Paenibacillus-based endospore display platform, products and methods

1. A nucleic acid molecule encoding a fusion protein, comprising (a) a first polynucleotide sequence encoding an N-terminal signal peptide, operably linked to (b) a second polynucleotide sequence encoding a polypeptide heterologous to the N-terminal signal peptide, wherein the first polynucleotide sequence comprises:

(i) a polynucleotide sequence comprising SEQ ID NO: 1; or

(ii) a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 2;

wherein the N-terminal signal peptide is capable of targeting the fusion protein to a spore surface of a Paenibacillus endospore.

2. The nucleic acid molecule of claim 1 , wherein the polypeptide heterologous to the N-terminal signal peptide comprises:

(a) a plant growth-stimulating protein;

(b) an enzyme;

(c) a protein;

(d) a polypeptide heterologous to Paenibacillus;

(e) a therapeutic protein; or

(f) a plant immune-stimulating protein.

3. The nucleic acid molecule of claim 1 , further comprising a third polynucleotide sequence, encoding:

(a) a polypeptide comprising one or more protease cleavage sites, wherein the polypeptide is positioned between the N-terminal signal peptide and the polypeptide heterologous to the N-terminal signal peptide;

(b) a polypeptide comprising a selectable marker;

(c) a polypeptide comprising a visualization marker;

(d) a polypeptide comprising a protein recognition/purification domain; or

(e) a polypeptide comprising a flexible linker element, which connects the N-terminal signal peptide and the polypeptide heterologous to the N-terminal signal peptide.

4. The nucleic acid molecule of claim 1 , wherein the first polynucleotide sequence comprises:

a codon-optimized polynucleotide sequence having at least 80% sequence identity with SEQ ID NO: 1 or a fragment thereof, which is expressed at a higher rate or level in the Paenibacillus endospore compared to the corresponding unoptimized sequence under identical conditions.

5. A recombinant Paenibacillus cell comprising a bacterial chromosome comprising the nucleic acid molecule of claim 1 .

6. A vector comprising the nucleic acid molecule of claim 1 , wherein the vector comprises a plasmid, an artificial chromosome, or a viral vector.

7. The vector of claim 6 , further comprising at least one of the following:

(a) an origin of replication that provides stable maintenance in a Paenibacillus cell;

(b) an origin of replication that provides selectively non-stable maintenance in a Paenibacillus cell;

(c) a temperature-sensitive origin of replication that provides selectively non-stable maintenance in a Paenibacillus cell;

(d) a polynucleotide encoding a selection marker, operably linked to an expression control sequence; and/or

(e) a polynucleotide encoding a plant growth stimulating protein, operably linked to an expression control sequence.

8. A recombinant Paenibacillus cell transformed with a vector comprising the nucleic acid molecule of claim 1 .

9. The recombinant Paenibacillus cell of claim 8 , wherein the Paenibacillus cell is a Paenibacillus species, comprising: Paenibacillus sp. NRRL B-50972, Paenibacillus terrae, Paenibacillus polymyxa , or Paenibacillus peoriae ; or a bacterium that possesses a 16S rRNA gene that shares at least 97, 98 or 99% identity with a 16S rRNA gene of a Paenibacillus species.

10. A fusion protein comprising an N-terminal signal peptide operably linked to a polypeptide heterologous to the N-terminal signal peptide, wherein the N-terminal signal peptide comprises:

(i) a polypeptide comprising an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 2;

wherein the N-terminal signal peptide is capable of targeting the fusion protein to the spore surface of a Paenibacillus endospore.

11. The fusion protein of claim 10 , wherein the polypeptide heterologous to the N-terminal signal peptide comprises:

(a) a plant growth-stimulating protein;

(b) an enzyme;

(c) a protein;

(d) a polypeptide heterologous to Paenibacillus;

(e) a therapeutic protein; or

(f) a plant immune-stimulating protein.

12. The fusion protein of claim 10 , wherein the fusion protein further comprises:

(a) a polypeptide containing one or more protease cleavage sites, positioned between the N-terminal signal peptide and the polypeptide heterologous to the N-terminal signal peptide;

(b) a polypeptide comprising a selectable marker;

(c) a polypeptide comprising a visualization marker;

(d) a polypeptide comprising at least one protein recognition/purification domain; or

(e) a polypeptide comprising a flexible linker element, connecting the signal peptide and the polypeptide heterologous to the N-terminal signal peptide.

13. A seed treated with the fusion protein of claim 10 .

14. A composition comprising an isolated and/or purified form of the fusion protein of claim 12 .

15. A method of delivering a protein of interest to a plant, seed or field, comprising:

applying a composition comprising a recombinant Paenibacillus endospore to a plant, seed, or field;

wherein the recombinant Paenibacillus endospore has been modified to express the fusion protein of claim 12 .

16. The method of claim 15 , wherein the composition is applied to a field:

a) pre- or post-planting;

b) pre- or post-emergence;

c) as a powder, suspension or solution; and/or

d) wherein the composition further comprises one or more additional compounds that stimulate plant growth or protect plants from pests.

17. A method of treating a plant, a seed, a plant part, or the soil surrounding the plant to enhance plant growth and/or promote plant health comprising the step of applying to the plant, seed, plant part, or soil a recombinant endospore-producing Paenibacillus cell that expresses the fusion protein of claim 10 .

18. The method of claim 17 , wherein the polypeptide heterologous to the N-terminal signal peptide comprises:

(a) a plant growth-stimulating protein;

(b) an enzyme;

(c) a protein;

(d) a polypeptide heterologous to Paenibacillus ; or

(e) a plant immune-stimulating protein.