Food-grade integration vectors for industrial bacterial strains
First Claim
1. An integration vector to be integrated into a microorganism'"'"'s chromosome comprising as operably joined components:
- (a) a first DNA sequence, wherein said first DNA sequence encodes a plasmid replication and maintenance sequence from plasmid pSK11, wherein the plasmid replication or maintenance sequence provides for temperature sensitive loss of the autonomously replicating plasmid and wherein said sequence includes at least a 2.3 kb ScaI-SpeI fragment; and
(b) a second DNA sequence, wherein said second DNA sequence is comprised of at least one selectable food grade marker gene; and
(c) a third DNA sequence, wherein said third DNA sequence encodes a fragment of an insertion sequence, and wherein said fragment of the insertion sequence has sufficient sequence identity to DNA sequences on the microorganism'"'"'s chromosome to provide for integration of the vector into the chromosome by homologous recombination, wherein the microorganism is selected from the group consisting of Pediococcus, Leuconostoc, Lactococcus, Lactobacillus, Streptococcus thermophilous, and Bacillus; and
(d) a fourth DNA sequence, wherein said fourth DNA sequence is comprised of at least one gene encoding an unstable trait.
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Abstract
The present invention is directed to integration vectors including food-grade integration vectors, a method of increasing the stability of the inheritance of genes in microorganisms and transformed microorganisms containing an integrated vector and which exhibit an increase in the stability of inheritance of a gene. The integration vector is comprised of a first DNA sequence encoding a temperature sensitive plasmid maintenance and replication region, a second DNA sequence encoding at least one selectable marker gene, a third DNA sequence encoding an insertion sequence, and a fourth DNA sequence which encodes an unstable gene. The method of increasing the stability of the inheritance of a gene includes introducing the integration vector into a host microorganism, selecting transformed microorganisms, and confirming that selected microorganisms exhibit an increase in the stability of inheritance of a gene encoding a trait. Transformed microorganisms are provided which have at least one integration vector integrated into the genome and which exhibit an increase in the stability of the inheritance of a gene encoding a trait. The invention also provides DNA sequences substantially corresponding to a temperature sensitive plasmid maintenance and replication sequence, a nisin resistance gene, and a novel lactococcal insertion sequence.
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Citations
28 Claims
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1. An integration vector to be integrated into a microorganism'"'"'s chromosome comprising as operably joined components:
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(a) a first DNA sequence, wherein said first DNA sequence encodes a plasmid replication and maintenance sequence from plasmid pSK11, wherein the plasmid replication or maintenance sequence provides for temperature sensitive loss of the autonomously replicating plasmid and wherein said sequence includes at least a 2.3 kb ScaI-SpeI fragment; and (b) a second DNA sequence, wherein said second DNA sequence is comprised of at least one selectable food grade marker gene; and (c) a third DNA sequence, wherein said third DNA sequence encodes a fragment of an insertion sequence, and wherein said fragment of the insertion sequence has sufficient sequence identity to DNA sequences on the microorganism'"'"'s chromosome to provide for integration of the vector into the chromosome by homologous recombination, wherein the microorganism is selected from the group consisting of Pediococcus, Leuconostoc, Lactococcus, Lactobacillus, Streptococcus thermophilous, and Bacillus; and (d) a fourth DNA sequence, wherein said fourth DNA sequence is comprised of at least one gene encoding an unstable trait. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A DNA sequence corresponding to the insertion sequence IS981 (SEQ ID NO:
- 5) and portions thereof including at least 700 base pairs.
- View Dependent Claims (13)
- 14. A DNA sequence corresponding to the 14.8 kilobase pair PvuII fragment of the pSK11L plasmid and portions thereof including at least a 2.3 kb ScaI-SpeI fragment.
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16. A method of increasing the stability of inheritance of genes encoding traits in a microorganism by integration of the genes into a microorganism'"'"'s chromosome comprising the steps of:
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(a) introducing an integration vector into a microorganism, wherein said integration vector is comprised as operably joined components a first DNA sequence wherein said first DNA sequence encodes a plasmid replication and maintenance sequence from pSK11, wherein the plasmid replication or maintenance sequence provides for temperature sensitive loss of the autonomously replicating plasmid, and wherein said sequence includes at least a 2.3 kb ScaI-SpeI fragment;
a second DNA sequence, wherein said second DNA sequence is comprised of at least one food grade selectable marker gene;
a third DNA sequence, wherein said third DNA sequence encodes a fragment of an insertion sequence, and wherein said fragment of the insertion sequence has sufficient sequence identity to DNA sequences on a bacterial chromosome to provide for integration of the vector into the chromosome by homologous recombination, wherein the bacteria is selected from the group consisting of Pediococcus Leuconostoc, Lactococcus Lactobacillus, Streptococcus thermophilous, and Bacillus; and
a fourth DNA sequence, wherein said fourth DNA sequence is comprised of at least one gene encoding an unstable trait to yield a transformed population of microorganisms; and(b) subjecting the population of transformed microorganisms to a selective condition to yield a selected population of microorganisms; and (c) growing the selected population of microorganisms through a plurality of growth cycles to confirm that a population of microorganisms exhibits an increase in the stability of inheritance of a gene encoding an unstable trait is obtained. - View Dependent Claims (17, 18, 19, 20, 21)
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22. A transformed microorganism which exhibits an increase in the stability of the inheritance of a gene encoding an unstable trait, wherein said microorganism has an integration vector integrated into a chromosome, and wherein said integration vector is comprised as operably joined components:
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a first DNA sequence, wherein said first DNA sequence encodes a plasmid replication and maintenance sequence from pSK11, wherein the plasmid replication or maintenance sequence provides for temperature sensitive loss of the autonomously replicating plasmids, wherein said sequence includes at least a 2.3 kb ScaI-SpeI fragment; a second DNA sequence, wherein said second DNA sequence is comprised of at least one selectable food grade marker gene; a third DNA sequence, wherein said third DNA sequence encodes a fragment of an insertion sequence and wherein said fragment of the insertion sequence has sufficient sequence identity to DNA sequences on a microorganism chromosome to provide for integration of the vector into the chromosome by homologous recombination, wherein the microorganism is selected from the group consisting of Pediococcus, Leuconostoc, Lactococcus, Lactobacillus, Streptococcus thermophilous, and Bacillus; and a fourth DNA sequence, wherein said fourth DNA sequence is comprised of at least one gene encoding an unstable trait. - View Dependent Claims (23, 24, 25)
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26. An integration vector to be integrated into a microorganism'"'"'s chromosome comprising as operably joined components:
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(a) a first DNA sequence, wherein said first DNA sequence encodes a plasmid replication and maintenance sequence from pSK11, wherein the plasmid replication or maintenance sequence provides for temperature sensitive loss of the autonomously replicating plasmid and wherein said sequence includes at least a 2.3 kb ScaI-SpeI fragment; and (b) a second DNA sequence, wherein said second DNA sequence is comprised of at least one food grade selectable marker gene; and (c) a third DNA sequence, wherein said third DNA sequence encodes a fragment of a lactococcal insertion sequence IS981, and wherein said fragment of the insertion sequence has sufficient sequence identity to DNA sequences on the lactococcal bacterial chromosome to provide for integration of the vector into the chromosome by homologous recombination, wherein the bacteria is a Lactococcal species; and (d) a fourth DNA sequence, wherein said fourth DNA sequence encodes an unstable trait selected from the group consisting of lactose metabolism genes, proteinase genes, citrate metabolism genes, bacteriocin production and immunity genes, nisin resistance genes, exopolymer genes, and bacteriophage resistance genes. - View Dependent Claims (27, 28)
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Specification