Pectate lyases, nucleic encoding them and methods for making and using them

1. An isolated, synthetic or recombinant nucleic acid molecule comprising

(a) a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or more, or 100% sequence identity to SEQ ID NO:77 or SEQ ID NO:131 and encoding a polypeptide having pectate lyase (EC 4.2.2.2) activity, or enzymatically active fragments thereof;

(b) a nucleic acid sequence comprising the sequence SEQ ID NO:77 or SEQ ID NO:131 and encoding a polypeptide having pectate lyase (EC 4.2.2.2) activity, or an enzymatically active fragment thereof;

(c) a nucleic acid sequence encoding a polypeptide having pectate lyase (EC 4.2.2.2) activity, wherein the polypeptide has at least 95%, 96%, 97%, 98%, 99%, or more, or 100% sequence identity to SEQ ID NO:78 or SEQ ID NO:132, or an enzymatically active fragment thereof;

(d) a nucleic acid sequence encoding a polypeptide having pectate lyase (EC 4.2.2.2) activity, wherein the polypeptide has the sequence SEQ ID NO:78 or SEQ ID NO:132, or an enzymatically active fragment thereof;

(e) the nucleic acid sequence of (c) or (d) further comprising a heterologous sequence encoding a signal sequence, a catalytic domain, and/or prepro domain from a different pectate lyase operably linked to the nucleic acid sequence or encoding a signal sequence and/or prepro domain from a non-pectate lyase protein linked to the nucleic acid sequence;

(f) a sequence complementary to the full-length nucleic acid of (a), (b), (c), (d), or (e).

2. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the pectate lyase activity comprises the breakup or dissolution of a plant fiber or a plant cell wall; beta-elimination (trans-elimination) cleavage of pectin, polygalacturonic acid (pectate), 1,4-linked alpha-D-galacturonic acid, and/or methyl-esterified galacturonic acid; and/or catalyzes the random cleavage of alpha-1,4-glycosidic linkages in pectic acid (polygalacturonic acid).

3. An isolated, synthetic or recombinant nucleic acid, comprising:

(a) a polynucleotide sequence that hybridizes under stringent conditions to the complete complement of a nucleic acid comprising SEQ ID NO:77 or SEQ ID NO:131,

wherein the polynucleotide sequence encodes a polypeptide having a pectate lyase activity and,

wherein the stringent conditions include a wash step comprising a wash in 0.2.times SSC at a temperature of about 65 degrees C. for about 15 minutes, or

(b) a nucleic acid sequence complementary to the full-length sequence of (a).

4. A nucleic acid probe for identifying a nucleic acid encoding a polypeptide with a pectate lyase activity, wherein the probe comprises at least 75, 100, 150, 200, 300, 400 or 500 consecutive bases of SEQ ID NO:77 or SEQ ID NO:131,

wherein the probe identifies the nucleic acid by binding or hybridization under stringent conditions, wherein the stringent conditions include a wash step comprising a wash in 0.2 times SSC at a temperature of about 65 degrees C. for about 15 minutes.

5. An amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide having a pectate lyase activity, wherein the primer pair is capable of amplifying the nucleic acid molecule of claim 1 or claim 3 , or a fragment thereof encoding a polypeptide with pectate lyase activity.

6. An expression cassette comprising a nucleic acid comprising the nucleic acid molecule as set forth in claim 1 or claim 3 .

7. A vector or cloning vehicle comprising a nucleic acid comprising the nucleic acid molecule as set forth in claim 1 or claim 3 .

8. A transformed cell comprising a nucleic acid comprising the nucleic acid molecule as set forth in claim 1 or claim 3 .

9. An antisense oligonucleotide consisting of a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to the nucleic acid sequence of claim 1 or claim 3 , or a fragment thereof

wherein the stringent conditions include a wash step comprising a wash in 0.2 times SSC at a temperature of about 65 degrees C. for about 15 minutes.

10. A double-stranded inhibitory RNA (RNAi) molecule comprising a fragment of the nucleic acid sequence of claim 1 or claim 3 .

11. An array comprising the nucleic acid molecule of claim 1 or claim 3 , wherein the nucleic acid molecule is immobilized.

12. A method of producing a recombinant polypeptide comprising the steps of:

(a) providing a nucleic acid operably linked to a promoter, wherein the nucleic acid comprises the nucleic acid molecule of claim 1 or claim 3 ; and

(b) expressing the nucleic acid of step (a) under conditions that allow expression of the encoded polypeptide, thereby producing the recombinant polypeptide.

13. A method for overexpressing a recombinant pectate lyase in a cell

wherein, the method comprises expressing a vector comprising the nucleic acid molecule of claim 1 or claim 3 ,

wherein, overexpression is achieved by use of a high activity promoter, a dicistronic vector, or by gene amplification of the vector.

14. The isolated, synthetic or recombinant nucleic acid molecule of claim 1 , wherein the pectate lyase activity is exo-acting or endo-acting.

15. The isolated, synthetic or recombinant nucleic acid molecule of claim 1 , wherein the pectate lyase activity is thermostable or thermotolerant.

16. The isolated, synthetic or recombinant nucleic acid molecule of claim 1 , wherein the nucleic acid sequence has at least 95% sequence identity to SEQ ID NO: 77 or SEQ ID NO: 131 and encodes a polypeptide having pectate lyase activity.

17. The isolated, synthetic or recombinant nucleic acid molecule of claim 1 , wherein the nucleic acid sequence has at least 97% sequence identity to SEQ ID NO: 77 or SEQ ID NO: 131 and encodes a polypeptide having pectate lyase activity.

18. The isolated, synthetic or recombinant nucleic acid molecule of claim 1 , wherein the nucleic acid sequence has at least 98% sequence identity to SEQ ID NO: 77 or SEQ ID NO: 131 and encodes a polypeptide having pectate lyase activity.

19. The isolated, synthetic or recombinant nucleic acid molecule of claim 1 , wherein the nucleic acid sequence has at least 99% sequence identity to SEQ ID NO: 77 or SEQ ID NO: 131 and encodes a polypeptide having pectate lyase activity.