Bacterial strains which overproduce riboflavin
First Claim
1. A method for production of riboflavin comprising, providing a recombinant bacterium comprising an exogenously introduced nucleic acid sequence, which sequence comprises a bacterial ORF3 structural gene and an exogenous promoter, said promoter being positioned to transcribe said ORF3 structural gene, said ORF3 structural gene encoding an amino acid sequence that has ORF3 activity, hybridizing to B. subtilis rib ORF3;
- culturing said recombinant bacterium under suitable culture conditions such that said ORF3 structural gene is expressed and the recombinant bacterium produces a riboflavin titre of at least 10 g/l when fermented using standard commercial batch and feed conditions; and
recovering riboflavin produced by said bacterium.
1 Assignment
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Accused Products
Abstract
Vectors and recombinant bacteria for overproducing riboflavin, in which nucleic acid overproducing riboflavin biosynthetic proteins is introduced in the chromosome of the host organism, e.g. at multiple sites and in multiple copies per site. A rib operon having at least five genes is used to make such recombinant bacteria.
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Citations
40 Claims
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1. A method for production of riboflavin comprising, providing a recombinant bacterium comprising an exogenously introduced nucleic acid sequence, which sequence comprises a bacterial ORF3 structural gene and an exogenous promoter, said promoter being positioned to transcribe said ORF3 structural gene, said ORF3 structural gene encoding an amino acid sequence that has ORF3 activity, hybridizing to B. subtilis rib ORF3;
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culturing said recombinant bacterium under suitable culture conditions such that said ORF3 structural gene is expressed and the recombinant bacterium produces a riboflavin titre of at least 10 g/l when fermented using standard commercial batch and feed conditions; and recovering riboflavin produced by said bacterium. - View Dependent Claims (3, 4, 5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 28, 29, 30, 36, 37, 38, 39, 40)
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2. A method for production of riboflavin comprising,
providing a recombinant bacterium comprising an exogenously introduced nucleic acid sequence, which sequence comprises a bacterial ORF5 structural gene and an exogenous promoter, said promoter being positioned to transcribe said ORF5 structural gene, said ORF5 structural gene encoding an amino acid sequence with ORF5 activity, hybridizing to B. subtilis rib ORF5; -
culturing said recombinant bacterium under suitable culture conditions such that said ORF5 structural gene is expressed and the recombinant bacterium produces at least 10 g/l of riboflavin when fermented using standard commercial batch and feed conditions; and recovering riboflavin produced by said bacterium. - View Dependent Claims (26)
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6. A method for production of riboflavin comprising,
providing a recombinant bacterium comprising an exogenously introduced nucleic acid sequence comprising at least one bacterial rib operon open reading frame selected from the group consisting of a first open reading frame which hybridizes with B. subtilis ORF2 and encodes a protein that has ORF2 activity, a second open reading frame which hybridizes with B. subtilis ORF3 and encodes a protein that has ORF3 activity, a third open reading frame which hybridizes with B. subtilis ORF4 and encodes a protein that has ORF4 activity, a fourth open reading frame which hybridizes with B. subtilis ORF5 and encodes a protein that has ORF5 activity, and a fifth open reading frame which hybridizes with the B. subtilis β - -riboflavin synthase-encoding ORF and encodes a protein that has β
-riboflavin synthase activity,at least two copies of said exogenously introduced nucleic acid being positioned in the chromosome of said recombinant bacterium; culturing said recombinant bacterium under suitable culture conditions wherein said exogenously introduced nucleic acid is expressed by said recombinant bacterium such that riboflavin biosynthesis by the recombinant bacterium is increased relative to biosynthesis by a bacterium that is identical to said recombinant bacterium except for the absence of said exogenously introduced sequence, and the recombinant bacterium produces a riboflavin titre of at least 10 g/l when fermented using standard commercial batch and feed conditions; and recovering riboflavin produced by said bacterium. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 24)
- -riboflavin synthase-encoding ORF and encodes a protein that has β
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27. A method for production of riboflavin comprising,
providing a Bacillus subtilis strain RB50, as deposited with the NRRL and assigned accession number B 18502, or strain RB58 as deposited with the ATCC and assigned accession number 55053, and riboflavin-producing variants of either strain thereof; -
culturing said recombinant bacterium under suitable culture conditions such that the recombinant bacterium produces a riboflavin titre of at least 10 g/l when fermented using standard commercial batch and feed conditions; and recovering riboflavin produced by said bacterium.
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31. A method for production of riboflavin comprising,
providing a recombinant bacterium comprising an exogenously introduced nucleic acid sequence, which sequence comprises a bacterial ORF3 structural gene and an exogenous promoter, said promoter being positioned to transcribe said ORF3 structural gene, said ORF3 structural gene: -
encoding an amino acid sequence that has ORF3 activity, hybridizing to B. subtilis rib ORF3 under conditions which do not permit hybridization of non-ORF3 DNA to B. subtilis rib ORF3; and culturing said recombinant bacterium under suitable culture conditions such that said ORF3 structural gene is expressed and the recombinant bacterium produces a riboflavin titre of at least 10 g/l when fermented using standard commercial batch and feed conditions, and recovering riboflavin produced by said bacterium.
