Bacterial strains which overproduce riboflavin

• US 5,925,538 A
• Filed: 08/24/1998
• Issued: 07/20/1999
• Est. Priority Date: 06/22/1989
• Status: Expired due to Fees

• Current Assignee: DSM Nutritional Products Incorporated (DSM-Firmenich AG)
• Original Assignee: Roche Vitamins, Inc. (Roche Holding AG)
• Inventors: Sloma, Alan, Hermann, Theron, Pero, Janice G., Perkins, John B., Erdenberger, Thomas, Hatch, Randolph T.
• Primary Examiner(s): RAILEY, JOHNNY F
• Application Number:
  US09/138,775
• Time in Patent Office: 330 Days
• Field of Search: 435/66, 435/69.1, 435/71.1, 435/71.2, 435/252.3, 435/252.31, 536/23.2, 536/23.7
• US Class Current: 435/66
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12P 25/00 Preparation of compounds containing alloxazine or isoalloxazine nucleus, e.g. riboflavin

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.

Claims

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.

3. The method of claim 1, wherein the recombinant bacterium comprising the exogenously introduced nucleic acid sequence further comprises a bacterial ORF5 structural gene, said promoter being positioned to transcribe said ORF5 structural gene, said ORF5 structural gene being specifically hybridizable to B. subtilis rib ORF5 and being expressed by said bacterium.

4. The method of claim 1, wherein the exogenously introduced nucleic acid sequence comprises a bacterial rib operon including at least three open reading frames selected from the group consisting of a first open reading frame which hybridizes with B. subtilis ORF2 and encodes a protein that has QRF2 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.

5. The method of any one of claims 1-4, wherein the exogenously introduced nucleic acid sequence is positioned in the chromosome of the recombinant bacterium.

15. The method of claim 1, claim 2, or claim 11 wherein said promoter is a constitutive promoter.

16. The method of claim 1, claim 2, or claim 11 wherein said promoter is a SPO1 promoter.

17. The method of claim 1, claim 2, or claim 6 wherein said exogenously introduced nucleic acid further comprises a ribosome binding site not naturally associated with a rib structural gene in said exogenously introduced nucleic acid.

18. The method of claim 1, claim 2 or claim 6 wherein said host bacterium is resistant to a purine analog selected from the group consisting of:

8-azaguanine;

decoyinine; and

methionine sulfoxide.

19. The method of claim 1, claim 2, or claim 6 in which the recombanant bacterium is resistant to roseoflavin.

20. The method of claim 1, claim 2, or claim 6 wherein said bacterium is a gram positive bacterium.

21. The method of claim 20 wherein the recombinant bacterium is a member of the genus Bacillus or Cornyebacterium.

22. The method of claim 1, claim 2, or claim 6 wherein the recombinant bacterium is a gram negative bacterium.

23. The method of claim 22, wherein the recombinant bacterium is a member of the genus Escherichia or Klebsiella.

25. The method of claim 1, wherein the exogenously introduced nucleic acid sequence is a rib operon sequence present on a plasmid chosen from the group consisting of pRF69, pRF71, pRF81 and pRF89.

28. The method of claim 1, wherein the exogenous promoter is a first exogenous promoter, and said exogenously introduced nucleic acid sequence further comprises a ORF4 structural gene and a second exogenous promoter,

a) said ORF4 structural gene being hybridizable with B. subtilis ORF4 and encoding a protein that has ORF4 activity,

b) said second exogenous promoter being positioned to transcribe said ORF4 structural gene, and

c) said first exogenous promoter being positioned between said ORF4 structural gene and said ORF3 structural gene to transcribe said ORF3 structural gene.

29. The method of claim 1, claim 2, or claim 6 in which each said bacterial rib operon open reading frame is a B. subtilis rib operon open reading frame.

30. The method of claim 29 wherein the recombinant bacterium is a Bacillus or an E. coli bacterium.

36. The method of claim 1, 2, 6, 27, 31, 32, 33 or 34, wherein the cells producing said riboflavin are grown under aerobic conditions with the level of dissolved oxygen maintained at a concentration between 5 and 30%;

and

the growth of said cells are limited by limiting the availability of a component in the growth medium such that said dissolved oxygen concentration is maintained at said level.

37. The method of claim 36, wherein said component is chosen from a carbon source, nitrogen source or a component required by said cells for their growth.

38. The method of claim 37, wherein said carbon source is glucose or a citric acid cycle acid.

39. The method of claim 36, wherein said limiting growth of cells comprises limiting introduction of said component in a feed medium.

40. The method of claim 39, wherein said component is glucose or a citric acid cycle acid.

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.

26. The method of claim 2, wherein the exogenously introduced nucleic acid sequence is a rib operon sequence present on a plasmid chosen from the group consisting of pRF50, pRF69, pRF78, pRF81, pRF85 and pRF89.

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.

7. The method of claim 6, wherein the exogenously introduced nucleic acid sequence comprises a bacterial rib operon of at least the following five open reading frames:

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 the B. subtilis ORF5 and encodes a protein that has QRF5 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

-ribofeavin synthase activity.

8. The method of claim 6, wherein the recombinant bacterium containing the second exogenously introduced nucleic acid sequence comprises at least one 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 QRF5 and encodes a protein that has ORF5 activity, and a fifth open reading frame which hybridizes with the B. subtilis β

-riboflavin synthase-encoding open reading frame and encodes a protein that has β

-riboflavin synthase activity,

said second exogenously introduced nucleic acid being positioned in the chromosome of said recombinant bacterium, said second exogenously introduced nucleic acid being expressed by said recombinant bacterium such that riboflavin biosynthesis by the bacterium is increased relative to a bacterium that is identical to said recombinant bacterium except for the absence of said second exogenously introduced nucleic acid sequence.

9. The method of claim 8, wherein the first exogenously introduced nucleic acid is present in multiple copies at said first site, and said second exogenously introduced nucleic acid is present in multiple copies at said second site.

10. The method of claim 9, wherein the first exogenously introduced nucleic acid is present in at least 5 copies at said first site, and said second exogenously introduced nucleic acid is present in at least 5 copies at said second site.

11. The method of claim 6, wherein the recombinant bacterium is further characterized in that the exogenously introduced nucleic acid comprises an exogenous promoter positioned to transcribe said open reading frame.

12. The method of claim 11, wherein said promoter is positioned in an intercistronic region of one of said bacterial rib open reading frames.

13. The method of claim 12, wherein said promoter is located between said ORF3 and said ORF4, in a region 5'"'"' upstream of ORF3.

14. The method of claim 11, said promoter is located 5'"'"' upstream of said ORF5, with no terminators intervening between said promoter and said ORFS.

24. The method of claim 6 wherein said bacterial rib operon is a Bacillus rib operon.

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.

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.

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.

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.

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.

35. The method of claim 34 in which said bacterium is a B. subtilis bacterium.