively the attP site can be cloned on a different plasmid vector, e.g. one with an origin which is not dependent on other plasmids for replication, and / or a thermosensitive origin. The attP site can also be included on the plasmid from which integrase is expressed.

[0168]The plasmid containing attP can be used as a vector for cloning genes in such a way, that integration of the plasmid in attB in the chromosome will lead to expression of the gene from a promoter present in the chromosome next to the attB site. We therefore included the mRNA-stabilizing cryIIIA region in pA...

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Abstract

Methods of constructing a cell comprising in its chromosome one or more copies of an open reading frame (ORF) or operon encoding at least one polypeptide of interest, each copy being under the transcriptional control of a heterologous promoter using a site specific recombinase and in vivo integration by recombination; means for Promoter carrying out the methods, resulting cells, and methods for producing a polypeptide of interest using the resulting cells.

Description

FIELD OF INVENTION[0001]A large number of naturally-occurring organisms have been found to produce useful polypeptide products, e.g., enzymes, the large scale production of which is desirable for research and commercial purposes. Once such product has been identified efforts are being made to develop production methods leading to a high production of the product. One widely used method, which is based on recombinant DNA techniques, is to clone a gene encoding the product, inserting the gene into a suitable expression system permitting the expression of the product and culturing a suitable host cell comprising the expression system, either integrated in the chromosome or as an extrachromosomal entity, under conditions conducive for the expression of the product.[0002]Irrespective of which production method is used, it is normally desirable to be able to increase the production level of a given polypeptide or protein. Thus, efforts are being made to increase the production, e.g. by in...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/74C12N1/20C12P21/04
CPCC12N15/1082C12N15/1086C12N2800/30C12N15/90C12N15/75C12N15/902C12N2830/50
Inventor BREUNER, ANNERASMUSSEN, MICHAEL DOLBERG
Owner NOVOZYMES AS
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