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32. A method for production of riboflavin comprising,
providing a recombinant bacterium comprising an exogenously introduced nucleic acid sequence, which sequence comprises a bacterial ORF5 structural gene and an exogenous promoter, said promoter being positioned to transcribe said ORF5 structural gene, said ORF5 structural gene: -
encoding an amino acid sequence that has ORF5 activity hybridizing to B. subtilis rib ORF5 under conditions which do not permit hybridization of non-ORF5 DNA to B. subtilis rib ORF5; and culturing said recombinant bacterium under suitable culture conditions such that said ORF5 structural gene is expressed and the recombinant bacterium produces a riboflavin titre of at least 10 g/l when fermented using standard commercial batch and feed conditions, and recovering riboflavin produced by said bacterium.
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33. A method for production of riboflavin comprising,
providing a recombinant bacterium comprising an exogenously introduced nucleic acid sequence comprising at least one bacterial rib operon open reading frame selected from the group consisting of: -
a first open reading frame which hybridizes with B. subtilis ORF2 under conditions which do not permit hybridization of non-ORF2 DNA to B. subtilis rib ORF2 and which encodes a protein that has ORF2 activity, a second open reading frame which hybridizes with B. subtilis ORF3 under conditions which do not permit hybridization of non-ORF3 DNA to B. subtilis rib ORF3 and which encodes a protein that has ORF3 activity, a third open reading frame which hybridizes with B. subtilis ORF4 under conditions which do not permit hybridization of non-ORF4 DNA to B. subtilis rib ORF4 and which encodes a protein that has ORF4 activity, a fourth open reading frame which hybridizes with B. subtilis ORF5 under conditions which do not permit hybridization of non-ORF5 DNA to B. subtilis rib ORF5 and which encodes a protein that has ORF5 activity, and a fifth open reading frame which hybridizes with the B. subtilis β
-riboflavin synthase-encoding ORF under conditions with do not permit hybridization to non-β
-riboflavin synthase-encoding ORF and which encodes a protein that has β
-riboflavin synthase activity,at least two copies of said exogenously introduced nucleic acid being positioned in the chromosome of said recombinant bacterium; culturing said recombinant bacterium under suitable culture conditions wherein said exogenously introduced nucleic acid is expressed by said recombinant bacterium such that riboflavin biosynthesis by the recombinant bacterium is increased relative to biosynthesis by a bacterium that is identical to said recombinant bacterium except for the absence of said exogenously introduced sequence, and the recombinant bacterium produces a riboflavin titre of at least 10 g/l when fermented using standard commercial batch and feed conditions; and recovering riboflavin produced by said bacterium.
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34. A method for production of riboflavin comprising,
providing a recombinant bacterium comprising an exogenously introduced nucleic acid sequence comprising at least one bacterial rib operon open reading frame selected from the group consisting of a first open reading frame which is B. subtilis ORF2, a second open reading frame which is B. subtilis ORF3, a third open reading frame which is B. subtilis ORF4, a fourth open reading frame which is B. subtilis ORF5, and a fifth open reading frame which is B. subtilis β - -riboflavin synthase-encoding ORF, at least two copies of said exogenously introduced nucleic acid being positioned at a first site in the chromosome of said recombinant bacterium, said bacterium further comprising a second exogenously introduced nucleic acid sequence comprising at least one open reading frame selected from the group consisting of a first open reading frame which is B. subtilis ORF2, a second open reading frame which is B. subtilis ORF3, a third open reading frame which is B. subtilis ORF4, a fourth open reading frame which is B. subtilis ORF5, and a fifth open reading frame which is the B. subtilis β
-riboflavin synthase-encoding open reading frame, said second exogenously introduced nucleic acid being positioned at a second site in said chromosome, said first site being different from said second site;culturing said recombinant bacterium under suitable culture conditions wherein each of first and second exogenously introduced nucleic acids are expressed under suitable culture conditions, the riboflavin biosynthesis by the recombinant bacterium is increased relative to biosynthesis by a bacterium that is identical to said recombinant bacterium except for the absence of said exogenously introduced sequence, and the recombinant bacterium produces a riboflavin titre of at least 10 g/l when fermented using standard commercial batch and feed conditions; and recovering riboflavin produced by said bacterium. - View Dependent Claims (35)
- -riboflavin synthase-encoding ORF, at least two copies of said exogenously introduced nucleic acid being positioned at a first site in the chromosome of said recombinant bacterium, said bacterium further comprising a second exogenously introduced nucleic acid sequence comprising at least one open reading frame selected from the group consisting of a first open reading frame which is B. subtilis ORF2, a second open reading frame which is B. subtilis ORF3, a third open reading frame which is B. subtilis ORF4, a fourth open reading frame which is B. subtilis ORF5, and a fifth open reading frame which is the B. subtilis β
Specification