INTEIN-MODIFIED ENZYMES, THEIR PRODUCTION AND INDUSTRIAL APPLICATION

• US 20160186157A1
• Filed: 01/20/2016
• Published: 06/30/2016
• Est. Priority Date: 11/06/2009
• Status: Active Grant

• Current Assignee: Agrivida, Inc. (Nippon Soda Company Limited)
• Original Assignee: Agrivida, Inc. (Nippon Soda Company Limited)
• Inventors: SHEN, BINZHANG, LAZAR, GABOR, DE LA VEGA, HUMBERTO, APGAR, JAMES, LESSARD, PHILIP, RAAB, R. MICHAEL
• Granted Patent: US 10,196,623 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8241 Phenotypically and genetically modified plants via recombinant DNA technology
• CPC Class Codes: C12N 15/8246 Non-starch polysaccharides, e.g. cellulose, fructans, levans
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12N 9/2482 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)

Abstract

A method of predicting an intein insertion site in a protein that will lead to a switching phenotype is provided. The method includes identifying a plurality of C/T/S sites within the protein; selecting from the plurality of C/T/S/sites those that are ranked 0.75 or higher by a support vector machine, within ten angstroms of the active site of the protein, and at or near a loop-β-sheet junction or a loop-α-helix junction. A method of controlling protein activity and hosts including proteins with controlled activity are also provided. Also, intein modified proteins and plants containing intein modified proteins are provided.

Claims

1. An intein modified protein comprising a target protein and an intein inserted into the target protein at a site i) immediately prior to a cysteine, threonine, or serine residue of the target protein, ii) within ten angstroms from an active site of the target protein, and iii) within 10 amino acids of a loop-β

-sheet junction or within 10 amino acids of a loop-α

-helix junction of the target protein, wherein the target protein is a xylanase or a cellulase.

2. The intein modified protein of claim 1, wherein the site includes an insertion site cassette having amino acid positions −

3, −

2, −

1, 0, 1, and 2 where the 0 position is the cysteine, theronine, or serine and the insertion site cassette has a −

3, −

2, −

1, 0, 1, 2 sequence selected from the group consisting of GGKCGG (SEQ ID NO;

     1513), GGKSGG (SEQ ID NO;

     1514), GGKTGG (SEQ ID NO;

     1515), PGATSP (SEQ ID NO;

     1516), PGATVP (SEQ ID NO;

     1517), GAKSLG (SEQ ID NO;

     1518), PGATSL (SEQ ID NO;

     1519), PGASPL (SEQ ID NO;

     1520), PGATGP (SEQ ID NO;

     1521), AQRSLG (SEQ ID NO;

1522), NQPSIV (SEQ ID NO;

     1523), NQASIV (SEQ ID NO;

     1524), PNMSSA (SEQ ID NO;

     1525), GNHSSG (SEQ ID NO;

     1526), PSHSAY (SEQ ID NO;

     1527), SLMSSC (SEQ ID NO;

     1528), TNTSNY (SEQ ID NO;

     1529), IDTSRN (SEQ ID NO;

     1530), PSTSAY (SEQ ID NO;

     1531), QIKSLG (SEQ ID NO;

     1532), FETCNY (SEQ ID NO;

     1533), AVLSVN (SEQ ID NO;

     1534), LVYSAH (SEQ ID NO;

     1535), AGYSSA (SEQ ID NO;

     1536), MWGTLR (SEQ ID NO;

     1537), LSASSY (SEQ ID NO;

     1538), FAQTQI (SEQ ID NO;

     1539), GGRSFV (SEQ ID NO;

     1540), SFVCGF (SEQ ID NO;

     1541), GFGSNP (SEQ ID NO;

     1542), NPPTRP (SEQ ID NO;

     1543), HHRSSS (SEQ ID NO;

     1544), HRSSSC (SEQ ID NO;

     1545), RSSSCP (SEQ ID NO;

     1546), DWNTFN (SEQ ID NO;

     1547), TFNSPD (SEQ ID NO;

     1548), DDRSDY (SEQ ID NO;

     1549), EVATDY (SEQ ID NO;

     1550), (SEQ ID NO;

NQVTEL

     1551), SSVTFW (SEQ ID NO;

     1552), LRESVW (SEQ ID NO;

     1553), RFHTLV (SEQ ID NO;

     1554), DLSSVT (SEQ ID NO;

     1555), DNHTWL (SEQ ID NO;

     1556), DYNTEV (SEQ ID NO;

     1557), LDVSLY (SEQ ID NO;

     1558), HYNSIV (SEQ ID NO;

