GB2389584A - A fusion protein - Google Patents

Abstract

A fusion protein comprising: a cellulose binding domain peptide; and a cell surface polypeptide. The cell surface polypeptide is capable of causing the cellulose binding domain peptide to be exposed on the surface of a cell.

Description

f) 1 ! A FUSION PROTEIN

The present invention relates to a fusion protein. The present invention also relates to a nucleic acid encoding the fusion protein and a vector that includes the nucleic acid. In addition, the present invention relates to a cell transformed with the nucleic 5 acid and to a method of increasing cellular adhesion of cells. Furthermore, the present invention relates to a method of improving plant growth or health.

Background

Plant growth is a very important consideration in agriculture. It is known that one of the factors that affects the growth of plants is the presence of plant growth promoting 10 rhizobacteria around the roots of a plant. Such rhizobacteria can assist plants by fixing nitrogen from the atmosphere and then transferring the nitrogen to the plant as ammonium. The bacteria can also provide certain sugars and amino acids and, more importantly, plant hormones, which assist in the growth of the plant. Furthermore, plant growth promoting rhizobacteria can improve the health of a plant by protecting 15 the plant roots from soil borne fungal pathogens, which are the primary cause of root diseases. This protection is mediated by such means as induced systemic resistance, competition for substrates, biosynthesis of anti-fungal compounds and physical shielding of plant roots.

The binding of bacterial cells to plant roots is mediated by microbial adhesins and 20 surface polysaccharides. The term Adhesion is a generic term referring to a proteinaceous determinant for adhesion. Thus, an adhesin is a (glyco) protein that is part of the outer membrane or appendages of bacterial cells (such as flagella, pill, fimbriae, etc).

The movement of plant growth promoting rhizobacteria towards the plant root surface 25 is triggered by the exudation of chemo-attractants by the plant roots (i.e. chemotaxis). It is known that there are then essentially two steps in the attachment of the plant growth rhizobacteria to the plant roots: adsorption and anchoring.

Adsorption involves a weak physical interaction between the bacteria and the plant root surface. Anchoring involves the fixing of the bacteria to the plant root surface by

t r ( a strong physical interaction so that the bacteria remain in position around the plant roots. Accordingly, it is desired to increase the colonization of plant roots by plant growth rhizobacteria in order to improve the growth and health of plants. The problem in 5 achieving this aim is that the quantity of bacteria colonising a plant root depends on such parameters as temperature and soil conditions which cannot easily be controlled in an agricultural environment.

To date, interest in cellulose binding domains (CBDs) has been a separate area of study. Cellulose binding domains are found incorporated into numerous enzymes 10 involved in the degradation of biomass. Typically these enzymes comprise a catalytic domain, which is responsible for hydrolysis of a particular substrate, connected by a flexible linker peptide to a cellulose binding domain. Thus the cellulose binding domain of the enzyme is able to bind tightly to a cellulose matrix and the catalytic domain of the enzyme is then able to effect hydrolysis. Although the 15 cellulose binding domain is not involved in the hydrolysis, if it is removed, then much of the desired enzyme activity towards cellulose is lost.

The presence of the two distinct domains (i.e. the catalytic domain and the cellulose binding domain) was first demonstrated for the cellulases from Tr/choderrna reesei 20 and Cellulomonas fmi by limited proteolysis (truncation) and cellulose binding assays. For these (and also other) CBDs, amino acids in the cellulose binding domain essential for the binding to insoluble cellulose have been identified by mutagenesis. (Mattinen et al. (1997), Interaction between cellohexaose and cellulose binding domains from Trichoderma reesei cellulases, FEBS Lett. 407, pp. 25 291-296. Linder et al., (1995), Identification of functionally important amino acids in the cellulose-binding domain of Trkhoderma reesei cellobiohydrolase 1, Prot. Sci. 4, pp. 1056-1064. Mattinen et al. (1997), Three-dimensional structures of three engineered cellulose- binding domains of cellobiohydrolase I from Tnchoderrna reesei, Prot. Sci. 6, pp. 294-303. Mattinen et a/. (1997), Cellulose-cellulase 30 interaction, Bull. Magn. Reson. 17: 268-269, each of which are hereby incorporated by reference.)

Over one hundred and twenty cellulose binding domains have been classified (Tomrne, P. et al "Cellulose-binding domains: Classification and Properties" American Chemical Society 1995) Into eleven different families on the basis of their structural similarity. Cellulose binding domains have been found in bacterial as well 5 as in some fungal enzymes.

The present invention has arisen from the finding that the presence of cellulose binding domains on the surface of bacteria Improves the binding of the bacteria to plant roots.

Summarv of the Invention 10 According to a first aspect of the present invention, there is provided a fusion protein comprising: a cellulose binding domain peptide; and a cell surface polypeptide capable of causing the cellulose binding domain peptide to be exposed on the surface of a cell.

Advantageously, the cell surface polypeptide comprises the sequence of SEQ ID 15 NO.:4, SEQ ID NO.: 6 or SEQ ID NO.: 8.

Conveniently the fusion protein comprises the sequence of SI5Q ID NO.: 12, SEQ ID NO.: 14, SEQ ID NO.: 16, SEQ ID NO.:18, SEQ ID NO.:20, SEQ ID NO. :22 or SEQ ID NO.:24.

Preferably the cell is a bacterium or fungal cell.

20 According to a second aspect of the present invention, there is provided a nucleic acid encoding a fusion protein as described above.

According to a third aspect of the present invention, there is provided a vector comprising the nucleic acid described above.

Advantageously the vector is a plasmid.

25 According to a fourth aspect of the present invention, there is provided a cell transformed with a nucleic acid encoding a cellulose binding domain peptide such

that, when the nucleic acid is expressed, the cellulose binding domain peptide is exposed on the surface of the cell.

Conveniently the cellulose binding domain peptide comprises the sequence BXQCGGX--XB-XXX)(-CXXX-XCXXX)(X--XXXQC

5 wherein "B" is an amino acid having an aromatic group; "X" is any amino acid and "-"

is any or no amino acid.

Preferably the cellulose binding domain peptide has a sequence comprising BXQCGGX--XB-XXXX-CXXX-XCXXXNX--BYXQC

wherein BIB" is an amino acid having an aromatic group; UX" is any amino acid and "-"

10 is any or no amino acid.

Advantageously the cellulose binding domain peptide comprises the following sequence YXXXX^-XX-XXXX-XXXX-X)O(^NX--WXQ

wherein UX" is any amino acid and U_n iS any or no amino acid.

15 Conveniently the cellulose binding domain peptide comprises the sequence TQSHYGQCGGIGYSGPTVCASG1lCQVLNPWSQCL Preferably the cell is transfommed with a nucleic acid sequence as described above Advantageously the cell is a microorganism, such as bacteria or fungi.

Conveniently the bacterium is a Plant-Growth Promoting Rhizobacterium.

20 Preferably the bacterium is Azospiri/lum brasilense.

According to a fifth aspect of the present invention there is provided a plant seed coated with one or more microorganisms described above.

( According to a sixth aspect of the present invention, there is provided a method of increasing cellular adhesion comprising transforming a cell with a cellulose binding domain peptide such that the cellulose binding domain peptide is exposed on the surface of the cell.

5 Advantageously, the method comprises transforming the cell with a nucleic acid as described above.

According to a seventh aspect of the present invention, there is provided the use of a cellulose binding domain peptide to improve cellular adhesion.

Conveniently, the cellulose binding domain peptide is part of a fusion protein as 10 described above.

According to an eighth aspect of the present invention, there is provided a method of improving plant growth or health comprising applying a cell as described above to the roots of a plant.

Descriotion In the detailed description of embodiments of the invention, reference is made to the

following figures.

Figure 1 is a schematic view of plasmid pFAJ92.

Figure 2 is a schematic view of an EcoRI-Hindlil fragment of plasmid pFAJ92fse1.

20 Figure 3 is a schematic view of an EcoRI-Hindlil fragment of plasmid pFAJ92fse2.

Figure 4 is a schematic view of an EcoRI-Hindlil fragment of plasmid pFAJ92fse3.

Figure 5 is a schematic view of plasmid pFAJ99.

Figure 6 is a schematic view of a nucleic acid fragment containing a cellulose binding domain. 25 Figure 7 is a schematic view of plasmid pFAJ99b.

( ( Figure 8 is a schematic view of plasmid pFAJ97-1 .

Figure 9 is a schematic view of plasmid pFAJ97-2 .

Figure 1 0 is a schematic view of plasmid pFAJ97-3 .

figure 11 is a schematic view of plasmid pFAJ97-1.

5 Figure 12 is a schematic view of plasmid pFAJ97-2.

Figure 1 3 is a schematic view of plasmid pFAJ97-3.

Figure 14 is a schematic view of a EcoRI-Hindlil fragment of plasmid pFAJ97-1.

Figure 15 is a schematic view of a EcoRI-Hindlil fragment of plasmid pFAJ97-2.

Figure 16 is a schematic view of a EcoRI-Hindlil fragment of plasmid pFAJ97-3.

10 Figure 17 is a schematic view of plasmid pFAJ98-1.

Figure 1 8 is a schematic view of plasmid pFAJ98-2.

Figure 19 is a schematic view of plasmid pFAJ98-3.

Figure 20 is a schematic view of plasmid pFAJ90.

Figure 21 is a schematic view of a Hindlil-EcoRI fragment of plasmid pFAJ9OFse1.

15 Figure 22 is a schematic view of a Hindlil-EcoRI fragment of plasmid pFAJ9Ofse2.

Figure 23 is a schematic view of plasmid pFAJ93-1 .

Figure 24 is a schematic view of plasmid pFAJ93-2 .

Figure 25 is a schematic view of plasmid pFAJ93-1.

Figure 26 is a schematic view of plasmid pFAJ93-2.

20 Figure 27 is a schematic view of a BamHI-Hindlil fragment of plasmid pFAJ93-1.

figure 28 is a schematic view of a BamHI-Hindlil fragment of plasmid pFAJ93-2.

Figure 29 is a schematic view of plasmid pFAJ94-1.

Figure 30 is a schematic view of plasmid pFAJ94-2.

Figure 31 is a schematic view of plasmid pFAJ91.

5 Figure 32 is a schematic view of a Hindlil-EcoRI fragment of plasmid pFAJ91fse2.

Figure 33 is a schematic view of a Hindlil-EcoRI fragment of plasmid pFAJ91fse3.

Figure 34 is a schematic view of plasmid pFAJ95-2 .

Figure 35 is a schematic view of plasmid pFAJ95-3 .

Figure 36 is a schematic view of plasmid pFAJ95-2.

10 Figure 37 is a schematic view of plasmid pFAJ95-3.

Figure 38 Is a schematic view of a EcoRI-Hindlil fragment of plasmid pFAJ95-2.

Figure 39 is a schematic view of a EcoRI-Hindlil fragment of plasmid pFAJ95-3.

Figure 40 is a schematic view of plasmid pFAJ96-2.

Figure 41 is a schematic view of plasmid pFAJ96-3.

15 In this specification, reference is also made to the attached sequence listings.

SEQ ID NO.:1 is the nucleotide sequence of the enzyme cellobiohydrolasel (CBHI) of Tnchoderma reesei.

SEQ ID NO:.2 is the amino acid sequence encoded by the coding sequence of SEQ ID NO:1.

20 SEQ ID NO.:3 is the nucleotide sequence of the flagellin protein of the lateral flagella (laf 1) of Azospirillum brasilense.

SEQID NO.: 4 is the amino acid sequence encoded by the coding sequence of SEQ ID NO.: 3.

SEQ ID NO.: 5 is the nucleotide sequence of the major outer membrane protein (OmaA) of Azospirillum brasilense.

5 SEQID NO.: 6 is the amino acid sequence encoded by the coding sequence of SEQ ID NO.: 5

SEQ ID NO.:7is the nucleotide sequence of the major outer membrane protein (OprF) of Pseudomonas fluorescens.

SEQ ID NO.: 8 is the amino acid sequence encoded by the coding sequence of SEQ 10 ID NO.: 7.

SEQ ID NO.: 9 is the nucleotide sequence encoding the CBD of CBHI of Trichoderrna reesei flanked at the 3' end by an Fsel site and the sequence GATC.

SEQIDNO.:10 is the complementary oligonucleotide of SEQID NO.: 9, flanked by the sequence AGCT.

15 SEQ ID.NO.:11is the nucleotide sequence encoding the major outer membrane protein (OmaA) - cellulose binding domain fusion protein, which nucleotide sequence forms part of plasmid pFAJ98-1, shown in Figure 17.

SEQIDNO.:12is the amino acid sequence encoded by the coding sequence of SEQ ID NO.:11.

20 SEQ ID NO.:13is the nucleotide sequence encoding the major outer membrane protein (OmaA) - cellulose binding domain fusion protein, which nucleotide sequence forms part of plasmid pFAJ98-2 shown in Figure 18.

SEQ IDNO.:14is the amino acid sequence encoded by the coding sequence of SEQ ID NO.:13.

( SEQ ID NO.:15 is the nucleotide sequence encoding the major outer membrane protein (OmaA) - cellulose binding domain fusion protein, which nucleotide sequence forms part of plasmid pFAJ98-3, shown in Figure 19.

SEQ ID NO.:16 is the amino acid sequence encoded by the coding sequence of SEQ 5 ID NO.: 15.

SEQ ID NO: 17 is the nucleotide sequence encoding the flagellin protein (laf1) -

cellulose binding domain fusion protein, which nucleotide sequence forms part of plasmid pFAJ94-1, shown in Figure 29.

SEQ ID NO.:18 is the amino acid sequence encoded by the coding sequence of SEQ 10 ID NO.: 17.

SEQ ID NO.: 19 is the nucleotide sequence encoding the flagellin protein (laf1) -

cellulose binding domain fusion protein, which nucleotide sequence forms part of plasmid pFAJ94-2, shown in Figure 30.

SEQ ID NO.: 20 is the amino acid sequence encoded by the coding sequence of 1 5 SEQ ID NO.: 1 9.

SEQ ID NO.: 21 is the nucleotide sequence encoding the major outer membrane protein (OprF) -cellulose binding domain fusion protein, which nucleotide sequence forms part of plasmid pFAJ96-2, shown in Figure 40.

SEQ ID NO.: 22 is the amino acid sequence encoded by the coding sequence of 20 SEQ ID NO.: 21.

SEQ ID NO.: 23 is the nucleotide sequence encoding the major outer membrane protein (OprF) -cellulose binding domain fusion protein, which nucleotide sequence forms part of plasmid pFAJ96-3, shown in Figure 41.

SEQ ID NO.: 24 is the amino acid sequence encoded by the coding sequence of 25 SEQ ID NO.: 23.

The invention will now be described, by way of example, as a series of embodiments.

( ! The present invention relates, in general, to a fusion protein comprising a cellulose binding domain peptide. The fusion protein, when exposed on the surface of a cell, causes the cell to have improved adherence to cellulose structures such as plant roots. 5 A cellulose binding domain peptide is one which is capable of binding tightly to a cellulose matrix. There are many different types of cellulose binding domain peptides, divided into eleven families (see Tomme, P. et a/., ibid. , each of the cellulose binding domains disclosed in which are hereby incorporated into the present specification by reference). Cellulose binding domain structures in newly

10 discovered hydrolases can be recognized by comparison of their primary structures with those of enzymes whose structural and functional organization has been defined. In a preferred embodiment the cellulose binding domain peptide is of Family lo and thus comprises the consensus sequence of this family, i.e. 1 5 BXQCGGX--XB-X)(XX-CXXX-XCXXXXX--XXXQC

wherein RIB" is an amino acid having an aromatic group; "X" is any amino acid and "-a is any or no amino acid. In this regard, an amino acid having an aromatic group is tyrosine, phenylalanine or tryptophan. A particularly preferred cellulose binding peptide is that from the enzyme cellobiohydrolasel of Trichodenna reesei which has 20 the sequence TQSHYGQCGGIGYSGPTVCASGTTCQVLNPWSQCL.

