METHOD OF INCREASING THE REPLICATION OF A CIRCULAR DNA MOLECULE
The present invention relates to a covalently closed circular recombinant DNA molecule comprising an origin of replication and an insert comprising a homopolymeric region, wherein the homopolymeric region is located at a distance of least 500 bp from the origin of replication in the direction of replication and/or wherein the insert comprising a homopolymeric region is oriented so that the direction of transcription of the insert is the same as the direction of replication of the origin of replication. The invention further relates to the use of the covalently closed circular recombinant DNA molecule for increasing the yield and/or shortening the fermentation time during fermentation.
1 . Covalently closed circular recombinant DNA molecule comprising:
an origin of replication,
an insert comprising a homopolymeric region,
wherein the homopolymeric region is located at a distance of at least 500 bp from the origin of replication in the direction of replication.
2 . Covalently closed circular recombinant DNA molecule of claim 1 , wherein the homopolymeric region comprises at least one poly(C) sequence.
3 . Covalently closed circular recombinant DNA molecule of claim 1 or 2 , wherein the insert comprising a homopolymeric region is oriented so that the direction of transcription of the insert is the same as the direction of replication of the origin of replication.
4 . Covalently closed circular recombinant DNA molecule according to any one of the previous claims, wherein the yield of the covalently closed circular recombinant DNA molecule in fermentative production is increased compared to the yield of a covalently closed circular recombinant DNA molecule in which the homopolymeric region is located at a distance of less than 500 bp from the origin of replication in the direction of replication.
5 . Covalently closed circular recombinant DNA molecule according to claim 3 , wherein the yield of the covalently closed circular recombinant DNA molecule in fermentative production is increased compared to the yield of a covalently closed circular recombinant DNA molecule in which the insert comprising the homopolymeric region is oriented so that the direction of transcription of the insert is opposite to the direction of replication of the origin of replication.
6 . Covalently closed circular recombinant DNA molecule according to any one of the previous claims, wherein the covalently closed circular recombinant DNA molecule is selected from the group consisting of plasmid, cosmid, bacterial artificial chromosome (BAC), bacteriophage, viral vector or hybrids thereof.
7 . Covalently closed circular recombinant DNA molecule according to any one of the previous claims, wherein the covalently closed circular recombinant DNA molecule is a plasmid.
8 . Covalently closed circular recombinant DNA molecule according to any one of the previous claims, wherein the origin of replication is of bacterial origin.
9 . Covalently closed circular recombinant DNA molecule according to any one of the previous claims, wherein the origin of replication is a high copy number origin.
10 . Covalently closed circular recombinant DNA molecule according to any one of the previous claims, wherein the origin of replication is derived from the pBR322 plasmid, pUC plasmid, pMB1 plasmid, ColE1 plasmid, R6K plasmid, p15A plasmid, pSC101 plasmid or F1 phagemid.
11 . Covalently closed circular recombinant DNA molecule according to any one of the previous claims, wherein the origin of replication is derived from the pUC plasmid.
12 . Covalently closed circular recombinant DNA molecule according to any one of the previous claims, wherein the covalently closed circular recombinant DNA molecule further comprises a primosome assembly site in the heavy strand (PAS-BH).
13 . Covalently closed circular recombinant DNA molecule according to any one of the previous claims, wherein the covalently closed circular recombinant DNA molecule further comprises a selection marker.
14 . Covalently closed circular recombinant DNA molecule according to claim 11 , wherein the selection marker is an antibiotic resistance gene.
15 . Covalently closed circular recombinant DNA molecule according to claim 11 , wherein the selection marker is a sucrose selectable marker.
16 . Covalently closed circular recombinant DNA molecule according to claim 7 , wherein the plasmid is derived from the backbone having the sequence identified in SEQ ID NO: 4 or having the sequence identified in SEQ ID NO: 5.
17 . Covalently closed circular recombinant DNA molecule according to claims 2 to 16 , wherein the homopolymeric region further comprises at least one poly(A) sequence.
18 . Covalently closed circular recombinant DNA molecule according to claim 17 , wherein the poly(A) sequence comprises a sequence of about 20 to about 400 adenosine nucleotides.
19 . Covalently closed circular recombinant DNA molecule according to claim 18 , wherein the poly(A) sequence comprises a sequence of about 60 to about 250 adenosine nucleotides.
20 . Covalently closed circular recombinant DNA molecule according to claims 16 to 19 , wherein the poly(C) sequence comprises a sequence of about 15 to 200 cytidine nucleotides.
21 . Covalently closed circular recombinant DNA molecule according to claim 20 , wherein the poly(C) sequence comprises a sequence of about 20 to 40 cytidine nucleotides.
22 . Use of a covalently closed circular recombinant DNA molecule, according to any one of claims 1 to 21 for increasing the yield of the covalently closed circular recombinant DNA in fermentative production compared to the yield of a covalently closed circular recombinant DNA molecule in which the insert comprising the homopolymeric region is located at a distance of less than 500 bp from the origin of replication in the direction of replication.
