Our product and project portfolio represents a huge diversity of different enzymes screened from a variety of microorganisms in nature or from molecular evolution in the laboratory. Our bacterial expression systems are based on the host strains from Bacillus species such as Bacillus subtilis and Bacillus licheniformis, which has a documented history of safe use in industrial processes over 30 years.
The ability to express enzymes of any class from any source is the key for unlocking the practical use of enzymes for industry. We produce the enzyme in yields often 1000 fold higher than what is achieved by the indigenous organism. Our product diversity and world class speed of bringing new recombinant proteins to the market is a solid prove of the versatility of our unique expression systems.
Below are a few of our patents covering the Prokaryotic expression area. If you are interested in one of the patents do not hesitate to contact us for more details.
Title of Technology: Production of translocated polypeptides
Abstract: The technology relates to a method of producing a translocated polypeptide comprising cultivating a cell of Bacillus which through conjugation has aquired a DNA construct encoding said translocated polypeptide.
The technology also relates to methods of introducing a DNA construct encoding a polypeptide of interest into recipient cells of Bacillus sp. by conjugation, in which methods a population of bacterial donor cells harboring i) a plasmid comprising the DNA construct and at least one cisacting DNA sequence required for the transfer of said plasmid by conjugation in the presence of a transacting mobilizing elem ent, and ii) at least one DNA sequence encoding said transacting mobilizing element, and a population of Bacillus sp. recipient cells are mixed under conditions allowing the plasmid to be transferred from the popu-lation of donor cells to the population of recipient cells by conjugation. The recipient cells may e.g. be cells of industrial Bacillus strains or alkalophilic Bacillus sp. for which known DNA introduction methods either are nonexisting or very laborious.
Detailed Description: Production of a Translocated Polypeptide: A translocated polypeptide is produced by cultivation of a cell of Bacillus which through conjugation has aquired a DNA construct encoding the translocated polypeptide in question.
The conjugation is preferably accomplished by use of a plasmid carrying the DNA construct and at least one cisacting sequence required for transfer of said plasmid by conjugation in the presence of at least one mobilizing element, the mobilizing element being provided in trans, i.e. by any of the methods of conjugation described in US 5,843,720.
The Bacillus cell (e.g. a cell of an alkalophilic Bacillus) is free from undesired selectable marker gene(s) such as a gene encoding an antibiotic resistance marker which has been used for selection of cells having received the DNA sequence encoding the translocated polypeptide. Methods of obtaining Bacillus cells free from undesired se-lectable marker gene(s) are described in US 5,843,720.
In order to improve the stability of the Bacillus cell to be used in the method it is desirable that the DNA construct encoding the translocated polypeptide is integrated into the genome of the Bacillus cell. This may be accomplished when the plasmid carries one or more DNA sequences which are sufficiently homologous to a part of the genome of the recipient cell to allow for homologous recombination. Suitable methods for obtai ning stable integration of the DNA construct in the genome of the reciepient cell are discussed in further details below.
The translocatable polypeptide to be produced is preferably a se-creted polypeptide or a polypeptide of the secretory pathway of a secreting cell. The secreted polypeptide may be an enzyme, e.g., selected from an amylolytic enzyme, a lipolytic enzyme, a proteolytic enzyme, a cellulytic enzyme, an oxidoreductase or a plant cellwall degrading enzyme.
Methods for DNA Introduction by Conjugation
Using an Industrial Bacillus or an Alkalophilic Bacillus Recipient Cell:
This method relates to a population of bacterial donor cells harboring i) a plasmid comprising the DNA construct and at least one cisacting DNA sequence required for the transfer of said plasmid by conjugation in the presence of a transacting mobilizing element, and ii) at least one DNA sequence encoding said transacting mobilizing element, and a population of Bacillus sp. recipient cells are mixed under conditions allowing the plasmid to be transferred from the population of donor cells to the population of recipient cells by conjugation, the Bacillus sp. being an alkalophilic Bacillus sp. and/or an industrial Ba-cillus sp.
This method is particularly advantageous since strains of alkalophilic Bacillus sp. and/or industrial Bacillus strain-in general - have been found to be non-transformable by competence and thus only subject to protoplast transformation which-as described above-is a very cumbersome method for introduction of DNA.
Examples of alkalophilic Bacillus sp. and industrial Bacillus strains are given in US 5,843,720. In particular, the Bacillus cell to be used in accordance with this aspect of the technology may be a high-yielding mutant.
Using an Auxotrophic Donor Cell:
This method relates to cons truction of a cell of a Bacillus sp. harboring a DNA construct encoding a polypeptide of interest, in which method a population of auxotrophic bacterial donor cells harboring i) a plasmid comprising the DNA construct and at least one cisacting DNA sequence required for the transfer of said plasmid by conjugation in the presence of a transacting mobilizing element, and ii) at least one DNA sequence encoding said transacting mobilizing element, and a population of unmarked Bacillus sp. recipient cells are mixed under conditions allowing the plasmid to be transferred from the population of the auxotrophic donor cells to the population of unmarked recipient cells by conjugation, and the auxotrophic property of the donor cell is exploited to select for recipient cells.