The complete sequence of the enzyme is shown in SEQ ID NO.: 2.

However, it is to be appreciated that, in other embodiments of the invention, the cellulose binding domain has a variety of different sequences and is from different 25 families, the important feature of the cellulose binding domain peptide being that it binds cellulose. In some embodiments of the invention, a cellulose binding domain is defined, more particularly, as a peptide having a partition coefficient of at least 1. 0g' at 22 C measured in accordance with the protocol used in Linder et a/. (The cellulose-binding domain of the major cellobiohydrolase of Trichodenna reesei 30 exhibits true reversibility and a high exchange rate on crystalline cellulose, Proc. Nail.

( ( Acad. Sci. USA, Vol. 93, pp.12251-12255, October 1996). In preferred embodiments, a cellulose binding domain peptide has a partition coefficient of at least 1.59-', and more preferably 1.99 i, at 22 C.

The fusion protein also comprises a cell surface polypeptide. These are externally 5 located proteins which are normally present on the cell surface such as flagellin proteins (such as that encoded by the laf1 gene of A. brasilense which is shown in SEQ ID NO.: 3), pill proteins and outer membrane proteins (such as that encoded by the omaA gene of A. brasilense, SEQ ID NO.: 5 and by the oprF gene of P. fluorescens, SEQ ID NO.: 7). There are many mechanisms by which cell surface 10 polypeptides are exposed on the cell surface as is known to those skilled in the art.

It is important that the cellulose binding domain peptide be exposed on the surface of the cell. In this regard, the word "exposed" means that the peptide is located externally on the ceil surface so that it is able to interact with other molecules outside the cell.

15 In order to locate the cellulose binding domain peptide so that it is exposed on the cell surface there are several alternative procedures. In a first procedure, the hydrophobicity of the other portions of the fusion protein is analysed and the cellulose binding domain peptide is located within portions of the fusion protein that are hydrophilic and are therefore predicted to be on the exterior of the protein.

20 Hydrophobicity analyses of proteins are done using the Kyte-Doolittle method (Kyte J and Doolittle RF A Simple Method for Displaying the Hydropathic Character of a Protein, Journal of Molecular Biology 157(6): 105-142, 1982). This can be accessed at http://bioinforrnatics.weizmann. ac.il/hydroph/hydroph help.html. Furthermore, different computer programs such as TopPred2 (Membrane Protein Structure 25 Prediction, Hydrophobicity Analysis and the Positive-inside Rule", Gunnar von Heijne, J. Mol. Biol. (1992) 225, 48794); TMPred (K. Hofmann & W. Stoffel (1993), TMbase - A database of membrane spanning proteins segments, Biol. Chem. Hoppe-Seyler 374,166); and TMHMM (A. Krogh et a/., Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes, 30 Joumal of Molecular Biology, 305(3):567-580, January 2001 and E. L.L.

Sonnhammer et a/., A hidden Markov model for predicting transmembrane helices in

( protein sequences, in J. Glasgow et al., editors, Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-

182, Menlo Park, CA, 1998. AAAI Press.) can be used to predict the topology of membrane proteins. These programs can be accessed via the website 5 http:l/us.expasy.org/tools/#transmem. It is to be understood that it is preferable to use a plurality of different programs in order to obtain a more reliable prediction of hydrophobicity. Alternatively, the homology of the other portions of the fusion protein with known 10 proteins is used to predict the topology of the fusion protein and thus those regions which are likely to be on the protein exterior. The cellulose binding domain peptide is then located in those regions.

In a second procedure, which can be used in conjunction with the first procedure, the cellulose binding domain peptide is located within the fusion protein (either randomly 15 or as explained in the first procedure) and improvement in adhesion of cells expressing the fusion protein is tested such as by using the assays explained in Examples 4 and 5. If the cells have increased adhesion then it is an indication that the cellulose binding domain peptide is located in the fusion protein so that it is exposed on the surface of the cell. If the cells do not have increased adhesion then 20 the procedure is repeated with the cellulose binding domain peptide in a different location in the fusion protein.

In a third procedure (which can be used in conjunction with the first and second procedures) the cellulose binding domain peptide is located within the fusion protein (either randomly or at positions predicted to be suitable using the first procedure) and 25 then it is experimentally confirmed whether or not the cellulose binding domain peptide is exposed by its ability to interact with other molecules. This is confirmed, for example, by producing antibodies to the cellulose binding domain peptide and detecting their binding to the fusion protein exposed on a cell surface using an ELISA as is discussed in Example 7.

30 In further embodiments of the present invention, a nucleic acid is provided which encodes a fusion protein as discussed above. The nucleic acid is, in some

( ( embodiments, part of a vector, such as plasmid. The nucleic acid is useful for transforming cells so as to express the fusion protein.

Accordingly, another embodiment of the present invention is a cell which is transformed with a nucleic acid encoding a cellulose binding domain peptide. The cell 5 is transformed using any of the techniques that are known in the art. When the nucleic acid is expressed, the cellulose binding domain peptide is exposed on the surface of the cell. In some embodiments, the cell is transformed using an exogenous plasmid but in preferred embodiments the cell is transformed using an endogenous plasmid or by chromosomal integration. The resulting cell has increased 10 cellular adhesion as can be tested, for instance, using the assays discussed in Example 4.

In some embodiments, the cell is a fungal cell, in particular plantgrowth promoting fungus. However, in preferred embodiments of the invention, the cell is a bacterial cell, preferably a Plant-Growth Promoting Rhizobacterium. Plant Growth Promoting 15 Rhizobacteria or PGPR are a group defined in Davison J. 1988. Plant beneficial bacteria. Biotechnology 6: 282-286, and in Kloepper, J.W. Lifshitz, R. and Zablotowicz, R.M. 1989, Free-living bacterial inocula for enhancing crop productivity, Trends Biotechnology 7: 39-43. The group includes different bacterial species and strains belonging to genera such as Acetobacter, Azospirillum, Azotobacter, Bacillus, 20 Burkholderia, Herbaspirillum and Pseudomonas.

In these embodiments, the cells are used in agriculture to improve the growth and/or health of plants, prophylactically or otherwise. The cells are applied to the roots of a plant by coating plant seeds with the transformed cells or by introducing the transformed cells into the soil, or other medium, around the plant roots. The 25 presence of the exposed cellulose binding domain peptide allows the cells to adhere to the plant roots more strongly than a wild type, untransformed cell. Thus the presence of the exposed cellular binding domain peptide minimises the effect of uncontrollable factors in the colonising of plant roots by the cells. This causes improved plant growth because of the release of plantgrowth promoting hormones 30 and results in a reduced potential of disease causing organisms because of, inter alla, shielding of the plant roots thus improving plant health. Furthermore, while plant

( growth promoting bacteria are typically specific for a particular plant (for example Azospinilum brasilense is usually specific for the roots of major cereal crops, maize and rice) the transformed cells of the present invention are able to adhere to any type of plant root.

5 In some embodiments, the plants to which the transformed cells are applied are located in an open field, but in other embodiments the plants are located in a

controlled environment such as a greenhouse. In a controlled environment, there is the additional advantage in using the transformed cells in that there is no requirement for the use of anti-fungal fumigants in order to protect the plants since the 10 transformed bacteria are able to protect the plant roots from disease causing fungi.

As has been explained above, in some embodiments of the invention, the use of the transformed cells is in an agricultural environment. However, in other embodiments, the transformed cells are used in industrial applications. For example, in one embodiment, the transformed cells are cells capable of breaking down effluent. A 15 membrane is provided having cellulose on its surface and the transformed cells are attached to the membrane using their exposed cellulose binding domain peptides to give improved adherence. This provides a "live" membrane, with the cells on its surface, which is effective at filtering and breaking down an effluent flow.

In another embodiment, the transformed cells are part of a biofilm, the presence of 20 the cellulose binding domain peptide exposed on their surface providing an improved ability for the biofilm to stick to surfaces. It is to be appreciated that cellulose is present in many common products such as paper. Therefore, in another embodiment, the transformed cells are used to bind to products such as paper, cardboard etc. In a further embodiment, the transformed cells are used for 25 experimental purposes in order to hold the cells in place while investigations are carried out. For example, in one embodiment, the transformed cells are subjected to atomic bombardment analysis which is assisted by their being held in place on a solid surface.

Thus the transformed cells are useful not only for improving plant growth and plant 30 health, but for increasing cellular adhesion in general.

l ( Examples

The present invention is now further illustrated by way of the following examples Example 1

The DNA encoding the cellulose binding domain of cellobiohydrolasel (CBHI) in Trichoderma reesei was inserted into the gene for the major outer membrane protein 10 (omaA or momp gene) of Azospirillum brasilense to generate a construct encoding a fusion protein. The nucleotide sequence of CBHI is shown in SEQ ID NO.: 1, the cellulose binding domain consisting of amino acid residues 478 to 513 of SEQ ID NO.: 2. The sequence of the omaA gene is shown in SEQ ID NO.: 5. The preparation of the DNA constructs used will now be described in detail.

Preparation began with the plasmid pFAJ92 shown in Figure 1. The plasmid contains the A. brasilense omaA gene inserted as a EcoRI-Hindlil fragment in the pUC19 plasmid. By means of site specific mutagenesis, three unique Fsel sites were created at 3 distinct sites in the omaA gene. The resulting plasmids were termed 20 pFAJ92fse1, pFAJ92fse2 and pFAJ92fse3, fragments of which are shown in Figures 2, 3 and 4 respectively.

Separately, the nptil gene was amplified by means of PCR using a forward primer containing the restriction sites EcoRI, Fsel and Pacl, and a reverse primer containing 25 the restriction sites BamHI and Pacl. This PCR amplification product was inserted into plasmid pUC18 as an EcoRIBamHI fragment to produce plasmid pFAJ99, which is shown in Figure 5.

An oligonucleotide (AZ0129) (reproduced as SEQ ID NO.: 9) encoding the cellulose 30 binding domain (CBD) of CBHI of Trichoderma reesei, flanked at the 3' end by an Fsel site and the sequence GATC, which is the sticky end of BamHI, was provided.

The complementary oligonucleotide (AZ0131, reproduced as SEQ ID NO.: 10) flanked by AGCT, which is the sticky end of Hindlil, was also provided. The two

oligonucleotides (AZ0129 and AZ0131), were annealed into the constructshown in Figure 6. The construct was subsequently cloned into plasmid pFAJ99, resulting in pFAJ99b which is shown in Figure 7.

5 The sequence encoding CBD and NPTII was then cloned as an Fsel fragment into the Psel sites of pFAJ92fse1, pFAJ92fse2 and pFAJ92fse3 resulting in the recombinant plasmids pFAJ97-1 , pFAJ97-2 and pFAJ97-3 , respectively. Plasmid pFAJ97-1 is shown in Figure 8; pFAJ97-2 is shown in Figure 9; and pFAJ97-3 is shown in Figure 10.

The npUI gene was removed from pFAJ97-1 , pFAJ97-2 and pFAJ97-3 by digestion with the Pacl enzyme and the remaining constructs were closed again by religation resulting in the plasmids pFAJ97-1, pFAJ97-2 and pFAJ97-3, respectively. Plasrnid pFAJ97-1 is shown in Figure 11, plasmid pFAJ97-2 is shown in Figure 12 and 15 plasmid pFAJ97-3 is shown in Figure 13.

The omaA-CBD fusion protein encoding sequences of pFAJ97-1 and pFAJ97-2 were cut out as EcoRI-Hindlil fragments shown in Figures 14 and 15 respectively. The fragments were then inserted into plasmid pLAFR3 (not shown) to give plasmids 20 pFAJ98-1 (Figure 17) and pFAJ98-2 (Figure 18) respectively. The omaA-CBD fusion protein encoding sequences are shown in SEQ ID NO.: 11 and 13, respectively. In SEQ ID NO.: 11, the CBD is present as nucleotides 157 to 264. In SEQ ID NO.: 13, the CBD is present as nucleotides 757 to 864.

25 The omaA-CBD fusion protein encoding sequence of pFAJ97-3 was also cut out as a EcoRI-Hindlil fragment which is shown in Figure 16. This fragment was inserted into plasmid pFAJ1700 (not shown) to produce plasmid pFAJ98-3 as shown in Figure 19.

The omaA-CBD fusion protein encoding sequence is shown as SEQ ID NO.: 15. In SEQ ID NO.: 15, the CBD is present as nucleotides 901 to 1008.

( ! Example 2

The DNA encoding the cellulose binding domain of cellobiohydrolasel (CBHI) in Trichoderma reesei was inserted into the gene for the flagellin protein of the lateral flagella (laf1 gene) of Azospirillum brasilense to generate a construct encoding a fusion protein. The nucleotide sequence of CBHI is shown in SEQ ID NO.: 1, the cellulose binding domain consisting of amino acid residues 478 to 513 of SEQ ID NO.: 2. The nucleotide sequence of the laf1 gene is shown in SEQ ID NO.: 3. The preparation of the DNA constructs used will now be described In detail.

Preparation began with the pUCBM20 plasmid in which was inserted the laf1 gene inserted as a Ncol-Sphl fragment to form plasmid pFAJ90, which is shown in Figure 20. By means of site specific mutagenesis, two unique Fsel sites were created at two distinct sites in the laf1 gene. The resulting plasmids were termed pFAJ9Ofse1 and 15 pFAJ9Ofse2 and Hindlil- EcoRI fragments thereof are shown in Figures 21 and 22 respectively. Plasmid pFAJ99b (see Figure 7) was then produced as described in Example 1.

20 The sequence encoding CBD and NPTII was then cloned as an Fsel fragment into the Fsel sites of plasmids pFAJ9Ofse1 and pFAJ9Ofse2 resulting in the recombinant plasrnids pFAJ93-1 , pFAJ93-2 , respectively. Plasmid pFAd93-1 is shown in Figure 23 and plasmid pFAJ93-2 is shown in Figure 24.

25 The nptl/ gene was removed from plasmids pFAJ93-1 and pFAJ93-2 by digestion with the restriction enzyme Pact and the remaining constructs were closed again by religation, resulting in the plasmids pFAJ93-1 (shown in Figure 25) and pFAJ93-2 (shown in Figure 26), respectively.

30 The laf1-CBD fusion protein encoding sequences of plasmids pFAJ93-1 and pFAJ93 2 were then cut out as BamHI-Hindlil fragments shown in Figures 27 and 28, respectively. The fragments were inserted in plasmid pLAFR3 (not shown) to produce plasmids pFAJ94-1 (shown in Figure 29) and pFAJ94-2 (shown in Figure

30) respectively. The laf1-CBD fusion protein encoding sequences are shown in SEQ ID NO.: 17 and 19, respectively. In SEQ ID NO.: 17, the CBD is present as nucleotides 679 to 786. In SEQ ID NO.: 19, the CBD is present as nucleotides 892 to 999. Example 3

The DNA encoding the cellulose binding domain of cellobiohydrolasel (CBHI) in 10 Tnchoderma reesei was inserted into the gene for the major outer membrane protein (OprF gene) of P. fluorescent to generate a construct encoding a fusion protein. The nucleotide sequence of CBHI is shown in SEQ ID NO.: 1, the cellulose binding domain consisting of amino acid residues 478 to 513 of SEQ ID NO.: 2. The nucleotide sequence of the OprF gene is shown in SEQ ID NO.: 7. The preparation 15 of the DNA constructs used will now be described in detail.

Preparation began with plasmid pUC18 (not shown) in which was inserted a BamHI Hindlil fragment containing the P. fluorescent oprF gene to form plasmid pFAJ91 which is shown in Figure 31. By means of site specific mutagenesis, two unique Fsel 20 sites were created at two distinct sites in the oprF gene. The resulting plasmids were termed pFAJ91fse2 and pFAJ91fse3 and Hindlil-EcoRI fragments of the plasmids are shown in Figures 32 and 33, respectively.

Plasmid pFAJ99b (see Figure 7) was then produced as described in Example 1.

