Hyaluronan synthase gene and uses thereof
View Patent ↗The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active Streptococcus equisimilis hyaluronate synthase (seHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product. Hyaluronate is also known as hyaluronic acid or hyaluronan.
1. A method of producing hyaluronic acid, comprising the steps of:
providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes at least two amino acid motifs selected from the group consisting of:
(i)
GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X) 3–7
(SEQ ID NO:11)
(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I)
DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE;
(ii)
(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)
(SEQ ID NO:12)
(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)
(I/V/C)(S/G)GPL(G/S)XY(K/R);
(iii)
(D/E)X(W/Y)(X) 2 QXF(L/M)G(X) 2 (C/V)(S/T)
(SEQ ID NO:13)
XGDDR(H/R/C)LTN;
and
(iv)
(E/D)(T/S/V)P(X) 5 (W/Y/F)(L/I)XQQ(T/N)
(SEQ ID NO:14)
RW(S/T/N)KS(Y/W/F)(F/C)RE,
with X being selected from the group consisting of any amino acid; and
wherein the at least two amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and
culturing the host cell under conditions appropriate for the production of hyaluronic acid.
2. The method of claim 1 , further comprising the step of separating the hyaluronic acid from the host cell.
3. The method of claim 1 , wherein the hyaluronan synthase gene is isolated from a prokaryotic source.
4. The method of claim 1 , wherein the hyaluronan synthase gene is isolated from a microbial source.
5. A method of producing hyaluronic acid, comprising the steps of:
providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes at least three amino acid motifs selected from the group consisting of:
(i)
GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X) 3–7
(SEQ ID NO:11)
(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I)
DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE;
(ii)
(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)
(SEQ ID NO:12)
(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)
(I/V/C)(S/G)GPL(G/S)XY(K/R);
(iii)
(D/E)X(W/Y)(X) 2 QXF(L/M)G(X) 2 (C/V)(S/T)
(SEQ ID NO:13)
XGDDR(H/R/C)LTN;
and
(iv)
(E/D)(T/S/V)P(X) 5 (W/Y/F)(L/I)XQQ(T/N)
(SEQ ID NO:14)
RW(S/T/N)KS(Y/W/F)(F/C)RE,
with X being selected from the group consisting of any amino acid; and
wherein the at least three amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and
culturing the host cell under conditions appropriate for the production of hyaluronic acid.
6. The method of claim 5 , further comprising the step of separating the hyaluronic acid from the host cell.
7. The method of claim 5 , wherein the hyaluronan synthase gene is isolated from a prokaryotic source.
8. The method of claim 5 , wherein the hyaluronan synthase gene is isolated from a microbial source.
9. A method of producing hyaluronic acid, comprising the steps of:
providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes the amino acid motifs:
(i)
GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X) 3–7
(SEQ ID NO: 11)
(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I)
DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE;
(ii)
(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)
(SEQ ID NO:12)
(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)
(I/V/C)(S/G)GPL(G/S)XY(K/R);
(iii)
(D/E)X(W/Y)(X) 2 QXF(L/M)G(X) 2 (C/V)(S/T)
(SEQ ID NO:13)
XGDDR(H/R/C)LTN;
and
(iv)
(E/D)(T/S/V)P(X) 5 (W/Y/F)(L/I)XQQ(T/N)
(SEQ ID NO:14)
RW(S/T/N)KS(Y/W/F)(F/C)RE,
with X being selected from the group consisting of any amino acid; and
wherein the amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and
culturing the host cell under conditions appropriate for the production of hyaluronic acid.
10. The method of claim 9 , further comprising the step of separating the hyaluronic acid from the host cell.
11. The method of claim 9 , wherein the hyaluronan synthase gene is isolated from a prokaryotic source.
12. The method of claim 9 , wherein the hyaluronan synthase gene is isolated from a microbial source.
13. A method of producing hyaluronic acid, comprising the steps of:
providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes at least two amino acid motifs selected from the group consisting of:
(i)
GKR(X) 6 F(X) 11–15 DSDT(X) 8 E;
(SEQ ID
NO:15)
(ii)
RY(X) 2 AF(X) 2 ER(X) 2 QS(X) 9 GPL(X) 2 Y;
(SEQ ID
NO:16)
(iii)
QXFXG(X) 5 GDDRXLTN;
(SEQ ID
and
NO:17)
(iv)
P(X) 8 QQXRWXKS(X) 2 RE,
(SEQ ID
NO:18)
with X being selected from the group consisting of any amino acid; and
wherein the at least two amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and
culturing the host cell under conditions appropriate for the production of hyaluronic acid.
14. The method of claim 13 , further comprising the step of separating the hyaluronic acid from the host cell.
15. The method of claim 13 , wherein the hyaluronan synthase gene is isolated from a prokaryotic source.
16. The method of claim 13 , wherein the hyaluronan synthase gene is isolated from a microbial source.
17. A method of producing hyaluronic acid, comprising the steps of:
providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes at least three amino acid motifs selected from the group consisting of:
(i)
GKR(X) 6 F(X) 11–15 DSDT(X) 8 E;
(SEQ ID
NO:15)
(ii)
RY(X) 2 AF(X) 2 ER(X) 2 QS(X) 9 GPL(X) 2 Y;
(SEQ ID
NO:16)
(iii)
QXFXG(X) 5 GDDRXLTN;
(SEQ ID
and
NO:17)
(iv)
P(X) 8 QQXRWXKS(X) 2 RE,
(SEQ ID
NO:18)
with X being selected from the group consisting of any amino acid; and
wherein the at least three amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and
culturing the host cell under conditions appropriate for the production of hyaluronic acid.
18. The method of claim 17 , further comprising the step of separating the hyaluronic acid from the host cell.
19. The method of claim 17 , wherein the hyaluronan synthase gene is isolated from a prokaryotic source.
20. The method of claim 17 , wherein the hyaluronan synthase gene is isolated from a microbial source.
21. A method of producing hyaluronic acid, comprising the steps of:
providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that includes the amino acid motifs:
(i)
GKR(X) 6 F(X) 11–15 DSDT(X) 8 E;
(SEQ ID
NO:15)
(ii)
RY(X) 2 AF(X) 2 ER(X) 2 QS(X) 9 GPL(X) 2 Y;
(SEQ ID
NO:16)
(iii)
QXFXG(X) 5 GDDRXLTN;
(SEQ ID
and
NO:17)
(iv)
P(X) 8 QQXRWXKS(X) 2 RE,
(SEQ ID
NO:18)
with X being selected from the group consisting of any amino acid; and
wherein the amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the enzymatically active hyaluronan synthase; and
culturing the host cell under conditions appropriate for the production of hyaluronic acid.
22. The method of claim 21 , further comprising the step of separating the hyaluronic acid from the host cell.
23. The method of claim 21 , wherein the hyaluronan synthase gene is isolated from a prokaryotic source.
24. The method of claim 21 , wherein the hyaluronan synthase gene is isolated from a microbial source.