This method is clearly advantageous in that no selection need to be made for recipient cells they are the only one remaining when exploiting the auxotrophic property of the donor cell.
The donor cell may, e.g., be auxotrophic for specific amino acids. A particular preferred donor to be used is a donor which is auxotrohic for D-alanine, i.e. a donor which is dal.sup.-. After the conjugation treatment has been accomplished the mixture of dal.sup.- donor cells and recipient cells is cultivated on or in a medium devoid of D-alanine, i.e. as medium in or on which the dal.sup.- donor cells are unable to grow. Thereby only recipient cells remain. The principle of using an auxotrophic marker is described in, e.g., US 4,920,048.
Subsequently, only selection for recipient cells having aquired the DNA construct of interest must be made, conveniently by use of a selection marker, e.g. an antibiotic resistance, encoded by the plasmid.
Using a Curable Plasmid:
This method relates to introduction of a DNA construct encoding a polypeptide of interest into a cell of a Bacillus sp., in which method a po pulation of bacterial donor cells harboring i) a curable plasmid comprising the DNA construct and at least one cis-acting DNA sequence required for the transfer of said plasmid by conjugation in the presence of a transacting mobilizing element, and ii) at least one DNA sequence encoding said trans-acting mobilizing element, and a population of Bacillus sp. recipient cells are mixed under conditions allowing the plasmid to be transferred from the population of donor cells to the population of recipient cells by conjugation.
The use of a curable plasmid is of particular relevance for the construction of recipient cells having received some elements of the plasmid which through conjugation has been transferred into the cell, but which is free from other elements such as the cis-acting DNA sequence required for the transfer of the plasmid by conjugation. In such cases it may be advantageous to integrate the DNA construct encoding a polypeptide of interest into the genome of the recipient cell, optionally together with other elements to be retained in the cell, whe-reas the elements which are not desired in the cell (such as the cis-acting DNA sequence) are retained on the plasmid. After genomic integration has taken place the cell is cured from the curable plasmid carrying the unwanted elements.
The curable plasmid to be used may be constructed by combining the respective elements (e.g. a temperature sensitive origin of replication, a cis-acting DNA sequence, a DNA construct of interest, a selectable marker gene, etc.) in accordance with methods known in the art, typically by modification of either a curable plasmid or a plasmid carrying the cis-acting DNA sequence.
Genomic Integration of a DNA Construct of Interest:
In a further aspect the technology relates to a method of introducing a DNA construct encoding a polypeptide of interest into a cell of a Bacillus sp. and obtai ning stable integration thereof into the genome of the recipient cell.
Amplification of Genomic DNA Sequences:
In a still further aspect the technology relates to a method for in vivo amplification of a DNA sequence B present in the genome of a recipient Bacillus sp. cell, in which method a) a population of bacterial donor cells harboring i) a plasmid comprising at least one cis-acting DNA sequence required for the transfer of said plasmid by conjugation in the presence of a trans-acting mobilizing element, and the following structure: C-M-A-D, in which A denotes a DNA sequence which is homologous with a genomic DNA fragment either flanking or overlapping the DNA sequence B to be amplified or being a subsequence of the DNA sequence B constituting one of the ends of said sequence B,
C denotes a DNA sequence which is homologous with a genomic DNA fragment either flanking or overlapping the DNA sequence B to be amplified or being a subsequence of the DNA sequence B constituting one of the ends of said sequence B, the sequence C being located in the opposite end of the sequence B as compared to A,
D denotes a DNA sequence which is homologous with a genomic DNA fragment located distal for C as compared to B, and
M denotes a DNA sequence encoding a selection marker,
and ii) at least one DNA sequence encoding said transacting mobilizing element, and a population of recipient Bacillus sp. cells, which in their genome harbors at least one copy of the DNA sequence B to be amplified, are mixed under conditions allowing the plasmid to be transferred from the population of donor cells to the population of recipient cells by conjugation,
b) recipient cells are selected in which the DNA sequence M has been integrated in the genome either upstream or downstream of the DNA sequence B together with the sequence A, which cell s comprise, in any orientation, the structure A-B-C-M-A-D, and
c) the cells selected in step b) are propagated under increasing selec-tion pressure for the selection marker encoding by the DNA sequence M so as to obtain a cell which has obtained an increased number of genomically integrated copies of the DNA sequences B and M as compared to the parent cell.
The plasmid is transferred into the recipient cell by conjugation medi-ated by a conjugative plasmid, after which cells are selected (step b)) in which the DNA sequence M has been integrated in the genome either upstream or downstream of the DNA sequence B together with the sequence A, which cells comprise, in any orientation, the structure A-B-C-M-A-D.
The cells selected in step b) are propagated under increasing selec-tion pressure for the selection marker encoding by the DNA sequence M so as to obtain a cell which has obtained an increased number of genomically integrated copies of the DNA sequences B and M as compared to the parent cell.