The sequence encoding CBD and NPTII was then cloned as an Fsel fragment into the Fsel sites of plasmids pFAJ91fse2 and pFAJ91fse3 resulting in the recombinant plasmids pFAJ95-2 (shown in Figure 34) and pFAJ95-3 (shown in Figure 35), respectively. The npUI gene was removed from plasmids pFAJ95-2 and pFAJ95-3 by digestion with restriction enzyme Pacl and the remaining constructs were closed again by

( religation, resulting in the plasmids pFAJ95-2 (shown in Figure 36) and pFAJ95-3 (shown in Figure 37), respectively.

The oprF-CBD fusion protein encoding sequences were cut out of plamids pFAJ95-2 5 and pFAJ95-3 as EcoRI-Hindlil fragments shown in Figures 38 and 39, respectively.

The fragments were inserted into plasmid pFAJ1700 (not shown) to form plasmids pFAJ96-2 (shown in Figure 40) and pFAJ96-3 (shown in Figure 41), respectively.

The oprF-CBD fusion protein encoding sequences are shown in SEQ ID NO.: 21 and 23, respectively. In SEQ ID NO.: 21, the CBD is present as nucleotides 274 to 381. In 1 0 SEQ ID NO.: 23, the CBD is present as nucleotides 622 to 729.

Example 4

15 In order to determine the effect on root adhesion of bacteria transformed with the constructs of Examples 1 to 3, the following wheat root adhesion assay was used.

One week before carrying out an adsorption assay, wheat seeds were sterilised as follows. The seeds were immersed in 70% ethanol for 3 minutes after which they 20 were washed twice in sterile water. The seeds were then incubated for 30 minutes in a NaOCI/SDS solution Comprising 50ml sterile water, 10ml commercial bleach and 0.6ml of 10% SDS. The solution was refreshed and the seeds incubated again for 30 minutes following which the solution was refreshed again and the seeds incubated for a further 30 minutes. The seeds were then washed twice with sterlie water and 25 afterwards suspended in sterile water and incubated for one hour.

The seeds were then transferred to petri dishes containing sterile filter paper that had previously been wetted with 3 ml of sterile water. The petri dishes were incubated in a plant incubation room at 26 C and were kept dark by wrapping the petri dishes in 30 aluminium foil.

Two days before the assay, A. brasilense strains were inoculated in 5ml nutrient broth (8g/l) and incubated at 30 C for 16 hours. One strain was the wild-type strain.

( ( Three strains were transformed, using standard procedures, with the plasmids pFAJ98-1, pFAJ98-2 and pFAJ98-3. Another strain was Sp7 p90D084, carrying a deletion in the endogenous p90 megaplasmid, which was shown previously to be a knock-out mutant for root attachment genes. This strain thus acted as a negative 5 control.

One day before the assay, 1ml of the preculture was transferred to 100ml nutrient broth and incubated for 16 hours.

10 The adsorption assay, itself, was carried out as follows. 5ml of each culture was centrifuged for 10 minutes at 4000 RPM and at TIC. The resultant pellets were resuspended in 0.05M PBS pH7 (5315 9/l K2HPO4; 2 65 g/l KHzPO4; 8 6 9/l NaCI) to an OD54o Of exactly 0.1. The bacterial suspensions were diluted 10 times in sterile PBS (OD540 = 0.01). Serial dilutions of the suspensions were made to count the 1 5 bacteria.

0.5 g of wheat roots were transferred to a Falcon tube, to which were added 9.8 ml PBS and 0.2 ml of the final bacterial suspensions. Four tubes were prepared for each strain (wild type or recombinant) to be tested. The Falcon tubes were attached to a 20 'hybridization tube' and incubated in a 'hybridization oven' at 25 C for 1 hour and were rotated very gently.

The roots were then transferred to a fresh Falcon tube containing 15ml PBS and were washed very gently by inverting the tubes upside down 10 times. This step was 25 repeated three times. Subsequently, the roots were transferred to a fresh Falcon tube containing 5ml PBS and were vortexed very heavily for 5 minutes to release the bacteria. Serial dilutions of the resultant solution were made and plated out to count the bacteria.

30 The adsorption assay was carried out five times, under the same conditions, although the strains used were varied as shown in Tables 1 to 4 (see the table headings).

( The results are shown in the following tables in which, except for Table 2, the adsorption capacity of the strains is expressed as a percentage of the initial inoculum attached, i.e.: 5 The number of bacteria released from the roots x 100 The number of bacteria added to the roots Table 2 shows the results calculated as a proportion of the adsorption of the control strain (Sp7) in each experiment (control strain adsorption = 1).

Table 1: Results of adsorption assay with omaA-CBD recombinants Expt. No. Sp7 Sp7(pFAJ98-1) I Sp7(pFAJ98-2) Sp7(pFAJ98-3) Sp7 p90D084 10.65 %, i 0. 00025 % 22.03 /O 5.30 % 0.05 %

34.22 % 31.94 % 11.77 % 2.08 % 1 0.01 %

4j 0.99 % 7.22 % 3.68 % 4.00 %, 51.32 % 8.75 % 8.80 %

"-" indicates that the experiment was not carried out.

Table 2: Results of adsorption assay with omaA-CBD recombinants Expt. No. 1 Sp7 Sp7(pFA.198-1) Sp7(pFAJ98-2) Sp7(pFAJ98-3) Sp7 p90D084 l 1 j 1 0. 000385 2 2.61 0.0246

3 1 7.57 2.79 0.493 0.00237

4 _ 7.29 3.72 4.04 3

5 1 1 6.63 6.67

"-" indicates that the experiment was not carried out.

Subsequently, assays were carried out using /af1-CBD recombinants. The protocol was the same as that described above except that the A. brasilense strains were transformed with plasmids pFAJ94-1 and pFAJ94-2. Furthermore, as a comparison, the experiments were also conducted using the strain FAJ0201, untransformed and

( transformed with pFAJ94-1 and pFAJ94-2. FAJ0201 is an A. brasilense Sp7 laf1 knock-out mutant. This mutant does not have lateral flagella.

In the assays using taf1-CBD recombinants (shown in Table 3), the bacterial strains 5 were grown beforehand on solid media to induce the expression of the laf1 gene.

Table 3: Results of adsorption assay with laf1-CBD recombinants Expt. No. Sp7 Sp7 (pF=94 1) Sp7F0201 F0201 F0201 (pFAJ94-2)_ (pFAJ94.1) (pF94-2) 1 0. 18% 0.20% 0.24%

2 0. 56% 1.06% 4.36% 2.74%

3 0.48% 0.46% 1. 15%

10 "-" indicates that the experiment was not carried out.

The table shows that modification of the flagella proteins with CBD generally results in increased adsorption.

15 Assays were also carried out using OprF-CBD recombinants and the results are shown in Table 4. The experimental protocol was the same as for the laf1-CBD recombinants except that the A.brasilense strains were transformed with plasmids pFAJ96-2 and pFAJ96-3. As a comparison, experiments were also carried out using a wild-type P.fluorescens strain, OE28.3.

Al f} ( Table 4:Results of adsorption assay with OprF-CBD recombinants _ Expt. No. Sp7 Sp7 Sp7 Ps. Sp7 (pee A") l (pFAJ96-2) (pFAJ96-3) Fluorescens OE28.3

1 10.22% 21.52% 0. 15%

l l 0.5% 0.72% 0 98% 1.09% r_' indicates that the experiment was not carried out.

The table shows that modification of the OprF protein with CBD generally results in increased adsorption Thus these experiments show that the Sp7(pFAJ98-1) strain consistently had 10 between 6 and 8 times the adsoption to wheat roots as the wild-type strain. The Sp7(pFAJ98-2) strain also showed 3 to 4 times the adsorption to wheat roots compared with the wild-type strain. The Sp7(pFAJ98-3) strain showed somewhat inconsistent performance in comparison to the wild-type strain. The results for the Sp7 p90-684 negative control strain confirmed that, without functional root 15 attachment genes, the level of adsorption of the bacteria to the wheat roots was very low indeed.

The experiments also show that bacteria transformed with the laf1-CBD constructs and OprF-CBD constructs had increased adsorption compared with wild-type 20 bacteria although the increase was less than for transformation with the omaA-CBD constructs. ExamoIe 5 In order to assess the practicality of adding bacteria transformed to express a 25 cellulose binding domain on their cell surface to soils containing wild-type bacteria, a competition experiment was carried out as follows.

( The protocol for the assay was similar to that of Example 4 except that, in the step in which 0.2 ml of the final bacterial suspensions were added to 0.5 9 of wheat roots, a mixture of 1001 of cell culture Sp7 and 100,u1 of cell culture Sp7(pFAJ98-1) was added. As in Example 4, the quantity of bacteria was measured both before exposure 5 to the wheat roots (i.e. the start inoculum) and after attachment to the wheat roots.

The results are shown in Table 5.

Table 5: Results of competition adsorption assay with omaA-CBD recombinants.

Strain 1 Proportion in Start Inoculum Proportion after Attachment _ Sp7 88.5% 78.7% _ _ Sp7(pFAJ98-1) 11.5% 21.3% Accordingly these experiments confirm that the addition of transformed bacteria to 10 plant roots in the presence of wild-type bacteria results in an increased proportion of the transformed bacteria being present after attachment to the plant roots than beforehand. Therefore, these experiments indicate that transformed bacteria could be added to plant roots in sffu and have increased adherence to the plant roots even in the presence of wild-type bacteria.

Example 6

In order to confirm the presence of the COD peptides in the proteins in which they were present, a Western Blot analysis was carried out.

Western analyses on total protein extracts of the Azospirillum wild type strain Sp7 20 and omaA recombinants (containing the fusion protein of Example 1) were performed. This experiment was carried out using a standard procedure described in Harlow, E. and Lane, D., 1988 'Antibodies: a laboratory manual', Cold Spring Harbor Laboratory, pp. 471- 510. Different amounts of the primary antibody against the COD peptide were used (i.e. dilutions: 1/100, 1/1000 and 1/10 000). Only dilution 1/10 000

( ( gave no signal in the Sp7 extract and a positive signal for a 40 kDa protein in the extracts of the recombinants.

Thus the Western Blot analysis confirms that the CBD peptides are present within the recombinant omaA gene.

Example 7

In order to ascertain whether the CBD peptides are located on the cell surface of the transformed cells, an ELISA analysis was conducted.

Intact cells of Sp7 and recombinants (containing the fusion protein of Example 1) 10 were incubated with the primary antibody against CBD, washed several times and subsequently incubated with the second antibody and evaluated for alkaline fosfatase activity. Alkaline fosfatase activity could be detected for the three omaA recombinants. No activity was observed for the wild type cells. In contrast to the Westem analyses of Example 6, in which the presence of CBD in the OMAN protein 15 is demonstrated, this Elisa experiment additionally proves the presence of the CBD on the cellular surface of the recombinants.

Accordingly, these experimental results show that the CBD peptide is present on the cell surface of the transfomned cells.

2b ( SEQUENCE LISTING

<110> Norsk Hydro ASA c120, A Fusion Protein c130> SJW/WA/53137 <160> 24

c170> Patentln version 3.1 <210> 1

c211> 3297 c212> DNA <213> Trichoderma reesei c220> c221> CDS c222> (532). .(992) c223> c2ZO> c221> CDS c222> (1061)..(1757) c223> c220> c221> CDS c222> (1821)..(2201) c223> c400> 1 gaeattcaag gagtatetag ccagggatgc ttgagtgtat cgtgtaagga gghttgtotg 60 3 ccgatacgac gaetactgta tagtcacttc tgatgeagtg gtccatattg aaatgtaagt 120 a cggoaetgaa caggcaaaeg attgagttga aactgcctaa gatctcgggc catagggcct 180 tcggcctttg ggtgtacatg tttgtgctcc gggoaaaLgc aaagtgtggt aggatcgaac 240 aeachgctgc ckttaccaag eagctgaggg taLgtgatag gcaaatgttc aggggccact 300 goatOghttc gaatagaaag agsagcttag ccaagaacaa tagccgataa agatagccte 360 attaaacgga atgagatagt aggcaaagtc agcgaetgtg tatatataaa ggetegaggt 420 ccgtgcctcc etcatgctat ccccatctac tcatcaactc agatcctcca ggagaettgt 480 aeaccatctt ttgaggcaca gaaacceaat agteaacogc ggaetggcat c atg tat 537 Met Tyr cgg aag ttg gcc gtc ate leg gcc ttc ttg gcc ace gct cgt gct cag 585 Arg Lys Leu Ala Val Ile Ser Ala Phe Leu Ala Thr Ala Arg Ala Gln 5 10 15

leg gee tgc act ctc caa leg gag act cac ccg cct ctg ace tgg cag 633 Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr Trp Gln 20 25 30

( ( ai tgc tcg tct ggt ggc acg tgc act caa cag aca ggc tcc gtg gtc 681 Lys Cys Ser Ser Gly Gly Thr Cy6 Thr Gln Gln Thr Gly Ser Val Val 35 40 45 50

atc gac gcc aac tgg cgc tgg act cac gct acg aac agc agc acg aac 729 Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr Asn 55 60 65

tgc tac gat gOc aac act tgg agc tcg acc cta tgt cat gac aac gag 777 Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn Glu 70 75 80

acc tgc gcg aag aac tgc tgt ctg gac ggt gcc gac tac gcg tcc acg 825 Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser Thr 85 90 95

tac gga gtt acc acg agc ggt aac agc ctc tcc att ggc ttt gtc acc 873 Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe Val Thr 100 105 110

cag tct gag cag aag aac gtt ggc gct cgc ctt tac ctt atg gcg apc 921 Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met Ala Ser 115 120 125 130

gac acg acc tac cag gaa ttc acc ctg cUt ggc aac gag ttc tct ttc 969 Asp Thr Thr Tyr Gln Glu Phe Thr Leu heu Gly Asn Glu Phe Ser Phe 135 140 145

gat ght gat gtt tcg cag ctg cc gtaagtgact taccatgaac ccctgacgat 1022 Asp Val Asp Val Ser Gln Leu Pro atothcttgt tggatcccag ctgactggcc aattcaag 9 tgc ggc ttg aac gga 1076 Cys Gly Leu Asn Gly gct ctc tac ttc gtg tcc atg gac gcg gat ggt ggc gtg agc aag tat 1124 Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr 160 165 170 175

ccc acc aac acc gct ggc gcc aag tac ggc acg gOg tac tOt gac agc 1172 Pro Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser 180 185 190

cag tgt ccc cgc gat ctg aag ttc atc ast ggc cag gcc aac gtt gag 1220 Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu 195 200 205

ggc tgg gag cag tca tcc aac aac gcg aac acg ggc att gga gga cac 1268 Gly Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His 210 215 220

gga agc tgc tgc tct gag atg gat atc tOg gag gcc aac tcc atc tcc 1316 Gly Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser 225 230 235

gag gct ctt acc ccc cac cct tgc acg act gtc ggc cag gag atc tgc 1364 Glu Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys 240 245 250 255

gag ggt gat ggg tgc ggc gga act tac tcc gat aac aga tat gOc gOc 1412

2s ( ( G1t ly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly 260 265 270

act tgc gat ccc gat ggc tgc gac tgg aac cca tac cgc ctg ggc aac 1460 Thr Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn.

