Compositions and methods for bacterial production of chondroitin

1. A construct comprising a gene cluster comprising kpsF, kpsE, kpsD, kpsU, kpsC, kpsS, kfoA, kfoC, kfoG, and kfoF, wherein the gene cluster does not contain a functional gene of one or more of kfoD, orf3 (kfoI), kfoE, or orf1 (kfoH), and wherein the construct is suitable for producing a chondroitin in a non-pathogenic bacterial host cell.

2. The construct of claim 1 , wherein the chondroitin is non-fructosylated.

3. The construct of claim 1 , wherein the chondroitin is secreted from the host cell.

4. The construct of claim 1 , wherein the gene cluster further comprises kfoB.

5. The construct of claim 1 , wherein the gene cluster further comprises kpsM and kpsT.

6. The construct of claim 1 , wherein the construct comprises pDD66, pDD67, pCX040, pCX042, pCX096, or pBR1052.

7. The construct of claim 1 , wherein one or more genes are modified for optimal codon usage in the bacterial host cell.

8. The construct of claim 1 , further comprising a promoter.

9. The construct of claim 8 , wherein the promoter is selected from the group consisting of Pm, Plac, Ptrp, Ptac, λpL, PT7, PphoA, ParaC, PxapA, Pcad, and PrecA.

10. The construct of claim 9 , wherein the promoter is Pm.

11. The construct of claim 1 , wherein the construct further comprises a second promoter.

12. The construct of claim 11 , wherein the second promoter is selected from the group consisting of Pm, Plac, Ptrp, Ptac, 80 pL, PT7, PphoA, ParaC, PxapA, Pcad, and PrecA.

13. The construct of claim 12 , wherein the second promoter is Pm.

14. The construct of claim 11 , wherein the second promoter is operably linked to one or more genes.

15. The construct of claim 14 , wherein the second promoter is operably linked to kpsFEDUCS.

16. The construct of claim 1 , wherein the construct further comprises a xylS regulatory gene.

17. The construct of claim 1 , further comprising an antibiotic resistance gene.

18. The construct of claim 17 , wherein the antibiotic resistance gene is selected from the group consisting of chloramphenicol (CamR), kanamycin (KanR), ampicillin (AmpR), tetracycline (TetR), bleomycin (BleR), spectinomycin (SpcR), sulfonamide (SuR), streptomycin (StrR), carbenicillin (CbR), and erythromycin (EryR).

19. The construct of claim 1 , comprising a K4 gene cluster.

20. The construct of claim 1 , wherein the bacterial host cell is or is derived from a non-pathogenic organism selected from the group consisting of Escherichia, Pseudomonas, Xanthomonas, Methylomonas, Acinetobacter , and Sphingomonas.

21. A non-pathogenic bacterial host cell comprising the construct of claim 1 .

22. The non-pathogenic bacterial host cell of claim 21 , wherein the bacterial host cell is a bacterial strain selected from the group consisting of MSC279, MSC280, MSC315, MSC316, MSC317, MSC319, MSC322, MSC324, MSC326, MSC328, MSC346, MSC347, MSC348, MSC350, MSC356, MSC359, MSC392, MSC402, MSC403, MSC404, MSC405, MSC410, MSC411, MSC436, MSC437, MSC438, MSC439, MSC458, MSC459, MSC460, MSC461, MSC466, MSC467, MSC469, MSC480, MSC494, MSC498, MSC499, MSC500, MSC510, MSC511, MSC522, MSC526, MSC537, MSC551, MSC561, MSC562, MSC563, MSC564, MSC566, MSC567, MSC619, MSC625, MSC627, MSC640, MSC641, MSC643, MSC646, MSC650, MSC656, MSC657, MSC658, MSC659, MSC660, MSC669, MSC670, MSC671, MSC672, MSC673, MSC674, MSC675, MSC676, MSC677, MSC678, MSC679, MSC680, MSC681, MSC682, MSC683, MSC684, MSC687, MSC688, MSC689, MSC690, MSC691, MSC692, MSC693, MSC694, MSC700, MSC701, MSC702, MSC722, MSC723 and MSC724.

