Compositions and methods for bacterial and genetically modified microorganism production of chondroitin
View Patent ↗The invention relates to the field of recombinant DNA technology for the production of chondroitin, including the production of chondroitin sulfate via a combination of recombinant bacterial fermentation and post-fermentation sulfation.
1. A construct comprising a kfoA, kfoC, and kfoF gene, wherein the construct 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 chondroitin in a non-pathogenic bacterial host cell.
2. The construct of claim 1 , wherein the construct further comprises a kfoG gene, a kfoB gene, or a combination thereof.
3. The construct of claim 1 , wherein the chondroitin is non-fructosylated.
4. The construct of claim 1 , wherein the construct further comprises a kpsF, kpsE, kpsD, kpsU, kpsC, and kpsS gene.
5. The construct of claim 4 , wherein the construct further comprises a kpsM and kpsT gene.
6. The construct of claim 4 , wherein the chondroitin is secreted from the host cell.
7. The construct of claim 1 , wherein the construct also does not contain a functional gene of one or more of kpsM, kpsT, kpsE, kpsD, kpsC, or kpsS.
8. The construct of claim 7 , wherein the chondroitin is not secreted from the host cell.
9. The construct of claim 1 , wherein one or more genes are modified for optimal codon usage in a bacterial host cell.
10. The construct of claim 1 , comprising a K4 gene cluster.
11. A non-pathogenic bacterial host cell comprising the construct of claim 1 .
12. The non-pathogenic bacterial host cell of claim 11 , wherein the non-pathogenic 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.
13. The non-pathogenic bacterial host cell of claim 12 , wherein the bacterial host cell is a bacterial strain selected from the group consisting of MSC279, MSC280, MSC315, MSC316, MSC317, MSC319, MSC322, MSC323, MSC324, MSC325, 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.
14. A method for producing a non-pathogenic bacterial host cell comprising the construct of claim 1 , comprising transferring the construct to a non-pathogenic bacterial host cell.
15. A method for producing a bacterial cell that is capable of producing non-fructosylated chondroitin, wherein the cell comprises a kfoA gene, a kfoC gene, and a kfoF gene, the method comprising inactivating a gene in the cell selected from the group consisting of: kfoD, orf3(kfoI), kfoE, orf1(kfoH), and combinations thereof.
16. A recombinant bacterial cell comprising a kfoA gene, a kfoC gene, and a kfoF gene, wherein the cell does not comprise a functional gene of one or more of kfoD, orf3(kfoI), kfoE, or orf1(kfoH), and wherein the cell is capable of producing chondroitin.
17. The recombinant bacterial cell of claim 16 , wherein the kfoA gene, kfoC gene, kfoF gene, or combinations thereof are expressed from a promoter selected from the group consisting of: Pm, Plac, Ptrp, Ptac, λpL, PT7, PphoA, ParaC, PxapA, Pcad, and PrecA.
18. A genetically modified microorganism comprising a kfoA gene, a kfoC gene, and a kfoF gene, wherein the microorganism has been genetically modified to delete or inactivate a gene of one or more of kfoD, orf3(kfoI), kfoE, or orf1(kfoH), and wherein the microorganism is capable of producing chondroitin.
19. A method for producing a chondroitin, comprising culturing the recombinant bacterial cell of claim 16 under fermentation conditions sufficient for production of the chondroitin.
20. A method for producing a chondroitin, comprising culturing the genetically modified microorganism of claim 18 under fermentation conditions sufficient for production of the chondroitin.
21. 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.
22. The method of claim 21 , wherein the genes of the construct are integrated into a chromosome of the bacterial host cell.
23. The method of claim 22 , wherein two or more copies of a gene of the construct are integrated the chromosome of the bacterial host cell.
24. The method of any one of claims 19 - 21 , wherein the chondroitin is non-fructosylated.
25. A method for producing a chondroitin sulfate, comprising:
(a) producing a chondroitin by the method of any one of claims 19 - 21 , and
(b) sulfating the chondroitin.
26. The method of claim 25 , wherein the sulfating comprises mixing sulfurtrioxide-triethylamine complex or chlorosulfonic acid with the chondroitin in formamide.