275 280 2B5:

acc agc ttc tac ggc cct ggc tca agc ttt acc ctc gat acc acc aag 1508 Thr Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys 290 295 300

aaa ttg acc gtt gtc acc cag ttc gag acg tcg ggt gcc atc aac cga 1556 Lys Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg; 305 310 315

tac tat gtc cag aat ggc gtc act ttc cag cag ccc aac gcc gag ckt 1604 Tyr Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu 320 325 330 335

ggt agt tac tct ggc aac gag ctc aac gat gat tac tgc aca gat gag 1652 Gly Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu 340 345 350

gag gca gaa tCc ggc gga tcc tat ttc tca gac aDg ggc ggc ctg act 1700 Glu Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr 355 360 365

cag thc aag aag gct acc tot ggc ggc atg gtt ctg gtc atg agt ctg 1748 Gln Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu 370 375 380

tOg gat gat gtgagtttga tggacaaaca tgcgcgttga caaagagtca 1797 Trp Asp Asp aBcagctgac tgagatgtta cag tac tac gcc aac atg ctg tgg ctg gac tcc 1850 Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser 390 395

acc tac ccg aca aac gag acc tcc tcc aca ccc ggt gcc gtg cgc gga 1898 Thr Tyr Pro Thr Asn Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly 400 405 410

agc tgc tcc acc agc tcc ggt gEc cct gct cag gtc gsa tct cag tct 1946 Ser Cys Ser Thr Ser Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser 415 420 425

ccc aac gcc aag gtc acc ttc tcc aac atc aag ttc gga ccc att ggc 1994 Pro Asn Ala Lys Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly -

430 435 440

* agc acc gOc aac cct agc ggc ggc aac cct coc ggc gga aac cgt ggc 2042 Ser Thr Gly Asn Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Arg Gly 445 450 455 460

acc acc acc acc cOc cDc cca gcc act acc act gga agc tct ccc gga 2090 Thr Thr Thr Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly 465 470 475

cct acc cag tat cac tac ggc cag tgc gOc ggt att ggc tac agc ggc 2138 Pro Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly

q 480 485 490

ccc acg gEc tgc gcc agc ggc aca act tgc cag gec ctg aac cct tac 2186 Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr 495 500 505

tac tct cag tgc ctg taaagctccg tgcgaaagcc tgacgoacog gtagattctt 2241 Tyr Ser Gln Cys Leu ggtgagcccg tatcatgacg gcggcgggag ctacatggcc ccgggtgatt tatttttttt 2301 gtatctactt ctgacccttt tcaaatatac ggtcaactca tatttcactg gagatgcggc 2361 ctgchtOgLa ttgcgatgtt gEcagattgg caaattOtOg ctetcOaaaa cacaaaacOa 2421 ttccttagLa gccatgcatt ttaagataac ggaatagaeg aaagaggaaa ttaaaaaaaa 2481 aaaaaaaaca aacatcccgt tcataacccg tagaatogcc gatcttcgtg tatcccagta 2541 ccacOgcaaa gOtattEcat gatcgttcaa tgttgatatt gthccagcca gLaLggctcc 2601 accacocatc tccgcgaatc tcctcttctc gaacgcgOtg tOgcgcgcca attggtaatg 2661 accccatagg gagacaaaca gcataatagc aacagtggaa attagtggag caateattga 2721 gaacacagtg agaccatagc tggcggcctg gaaagoactg ttggagacca acttgtccgt 2781 tgagaggcca acttgcattg ctgtcaagac gatgacaaeg tagocgagga cagtcacaag 2841 ggacgcaaag ttgtcgcgga tgaggtctcc gtagatggca tagccggcaa tccgagagta 2901 gcctotcaac aggtggoctt ttcgaaacag gtaaaccttg tCcagacgtc ctagccgcag 2961 ctcaccgtac cagtatcgag gattgacggc agaatagcag tggctctcca ggatttgact 3021 ggacaaaatc ttccagtatt cccaggtcac agtgtatggc agaagtcoct tctcgagtgc 3081 gagtcgaaag tcgctatagt gcgcaatgag agcacagLag gagaatagga acccgcgagc 3141 acattgttca atctccacat gaattggatg actgctgggc agaaLgugct gcctccaaaa 3201 tcctgcOtcc aacagatact ctggcagggg cttcagatga atgcatctgg gcccccagat 3261 aagatgcagc tctggattCt cggttacgat gatatc 3297 <210> 2

<211> 513

<212> PRT

<213> Trichoderma reesei <400> 2

Met Tyr Arg Lys Leu Ala Val Ile Ser Ala Phe Leu Ala Thr Ala Arg 1 5 10 15

Ala Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr 20 25 30

f ( ( Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser Val Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala Ser Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe hr 1Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Le M t Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 185 190

Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser 11e Ser Glu Ala Leu Thr Pro Hls Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr

( ( Cy( sp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr 275 2B0 285

Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys 290 295 300

Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr 305 310 315 320

Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly 325 330 335

Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu 340 345 350

Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln 355 360 365

Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp 370 375 380

Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr 385 390 395 400

Asn Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr 405 410 415

Ser Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys 420 425 430

Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn 435 440 445

Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Arg Gly Thr Thr Thr Thr 450 455 460

Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser 465 470 475 480

His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys 485 490 495

Ala Ser Gly Thr Thr Cy6 Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys 500 505 510

Leu

c210> 3 <211> 1573

<212> DNA

c213> Azospirillum brasilense <220> <221> CDS

<222> (214)..(1458)

<223> <400> 3

atttttataa aatgcgcgat tactUttaaa aaccgcacgg ctttaaaaga tactgagtca60 ccctggaaag gctgcggttt tettactccg gtttaaacOg gOtcggtcac agtcgaatgg120 tcggttgcgc gcccataaaa accaagtggg aaggcccgcg cacccggccc cagcgtcaga180 aaggagctat goatatagcg aatggagata cac atg gcc tcg atc atg acc aac234 Met Ala Ser Ile Met Thr Asn 1 5 acc tcg gcg atg acc gcg ckg cag acc gtg cgc cgc gtg acc gac gat282 Thr Ser Ala Met Thr Ala Leu Gln Thr Val Arg Arg Val Thr Asp Asp 10 15 20

ctg gcg acc acc cag gat cgc atc tag acc ggt ctc aag gtc aac aac330 Leu Ala Thr Thr Gln Asp Arg Ile Ser Thr Gly Leu Lys Val Asn Asn 25 30 35

gcc aag gac aac gcc gcc tac tgg tcg atc gcc acg acc aLg cOc gcc378 Ala Lys Asp Asn Ala Ala Tyr Trp Ser Ile Ala Thr Thr Met Arg Ala 40 45 50 55

gac gtc gcc ggc ttc aag gcg gtg aag gaa tcg ctg gaa ctg ggt tcg426 Asp Val Ala Gly Phe Lys Ala Val Lys Glu Ser Leu Glu Leu Gly Ser 60 65 70

ggc acg acc aac acc gcg tcg gtg gag tcc aag aac atc gtc gag aac474 Gly Thr Thr Asn Thr Ala Ser Val Ala Ser Lys Asn Ile Val Glu Asn 75 80 85

ctg cag acg ctg aag gcc cgc gtc atc gcc ggc cag acc aac ggc gtc522 eu Gln Thr Leu Lys Ala Arg Val Ile Ala Gly Gln Thr Asn Gly Val 90 95 100

gac aag tcg ctg atc cag aac gac atc gac cag ctc gtc aag ctg gtg570 Asp Lys Ser Leu Ile Gln Asn Asp Ile Asp Gln Leu Val Lys Leu Val 105 110 115

aag ggc gcc gcc gcc gac gcc tcc ttc aac ggc gac aac ctg ctg cgc618 Lys Gly Ala Ala Ala Asp Ala Ser Phe Asn Gly Asp Asn Leu Leu Arg 120 125 130 135

atc acc tac tcc aac gac gOc acg gcc aag gac cag aac gtc gac atc666 Ile Thr Tyr Ser Asn Asp Gly Thr Ala Lys Asp Gln Asn Val Asp Ile 140 145 150

ctg gcg tcg ctg aSc cOc agc gcc gOc acg gtc gat ccg agc tac atc714

( ( ( Le" Ala Ser Leu Ser Arg Ser Ala Gly Thr Val Asp Pro Ser Tyr Ile 155 160 165

agc ttc cag cOc cag gac at cag gtc acc tcg atc gtc gOc aag goc 762 Ser Phe Gln Arg Gln Asp Met Gln Val Thr Ser Ile Val Gly Lys Ala 170 175 180

acg atc gag cag cag gUg gac tcc acc aac gac ctg aag gcc agc gtc 810 Thr Ile Glu Cln Gln Val Asp Ser Thr Asn Asp Leu Lys Ala Ser Val 185 190 195

ggc atc gcc atc ggc gcg ccg gac acc acc ttc atc gac ggc cag aac 858 Gly Ile Ala Ile Gly Ala Pro Asp Thr Thr Phe Ile Asp Gly Gln Asn 200 205 210 215

ctg ggc ctg ggc aac ckg acc ctg aac gtc acc aac gag gag ggc acc 906 LeuGly Leu Gly Asn Leu Thr Leu Asn Val Thr Asn Glu Ala Gly Thr 220 225 230

aag acc gcc gtg aca gtg gac ctg tag ggc atc gac tac acc acc gac 954 Lys Thr Ala Val Thr Val Asp Leu Ser Gly Ile Asp Tyr Thr Thr Asp 235 240 245

ctg gcg acc acc cag ggc ctg atc gag acc gcg gtc aac gcg gcc ctg 1002 Leu Ala Thr Thr Gln Gly Leu Ile Glu Thr Ala Val Asn Ala Ala Leu 250 255 260

acc acg gcc gOc gOc gac ttc cag gtg gcc ttc aac ggc gac aag ctg lOSO Thr Thr Ala Gly Gly Asp Phe Gln Val Ala Phe Asn Gly Asp Lys Leu 265 270 275

gaa ttc acc gac cag gac ctg aac acc gac ggc aac ttc acc gcc aag 1098 Glu Phe Thr Asp Gln Asp Leu Asn Thr Asp Gly Asn Phe Thr Ala Lys 280 285 290 295

gtg gac agc ctg tgg gtc ggc tag aag gaa agc gac gac ttc ggc ggc 1146 Val Asp Ser Leu Trp Val Gly Ser Lys Glu Ser Asp Ala Phe Gly Gly 300 305 310

ctg gcc gac ctg acg cag atc gac gtg acc tcc aac gac acc aag tcg 1194 Leu Ala Asp Leu Thr Gln Ile Asp Val Thr Ser Asn Ala Thr Lys Ser 315 320 325

ctc gag gtc atc aac ggc ctg ctc gac aag gcc atc ggc aag gcc gcg 1242 Leu Glu Val Ile Asn Gly Leu Leu Asp Lys Ala Ile Gly Lys Ala Ala 330 335 340

gtg atc ggt tcc atc gag aac cgc gtg tcg gtg cag aac gac ttc gtg 1290 Val Ile Gly Ser Ile Glu Asn Arg Val Ser Val Gln Asn Asp Phe Val 345 350 355

tcg aag ctg acc gac tcg aLg aac aag gOc atc ggc tcc ctg gtg gac 1338 Ser Lys Leu Thr Asp Ser Met Asn Lys Gly Ile Gly Ser Leu Val Asp 360 365 370 375

goc gac aLg aac gag gaa tcg agc cgc ctg cag goc ctc cag gtc cag 138G Ala Asp Met Asn Glu Glu Ser Ser Arg Leu Gln Ala Leu Gln Val Gln 380 385 390

cag cag ctc gcc acc cag gcg ctg tcg atc gcc aac cag ggt ccg cag 1434 Gln Gln Leu Ala Thr Gln Ala Leu Ser Ile Ala Asn Gln Gly Pro Gln

( (' 395 400 405

Asn lt Lctc leg ctg tEc cgc tea cccggtccgg ccgg t 1488 gggagccggc ctctttttcc ttattogatc cagcoccagc cccattccgg cgccccgoag 1548 acgatggccg agtgcgaaga cccgc 1573 c210> 4 c211> 414 <212> PRT

c213> Azospirillum brasilense c400> 4 Met Ala Ser Ile Met Thr Asn Thr Ser Ala Met Thr Ala Leu Gln Thr 5 10 15

Val Arg Arg Val Thr Asp Asp Leu Ala Thr Thr Gln Asp Arg Ile Ser Thr Gly Leu Lys Val Asn Asn Ala Lys Asp Asn Ala Ala Tyr Trp Ser Ile Ala Thr Thr Met Arg Ala Asp Val Ala Gly Phe Lys Ala Val Lys Glu Ser Leu Glu Leu Gly Ser Gly Thr Thr Asn Thr Ala Ser Val Ala Ser Lys Asn Ile Val Glu Asn Leu Gln Thr Leu Lys Ala Arg Val Ile Ala Gly Gln Th 1OO n Gly Val Asp Lys Ser Leu {le Gln Asn A I1 ABP Gln Leu Val Lys Leu Val Lys Gly Ala Ala Ala Asp Ala Ser Phe Asn Gly Asp Asn Leu Leu Arg Ile Thr Tyr Ser Asn Asp Gly Thr Ala 145 P sn Val Asp Ile Leu Ala Ser Leu Ser Ar S Thr Val Asp Pro Ser Tyr Ile Ser Phe Gln Arg Gln Asp Met Gln Val

( ( ( Thr Ser Ile Val Gly Lys Ala Thr Ile Glu Gln Gln Val Asp Ser Thr 180 185 190

Asn Asp Leu Lys Ala Ser Val Gly Ile Ala Ile Gly Ala Pro Asp Thr 195 200 205

Thr Phe Ile Asp Gly Gln Asn Leu Gly Leu Gly Asn Leu Thr Leu sn 210 215 220

Val Thr Asn Glu Ala Gly Thr Lys Thr Ala Val Thr Val Asp Leu Ser 225 230 235 240

Gly Ile Asp Tyr Thr Thr Asp Leu Ala Thr Thr Gln Gly Leu Ile Glu 245 250 255

Thr Ala Val Asn Ala Ala Leu Thr Thr Ala Gly Gly Asp Phe Gln Val 260 265 270

Ala Phe Asn Gly Asp Lys Leu Glu Phe Thr Asp Gln Asp Leu Asn Thr 275 280 285

Asp Gly Asn Phe Thr Ala Lys Val Asp Ser Leu Trp Val Gly Ser Lys 290 295 300

Glu Ser Asp Ala Phe Gly Gly Leu Ala Asp Leu Thr Gln Ile Asp Val 305 310 315 320

Thr Ser Asn Ala Thr Lys Ser Leu Glu Val Ile Asn Gly Leu Leu Asp 325 330 335

Lys Ala Ile Gly Lys Ala Ala Val Ile Gly Ser Ile Glu Asn Arg Val 340 345 350

Ser Val Gln Asn Asp Phe Val Ser Lys Leu Thr Asp Ser Met Asn Lys 355 360 365

Gly Ile Gly Ser Leu Val Asp Ala Asp Met Asn Glu Glu Ser Ser Arg 370 375 380

Leu Gln Ala Leu Gln Val Gln Gln Gln Leu Ala Thr Gln Ala Leu Ser 385 390 395 400

Ile Ala Asn Gln Gly Pro Gln Asn Ile Leu Ser Leu Phe Arg 405 410

( \ <21, 5

c211> 1435 c212> DNA c213> Azospirillum brasilense c220> c221> CDS <222> (238)..(1410)