     1559), ADLSSV (SEQ ID NO;

     1560), NIITEL (SEQ ID NO;

     1561), GHQTHI (SEQ ID NO;

     1562), MRNSPW (SEQ ID NO;

     1563), RFHTLV (SEQ ID NO;

     1564), DYNTDD (SEQ ID NO;

     1565), DKYSWL (SEQ ID NO;

     1566), LDMSIY (SEQ ID NO;

     1567), HNQTPT (SEQ ID NO;

     1568), DIKSWD (SEQ ID NO;

     1569), WGISDK (SEQ ID NO;

     1570), SGATDL (SEQ ID NO;

     1571), YYYSWW (SEQ ID NO;

     1572), SWWSDG (SEQ ID NO;

     1573), NFGTYD (SEQ ID NO;

     1574), GKTTRV (SEQ ID NO;

     1575), NAPSID (SEQ ID NO;

     1576), GTQTFD (SEQ ID NO;

     1577), QYWSVR (SEQ ID NO;

     1578), IVATEG (SEQ ID NO;

     1579), GYFSSG (SEQ ID NO;

     1580), NGNSYL (SEQ ID NO;

     1581), YGWTRN (SEQ ID NO;

     1582), YDPSSG (SEQ ID NO;

     1583), LGKTTR (SEQ ID NO;

     1584), YFSSGY (SEQ ID NO;

     1585), IDHTDS (SEQ ID NO;

     1586), SWSTNE (SEQ ID NO;

     1587), HTDSWS (SEQ ID NO;

     1588), NEITIN (SEQ ID NO;

     1589), DSWSTN (SEQ ID NO;

     1590), LDQSYV (SEQ ID NO;

     1591), EDPTIT (SEQ ID NO;

     1592), SYVTGY (SEQ ID NO;

     1593), PWGSNS (SEQ ID NO;

     1594), GSNSFI (SEQ ID NO;

     1595), TPGSGG (SEQ ID NO;

     1596), TNYSHP (SEQ ID NO;

     1597), DGMSYL (SEQ ID NO;

     1598), PQKCYI (SEQ ID NO;

     1599), DLISLM (SEQ ID NO;

     1600), LMSSCM (SEQ ID NO;

     1601), AGSSQA (SEQ ID NO;

     1602), AGHSAW (SEQ ID NO;

     1603), GIATNT (SEQ ID NO;

     1604), ATNTSN (SEQ ID NO;

     1605), CDPSGR (SEQ ID NO;

     1606), PQGTWF (SEQ ID NO;

     1607), VIDTSR (SEQ ID NO;

     1608), QGLTSL (SEQ ID NO;

     1609), SGQSAL (SEQ ID NO;

     1610), NGDSYW (SEQ ID NO;

     1611), SGDTGG (SEQ ID NO;

     1612), GVQSYN (SEQ ID NO;

     1613), LVYSAH (SEQ ID NO;

     1614), EFGTTL (SEQ ID NO;

     1615), FQWTFW (SEQ ID NO;

     1616), TFWSWN (SEQ ID NO;

     1617), NPDSGD (SEQ ID NO;

     1618), GYQSSG (SEQ ID NO;

     1619), IVESWG (SEQ ID NO;

     1620), GWSTNP (SEQ ID NO;

     1621), NLGTID (SEQ ID NO;

     1622), TGNTTM (SEQ ID NO;

     1623), NGNSYL (SEQ ID NO;

     1624), YGWSTN (SEQ ID NO;

     1625), YQSSGS (SEQ ID NO;

     1626), SNASGT (SEQ ID NO;

     1627), and DGGTYD (SEQ ID NO;

     1628).

3. The intein modified protein of claim 1, wherein the intein has a sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

3, 88, 91-96, 98, 99 and 101.

4. The intein modified protein of claim 3, wherein the intein modified protein has a switching phenotype and switching is inducible upon exposure to temperatures of 48°

C. or higher.

5. The intein modified protein of claim 1, wherein the target protein is a xylanase having a sequence with at least 90% identity with a sequence selected from the group consisting of SEQ ID NOS:

104-108.

6. The intein modified protein of claim 5, wherein the intein has a sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

3, 88, 91-96, 98, 99 and 101.

7. The intein modified protein of claim 1, wherein the intein modified protein includes a sequence having at least 90% identity to one selected from the group consisting of SEQ ID NOS:

1701-1703 and 1711.

8. The intein modified protein of claim 1, wherein the target protein is a cellulase having a sequence with at least 90% identity with a sequence selected from the group consisting of SEQ ID NOS:

109-112.