23 . Use of a covalently closed circular recombinant DNA molecule, according to any one of claims 2 and 4 to 21 for increasing the yield of the covalently closed circular recombinant DNA in fermentative production compared to the yield of a covalently closed circular recombinant DNA molecule in which the insert comprising the homopolymeric region is oriented so that the direction of transcription of the insert is opposite to the direction of replication of the origin of replication.
24 . Use according to claim 22 , wherein the fermentation time is shortened compared to the fermentation time of a covalently closed circular recombinant DNA molecule in which the insert comprising the homopolymeric region is located at a distance of less than 500 bp from the origin of replication in the direction of replication.
25 . Use according to claim 23 , wherein the fermentation time is shortened compared to the fermentation time of a covalently closed circular recombinant DNA molecule in which the insert comprising the homopolymeric region is oriented so that the direction of transcription of the insert is opposite to the direction of replication of the origin of replication.
26 . Method for fermentative production of a covalently closed recombinant DNA molecule comprising the steps of:
(a) providing a microorganism comprising the covalently closed circular recombinant DNA molecule according to claims 1 to 21 ;
(b) fermenting the microorganism of step (a).
27 . The method according to claim 26 , further comprising a step of
(c) isolating the covalently closed circular recombinant DNA molecule from the microorganism of step(b).
28 . Method of claim 26 or 27 , wherein the microorganism is a bacterium containing a covalently closed circular recombinant temperature inducible high copy DNA plasmid and step (b) comprises the following steps:
(i) growing the bacteria containing a covalently closed circular recombinant temperature inducible high copy DNA plasmid at a temperature in the range of 25° C. to 32° C. during the growth phase of the fed-batch phase wherein the substrate is supplied at a rate such that the growth rate is μ=0.05 to 0.3 hr −1 during the fed-batch phase,
(ii) inducing production of said DNA plasmid after the growth phase by increasing the temperature to 36° C. to 45° C.; and
(iii) continuing fermentation at the temperature applied (ii) to accumulate the said DNA plasmid.
29 . Method according to claim 28 , wherein the temperature applied in step (i) is about 30° C. and the temperature applied in step (ii) is about 42° C.
30 . Method according to any one of claims 26 to 29 , wherein the yield of the covalently closed circular recombinant DNA molecule according to claims 1 to 21 is increased compared to the yield of a covalently closed circular recombinant DNA molecule in which the insert comprising homopolymeric region is located at a distance of less than 500 bp from the origin of replication in the direction of replication.
31 . Method according to any one of claims 26 to 29 , wherein the yield of the covalently closed circular recombinant DNA molecule according to claims 1 to 21 is increased compared to the yield of a covalently closed circular recombinant DNA molecule in which the insert comprising the homopolymeric region is oriented so that the direction of transcription of the insert is opposite to the direction of replication of the origin of replication.
32 . Method according to claim 30 , wherein the fermentation time is shortened, compared to the fermentation time of a covalently closed circular recombinant DNA molecule in which the insert comprising homopolymeric region is located at a distance of less than 500 bp from the origin of replication in the direction of replication.
33 . Method according to claim 31 , wherein the fermentation time is shortened compared to the fermentation time of a covalently closed circular recombinant DNA molecule in which the insert comprising the homopolymeric region is oriented so that the direction of transcription of the insert is opposite to the direction of replication of the origin of replication.
34 . Method according to any one of claims 26 to 33 , wherein the homopolymeric region sequence remains stable during the fermentation production.
35 . Method according to any one of claims 26 to 34 , wherein the sequence of the covalently closed circular recombinant DNA molecule remains stable during fermentation production.
36 . Method for improving the yield of a covalently closed circular recombinant DNA molecule comprising the following steps:
(a) providing a covalently closed circular recombinant DNA molecule having
an origin of replication, and
an insert comprising a homopolymeric region sequence, wherein the homopolymeric region is located at a distance of less than 500 bp from the origin of replication in the direction of replication;
(b) modifying the covalently closed circular recombinant DNA molecule of (a) so that the insert comprising a homopolymeric region sequence is located at a distance of at least 500 bp from the origin of replication in the direction of replication.
37 . Method for improving the yield of a covalently closed circular recombinant DNA molecule comprising the following steps:
(a) providing a covalently closed circular recombinant DNA molecule having
an origin of replication, and
an insert comprising a homopolymeric region sequence, wherein the insert comprising the homopolymeric region sequence is oriented so that the direction of transcription of the insert is opposite to the direction of replication of the origin of replication;
(b) modifying the covalently closed circular recombinant DNA molecule of (a) so that the insert comprising a homopolymeric region is oriented so that the direction of transcription of the insert is the same as the direction of replication of the origin of replication.
38 . Use of the covalently closed circular recombinant DNA molecule according to claims 1 to 21 for the in vitro transcription of