23. A method for producing a chondroitin, comprising:

(a) transferring the construct of claim 1 to a non-pathogenic bacterial host cell, and

(b) culturing the bacterial host cell under fermentation conditions wherein the chondroitin is produced by the bacterial host cell.

24. A method for producing a chondroitin, comprising culturing a non-pathogenic bacterial host cell comprising the construct of claim 1 under fermentation conditions sufficient for production of the chondroitin.

25. A method for producing a non-pathogenic bacterial host cell comprising the construct of claim 1 , comprising transferring the construct of claim 1 to a non-pathogenic bacterial host cell.

26. The method of claim 24 , wherein the gene cluster or gene of the construct is integrated into a chromosome of the bacterial host cell.

27. The method of claim 26 , wherein two or more copies of the gene cluster or gene are integrated into the chromosome of the bacterial host cell.

28. The method of claim 24 , wherein the chondroitin is non-fructosylated.

29. The method of claim 24 , further comprising sulfating the chondroitin.

30. A method for producing a chondroitin sulfate, comprising:

(a) producing a chondroitin by the method of claim 24 , and

(b) sulfating the chondroitin.

31. The method of claim 30 , wherein the sulfating comprises mixing sulfurtrioxide-triethylamine complex or chlorosulfonic acid with the chondroitin in formamide.

32. The method of claim 24 , wherein the bacterial host cell is or is derived from a non-pathogenic organism selected from the group consisting of Escherichia, Pseudomonas, Xanthomonas, Methylomonas, Acinetobacter , and Sphingomonas.

33. The method of claim 24 , wherein the bacterial host cell is or is derived from a gram-negative organism.

34. The method of claim 32 , wherein the bacterial host cell is a Xanthomonas campestris.

35. The method of claim 34 , wherein the X. campestris is a bacterial strain selected from the group consisting of MSC255, MSC256, MSC257, MSC225, and MSC226.

36. The method of claim 32 , wherein the bacterial host cell is a non-pathogenic E. coli.

37. The method of claim 36 , wherein the non-pathogenic E. coli is selected from the group consisting of E. coli K-12 and E. coli B.

38. The method of claim 37 , wherein the E. coli K-12 is a bacterial strain selected from the group consisting of MSC188 and MSC175.

39. The method of claim 37 , wherein the E. coli B is bacterial strain MSC364.

40. The method of claim 24 , wherein an endogenous gene of the bacterial host cell is deleted or inactivated by homologous recombination.

41. The method of claim 24 , wherein the bacterial host cell does not express extracellular polysaccharides endogenous to the host cell.

42. The method of claim 24 , wherein the bacterial host cell is suitable for conjugal transfer from a laboratory cloning strain.

43. The method of claim 24 , further comprising recovering the chondroitin from the bacterial host cell.

44. The method of claim 24 , further comprising recovering the chondroitin from extracellular culture medium.

45. The method of claim 24 , wherein 1 g/L to 50 g/L of the chondroitin is secreted from the bacterial host cell in 24 hours to 72 hours.

46. The method of claim 45 , wherein 5 g/L to 50 g/L of the chondroitin is secreted from the bacterial host cell in 24 hours to 72 hours.

47. The method of claim 46 , wherein 15 g/L to 50 g/L of the chondroitin is secreted from the bacterial host cell in 24 hours to 72 hours.

48. The method of claim 24 , further comprising purifying the chondroitin.

49. The method of claim 24 , wherein the bacterial host cell is cultured at 25° C. to 37° C.

50. The method of claim 24 , wherein the bacterial host cell is cultured in a medium comprising glycerine.

51. The chondroitin produced by the method of claim 24 .

52. A composition comprising the chondroitin of claim 51 .