9. The intein modified protein of claim 8, wherein the intein has a sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

3, 88, 91-96, 98, 99 and 101.

10. The intein modified protein of claim 1, wherein the intein modified protein has a sequence with at least 90% identity to one selected from the group of SEQ ID NOS:

1744, 1751, and 1752.

11. The intein modified protein of claim 1, wherein the intein modified protein has a sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:

1629-1691, 1696, 1697, 1699-1712, 1713-1718, 1720-1731, 1733-1738, 1743, 1744, 1751, 1752, 1767, 1770-1771, and 1774-1784.

12. An intein modified protein having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:

1692-1695, 1698, 1719, 1732, 1739-1742, 1745-1750, 1753-1766, 1768-1769, 1772-1773.

13. A transgenic plant including an expression construct encoding an intein modified protein comprising a target protein and an intein inserted into the target protein at a site i) immediately prior to a cysteine, threonine, or serine residue of the target protein, ii) within ten angstroms from an active site of the target protein, and iii) within 10 amino acids of a loop-α

-sheet junction or within 10 amino acids of a loop-β

-helix junction of the target protein, wherein the target protein is a xylanase or a cellulase.

14. The transgenic plant of claim 13, wherein the site includes an insertion site cassette having amino acid positions −

3, −

2, −

1, 0, 1, and 2 where the 0 position is the cystein, theronine, or serine and the insertion site cassette has a −

3, −

2, −

1, 0, 1, 2 sequence selected from the group consisting of GGKCGG (SEQ ID NO;

     1513), GGKSGG (SEQ ID NO;

     1514), GGKTGG (SEQ ID NO;

     1515), PGATSP (SEQ ID NO;

     1516), PGATVP (SEQ ID NO;

     1517), GAKSLG (SEQ ID NO;

     1518), PGATSL (SEQ ID NO;

     1519), PGASPL (SEQ ID NO;

     1520), PGATGP (SEQ ID NO;

     1521), AQRSLG (SEQ ID NO;

1522), NQPSIV (SEQ ID NO;

     1523), NQASIV (SEQ ID NO;

     1524), PNMSSA (SEQ ID NO;

     1525), GNHSSG (SEQ ID NO;

     1526), PSHSAY (SEQ ID NO;

     1527), SLMSSC (SEQ ID NO;

     1528), TNTSNY (SEQ ID NO;

     1529), IDTSRN (SEQ ID NO;

     1530), PSTSAY (SEQ ID NO;

     1531), QIKSLG (SEQ ID NO;

     1532), FETCNY (SEQ ID NO;

     1533), AVLSVN (SEQ ID NO;

     1534), LVYSAH (SEQ ID NO;

     1535), AGYSSA (SEQ ID NO;

     1536), MWGTLR (SEQ ID NO;

     1537), LSASSY (SEQ ID NO;

     1538), FAQTQI (SEQ ID NO;

     1539), GGRSFV (SEQ ID NO;

     1540), SFVCGF (SEQ ID NO;

     1541), GFGSNP (SEQ ID NO;

     1542), NPPTRP (SEQ ID NO;

     1543), HHRSSS (SEQ ID NO;

     1544), HRSSSC (SEQ ID NO;

     1545), RSSSCP (SEQ ID NO;

     1546), DWNTFN (SEQ ID NO;

     1547), TFNSPD (SEQ ID NO;

     1548), DDRSDY (SEQ ID NO;

     1549), EVATDY (SEQ ID NO;

     1550), (SEQ ID NO;

NQVTEL

     1551), SSVTFW (SEQ ID NO;

     1552), LRESVW (SEQ ID NO;

     1553), RFHTLV (SEQ ID NO;

     1554), DLSSVT (SEQ ID NO;

     1555), DNHTWL (SEQ ID NO;

     1556), DYNTEV (SEQ ID NO;

     1557), LDVSLY (SEQ ID NO;

     1558), HYNSIV (SEQ ID NO;

     1559), ADLSSV (SEQ ID NO;

     1560), NIITEL (SEQ ID NO;

     1561), GHQTHI (SEQ ID NO;

     1562), MRNSPW (SEQ ID NO;

     1563), RFHTLV (SEQ ID NO;

     1564), DYNTDD (SEQ ID NO;

     1565), DKYSWL (SEQ ID NO;

     1566), LDMSIY (SEQ ID NO;

     1567), HNQTPT (SEQ ID NO;

     1568), DIKSWD (SEQ ID NO;

     1569), WGISDK (SEQ ID NO;