<223> c400> 5 cagcctgtgc ctttgchgtg tccggggogt cgccggagoc ctgcggacgg gacggogcgg 60 gcggcggaat ttgaacggag gtgcacaatc aaggtgtgtt ccgtcggaca cactgttcat 120 tcattgctgc aacttttcga tttccggctt gctggcgtcc ccgcatctgc gtaccacaga 180 gccagccagg acgttaaccg gOctgaaaat tctgctgcca ggatOgagOc aacaatc 237 atg aac cgt tat ctg ctc gcg ggc tgc gcc gct gcc gct ctg gct ctg 285 Met Asn Arg Tyr Leu Leu Ala Gly Cys Ala Ala Ala Ala Leu Ala Leu 1 5 10 15

ggt gcc ggc gcc gcc aac gct cag gcc aag ttt gaa gtc aag gtc ggc 333 Gly Ala Gly Ala Ala Asn Ala Gln Ala Lys Phe Glu Val Lys Val Gly 20 25 30

ggc gac gcc tac ttc gaa ggc ggc tac gEc aac gea gac cgc gac gag 381 Gly Asp Ala Tyr Phe Glu Gly Gly Tyr Val Asn Glu Asp Arg Asp Glu 35 40 45

ggt gcc cgt tcg gtt gac ttc cgc aac cgc ttc cgc gtc atc gtg acc 429 Gly Ala Arg Ser Val Asp Phe Arg Asn Arg Phe Arg Val Ile Val Thr 50 55 60

ccg acc gcc aag gcc gac aac ggc ctg gag tac ggc gOc cOc atc cgt 477 Pro Thr Ala Lys Ala Asp Asn Gly Leu Glu Tyr Gly Gly Arg Ile Arg 65 70 75 80

atg cgc gcc aac tcc ggc gcc ggc aac gac cgc acg atg gac gcc gac 525 Met Arg Ala Asn Ser Gly Ala Gly Asn Ala Arg Thr Met Asp Ala Asp 85 90 95

cgc ggc tac atc ttc gcg cag ggc gag tCc ggt cag gtc cag atg ggc 573 Arg Gly Tyr Ile Phe Ala Gln Gly Ala Phe Gly Gln Val Gln Met Gly 100 105 110

gtg acc aac tcc tCc aac gac gcc acc tac gtc acg gcc ccg cag gat 621 Val Thr Asn Ser Phe Asn Asp Ala Thr Tyr Val Thr Ala Pro Gln Asp 115 120 125

tac ctg ccg ctg gcc att tat gac ggc gtc acc gcc tgg atc ggc cag 669 Tyr Leu Pro Deu Ala Ile Tyr Asp Gly Val Thr Ala Trp Ile Gly Gln 130 135 140

acc tcc gac atc ggc ggc gOc ctc gcc gOt ctg aac ggc tcg atc chg 717 Thr Ser Asp Ile Gly Gly Gly Leu Ala Gly Leu Asn Gly Ser Ile Leu 145 150 155 160

aac cag tcg ctg gtc gtc gag aac aac gag aeg aag atc gtg tac ttc 765 Asn Gln Ser Leu Val Val Glu Asn Asn Ala Thr Lys Ile Val Tyr Phe

( 165 170 175

leg ccg cgc ttc gcc ggc ckg cag gtg gOc gcc aBc tac acc cog cgc 813 Ser Pro Arg Phe Ala Gly Leu Gln Val Gly Ala Ser Tyr Thr Pro Arg 180 185 190

aac gac gac leg cac gcc tag gtc aac cOc gct gac ctg acc ccg gtc 861 Asn Asp Asp Ser His Ala Ser Val Asn Arg Ala Asp Leu Thr Pro Val 195 200 205

acc gcc acc ggc cOc ggc tCc leg acc acg ttc gag get ctg gtc gaa 909 Thr Ala Thr Gly Arg Gly Phe Ser Thr Thr Phe Glu Asp Leu Val Glu 210 215 220

gtc ggc gcc aac tac agc aac acc ttc ggt ggt gtc gct ctg aag gag 957 Val Gly Ala Asn Tyr Ser Asn Thr Phe Gly Gly Val Ala Leu Lys Ala 225 230 235 240

agc get ggc tac tac tgg ggc aac gcc gcc ggc acg acc aeg gtc ggc 1005 Ser Ala Gly Tyr Tyr Trp Gly Asn Ala Ala Gly Thr Thr Thr Val Gly 245 250 255

gcc agc gtc gag gac ctg aac gcc tgg cag gtt ggc gcc cag get ggc 1053 Ala Ser Val Glu Asp Leu Asn Ala Trp Gln Val Gly Ala Gln Val Gly 260 265 270

tac gcc ggc tCc leg ctc ggt ggt tog tac acc gac ttc ggt gag leg llO1 Tyr Ala Gly Phe Ser Leu Gly Gly Ser Tyr Thr Asp Phe Gly Glu Ser 275 280 285

ggc cag ale cag acc gcg acc leg gac aac gag aag agc cgc ctc tgg 1149 Gly Gln Ile Gln Thr Ala Thr Ser Asp Asn Glu Lys Ser Arg Leu Trp 290 295 300

gtt gtc ggc gct cag tac acc gcc gOc ccg ale gtc gtc ggc gcc aac 1197 Val Val Gly Ala Gln Tyr Thr Ala Gly Pro Ile Val Val Gly Ala Asn 305 310 315 320

tac aag aac ggc aag gac gct ggc aSc ctc tac acc gcc ggc agc cgc 1245 Tyr Lys Asn Gly Lys Asp Ala Gly Ser Leu Tyr Thr Ala Gly Ser Arg 325 330 335

aag ctg gaa gtc tac gag tUc ggc gcg gOc tac acc gtc gct cog ggc 1293 Lys Leu Glu Val Tyr Glu Phe Gly Ala Gly Tyr Thr Val Ala Pro Gly 340 345 350

ctg acc ctg cag gct cag tac gac tac ttc aeg gcc gac agc gac ctg 1341 Leu Thr Leu Gln Ala Gln Tyr Asp Tyr Phe Lys Ala Asp Ser Asp Leu 355 360 365

agc acg gcc gct gcg gac chg gac aac gac ggt cac gtc gtc ctg ctc 1389 Ser Thr Ala Ala Ala Asp Leu Asp Asn Asp Gly His Val Val Leu Leu 370 375 380

cgt acg gtc ctg gag ttc tea tctgcggtca tcogtOgatg gagaa 1435 Arg Thr Val Leu Ala Phe 385 390

<210> 6

c211> 390

3t 1 ( ( / (

c2> PRT <213> Azospirillum brasilense c400> 6 Met Asn Arg Tyr Leu Leu Ala Gly Cys Ala Ala Ala Ala Leu Ala Leu 1 5 10 15

Gly Ala Gly Ala Ala Asn Ala Gln Ala Lys Phe Glu Val Lys Val Gly 20 25 30

Gly Asp Ala Tyr Phe Glu Gly Gly Tyr Val Asn Glu Asp Arg Asp Glu 35 40 45

Gly Ala Arg Ser Val Asp Phe Arg Asn Arg Phe Arg Val Ile Val Thr 50 55 60

Pro Thr Ala Lys Ala Asp Asn Gly Leu Glu Tyr Gly Gly Ary Ile Arg 65 70 75 80

Met Arg Ala Asn Ser Gly Ala Gly Asn Ala Arg Thr Met Asp Ala Asp 85 90 95

Arg Gly Tyr Ile Phe Ala Gln Gly Ala Phe Gly Gln Val Gln Met Gly 100 105 110

Val Thr Asn Ser Phe Asn Asp Ala Thr Tyr Val Thr Ala Pro Gln Asp 115 120 125

Tyr Leu Pro Leu Ala Ile Tyr Asp Gly Val Thr Ala Trp Ile Gly Gln 130 135 140

Thr Ser Asp Ile Gly Gly Gly Leu Ala Gly Leu Asn Gly Ser Ile Leu 145 150 155 160

Asn Gln Ser Leu Val Val Glu Asn Asn Ala Thr Lys Ile Val Tyr Phe 165 170 175

Ser Pro Arg Phe Ala Gly Leu Gln Val Gly Ala Ser Tyr Thr Pro Arg 180 185 190

Asn Asp Asp Ser His Ala Ser Val Asn Arg Ala Asp Leu Thr Pro Val 195 200 205

Thr Ala Thr Gly Arg Gly Phe Ser Thr Thr Phe Glu Asp Leu Val Glu 210 215 220

Va1 ly Ala Asn Tyr Ser Asn Thr Phe Gly Gly Val Ala Leu Lys Ala 225 230 Z35 240

Ser Ala Gly Tyr Tyr Trp Gly Asn Ala Ala Gly Thr Thr Thr Val Gly 245 250 255

Ala Ser Val Glu Asp Leu Asn Ala Trp Gln Val Gly Ala Gln Val Gly 260 265 270

Tyr Ala Gly Phe Ser Leu Gly Gly Ser Tyr Thr Asp Phe Gly Glu Ser 275 280 285

Gly Gln Ile Gln Thr Ala Thr Ser Asp Asn Glu Lys Ser Arg Leu Trp 290 295 300

Val Val Gly Ala Gln Tyr Thr Ala Gly Pro Ile Val Val Gly Ala Asn 305 310 315 320

Tyr Lys Asn Gly Lys Asp Ala Gly Ser Leu Tyr Thr Ala Gly Ser Arg 325 330 335

Lys Leu Glu Val Tyr Glu Phe Gly Ala Gly Tyr Thr Val Ala Pro Gly 340 345 350

Leu Thr Leu Gln Ala Gln Tyr Asp Tyr Phe Lys Ala Asp Ser Asp Leu 355 360 365

Ser Thr Ala Ala Ala Asp Leu Asp Asn Asp Gly His Val Val Leu Leu 370 375 380

Arg Thr Val Leu Ala Phe 385 390

c210> 7 c211> 951 c212> DNA c213> Pseudomonas fluorescens c220> c221> CDS <222> (1)..(951)

c223> <400> 7

aaa aac acc ttg ggc ttg gcc att ggt act ctt att gac gct act let 48 Lys Asn Thr Leu Gly Leu Ala Ile Gly Thr Leu Ile Ala Ala Thr Ser 1 5 10 15

ttc gOc gcg ctg gca caa ggc caa ggc gca gtt gaa gOc cag ctg tec 96 Phe Gly Ala Leu Ala Gln Gly Gln Gly Ala Val Glu Gly Gln Leu Phe

o ( ( 20 25 30

tac aaa aag cag ttc aac get agc gtc aag cac ale gaa gac ggc tat 144 Tyr Lys Lys Gln Phe Asn Asp Ser Val Lys His lle Glu Asp Gly Tyr 35 40 45

aac cct ggc gct agc ale ggt tac ttc att acc gac gac gtg leg gtt 192 Asn Pro Gly Ala Ser {le Gly Tyr Phe Ile Thr Asp Asp Val Ser Val 50 55 60

aac ctg aac tac gac acc acc aac cac acc cgt leg aac gac ggt act 240 Asn Leu Asn Tyr Asp Thr Thr Asn His Thr Arg Ser Asn Asp Gly Thr 65 70 75 80

ggc aac cag aag ale aaa ggt get act ggc agc gtg act get cag tac 288 Gly Asn Gln Lys Ile Lys Gly Asp Thr Gly Ser Val Thr Ala Gln Tyr 85 90 95

cac ttc ggt cag gcc ggt gtt gac tcc ctg cgt cca tac gta gaa ggt 336 His Phe Gly Gln Ala Gly Val Asp Ser Leu Arg Pro Tyr Val Glu Gly 100 105 110

ggt ttc ggt cac cag agc cgt acc aac gta cag got gac ggc cac agc 384 Gly Phe Gly His Gln Ser Arg Thr Asn Val Gln Ala Asp Gly His Ser 115 120 125

ggt cgc gac cag leg acc ctg gct ale gct ggc gct gOt gtg aag tac 432 Gly Arg Asp Gln Ser Thr Leu Ala Ile Ala Gly Ala Gly Val Lys Tyr 130 135 140

tac ttc acc aac aac ctg ttc gct cgt gct ggc gtt gag let gac tac 4BO Tyr Phe Thr Asn Asn Leu Phe Ala Arg Ala Gly Val Glu Ser Asp Tyr 145 150 155 160

aac ctg gac aac gOc aeg tOg gac tac tcc gca ctg gtt ggc ctg ggc 528 Asn Leu Asp Asn Gly Lys Trp Asp Tyr Ser Ala Leu Val Gly Leu Gly 165 170 175

gtg aac ttc ggc gOt aac gct gOc gca gct gct cca gct cot acc cca 576 Val Asn Phe Gly Gly Asn Ala Gly Ala Ala Ala Pro Ala Pro Thr Pro 180 185 190

gca cca gct cca gag cca act cca gag cca gaa get cca gtt gct cag 624 Ala Pro Ala Pro Glu Pro Thr Pro Glu Pro Glu Ala Pro Val Ala Gln 195 200 205

gtt gtt cOt gtt gag ctg gac gtt aeg thc gac ttc gac aag leg gtt 672 Val Val Arg Val Glu Leu Asp Val Lys Phe Asp Phe Asp Lys Ser Val 210 215 220

gtt aag cct aac agc tac gOc gac gtg aaa aac ctg gcc gac ttc aLg 720 Val Lys Pro Asn Ser Tyr Gly Asp Val Lys Asn Leu Ala Asp Phe Met 225 230 235 240

gct cag tac cca gct acc aac gta gee gtt gct gOt cac act gac tcc 768 Ala Gln Tyr Pro Ala Thr Asn Val Glu Val Ala Gly His Thr Asp Ser 245 250 255

ale gOt cca gac gcc tac aac cag aag chg tcc cag cOt cSt gct gac 816 Ile Gly Pro Asp Ala Tyr Asn Gln Dys Leu Ser Gln Arg Arg Ala Asp 260 265 270

( (] i get gtt aag caa gtc ctg gtt aaa gac ggc gtt gct cct agc cgc ale 864 Ala Val Lys Gln Val Leu Val Lys Asp Gly Val Ala Pro Ser Arg Ile 275 280 285

acc gcg gLa gUt tac ggc gaa tcc cgc cca gtt gct gac aac gca act 912 Thr Ala Val Gly Tyr Gly Glu Ser Arg Pro Val Ala Asp Asn Ala Thr 290 295 300

gaa gct gOt cgc gct ght aac cgt cgc gta gaa gcg tag 951 Glu Ala Gly Arg Ala Val Asn Arg Arg Val Glu Ala Ser 305 310 315

c210> 8 <211> 317

<212> PRT

c213> Pseudomonas fluorescens <400> 8

Lys Asn Thr Leu Gly Leu Ala Ile Gly Thr Leu Ile Ala Ala Thr Ser 1 5 10 15

Phe Gly Ala Leu Ala Gln Gly Gln Gly Ala Val Glu Gly Gln Leu Phe 20 25 30

Tyr Lys Lys Gln Phe Asn Asp Ser Val Lys His Ile Glu Asp Gly Tyr 35 40 45

Asn Pro Gly Ala Ser Ile Gly Tyr Phe Ile Thr Asp Asp Val Ser Val 50 55 60

Asn Leu Asn Tyr Asp Thr Thr Asn His Thr Arg Ser Asn Asp Gly Thr 65 70 75 80

Gly Asn Gln Lys Ile Lys Gly Asp Thr Gly Ser Val Thr Ala Gln Tyr 85 90 95

His Phe Gly Gln Ala Gly Val Asp Ser Leu Arg Pro Tyr Val Glu Gly 100 105 110

Gly Phe Gly His Gln Ser Arg Thr Asn Val Gln Ala Asp Gly His Ser 115 120 125

Gly Arg Asp Gln Ser Thr Leu Ala Ile Ala Gly Ala Gly Val Lys Tyr 130 135 140

Tyr Phe Thr Asn Asn Leu Phe Ala Arg Ala Gly Val Glu Ser Asp Tyr 145 150 155 160

Asr. eu Asp Asn Gly Lys Trp Asp Tyr Ser Ala Leu Val Gly Leu Gly 165 170 175

Val Asn Phe Gly Gly Asn Ala Gly Ala Ala Ala Pro Ala Pro Thr Pro 180 185 190

Ala Pro Ala Pro Glu Pro Thr Pro Glu Pro Glu Ala Pro Val Ala Gln 195 200 205

Val Val Arg Val Glu Leu Asp Val Lys Phe Asp Phe Asp Lys Ser Val 210 215 220

Val Lys Pro Asn Ser Tyr Gly Asp Val Lys Asn Leu Ala Asp Phe Met 225 230 235 240

Ala Gln Tyr Pro Ala Thr Asn Val Glu Val Ala Gly His Thr Asp Ser 245 250 255

Ile Gly Pro Asp Ala Tyr Asn Gln Lys Leu Ser Gln Arg Arg Ala Asp 260 265 270

Ala Val Lys Gln Val Leu Val Lys Asp Gly Val Ala Pro Ser Arg Ile 275 280 285

Thr Ala Val Gly Tyr Gly Glu Ser Arg Pro Val Ala Asp Asn Ala Thr 290 295 300

Glu Ala Gly Arg Ala Val Asn Arg Arg Val Glu Ala Ser 305. 310 315

c210> 9 c211> 120 c212> DNA c213> Trichoderma reesei -

<400> 9

gatcacccag tcgcactacg gccagtgcOg cOgcatcggc tacaBcOgcc ccaccgtOtg60 cgcotcgggc accacgtgcc aagtcctgaa cccgtactac tcgoagtgoc tgggcaggoc120 c210> 10 <211> 120

