METHODS AND STRAINS FOR THE PRODUCTION OF SARCINAXANTHIN AND DERIVATIVES THEREOF

20130130312

13/701344

June 01, 2011

The present invention relates to a new strain of Micrococcus luteus, named Otnes7, which is superior to known strains in its ability to synthesise the carotenoid sarcinaxanthin and a method of producing sarcinaxanthin or a derivative thereof, said method comprising introducing into and expressing in a host cell one or more nucleic acid molecules encoding an activity in the sarcinaxanthin biosynthetic pathway.

Promar AS (Fornebu, NO)

1. A method of producing sarcinaxanthin or a derivative thereof, said method comprising introducing into and expressing in a host cell one or more nucleic acid molecules encoding an activity in the sarcinaxanthin biosynthetic pathway, wherein said one or more nucleic acid molecules comprise: (i) a nucleotide sequence as set forth in SEQ ID NO: 37 or a part thereof; (ii) a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 37, or a part thereof; or (iii) a nucleotide sequence complementary to (i) or (ii).

2. The method of claim 1, wherein said one or more nucleic acid molecules comprise: (i) a nucleotide sequence as set forth in SEQ ID NO: 26 or a part thereof; (ii) a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 26, or a part thereof; or (iii) a nucleotide sequence complementary to (i) or (ii).

3. The method of claim 1, wherein said one or more nucleic acid molecules encode the sarcinaxanthin biosynthetic pathway.

4. The method of claim 1, further comprising the step of isolating the sarcinaxanthin or derivative thereof from the host cell.

5. The method of claim 1, wherein said method comprises introducing into and expressing in a host cell: (a) one or more nucleic acid molecules comprising nucleotide sequences encoding one or more proteins capable of synthesising flavuxanthin; and (b) one or more nucleic acid molecules comprising nucleotide sequences encoding one or more proteins having or contributing to C50 carotenoid γ-cyclase activity, wherein said one or more proteins of (b) are capable of catalysing the conversion of flavuxanthin to sarcinaxanthin.

6. The method of claim 5, wherein said host cell is a lycopene-producing host cell, preferably wherein said lycopene-producing host cell is capable of producing lycopene at levels of at least 0.5 mg/g CDW, further preferably, wherein the lycopene producing host cell comprises the plasmid pAC-LYC.

7. The method of claim 6, wherein said one or more proteins of (a) are capable of catalysing the conversion of lycopene to flavuxanthin.

8. The method of claim 7, wherein said one or more proteins have lycopene elongase activity.

9. The method of claim 5, wherein said one or more nucleic acid molecule of (b) comprises: (1) a nucleic acid molecule encoding a C50 carotenoid γ-cyclase subunit and comprising: (i) a nucleotide sequence as set forth in all or part of SEQ ID NO: 12 or SEQ ID NO: 2, or which is degenerate therewith, or which has at least 90% sequence identity to SEQ ID NO: 12 or 2; or (ii) a nucleotide sequence encoding a protein having all or part of an amino acid sequence as set forth in SEQ ID NO: 13 or 3 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 13 or 3; and (2) a nucleic acid molecule encoding a C50 carotenoid γ-cyclase subunit and comprising: (i) a nucleotide sequence as set forth in all or part of SEQ ID NO: 14 or 4, or which is degenerate therewith, or which has at least 90% sequence identity to SEQ ID NO: 14 or 4; or (ii) a nucleotide sequence encoding a protein having all or part of an amino acid sequence as set forth in SEQ ID NO: 15 or 5 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 15 or 5.

10. The method of claim 5, wherein said one or more nucleic acid molecules of (a) comprise: (i) a nucleotide sequence as set forth in all or part of SEQ ID NO: 10, 6 or 7, or which is degenerate therewith, or which has at least 90% sequence identity to SEQ ID NO: 10, 6 or 7; or (ii) a nucleotide sequence encoding a protein having all or part of an amino acid sequence as set forth in SEQ ID NO: 11, 8 or 9, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 11, 8 or 9.

11. The method of claim 5 any one of claims of claims 5 to 8, wherein said one or more nucleic acid molecule comprises a nucleotide sequence encoding all or part of a protein having an amino acid sequence selected from the sequences as set forth in any one of SEQ ID NO: 11, 13 and 15 or an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 11, 13 or 15.