     1570), SGATDL (SEQ ID NO;

     1571), YYYSWW (SEQ ID NO;

     1572), SWWSDG (SEQ ID NO;

     1573), NFGTYD (SEQ ID NO;

     1574), GKTTRV (SEQ ID NO;

     1575), NAPSID (SEQ ID NO;

     1576), GTQTFD (SEQ ID NO;

     1577), QYWSVR (SEQ ID NO;

     1578), IVATEG (SEQ ID NO;

     1579), GYFSSG (SEQ ID NO;

     1580), NGNSYL (SEQ ID NO;

     1581), YGWTRN (SEQ ID NO;

     1582), YDPSSG (SEQ ID NO;

     1583), LGKTTR (SEQ ID NO;

     1584), YFSSGY (SEQ ID NO;

     1585), IDHTDS (SEQ ID NO;

     1586), SWSTNE (SEQ ID NO;

     1587), HTDSWS (SEQ ID NO;

     1588), NEITIN (SEQ ID NO;

     1589), DSWSTN (SEQ ID NO;

     1590), LDQSYV (SEQ ID NO;

     1591), EDPTIT (SEQ ID NO;

     1592), SYVTGY (SEQ ID NO;

     1593), PWGSNS (SEQ ID NO;

     1594), GSNSFI (SEQ ID NO;

     1595), TPGSGG (SEQ ID NO;

     1596), TNYSHP (SEQ ID NO;

     1597), DGMSYL (SEQ ID NO;

     1598), PQKCYI (SEQ ID NO;

     1599), DLISLM (SEQ ID NO;

     1600), LMSSCM (SEQ ID NO;

     1601), AGSSQA (SEQ ID NO;

     1602), AGHSAW (SEQ ID NO;

     1603), GIATNT (SEQ ID NO;

     1604), ATNTSN (SEQ ID NO;

     1605), CDPSGR (SEQ ID NO;

     1606), PQGTWF (SEQ ID NO;

     1607), VIDTSR (SEQ ID NO;

     1608), QGLTSL (SEQ ID NO;

     1609), SGQSAL (SEQ ID NO;

     1610), NGDSYW (SEQ ID NO;

     1611), SGDTGG (SEQ ID NO;

     1612), GVQSYN (SEQ ID NO;

     1613), LVYSAH (SEQ ID NO;

     1614), EFGTTL (SEQ ID NO;

     1615), FQWTFW (SEQ ID NO;

     1616), TFWSWN (SEQ ID NO;

     1617), NPDSGD (SEQ ID NO;

     1618), GYQSSG (SEQ ID NO;

     1619), IVESWG (SEQ ID NO;

     1620), GWSTNP (SEQ ID NO;

     1621), NLGTID (SEQ ID NO;

     1622), TGNTTM (SEQ ID NO;

     1623), NGNSYL (SEQ ID NO;

     1624), YGWSTN (SEQ ID NO;

     1625), YQSSGS (SEQ ID NO;

     1626), SNASGT (SEQ ID NO;

     1627), and DGGTYD (SEQ ID NO;

     1628).

15. The transgenic plant of claim 13, wherein the intein has a sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

3, 88, 91-96, 98, 99 and 101.

16. The transgenic plant of claim 15, wherein the intein modified protein has a switching phenotype and switching is inducible upon exposure to temperatures of 48°

C. or higher.

17. The transgenic plant of claim 13, wherein the target protein is a xylanase having a sequence with at least 90% identity with a sequence selected from the group consisting of SEQ ID NOS:

104-108.

18. The transgenic plant of claim 17, wherein the intein has a sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

3, 88, 91-96, 98, 99 and 101.

19. The transgenic plant of claim 13, wherein the intein modified protein includes a sequence having at least 90% identity to one selected from the group consisting of SEQ ID NOS:

1701-1703 and 1711.

20. The transgenic plant of claim 13, wherein the target protein is a cellulase having a sequence with at least 90% identity with a sequence selected from the group consisting of SEQ ID NOS:

109-112.

21. The transgenic plant of claim 20, wherein the intein has a sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS:

3, 88, 91-96, 98, 99 and 101.

22. The transgenic plant of claim 13, wherein the intein modified protein has a sequence with at least 90% identity to one selected from the group of SEQ ID NOS:

1744, 1751, and 1752.

23. The transgenic plant of claim 13, wherein the intein modified protein has a sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:

1629-1691, 1696, 1697, 1699-1712, 1713-1718, 1720-1731, 1733-1738, 1743, 1744, 1751, 1752, 1767, 1770-1771, and 1774-1784.