<212> DNA

<213> Trichoderma reesei <400> 10

agctOgcogg cacaggoact gcgagtagta cgggttcagg acttOgeacg tOgUgccoga60 gOcgcacacg gtggggccgc tgtagccgat gccgccOcac tOgccOtagt gcOactOggt 120

k3 <210> 11

<211> 1305

<212> DNA

<213> Artificial <220> <223> The sequence is a combination of elements from Azospirillum brasi lense and Trichoderma reesei <220> <221> CDS

c222> (1)..(1305) c223> <400> 11

atg aac cgt tat ctg ctc gcg gOc tgc gcc gct gcc gct ctg gct ctg 48 Met Asn Arg Tyr Leu Leu Ala Gly Cys Ala Ala Ala Ala Leu Ala Leu 1 5 10 15

ggt gac ggc gcc gcc aac got cag goc aag ttt gaa gtc aag gtc ggc 96 Gly Ala Gly Ala Ala Asn Ala Gln Ala Lys Phe Glu Val Lys Val Gly 20 25 30

gOc gac gcc tac ttc gaa ggc gOc tac gtc aac gaa ggc cgg cca tta 144 Gly Asp Ala Tyr Phe Glu Gly Gly Tyr Val Asn Glu Gly Arg Pro Leu 35 40 45

att aac ggg ale acc cag tag cac tac ggc cag tgc gOc gOc ale gOc 192 Ile Asn Gly Ile Thr Gln Ser Bis Tyr Gly Gln Cys Gly Gly Ile Gly 50 55 60

tac agc ggc ccc acc gtg tgc gcc leg ggc acc aeg tgc caa gtc ctg 240 Tyr Ser Gly Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu 65 70 75 80

aac ccg tac tac leg cag tgc ckg ggc cOg ccc gag ggt gac cgt leg 288 Asn Pro Tyr Tyr Ser Gln Cys Leu Gly Arg Pro Glu Gly Ala Arg Ser 85 90 95

gtt gac ttc cgc aac cgc ttc cgc gtc ale gtg acc ccg acc goc aag 336 Val Asp Phe Arg Asn Arg Phe Arg Val Ile Val Thr Pro Thr Ala Lys 100 105 110

gac gac aac ggc ctg gag tac ggc ggc cgc ale cOt atg cgc gcc aac 384 Ala Asp Asn Gly Leu Glu Tyr Gly Gly Arg Ile Arg Met Arg Ala Asn 115 120 125

tcc gOc gcc ggc aac gcc cSc acg atg gac gcc gac cgc ggc tac ale 432 Ser Gly Ala Gly Asn Ala Arg Thr Met Asp Ala Asp Arg Gly Tyr Ile 130 135 140

ttc gcg cag gOc gcg ttc gOt cag gtc cag atg gOc gtg acc aac tcc 480 Phe Ala Gln Gly Ala Phe Gly Gln Val Gln Met Gly Val Thr Asn Ser 145 150 155 160

thc aac gac goc acc tac gtc acg gcc ccg cag get tac ctg ccg ctg 528 Phe Asn Asp Ala Thr Tyr Val Thr Ala Pro Gln Asp Tyr Leu Pro Leu 165 170 175

gac att tat gac ggc gtc acc gcc tgg ale ggc cag acc tcc gac ale 576

( A1" lle Tyr Asp Gly Val Thr Ala Trp Ile Gly Gln Thr Ser Asp Ile 180 185 190

ggc ggc ggc ctc gcc ggt ctg aac ggc tcg atc ctg aac cag tog ctg 624 Gly Gly Gly Leu Ala Gly Leu Asn Gly Ser Ile Leu Asn Gln Ser Leu 195 200 205

gtc gtc gag aac aac gag acg aag atc gtg tac ttc tog ccg cgc ttc 672 Val Val Glu Asn Asn Ala Thr Lys Ile Val Tyr Phe Ser Pro Arg Phe 210 215 220

gcc ggc ctg cag gtg ggc goc aDc tac acc ccg cOc aac gac gac tag 720 Ala Gly Leu Gln Val Gly Ala Ser Tyr Thr Pro Arg Asn Asp Asp Ser 225 230 235 240

cac gcc tog gtc aac cgc gct gac ctg acc cog gtc acc gac acc ggc 768 His Ala Ser Val Asn Arg Ala Asp Leu Thr Pro Val Thr Ala Thr Gly 245 250 255

cgc ggc ttc tcg acc acg tCc gag gat ctg gtc gaa gtc ggc gcc aac 816 Arg Gly Phe Ser Thr Thr Phe Glu Asp Leu Val Glu Val Gly Ala Asn 260 265 270

tac agc aac acc ttc ggt ggt gtc gct ctg aeg gcg agc gct ggc tac 864 Tyr Ser Asn Thr Phe Gly Gly Val Ala Leu Lys Ala Ser Ala Gly Tyr 275 280 285

tac tgg gOc aac gcc gcc ggc acg acc acg gtc gOc gcc aSc gtc gag 912 Tyr Trp Gly Asn Ala Ala Gly Thr Thr Thr Val Gly Ala Ser Val Glu 290 295 300

gac ctg aac gcc tgg cag gtt ggc gcc cag gtt ggc tac gcc ggc ttc 960 Asp Leu Asn Ala Trp Gln Val Gly Ala Gln Val Gly Tyr Ala Gly Phe 305 310 315 320

tcg ctc ggt ggt tag tac acc gac ttc ggt gag tcg ggc cag atc cag 1008 Ser Leu Gly Gly Ser Tyr Thr Asp Phe Gly Glu Ser Gly Gln Ile Gln 325 330 335

acc gcg acc tag gac aac gag aag agc cgc ctc tgg gtt gtc ggc gct 1056 Thr Ala Thr Ser Asp Asn Glu Lys Ser Arg Leu Trp Val Val Gly Ala 340 345 350

cag tac acc gcc ggc ccg atc gtc gtc ggc gcc aac tac aag aac ggc 1104 Gln Tyr Thr Ala Gly Pro Ile Val Val Gly Ala Asn Tyr Lys Asn Gly 355 360 365

aag gac gct ggc aSc ctc tac acc gac ggc agc cgc aag ctg gaa gtc 1152 Lys Asp Ala Gly Ser Leu Tyr Thr Ala Gly Ser Arg Lys Leu Glu Val 370 375 380

tac gag ttc ggc gcg ggc tac acc gtc gct ccg ggc ctg acc ctg cag 1200 Tyr Glu Phe Gly Ala Gly Tyr Thr Val Ala Pro Gly Leu Thr Leu Gln 385 390 395 400

gct cag tac gac tac ttc aag gcc gac aSc gac ctg aBc acg gcc gat 1248 Ala Gln Tyr Asp Tyr Phe Lys Ala Asp Ser Asp Leu Ser Thr Ala Ala 405 410 415

gcg gac ctg gac aac gac gOt cac gtc gtc chg ctc cOt acg gtc ctg 1296 Ala Asp Leu Asp Asn Asp Gly His Val Val Leu Leu Arg Thr Val Leu

( 420 425 430

gcg ttc tea 1305 Ala Phe <210> 12

<211> 434

<212> PRT

<213> Artificial <220> c223> The sequence is a combination of elements from Azospirillum brasi lense and Trichoderma reesei <400> 12

Met Asn Arg Tyr Leu Leu Ala Gly Cys Ala Ala Ala Ala Leu Ala Leu 1 5 10 15

Gly Ala Gly Ala Ala Asn Ala Gln Ala Lys Phe Glu Val Lys Val Gly 20 25 30

Gly Asp Ala Tyr Phe Glu Gly Gly Tyr Val Asn Glu Gly Arg Pro Leu 35 40 45

Ile Asn Gly Ile Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly 50 55 60

Tyr Ser Gly Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu 65 70 75 80

Asn Pro Tyr Tyr Ser Gln Cys Leu Gly Arg Pro Glu Gly Ala Arg Ser 85 90 95

Val Asp Phe Arg Asn Arg Phe Arg Val Ile Val Thr Pro Thr Ala Lys 100 105 110

Ala Asp Asn Gly Leu Glu Tyr Gly Gly Arg Ile Arg Met Arg Ala Asn 115 120 125

Ser Gly Ala Gly Asn Ala Arg Thr Met Asp Ala Asp Arg Gly Tyr Ile 130 135 140

Phe Ala Gln Gly Ala Phe Gly Gln Val Gln Met Gly Val Thr Asn Ser 145 150 155 160

Phe Asn Asp Ala Thr Tyr Val Thr Ala Pro Gln Asp Tyr Leu Pro Leu 165 170 175

Ala Ile Tyr Asp Gly Val Thr Ala Trp Ile Gly Gln Thr Ser Asp Ile 180 185 190

Gly Gly Gly Leu Ala Gly Leu Asn Gly Ser Ile Leu Asn Gln Ser Leu 195 200 205

Val Val Glu Asn Asn Ala Thr Lys Ile Val Tyr Phe Ser Pro Arg Phe 210 215 220

Ala Gly Leu Gln Val Gly Ala Ser Tyr Thr Pro Arg Asn Asp Asp Ser 225 230 235 240

His Ala Ser Val Asn Arg Ala Asp Leu Thr Pro Val Thr Ala Thr Gly 245 250 255

Arg Gly Phe Ser Thr Thr Phe Glu Asp Leu Val Glu Val Gly Ala Asn 260 265 270

Tyr Ser Asn Thr Phe Gly Gly Val Ala Leu Lys Ala Ser Ala Gly Tyr 275 280 285

Tyr Trp Gly Asn Ala Ala Gly Thr Thr Thr Val Gly Ala Ser Val Glu 290 29S 300

Asp Leu Asn Ala Trp Gln Val Gly Ala Gln Val Gly Tyr Ala Gly Phe 305 310 315 320

* Ser Leu Gly Gly Ser Tyr Thr Asp Phe Gly Glu Ser Gly Gln Ile Gln 325 330 335

Thr Ala Thr Ser Asp Asn Glu Lys Ser Arg Leu Trp Val Val Gly Ala 340 345 350

Gln Tyr Thr Ala Gly Pro Ile Val Val Gly Ala Asn Tyr Lys Asn Gly 355 360 365

Lys Asp Ala Gly Ser Leu Tyr Thr Ala Gly Ser Arg Lys Leu Glu Val 370 375 380

Tyr Glu Phe Gly Ala Gly Tyr Thr Val Ala Pro Gly Leu Thr Leu Gln 385 390 395 400

Ala Gln Tyr Asp Tyr Phe Lys Ala Asp Ser Asp Leu Ser Thr Ala Ala 405 410 415

A]= Asp Leu Asp Asn Asp Gly His Val Val Leu Leu Arg Thr Val Leu 420 425 430

Ala Phe <210> 13

c211> 1305 c212> DNA c213> Artificial c220> c223> The sequence is a combination of elements from Azospirillum brasi lense and Trichoderma reesei c220> <221> CDS

c222> (1)..(1305) c223> <400> 13

atg aac cgt tat ctg ctc gag ggc tgc gac gct gac gct ctg gct ctg 48 Met Asn Arg Tyr Leu Leu Ala Gly Cys Ala Ala Ala Ala Leu Ala Leu 1 5 10 15

ggt gcc ggc gcc gcc aac get cag gcc aag ttt gaa gtc aag gtc ggc 96 Gly Ala Gly Ala Ala Asn Ala Gln Ala Lys Phe Glu Val Lys Val Gly 20 25 30

ggc gac gcc tac ttc gaa gOc ggc tac gtc aac gaa gac cgc gac gag 144 Gly Asp Ala Tyr Phe Glu Gly Gly Tyr Val Asn Glu Asp Arg Asp Glu 35 40 45

ggt gcc cgt tag gtt gac ttc cgc aac cgc ttc cgc gtc ale gtg acc 192 Gly Ala Arg Ser Val Asp Phe Arg Asn Arg Phe Arg Val Ile Val Thr 50 55 60

cog acc gcc aag gcc gac aac ggc ctg gag tac ggc ggc cgc ale cOt 240 Pro Thr Ala Lys Ala Asp Asn Gly Leu Glu Tyr Gly Gly Arg Ile Arg 65 70 75 80

atg cgc gec aac tcc ggc gcc ggc aac gcc cgc acg atg gac gcc gac 288 Met Arg Ala Asn Ser Gly Ala Gly Asn Ala Arg Thr Met Asp Ala Asp 85 90 95

cgc ggc tac ale ttc gcg cag ggc gcg ttc ggt cag gtc cag atg ggc 336 Arg Gly Tyr Ile Phe Ala Gln Gly Ala Phe Gly Gln Val Gln Met Gly 100 105 110

gtg acc aac tcc ttc aac gac gcc acc tac gtc acg gcc ccg cag get 384 Val Thr Asn Ser Phe Asn Asp Ala Thr Tyr Val Thr Ala Pro Gln Asp 115 120 125

tac ctg ccg ctg gcc att tat gac ggc gtc acc gcc tgg ale ggc cag 432 Tyr Leu Pro Leu Ala Ile Tyr Asp Gly Val Thr Ala Trp Ile Gly Gln 130 135 140

acc tcc gac ale ggc ggc ggc ctc gac ggt ctg aac ggc leg ale ctg 480 Thr Ser Asp Ile Gly Gly Gly Leu Ala Gly Leu Asn Gly Ser Ile Leu

( 146 150 155 160

aac cag tcg ctg gtc gtc gag aac aac gcg acg aag atc gUg tac ttc 528 Asn Gln Ser Leu Val Val G]u Asn Asn Ala Thr Lys Ile Val Tyr Phe 165 170 175

tog cog cgc ttc gcc ggc ctg cag gtg ggc goc agc tac acc ccg cgc 576 Ser Pro Arg Phe Ala Gly Leu Gln Val Gly Ala Ser Tyr Thr Pro Arg 180 185 190

aac gac gac tcg cac gac tcg gtc aac cgc gct gac ctg acc ccg gtc 624 Asn Asp Asp Ser His Ala Ser Val Asn Arg Ala Asp Leu Thr Pro Val 195 200 205

acc gcc acc ggc cOc ggc ttc tcg acc acg thc gag gat chg gtc gsa 672 Thr Ala Thr Gly Arg Gly Phe Ser Thr Thr Phe Glu Asp beu Val Glu 210 215 220

gtc ggc gcc aac tac agc aac acc ttc ggt gOt gtc gct ctg aag gcg 720 Val Gly Ala Asn Tyr Ser Asn Thr Phe Gly Gly Val Ala Leu Lys Ala 225 230 235 240

agc gct ggc tac ggc cgg cca tta att aac gOg atc acc cag tcg cac 768 Ser Ala Gly Tyr Gly Arg Pro Leu Ile Asn Gly Ile Thr Gln Ser His 245 250 255

tac ggc cag tgc ggc gOc atc gOc tac aDc gOc ccc acc gtg tgc gcc 816 Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala 260 265 270

tcg ggc acc acg tgc caa gtc ctg aac ccg tac tac tcg cag tgc ctg 864 Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu 27S 280 285

ggc cgg coc aac gcc gcc ggc acg acc acg gec ggc gcc agc gtc gag 912 Gly Arg Pro Asn Ala Ala Gly Thr Thr Thr Val Gly Ala Ser Val Glu 290 295 300

gac ctg aac gcc tOg cag gtt gOc gcc cag gtt ggc tac gcc ggc tCc 960 Asp Leu Asn Ala Trp Gln Val Gly Ala Gln Val Gly Tyr Ala Gly Phe 305 310 315 320

tcg ctc ggt gOt tcg tac acc gac ttc ggt gag tag gOc cag atc cag 1008 Ser Leu Gly Gly Ser Tyr Thr Asp Phe Gly Glu Ser Gly Gln Ile Gln 325 330 335

acc gcg acc tcg gac aac gag aag agc cgc ctc tOg gtt gtc ggc gct 1056 Thr Ala Thr Ser Asp Asn Glu Lys Ser Arg Leu Trp Val Val Gly Ala 340 345 350

cag tac acc gcc gOc ccg atc gtc gtc ggc gec aac tac aag aac ggc 1104 Gln Tyr Thr Ala Gly Pro Ile Val Val Gly Ala Asn Tyr LYS Asn Gly 355 360 365

aag gac gct gOc aSc ctc tac acc gcc gOc agc cSc aag ctg gaa gtc 1152 Lys Asp Ala Gly Ser Leu Tyr Thr Ala Gly Ser Arg LYB Leu Glu Val 370 375 380

tac gag ttc ggc gcg ggc tac acc gtc gct ccg gOc ctg acc ctg cag 1200 Tyr Glu Phe Gly Ala Gly Tyr Thr Val Ala Pro Gly Leu Thr Leu Gln 385 390 395 400