12. The method of claim 11, wherein said nucleotide sequence encodes a protein which when expressed in a lycopene-producing host cell together with each of the other said proteins results in at least 91% of the total carotenoids produced being sarcinaxanthin, or a nucleic acid molecule which comprises a nucleotide sequence which is the complement of any aforesaid sequence.

13. The method of claim 11, wherein said nucleotide sequence encodes a protein which when expressed in a lycopene-producing host cell together with each of the other said proteins results in sarcinaxanthin production to a level of at least 150 μg/g of cell dry weight (CDW).

14. The method of claim 1, wherein said one or more nucleic acid molecules comprise: (i) a nucleotide sequence selected from sequences as set forth in SEQ ID NO: 10, 12 and 14; (ii) a nucleotide sequence which is degenerate with the sequence of any one of SEQ ID NOs: 10, 12 or 14; (iii) a nucleotide sequence which has at least 90% sequence identity to any one of SEQ ID NOs: 10, 12 or 14; (iv) a nucleotide sequence which is a part of the nucleotide sequence of any one of SEQ ID NOs: 10, 12 or 14 or of a nucleotide sequence which is degenerate therewith; or (v) a nucleotide sequence which is complementary to any of (i) to (iv) above.

15. The method of claim 14, wherein said one or more nucleic acid molecules comprises a nucleotide sequence encoding a protein having lycopene elongase activity and an amino acid sequence as set forth in all or part of SEQ ID NO: 11 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 11, wherein said amino acid sequence comprises one or more of the following: (a) alanine at position 8; (b) valine at position 88; (c) valine at position 158; or a nucleotide sequence which is the complement of any aforesaid sequence, wherein the position numbers are stated with reference to SEQ ID NO. 11, preferably wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 10 or a part of variant thereof, or a complement thereof.

16. The method of claim 14, wherein said one or more nucleic acid molecules comprises a nucleotide sequence encoding a protein which contributes to C50 carotenoid γ-cyclase activity and which has an amino acid sequence as set forth in all or part of SEQ ID NO: 13 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 13, wherein said amino acid sequence comprises one or more of the following: (a) valine at position 44; (b) valine at position 64; (c) glycine at position 103; (d) arginine at position 104; (e) proline at position 111; (f) glycine at position 117; or a nucleotide sequence which is the complement of any aforesaid sequence, wherein the position numbers are stated with reference to SEQ ID NO. 13, preferably wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 12 or a part of variant thereof, or a complement thereof.

17. The method of claim 14, wherein said one or more nucleic acid molecules comprises a nucleotide sequence encoding a protein which contributes to C50 carotenoid γ-cyclase activity and which has an amino acid sequence as set forth in all or part of SEQ ID NO: 15 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 15, wherein said amino acid sequence comprises one or more of the following: (a) a glycine residue at position 100; (b) a glycine residue at position 103; (c) a proline residue at position 107; or a nucleotide sequence which is the complement of any aforesaid sequence, wherein the position numbers are stated with reference to SEQ ID NO. 15, preferably wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 14 or a part of variant thereof, or a complement thereof.

18. The method of claim 1 comprising the introduction of a further nucleic acid molecule into said host cell, wherein said nucleic acid molecule encodes an enzyme capable of glycosylating sarcinxanthin.

19. The method of claim 18, wherein said further nucleic acid molecule encodes crtX from M. luteus or a functional equivalent thereof, preferably wherein the nucleic acid comprises: (i) a nucleotide sequence as set forth in all or part of SEQ ID NO: 33 or 16, or which is degenerate therewith, or a nucleotide sequence with at least 70% sequence identity to SEQ ID NO: 33 or 16; (ii) a nucleotide sequence which hybridizes to SEQ ID NO: 33 or 16 under non-stringent binding conditions of 6×SSC/50% formamide at room temperature and washing under conditions of high stringency, e.g. 2×SSC, 65° C., where SSC=0.15 M NaCl, 0.015M sodium citrate, pH 7.2; or (iii) a nucleotide sequence encoding a protein having all or part of an amino acid sequence as set forth in SEQ ID NO: 34 or 17 or which comprises an amino acid sequence which is at least 70% identical to SEQ ID NO: 34 or 17.

