Multi-species polynucleotide control sequences

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 ...

AI Technical Summary

Benefits of technology

[0021]These polynucleotide sequences of the invention have the great advantage that when present in a polynucleotide control sequence, such as a promoter, the control sequence will direct expression in a wide range of industrially relevant species, in both prokaryotes and eukaryotes. Moreover when applied in combination with selection marker genes one can perform selectable cloning in a laboratory host (for example making deletion constructs in E. coli) and use the same construct in the final host. Also, for genes encoding enzymes like acetamidase (for example the Aspergillus nidulans amdS gene), which enzyme enables both forward and backward selection (i.e. selection for the presence or the absence of the enzyme), the polynucleotide control sequences of the present invention provide for a very efficient selection marker cassette which can be used in a wide range of industrially relevant species.

[0041]The term “operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the sequence gene-of-interest such that the control sequence directs the production of a polypeptide. It should be clear to those skilled in the art that transcriptional and translational stop signals are not always well defined and also not need to be specifically added to the gene-of-interest sequence, although specific addition can increase the overall efficiency polypeptide production. The polynucleotide control sequence preferably contains part of or the complete polynucleotide sequence of the invention. The polynucleotide control sequence may be any nucleic acid sequence, which shows transcription regulatory activity in the cell including mutant, truncated, and hybrid promoters, and may be obtained from any source, including but not limited to genomic DNA, synthetic DNA, copy DNA. The polynucleotide control sequence may be either homologous or heterologous to the cell or to the gene-of-interest.

[0065]The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequences disclosed herein can be readily used to isolate the complete polynucleotide or nucleic acid sequence, which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.

[0069]In an even more preferred embodiment, Micro Array data is used to select genes, and thus polynucleotide control sequences of those genes, that have a certain transcriptional level and regulation. In this way one can adapt the gene expression cassettes optimally to the conditions it should function in.

[0070]Alternatively, random DNA fragments may be cloned in front of the selectable marker genes, for example an antibiotic resistance marker gene like the ble gene, encoding for a protein that provides resistance towards compounds like zeocin, bleomycin and phleomycin. This is a selection marker gene which is used in several species (fungi, yeasts, bacteria), although with species specific promoters. Using selective growth conditions one can easily select for active polynucleotide control sequences, as these should facilitate growth on media containing zeocin (or phleomycin or bleomycin or any suitable alternative compound) in such a concentration that it will inhibit growth of the parent cell or cells with a non-functional promoter. These DNA fragments can be derived from many sources, i.e. different species, PCR amplified, synthetically and the like.

[0077]The polynucleotide molecules and the host cell of the invention may advantageously be used in a method for in vitro or in vivo cloning experiments. The combination of expression cassettes consisting of polynucleotide control sequences fused to the selection marker genes has many advantages. It significantly enhances the success rate of classical restriction enzyme and ligation cloning, it enhances the success rate single and multiple fragment Gateway recombination reactions, enables efficient multiple fragment STABY cloning reactions.

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 practice this involves still quite some laboratory procedures, especially when one want to test hundreds to thousands genes of interest.

Moreover, technologies like Gateway insert a stretch of nucleotides (20-30 bp) between promoter and gene-of-interest, which can severely hamper the transcription and / or translation of the gene.

However, there seem to be some crucial differences in promoter organization between species; for example between eukaryotes and prokaryotes.

But species like Bacillus are much more stringent, while the wide metabolic diversity of Streptomyces might have evolved into an extremely wide range of promoter structures making it impossible to extract common (and predictive) features in this group.

So, in practice there might be sequences in a promoter which are not recognized by any of the cell's machinery in one host, while in another host this would be the basis of a very strong and unwanted transcriptional regulation.