4? ( gct cag tac gac tac ttc aag gcc gac agc gac ctg agc acg gcc gct 1248 Ala Gln Tyr Asp Tyr Phe Lys Ala Asp Ser Asp Leu Ser Thr Ala Ala 405 410 415

gog gac ctg gac aac gac ggt cac gtc gtc ctg ctc cSt acg gtc ctg 1296 Ala Asp beu Asp Asn Asp Gly His Val Val Leu Leu Arg Thr Val Leu 420 425 430

gag ttc tea 130S Ala Phe c210> 14 c211> 434 c2127 PRT <213> Artificial c220> <223> The sequence is a combination of elements from Azospirillum brasi lense and Trchoderma reesei c400> 14 Met Asn Arg Tyr Leu Leu Ala Gly Cys Ala Ala Ala Ala Leu Ala Leu 1 5 10 15

Gly Ala Gly Ala Ala Asn Ala Gln Ala Lys Phe Glu Val Lys Val Gly 20 25 30

Gly Asp Ala Tyr Phe Glu Gly Gly Tyr Val Asn Glu Asp Arg Asp Glu 35 40 45

Gly Ala Arg Ser Val Asp Phe Arg Asn Arg Phe Arg Val Ile Val Thr -

50 55 60

Pro Thr Ala Lys Ala Asp Asn Gly Leu Glu Tyr Gly Gly Arg Ile Arg 65 70 75 80

Met Arg Ala Asn Ser Gly Ala Gly Asn Ala Arg Thr Met Asp Ala Asp 85 90 95

Arg Gly Tyr Ile Phe Ala Gln Gly Ala Phe Gly Gln Val Gln Met Gly 100 105 110

Val Thr Asn Ser Phe Asn Asp Ala Thr Tyr Val Thr Ala Pro Gln Asp 115 120 125

Tyr Deu Pro Leu Ala Ile Tyr Asp Gly Val Thr Ala Trp Ile Gly Gln 130 135 140

Thr er Asp Ile Gly Gly Gly Leu Ala Gly Leu Asn Gly Ser Ile Leu 145 150 155 160

Asn Gln Ser beu Val Val G1u Asn Asn Ala Thr Lys Ile Val Tyr Phe 165 170 175

Ser Pro Arg Phe Ala Gly Leu Gln Val Gly Ala Ser Tyr Thr Pro Arg 180 185 190

Asn Asp Asp Ser His Ala Ser Val Asn Arg Ala Asp Leu Thr Pro Val 195 200 205

Thr Ala Thr Gly Arg Gly Phe Ser Thr Thr Phe Glu Asp Leu Val Glu 210 215 220

Val Gly Ala Asn Tyr Ser Asn Thr Phe Gly Gly Val Ala Leu Lys Ala 225 230 235 240

Ser Ala Gly Tyr Gly Arg Pro Leu Ile Asn Gly Ile Thr Gln Ser His 245 250 255

Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala 260 265 270

Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu 275 280 285

Gly Arg Pro Asn Ala Ala Gly Thr Thr Thr Val Gly Ala Ser Val Glu 290 295 300

Asp Leu Asn Ala Trp Gln Val Gly Ala Gln Val Gly Tyr Ala Gly Phe 305 310 315 320

Ser Leu Gly Gly Ser Tyr Thr Asp Phe Gly Glu Ser Gly Gln Ile Gln 325 330 335

Thr Ala Thr Ser Asp Asn Glu Lys Ser Arg Leu Trp Val Val Gly Ala 340 345 350 Gln Tyr Thr Ala Gly Pro Ile Val Val Gly Ala Asn Tyr Lys Asn Gly 355 360

365

Lys Asp Ala Gly Ser beu Tyr Thr Ala Gly Ser Arg Lys Leu Glu Val 370 375 380

Tyr Glu Phe Gly Ala Gly Tyr Thr Val Ala Pro Gly Leu Thr Leu Gln

3B, 390 395 400

Ala Gln Tyr ASp Tyr Phe Lys Ala Asp Ser Asp Leu Ser Thr Ala Ala 405 410 415

Ala Asp beu Asp Asn Asp Gly His Val Val Leu Leu Arg Thr Val Leu 420 425 430

Ala Phe c210> 15 c211> 1305 -

<212> DNA

<213> Artificial c220> <223> The sequence is a combination of elements from Azespirillum brasi lense and Trichoderma reesei c220> <221> CDS

c222> (1)..(1305) <223> <400> 15

atg aac cgt tat ctg ctc gcg ggc tgc gcc gct gcc gct ctg gct ctg48 Met Asn Arg Tyr Leu Leu Ala Gly Cys Ala Ala Ala Ala Leu Ala Leu 1 5 10 15

ggt gcc gOc gac gcc aac got cag gcc aag ttt gaa gec aeg gtc ggc96 Gly Ala Gly Ala Ala Asn Ala Gln Ala Lys Phe Glu Val Lys Val Gly 20 25 30

gOc gac gac tac ttc gaa ggc ggc tac gtc aac gaa gac cOc gac gag144 Gly Asp Ala Tyr Phe Glu Gly Gly Tyr Val Asn Glu Asp Arg Asp Glu 35 40 45

ggt gcc cgt tog gtt gac ttc cgc aac cgc ttc cgc gtc ale gtg acc192 Gly Ala Arg Ser Val Asp Phe Arg Asn Arg Phe Arg Val Ile Val Thr 50 55 60

ccg acc gcc aeg gcc gac aac ggc ctg gag tac ggc ggc cgc ale cOt240 Pro Thr Ala Lys Ala Asp Asn Gly Leu Glu Tyr Gly Gly Arg Ile Arg 65 70 75 80

aLg cgc gcc aac tcc ggc gcc ggc aac gac cgc acg atg gac gcc gac288 Met Arg Ala Asn Ser Gly Ala Gly Asn Ala Arg Thr Met Asp Ala Asp 85 90 95

cgc gOc tac ale ttc gcg cag ggc gcg ttc ggt cag gtc cag atg ggc336 Arg Gly Tyr Ile Phe Ala Gln Gly Ala Phe Gly Gln Val Gln Met Gly 100 105 110

gtg acc aac tcc tec aac gac gcc acc tac gtc acg gcc ccg cag get384 Val Thr Asn Ser Phe Asn Asp Ala Thr Tyr Val Thr Ala Pro Gln Asp 115 120 125

tac ctg ccg ctg gcc att tat gac ggc gtc acc gcc tgg atc ggc cag 432 Tyr Leu Pro Leu Ala Ile Tyr Asp Gly Val Thr Ala Trp Ile Gly Gln 130 135 140

acc tcc gac atc ggc ggc ggc ctc gcc ggt ctg aac ggc tcg atc ctg 480 Thr Ser Asp Ile Gly Gly Gly Leu Ala Gly Leu Asn Gly Ser Ile Leu 145 150 155 160

aac cag tcg ctg gtc gtc gag aac aac gcg acg aag atc gtg tac ttc 528 Asn Gln Ser Leu Val Val Glu Asn Asn Ala Thr Lys Ile Val Tyr Phe 165 170 175

tcg ccg cgc ttc gcc ggc ckg cag gtg ggc gcc agc tac acc ccg cgc 576 Ser Pro Arg Phe Ala Gly Leu Gln Val Gly Ala Ser Tyr Thr Pro Arg 180 185 190

aac gac gac tog cac gcc tcg gtc aac cgc gct gac ctg acc ccg gtc 624 Asn Asp Asp Ser His Ala Ser Val Asn Arg Ala Asp Leu Thr Pro Val 195 200 205

acc gcc acc ggc cgc ggc ttc tcg acc acg ttc gag gat ctg gtc gaa 672 Thr Ala Thr Gly Arg Gly Phe Ser Thr Thr Phe Glu Asp Leu Val Glu 210 215 220

gtc ggc gac aac tac agc aac acc ttc gOt ggt gtc gct ctg aag gcg 720 Val Gly Ala Asn Tyr Ser Asn Thr Phe Gly Gly Val Ala Leu Lys Ala 225 230 235 240

agc gct ggc tac tac tgg ggc aac gcc gcc ggc acg acc acg gtc ggc 768 Ser Ala Gly Tyr Tyr Trp Gly Asn Ala Ala Gly Thr Thr Thr Val Gly 245 250 255

gcc agc gtc gag gac chg aac gcc tOg cag gtt ggc gcc cag gtt ggc 816 Ala Ser Val Glu Asp Leu Asn Ala Trp Gln Val Gly Ala Gln Val Gly 260 265 270

tac gcc ggc ttc tog ctc ggt ggt tcg tac acc gac ttc gOt gag tog 864 Tyr Ala Gly Phe Ser Leu Gly Gly Ser Tyr Thr Asp Phe Gly Glu Ser 275 280 285

ggc cag atc cag gOc cOg cca tta att aac ggg atc acc cag tcg cac 912 Gly Gln Ile Oln Gly Arg Pro Leu Ile Asn Gly Ile Thr Gln Ser His 290 295 300

tac ggc cag tgc ggc ggc atc ggc tac agc ggc ccc acc gtg tgc gcc 960 Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala 305 310 315 320

tcg ggc acc acg tgc caa gtc ctg aac ccg tac tac tcg cag tgc ctg 1008 Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu 325 330 335

ggc cgg ccc tcg gac aac gag aag agc cOc ctc tgg gtt gtc gOc gct 1056 Gly Arg Pro Ser Asp Asn Glu Lys Ser Arg Leu Trp Val Val Gly Ala 340 345 350

cag tac acc gcc gOc ccg atc gtc gtc gOc gcc aac tac aag aac ggc 1104 Gln Tyr Thr Ala Gly Pro Ile Val Val Gly Ala Asn Tyr Lys Asn Gly; 355 360 365

( ( aag gac gct ggc agc ctc tac acc gcc ggc agc cgc aeg ctg gaa gec 1152 Lys Asp Ala Gly Ser Leu Tyr Thr Ala Gly Ser Arg Lys Leu Glu Val 370 375 380

tac gag ttc ggc gcg ggc tac acc gtc gct ccg ggc ctg acc ctg cag 1200 Tyr Glu Phe Gly Ala Gly Tyr Thr Val Ala Pro Gly Leu Thr Leu Gln 385 390 395 400

gct cag tac gac tac ttc aag gcc gac agc gac ctg agc acg gcc gct 1248 Ala Gln Tyr Asp Tyr Phe Lys Ala Asp Ser Asp Leu Ser Thr Ala Ala 405 410 415

gcg gac ctg gac aac gac ggt cac gtc gtc ctg ctc cOt acg gtc ctg 1296 Ala Asp Leu Asp Asn Asp Gly His Val Val Leu Leu Arg Thr Val Leu 420 425 430

gcg ttc tsa 1305 Ala Phe c210> 16 c211> 434 <212> PRT

c213> Artificial c220> c223> The sequence is a combination of elements from Azospirillum brasi lense and Trichoderma reesei c400> 16 Met Asn Arg Tyr Leu Leu Ala Gly Cys Ala Ala Ala Ala Leu Ala Leu 1 5 10 15

Gly Ala Gly Ala Ala Asn Ala Gln Ala Lys Phe Glu Val Lys Val Gly 20 25 30

Gly Asp Ala Tyr Phe Glu Gly Gly Tyr Val Asn Glu Asp Arg Asp Glu 3S 40 45

Gly Ala Arg Ser Val Asp Phe Arg Asn Arg Phe Arg Val Ile Val Thr 50 55 60

Pro Thr Ala Lys Ala Asp Asn Gly Leu Glu Tyr Gly Gly Arg Ile Arg 65 70 75 80

Met Arg Ala Asn Ser Gly Ala Gly Asn Ala Arg Thr Met Asp Ala Asp 85 90 95

Arg Gly Tyr Ile Phe Ala Gln Gly Ala Phe Gly Gln Val Gln Met Gly 100 105 110

Val Thr Asn Ser Phe Asn Asp Ala Thr Tyr Val Thr Ala Pro Gln Asp

s 115 120 12S

Tyr Leu Pro Leu Ala Ile Tyr Asp Gly Val Thr Ala Trp Ile Gly Gln 130 135 140

Thr Ser Asp Ile Gly Gly Gly Leu Ala Gly Leu Asn Gly Ser Ile Leu 145 150 155 160

Asn Gln Ser Leu Val Val Glu Asn Asn Ala Thr Lys Ile Val Tyr Phe 165 170 175

Ser Pro Arg Phe Ala Gly Leu Gln Val Gly Ala Ser Tyr Thr Pro Arg 180 185 190

Asn Asp Asp Ser His Ala Ser Val Asn Arg Ala Asp Leu Thr Pro Val 195 200 205

Thr Ala Thr Gly Arg Gly Phe Ser Thr Thr Phe Glu Asp Leu Val Glu 210 215 220

Val Gly Ala Asn Tyr Ser Asn Thr Phe Gly Gly Val Ala Leu Lys Ala 225 230 235 240

Ser Ala Gly Tyr Tyr Trp Gly Asn Ala Ala Gly Thr Thr Thr Val Gly 245 250 255

Ala Ser Val Glu Asp Leu Asn Ala Trp Gln Val Gly Ala Gln Val Gly 260 2G5 270

Tyr Ala Gly Phe Ser Leu Gly Gly Ser Tyr Thr Asp Phe Gly Glu Ser 275 280 285

Gly Gln Ile Gln Gly Arg Pro Leu Ile Asn Gly Ile Thr Gln Ser His 290 295 300

Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala 305 310 315 320

Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu 325 330 335

Gly Arg Pro Ser Asp Asn Glu Lys Ser Arg Leu Trp Val Val Gly Ala 340 345 350

Gln Tyr Thr Ala Gly Pro Ile Val Val Gly Ala Asn Tyr Lys Asn Gly; 355 360 365

s Lys Asp Ala Gly Ser Leu Tyr Thr Ala Gly Ser Arg Dys Leu Glu Val 370 375 380

Tyr Glu Phe Gly Ala Gly Tyr Thr Val Ala Pro Gly Deu Thr Leu Gln 385 390 395 400

Ala Gln Tyr Asp Tyr Phe Lys Ala Asp Ser Asp Leu Ser Thr Ala Ala 405 410 415

Ala Asp Leu Asp Asn Asp Gly His Val Val Leu Leu Arg Thr Val Leu 420 425 430

Ala Phe c210> 17 <211> 1377

<212> DNA

c213> Artificial <220> c223> The sequence is a combination of elements from Azospirillum brasi lense and Trichoderma reesei <220> <221> CDS

c222> (1)..(1377) <223> c400> 17 atg gcc leg ale atg acc aac acc leg gcg atg acc gcg ctg cag acc48 Met Ala Ser Ile Met Thr Asn Thr Ser Ala Met Thr Ala Leu Gln Thr 1 5 10 15

gtg cgc cgc gtg acc gac get ctg gcg acc acc cag get cgc ale tag96 Val Arg Arg Val Thr Asp Asp Leu Ala Thr Thr Gln Asp Arg Ile Ser 20 25 30

acc ggt ctc aag gtc aac aac gcc aag gac aac gcc gcc tac tgg leg144 Thr Gly Leu Lys Val Asn Asn Ala Lys Asp Asn Ala Ala Tyr Trp Ser 35 40 45

ale gcc acg acc atg cgc gcc gac gtc gcc ggc ttc aag gcg gtg aag192 Ile Ala Thr Thr Met Arg Ala Asp Val Ala Gly Phe Lys Ala Val Lys 50 55 60

gee leg ctg gaa ctg ggt tog ggc acg acc aac acc gcg tag gtg gcg240 Glu Ser Leu Glu Leu Gly Ser Gly Thr Thr Asn Thr Ala Ser Val Ala 65 70 75 80

tcc aag aac ale gtc gag aac ctg cag acg ctg aag gcc cgc gtc ale288 Ser Lys Asn Ile Val Glu Asn Leu Gln Thr Leu Lys Ala Arg Val Ile 85 90 95