20. The method of claim 19, wherein said further nucleic acid molecule comprises a nucleotide sequence encoding a protein having sarcinaxanthin glycosylase activity and an amino acid sequence as set forth in all or part of SEQ ID NO: 34 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 34, wherein said amino acid sequence comprises one or more of the following: (a) histidine at position 62; (b) serine at position 109; (c) arginine at position 129; (d) alanine at position 138; (e) arginine at position 248; (f) proline at position 251; or a nucleotide sequence which is the complement of any aforesaid sequence, wherein the position numbers are stated with reference to SEQ ID NO. 34, preferably wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 33 or a part of variant thereof, or a complement thereof.

21. The method of claim 1, wherein the expression of one or more said nucleic acid molecules is inducible.

22. The method of claim 1, wherein said host cell is a microorganism particularly a bacterium.

23. The method of claim 22, wherein said bacterium is selected from Escherichia sp., Salmonella, Klebsiella, Proteus, Yersinia, Azotobacter sp., Pseudomonas sp., Xanthomonas sp., Agrobacterium sp., Alcaligenes sp., Bordatella sp., Haemophilus influenzae, Methylophilus methylotrophus, Rhizobium sp., Thiobacillus sp. and Clavibacter sp., preferably wherein the host cell is an Escherichia coli cell or a Corynebacterium glutamicum cell.

24. An isolated nucleic acid molecule comprising or consisting of all or a part of a nucleotide sequence as set forth in SEQ ID NO: 37 or which has at least 90% sequence identity to SEQ ID NO. 37, which molecule encodes one or more proteins having activity in the biosynthesis of sarcinaxanthin, and wherein any nucleic acid molecule which comprises a nucleotide sequence which is a part of SEQ ID NO. 37 or which is at least 90% identical to SEQ ID NO. 37 encodes proteins which are able to synthesise sarcinaxanthin at substantially the same level as the proteins encoded by SEQ ID NO: 37 when expressed in a host cell.

25. The nucleic acid molecule of claim 24, wherein said part of said nucleic acid molecule comprises or consists of all or a part of a nucleotide sequence as set forth in SEQ ID NO: 26 or which has at least 90% sequence identity to SEQ ID NO. 26, which molecule encodes one or more proteins having activity in the biosynthesis of sarcinaxanthin, and wherein any nucleic acid molecule which comprises a nucleotide sequence which is a part of SEQ ID NO. 26 or which is at least 90% identical to SEQ ID NO. 26 encodes proteins which are able to synthesise sarcinaxanthin at substantially the same level as the proteins encoded by SEQ ID NO: 26 when expressed in a host cell.

26. The nucleic acid molecule of claim 24, wherein said part of said nucleic acid molecule comprises a nucleotide sequence encoding all or part of a protein having an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 11 and wherein said nucleotide sequence encodes a lycopene elongase with a lycopene to flavuxanthin conversion efficiency of at least 30%, when expressed in a host cell, or a nucleic acid molecule which comprises a nucleotide sequence which is the complement of any aforesaid sequence.

27. The nucleic acid molecule of claim 26, wherein said part of said nucleic acid molecule comprises: (i) a nucleotide sequence as set forth in SEQ ID NO: 10; (ii) a nucleotide sequence which is degenerate with the sequence of SEQ ID NO: 10; (iii) a nucleotide sequence which has at least 90% sequence identity to SEQ ID NO: 10; (iv) a nucleotide sequence which is a part of the nucleotide sequence of SEQ ID NO: 10 or of a nucleotide sequence which is degenerate therewith; or (v) a nucleotide sequence which is complementary to any of (i) to (iv) above.

28. The nucleic acid molecule of claim 24, wherein said part of said nucleic acid molecule comprises a nucleotide sequence encoding all or part of a protein having an amino acid sequence selected from the sequences as set forth in any one of SEQ ID NO: 11, 13 and 15 or an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 11, 13 or 15, and wherein said nucleotide sequence encodes a protein which when expressed in a lycopene-producing host cell together with each of the other said proteins results in at least 91% of the total carotenoids produced being sarcinaxanthin, or a nucleic acid molecule which comprises a nucleotide sequence which is the complement of any aforesaid sequence.

29. The nucleic acid molecule of claim 24, wherein said part of said nucleic acid molecule comprises a nucleotide sequence encoding all or part of a protein having an amino acid sequence selected from the sequences as set forth in any one of SEQ ID NO: 11, 13 and 15 or an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 11, 13 or 15, wherein said nucleotide sequence encodes a protein which when expressed in a lycopene-producing host cell together with each of the other said proteins results in sarcinaxanthin production to a level of at least 150 μg/g of cell dry weight (CDW).

