Multi-species polynucleotide control sequences

a polynucleotide control and multi-species technology, applied in the field of new nucleic acid sequences, can solve the problems of too much cloning work, high cost of synthetic dna, and 1000 synthetic genes costing 1 million us dollars

Inactive Publication Date: 2011-07-07
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although fairly efficient there is a common drawback in all these methods: they use host species-specific regulation systems.
There are however cases where this will not help: (i) if the gene sequence/s is/are unknown, i.e. in metagenomic screening projects; (ii) if the protein needs donor-specific chaperones or helper enzymes, like P450 enzymes; (iii) if the DNA, RNA-intermediates and/or enzyme is toxic to the new host; (iv) if the folding of the enzyme is crucial for the activity and not possible by the new host; (v) if one wants to compare many known enzymes the total costs of synthetic DNA will become very high again, i.e. 1000 synthetic genes cost 1 million US dollar.
However, current state-of-the-art tools allow only for species-specific expression cassettes.
For each host a species-specific promoter is used and this leads to too much cloning work if one wants to evaluate the same enzyme(s) in multiple organisms.
However, in pra

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 4

Multisite Insertion of Bacterial Sequences in Fungal Promoters Drive Efficient Gene Expression in Escherichia coli

[0112]In a further attempt to find a promoter which is active in both eukaryotes and prokaryotes, consensus sequences in the last 84 basepairs of the PE4 promoter of Bacillus subtilis (Stewart et al., 1998) were inserted at several positions in the glucose-repression-insensitive pcbC promoter of Penicillium chrysogenum (SEQ ID NO 67) and in the gpdA promoter of Aspergillus (SEQ ID NO 70). This lead to the polynucleotide sequences of SEQ ID NO 46 and 48, respectively, embedded in the polynucleotide control sequences of SEQ ID No 72 and 74. All constructs were made synthetically in front of the ble gene (DNA2.0, Menlo Park, Calif. 94025, USA) followed by fungal terminators (yielding plasmids p3306ble and p3310ble, respectively). In some cases the bacterial sequences replaced the fungal sequences at the site of insertion. This lead to the polynucleotide sequences of SEQ ID...

example 5

Fungal Sterol Transmethylase Like Promoter Drives Expression in E. coli

[0117]To verify the general applicability of the consensus sequence, the genomic sequences of Aspergillus niger and Penicillium chrysogenum were screened for promoter regions which followed the consensus sequence of Example 4 and could be used in E. coli.

[0118]As an example, one promoter of Penicillium chrysogenum was identified that was actively transcribed under standard growth conditions (verified by Affymetrix MicroArrays) and followed the consensus sequence (see FIG. 5A). The promoter is in front of a gene with strong similarity to the sterol transmethylase ERG6 of Candida albicans. Two constructs were made synthetically in front of the ble gene (Codon Devices, Cambridge, Mass., USA) and followed by termination sequences. One synthetic construct contains one modification in the promoter sequence: CAT in front of the startcodon to introduce an NdeI site (the polynucleotide sequence of SEQ ID NO 50, embedded...

example 6

Use of the amdS Gene and Acetamide Selection in Escherichia coli

[0121]As the amdS gene encoding acetamidase (for example the Aspergillus nidulans amdS gene), is a useful selectable marker gene in yeast and fungi from which transformants can be readily selected for the presence or the absence, it would be very useful if such a marker would function in prokaryotes, as they are often incapable of growing on acetamide (see for example the results below) and the gene is not part of general metabolism. To this end the cDNA of the Aspergillus nidulans amdS gene was PCR amplified from mRNA isolated from amdS positive Penicillium transformants of Example 2. Total RNA was isolated from approximately 106 cells using the StrataPrep Total RNA MicroPrep Kit (Stratagene). One-tenth of the total RNA was used for oligo-dT directed cDNA synthesis (Thermoscript RT-PCR, Life Technologies), which was used as template for 30 cycles of PCR (using a proofreading enzyme) using the specific oligonucleotides...

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Abstract

The present invention relates to polynucleotide sequences which enable a polynucleotide control sequence, such as a promoter, to direct expression in a wide range of industrially relevant species, both prokaryotes and eukaryotes. When the polynucleotide sequences of the invention are applied in combination with selection marker genes it is possible to perform selectable cloning in a laboratory host and use the same construct in the final host

Description

FIELD OF THE INVENTION[0001]The present invention relates to new nucleic acid sequences which are able to drive gene expression in several industrially relevant species.BACKGROUND OF THE INVENTION[0002]To optimize the production of various compounds of interest recombinant DNA technologies provide for a very relevant toolbox. These tools allow for the efficient modification of genomic DNA in such way that various alterations are possible. Among these are: overexpression of a homologous gene, overexpression of a heterologous gene, deletion of a homologous gene, block a metabolic route to a unwanted side product, diversion of a metabolic route. Although fairly efficient there is a common drawback in all these methods: they use host species-specific regulation systems. If one wants to test the properties of an enzyme encoded by a certain gene this is most often done in one species; the favorite species of the laboratory. If such a gene is obtained from a different donor species, quite ...

Claims

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Application Information

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IPC IPC(8): C12P1/00C07H21/00C12N15/63C12N1/00C12Q1/68C12P21/00C12N15/00
CPCC12N15/70C12N15/81C12N15/80C12N15/63C12N15/75
Inventor VAN DEN BERG, MARCO ALEXANDER
Owner DSM IP ASSETS BV
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