By use of this method for amplification of a genomic DNA sequence integration of the cis-acting and trans-acting conjugative elements may be avoided.
Patent References
| Patent Number: |
Title of Patent: |
Year of Issue: |
| US 5,843,720 |
Introduction of DNA into bacillus strains by conjugation |
1998
|
|
US 6,066,473
WO 9629418 |
Introduction of DNA into baci llus strains by conjugation
Introduction of DNA into bacillus strains by conjugation |
2000
International publication date: 26 September 1996 |
2000
Title of Technology: Prokaryotic multicopy production cells
Abstract: The technology relates to a prokaryotic cell expressing a gene of interest and comprising at least two copies of said gene on the chromosome.
One of the advantages of a prokaryotic cell, as described herein and in US 6,100,063, is that said two copies of a gene are antiparallelly transcribed (i.e. either convergently or divergently transcribed), which minimises the risk that one copy of said gene will be lost from the cell by homologous recombination, as compared to when the genes are parallelly transcribed.
This implies a further advantage of said prokaryotic cell, since it is then possible to have a stable integration of said two copies of a gene, without having a DNA segment situated between the two copies of said gene, which are essential for the growth of the cell.
Accordingly, there is no need for integrating the individual copies of the gene of interest at very distant places on the chromosome. This makes it relatively simpler to construct a prokaryotic cell.
A further advantage of a method for constructing the prokaryotic cell is that it provides a simple method of producing a prokaryotic cell which expresses a gene from two copies of said gene, without the cell comprising an introduced antibiotic resistance gene.
A yet further advantage of the prokaryotic cell is that it is stable in the sense that it during fermentation is able to maintain the two copies of said gene on the chromosome. Accordingly, it is especially suitable for largescale industrial fermentation's.
Detailed Description: Prokaryotic multicopy production cells, i.e. cells comprising more than one copy of a gene of interest, have been used for production of proteins of interest at industrial scale. Preferred multicopy production cells are cells which stabile maintain the individual copies of the genes of interest during fermentation.
Further, due to environmental concerns there is an increasing desire for production cells which do not comprise any integrated antibiotic resistance genes on the chromosome and according to this line production cells which are capable of stabile maintaining the copies of the gene of interest in a fermentation medium NOT comprising an antibiotic.
The individual copies of the gene of interest are stabile maintained in a fermented cell population due to the essential DNA. If a cell crosses out this vital DNA by homologous recombination between the two copies of the gene of interest, the cell looses vital DNA and this specific cell will die.
Thereby it is possible to maintain a stable cell population comprising the copies of the gene of interest.
The problem to be solved by the present technology is to provide a prokaryotic cell expressing a gene of interest and comprising at least two copies of said gene on the chromosome.
Further, said prokaryotic cell should be:
a) stable in the sense that during fermentation it is able to maintain two copies of the gene on the chromosome; and
b) able to stably maintain the two copies of the gene without having a DNA segment, situated b etween the two copies of the gene, which segment is essential for the growth of the cell as described in e.g. EP 284126.
The solution is based on that the present inventor has identified that it is possible during fermentation to stably maintaining two antiparallelly transcribed copies of a gene on the chromosome of a cell and the two antiparallelly transcribed genes can be stably maintained even without having a DNA segment, situated between the two copies of the gene, which is essential for the growth of the cell.
The gene of interest is a gene, which is capable of expressing a polypeptide which are secreted from the cell. Such a gene may preferably encode a fusion polypeptide comprising a signal peptide and the secreted mature polypeptide. Such signal peptides are well known in the art.
The gene may also encode an enzyme which may be an protease, amylase, cellulase, lipase, xylanase, phytase, and other enzymes known in the art.
The prokaryotic cell may be is a gram positive prokaryotic cell, such as a Bacillus cell or a Streptomyces cell. Preferred Bacillus cells are species such as Bacillus subtilis, Bacillus licheniformis, Bacillus len-tus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus circulans, and a Bacillus thuringiensis cell.
Glossary of Terms
| Term: |
Definition: |
| Gene |
The term "a gene" indicates herein a gene (a DNA sequence) will is capable of being expressed into a polypeptide within said cell. Accordingly, said gene sequence will be defined as an open reading frame starting from a start codon (normally "ATG", "G TG", or "TTG") and ending at a stop codon (normally "TAA", "TAG" or "TGA"). |
| Two anti-parallelly transcribed copies of said gene |
The term "two anti-parallelly transcribed copies of said gene" denotes herein that said the genes is either convergently or divergently tran-scribed, i.e. present in opposite orientation relative to each other. |
Patent References
Patent Number:
|
Title of Patent: |
Year of Issue: |
| US 6,100,063 |
Procaryotic cell comprising at least two cop-ies of a gene |
2000
|
| WO 9941358 |
A prokaryotic cell comprising two copies of a gene transcribed in different directions |
International publication date: 19 August 1999 |