30. The nucleic acid molecule of claim 28, wherein said nucleic acid molecule comprises: (i) a nucleotide sequence selected from sequences as set forth in SEQ ID NO: 10, 12 and 14; (ii) a nucleotide sequence which is degenerate with the sequence of any one of SEQ ID NOs: 10, 12 or 14; (iii) a nucleotide sequence which has at least 90% sequence identity to any one of SEQ ID NOs: 10, 12 or 14; (iv) a nucleotide sequence which is a part of the nucleotide sequence of any one of SEQ ID NOs: 10, 12 or 14 or of a nucleotide sequence which is degenerate therewith; or (v) a nucleotide sequence which is complementary to any of (i) to (iv) above.

31. The nucleic acid molecule of claim 30, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a protein having lycopene elongase activity and an amino acid sequence as set forth in all or part of SEQ ID NO: 11 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 11, wherein said amino acid sequence comprises one or more of the following: (a) alanine at position 8; (b) valine at position 88; (c) valine at position 158; or a nucleotide sequence which is the complement of any aforesaid sequence, wherein the position numbers are stated with reference to SEQ ID NO. 11, preferably wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 10 or a part of variant thereof, or a complement thereof.

32. The nucleic acid molecule of claim 30, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a protein which contributes to C50 carotenoid γ-cyclase activity and which has an amino acid sequence as set forth in all or part of SEQ ID NO: 13 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 13, wherein said amino acid sequence comprises one or more of the following: (a) valine at position 44; (b) valine at position 64; (c) glycine at position 103; (d) arginine at position 104; (e) proline at position 111; (f) glycine at position 117; or a nucleotide sequence which is the complement of any aforesaid sequence, wherein the position numbers are stated with reference to SEQ ID NO. 13, preferably wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 12 or a part of variant thereof, or a complement thereof.

33. The nucleic acid molecule of claim 30, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a protein which contributes to C50 carotenoid γ-cyclase activity and which has an amino acid sequence as set forth in all or part of SEQ ID NO: 15 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 15, wherein said amino acid sequence comprises one or more of the following: (a) a glycine residue at position 100; (b) a glycine residue at position 103; (c) a proline residue at position 107; or a nucleotide sequence which is the complement of any aforesaid sequence, wherein the position numbers are stated with reference to SEQ ID NO. 15, preferably wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 14 or a part of variant thereof, or a complement thereof.

34. The nucleic acid molecule of claim 24, wherein said part of said nucleic acid molecule comprises a nucleotide sequence encoding all or part of a protein having an amino acid sequence as set forth in SEQ ID NO: 34 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 34 and wherein said nucleotide sequence encodes a sarcinaxanthin glycosylase enzyme, which activity results in the production of both sarcinaxanthin mono- and diglucosides, when expressed in a host cell, or a nucleic acid molecule which comprises a nucleotide sequence which is the complement of any aforesaid sequence.

35. The nucleic acid molecule of claim 34, wherein said part of said nucleic acid molecule comprises: (i) a nucleotide sequence as set forth in SEQ ID NO: 33; (ii) a nucleotide sequence which is degenerate with the sequence of SEQ ID NO: 33, (iii) a nucleotide sequence which has at least 90% sequence identity to SEQ ID NO: 33; (iv) a nucleotide sequence which is a part of the nucleotide sequence of SEQ ID NO: 33 or of a nucleotide sequence which is degenerate therewith; or (v) a nucleotide sequence which is complementary to any of (i) to (iv) above.

36. The nucleic acid molecule of claim 35, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a protein having sarcinaxanthin glycosylase activity and an amino acid sequence as set forth in all or part of SEQ ID NO: 34 or an amino acid sequence which is at least 90% identical to SEQ ID NO: 34, wherein said amino acid sequence comprises one or more of the following: (a) histidine at position 62; (b) serine at position 109; (c) arginine at position 129; (d) alanine at position 138; (e) arginine at position 248; (f) proline at position 251; or a nucleotide sequence which is the complement of any aforesaid sequence, wherein the position numbers are stated with reference to SEQ ID NO. 34, preferably wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 33 or a part of variant thereof, or a complement thereof.

37. A vector comprising the isolated nucleic acid molecule of claim 24.

38. An isolated protein encoded by the nucleic acid molecule of claim 24.

39. A strain of Micrococcus luteus as deposited under number DSM 23579 at the DSMZ, or a mutant or modified strain thereof which produces sarcinaxanthin or a derivative thereof.

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