5b gcc ggc cag acc aac ggc gtc gac aag tcg ctg atc cag aac gac atc 336 Ala Gly Gln Thr Asn Gly Val Asp Lys Ser Leu Ile Gln Asn Asp Ile 100 105 110

gac cag ctc gtc aag ctg gtg aag ggc gcc gcc gcc gac gcc tcc tCc 384 Asp Gln Leu Val Lys Leu Val Lys Gly Ala Ala Ala Asp Ala Ser Phe 115 120 125

aac ggc gac aac chg ctg cgc atc acc tac tcc aac gac ggc acg gcc 432 Asn Gly Asp Asn Leu Leu Arg Ile Thr Tyr Ser Asn Asp Gly Thr Ala 130 135 140

aag gac cag aac gec gac atc ctg gcg tag ctg agc cgc agc gcc ggc 480 Lys Asp Gln Asn Val Asp Ile Leu Ala Ser Leu Ser Arg Ser Ala Gly 145 150 155 160

acg gtc gat ccg agc tac atc agc ttc cag cgc cag gac atg cag gtc 528 Thr Val Asp Pro Ser Tyr Ile Ser Phe Gln Arg Gln Asp Met Gln Val 165 170 175

acc teg atc gtc ggc aeg gcc acg atc gag cag cag gtg gac tcc acc 576 Thr Ser Ile Val Gly Lys Ala Thr Ile Glu Gln Gln Val Asp Ser Thr 180 185 190

aac gac ctg aeg gcc agc gtc ggc atc gcc atc ggc gcg ccg gac acc 624 Asn Asp Leu Lys Ala Ser Val Gly Ile Ala Ile Gly Ala Pro Asp Thr 195 200 205

acc ttc atc gac ggc cag aac ctg ggc ctg ggc cgg cca tta att aac 672 Thr Phe Ile Asp Gly Gln Asn Leu Gly Leu Gly Arg Pro Leu Ile Asn 210 215 220

ggg atc acc cag tcg cac tac ggc cag tgc ggc ggc atc ggc tac agc 720 Gly Ile Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser 225 230 235 240

ggc ccc acc gtg tgc gcc tcg ggc acc acg tgc caa gtc ctg aac ccg 768 Gly Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro a 24S 250 255 -

tac tac tcg cag tgc ctg gOc cgg cag acc ctg aac gtc acc aac gag el6 Tyr Tyr Ser Gln Cys Leu Gly Arg Pro Thr Leu Asn Val Thr Asn Glu -

260 265 270 -

gcg ggc acc aag acc gcc gtg aca gtg gac ctg tcg ggc atc gac tac 864 Ala Gly Thr Lys Thr Ala Val Thr Val Asp Leu Ser Gly Ile Asp Tyr 275 280 285

acc acc gac chg gag acc acc cag ggc ctg atc gag acc gcg gtc aac 912 Thr Thr Asp Leu Ala Thr Thr Gln Gly Leu Ile Glu Thr Ala Val Asn -

2gO 295 300 gcg gcc ctg acc acg gcc ggc ggc gac thc cag gtg gac ttc aac ggc 960 Ala Ala Leu Thr Thr Ala Gly Gly Asp Phe Gln Val Ala Phe Asn Gly 30s 310 315 320 gac aeg ctg gaa ttc acc gac cag gac ctg aac acc gac gOc aac ttc 1008 Asp Lys Leu Glu Phe Thr Asp Gln Asp Leu Asn Thr Asp Gly Asn Phe 325 330 335

acc gec aag gtg gac agc ctg tgg gtc gOc tcg aag gaa agc gac gac 1056

Th: la Lys Val Asp Ser Leu Trp Val Gly Ser Lys Glu Ser Asp Ala 340 345 350

tCc gOc ggc ctg gcc gac ctg acg cag ale gac gtg acc tcc aac gcc 1104 Phe Gly Gly Leu Ala Asp Leu Thr Gln Ile Asp Val Thr Ser Asn Ala 355 360 365

acc aeg leg ctc gag gtc ale aac ggc ctg ctc gac aag gcc ale ggc 1152 Thr Lys Ser Leu Glu Val Ile Asn Gly Leu Leu Asp Lys Ala Ile Gly 370 375 380

aag gcc gag gtg ale gOt tcc ale gag aac cgc gtg leg gtg cag aac 1200 Lys Ala Ala Val Ile Gly Ser Ile Glu Asn Arg Val Ser Val Gln Asn 385 39O 395 400

gac ttc gtg leg aag ctg acc gac tag atg aac aag ggc ale ggc tcc 1248 Asp Phe Val Ser Lys Leu Thr Asp Ser Met Asn Lys Gly Ile Gly Ser 405 410 415

ckg gtg gac gac gac atg aac gag gee leg agc cOc ctg cag gcc ctc 1296 Leu Val Asp Ala Asp Met Asn Glu Glu Ser Ser Arg Leu Gln Ala Leu 420 425 430

cag gtc cag cag cag ctc gac acc cag gcg ctg leg ale gcc aac cag 1344 Gln Val Gln Gln Gln Leu Ala Thr Gln Ala Leu Ser Ile Ala Asn Gln 435 440 445

ggt ccg cag aac att ctc leg ctg ttc cgc tea 1377 Gly Pro Gln Asn Ile Leu Ser Leu Phe Arg 450 455

c210> 18 c211> 458 c212> PRT c213> Artificial c220> a c223> The sequence is a combination of elements from Azaspirillum brasi lense and Trichoderma reesei c400> 18 Met Ala Ser Ile Met Thr Asn Thr Ser Ala Met Thr Ala Leu Gln Thr 1 5 10 15

Val Arg Arg Val Thr Asp Asp Leu Ala Thr Thr Gln Asp Arg Ile Ser 20 25 30

Thr Gly Leu Lys Val Asn Asn Ala Lys Asp Asn Ala Ala Tyr Trp Ser 35 40 45

le Ala Thr Thr Met Arg Ala Asp Val Ala Gly Phe Lys Ala Val Lys 50 55 60

Glu Ser Leu Glu Leu Gly Ser Gly Thr Thr Asn Thr Ala Ser Val Ala 65 70 75 80

ss ( Ser Lys Asn Ile Val Glu Asn Leu Gln Thr Leu Lys Ala Arg Val Ile Ala Gly Gln Thr Asn Gly Val Asp Lys Ser Leu Ile Gln Asn Asp Ile Asp Gln Leu Val Lys Leu Val Lys Gly Ala Ala Ala Asp Ala Ser Phe 115 120 125

Asn Gly Asp Asn Leu Leu Arg Ile Thr Tyr Ser Asn Asp Gly Thr Ala Lys Asp Gln Asn Val Asp Ile Leu Ala Ser Leu Ser Arg Ser Ala Gly Thr Val Asp Pro Ser Tyr Ile Ser Phe Gln Arg Gln Asp Met Gln Val 165 170 175

Thr Ser Ile Val Gly Lys Ala Thr Ile Glu Gln Gln Val Asp Ser Thr Asn Asp Leu Lys Ala Ser Val Gly Ile Ala Ile Gly Ala Pro Asp Thr Thr Phe Ile Asp Gly Gln Asn Leu Gly Leu Gly Arg Pro Leu Ile Asn Gly Ile Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Se!r Gly Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro 245 250 255

Tyr Tyr Ser Gln Cys Leu Gly Arg Pro Thr Leu Asn Val Thr Asn Glu Ala Gly Thr Lys Thr Ala Val Thr Val Asp Leu Ser Gly Ile Asp Tyr Thr Thr Asp Leu Ala Thr Thr Gln Gly Leu Ile Glu Thr Ala Val Asn 3AOa Ala Leu Thr Thr Ala Gly Gly Asp Phe Gln Val Ala Phe Asn Gly

Asp Lys Leu Glu Phe Thr Asp Gln Asp geu Asn Thr Asp Gly Asn Phe 325 330 33S

Thr Ala Lys Val Asp Ser Leu Trp Val Gly Ser Lys Glu Ser Asp Ala 340 345 350

Phe Gly Gly Leu Ala Asp Leu Thr Gln Ile Asp Val Thr Ser Asn Ala 355 360 365

Thr Lys Ser Leu Glu Val Ile Asn Gly Leu Leu Asp Lys Ala Ile Gly 370 375 380

Lys Ala Ala Val Ile Gly Ser Ile Glu Asn Arg Val Ser Val Gln Asn 385 390 395 400

Asp Phe Val Ser bys Leu Thr Asp Ser Met Asn Lys Gly Ile Gly Ser 405 410 415

Leu Val Asp Ala Asp Met Asn Glu Glu Ser Ser Arg Leu Gln Ala Leu 420 425 430

Gln Val Gln Gln Gln Leu Ala Thr Gln Ala Leu Ser Ile Ala Asn Gln 435 440 445

Gly Pro Gln Asn Ile Leu Ser Leu Phe Arg 450 455

<210> 19

<211> 1377 a <212> DNA -

<213> Artificial c220> c223> The sequence is a combination of elements from Azospirillum brasi lens e and Tri choderma ree se i = <220> <221> CDS

<222> (1)..(1377)

c223> <400> 19

atg gcc leg ale atg acc aac acc leg gag atg acc gcg ctg cag acc 48 Met Ala Ser Ile Met Thr Asn Thr Ser Ala Met Thr Ala Leu Gln Thr 1 5 10 15

gtg cgc cgc gtg acc gac get ctg gcg acc acc cag get cgc ale leg 96 Val Arg Arg Val Thr Asp Asp Leu Ala Thr Thr Gln Asp Arg Ile Ser 20 25 30

acc ggt ctc aag gtc aac aac gcc aag gac aac gcc gcc tac tgg leg 144

o ( Th: Gly Leu Lys Val Asn Asn Ala Lys Asp Asn Ala Ala Tyr Trp Ser 35 40 45

atc gcc acg acc atg cgc gcc gac gtc gcc ggc tec aag gcg gtg aag 192 Ile Ala Thr Thr Met Arg Ala Asp Val Ala Gly Phe Lys Ala Val LYB 50 55 60

gea tcg ctg gea ctg ggt tcg ggc acg acc aac acc gcg tcg gtg gog 240 Glu Ser Leu Glu Leu Gly Ser Gly Thr Thx Asn Thr Ala Ser Val Ala 65 70 75 80

tcc aag aac atc gtc gag aac ctg cag acg ctg aag gcc cgc gtc atc 288 Ser Lys Asn Ile Val Glu Asn Leu Gln Thr Leu Lys Ala Arg Val Ile 85 90 95

goc ggc cag acc aac ggc gtc gac aag tcg ctg atc cag aac gac atc 336 Ala Gly Gln Thr Asn Gly Val Asp Lys Ser Leu Ile Gln Asn Asp Ile 100 105 110

gac cag ctc gtc aag ctg gtg aag ggc gcc gcc gcc gac gcc tcc ttc 384 Asp Gln Leu Val Lys Leu Val Lys Gly Ala Ala Ala Asp Ala Ser Phe 115 120 125

aac ggc gac aac ctg ctg cgc atc acc tac tcc aac gac ggc acg gac 432 Asn Gly Asp Asn Leu Leu Arg Ile Thr Tyr Ser Asn Asp Gly Thr Ala 130 135 140

aag gac cag aac gtc gac atc ctg gcg tcg ctg agc cgc agc gcc ggc 480 Lys Asp Gln Asn Val Asp Ile Leu Ala Ser Leu Ser Arg Ser Ala Gly 145 150 155 160

acg gtc gat cog agc tac atc agc ttc cag cgc cag gac aLg cag gtc 528 Thr Val Asp Pro Ser Tyr Ile Ser Phe Gln Arg Gln Asp Met Gln Val 165 170 175

acc tcg atc gtc gOc aag gcc acg atc gag cag cag gtg gac tcc acc 576 Thr Ser Ile Val Gly Lys Ala Thr Ile Glu Gln Gln Val Asp Ser Thr 180 185 190 a aac gac ctg aag gcc agc gtc ggc atc goc atc ggc gcg cag gac acc 624 Asn Asp Leu Lys Ala Ser Val Gly Ile Ala Ile Gly Ala Pro Asp Thr 195 200 205

acc ttc atc gac gOc cag aac ctg ggc ctg ggc aac ctg acc ctg aac 672 Thr Phe Ile Asp Gly Gln Asn Leu Gly Leu Gly Asn Leu Thr Leu Asn 210 215 220 -

gtc acc aac gag gag ggc acc aag acc goc gtg aca gtg gac ctg teg 720 Val Thr Asn Glu Ala Gly Thr Lys Thr Ala Val Thr Val Asp Leu Ser 225 230 235 240

ggc atc gac tac acc acc gac ctg gcg acc acc cag ggc ctg atc gag 768 Gly Ile Asp Tyr Thr Thr Asp Leu Ala Thr Thr Gln Gly Leu Ile Glu 245 250 255

acc gcg gtc aac gcg gac ctg acc acg gcc ggc ggc gac ttc cag gtg 81G Thr Ala Val Asn Ala Ala Leu Thr Thr Ala Gly Gly Asp Phe Gln Val 260 2G5 270 -

gcc thc aac ggc gac aag ctg gaa ttc acc gac cag gac ctg aac acc 864 Ala Phe Asn Gly Asp Lys Leu Glu Phe Thr Asp 61n Asp Leu Asn Thr

275 280 285

gac ggc cgg cca tta att aac ggg ale acc cag leg cac tac ggc cag 912 Asp Gly Arg Pro Leu Ile Asn Gly Ile Thr Gln Ser His Tyr Gly Gln 290 295 300

tgc ggc ggc ale ggc tac agc ggc ccc acc gtg tgc gcc leg ggc acc 960 Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala Ser Gly Thr 305 310 315 320

acg tgc caa gtc ctg aac ccg tac tac tag cag tgc ctg ggc cgg cac 1008 Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu Gly Arg Pro 325 330 335

acc gcc aag gtg gac agc ctg tgg gtc ggc leg aag gaa agc gac gcc 1056 Thr Ala Lys Val Asp Ser Leu Trp Val Gly Ser Lys Glu Ser Asp Ala = 340 345 350

ttc ggc ggc ctg gcc gac ctg aeg cag ale gac gtg acc tcc aac gac 1104 Phe Gly Gly Leu Ala Asp Leu Thr Gln Ile Asp Val Thr Ser Asn Ala 355 360 365

acc aag leg ctc gag gec ale aac ggc ctg ctc gac aag gcc ale ggc 1152 Thr Lys Ser Leu Glu Val Ile Asn Gly Leu Leu Asp Lys Ala Ile Gly 370 375 380

aag gcc gcg gtg ale ggt tcc ale gag aac cgc gtg leg gtg cag aac 1200 Lys Ala Ala Val Ile Gly Ser Ile Glu Asn Arg Val Ser Val Gln Asn 385 390 395 400

gac ttc gtg leg aag ctg acc gac tag atg aac aag ggc ale ggc tcc 1248 Asp Phe Val Ser Lys Leu Thr Asp Ser Met Asn Lys Gly Ile Gly Ser 405 410 415

ctg gtg gac gcc gac atg aac gag gaa leg agc cgc ctg cag gcc ctc 1296 Leu Val Asp Ala Asp Met Asn Glu Glu Ser Ser Arg Leu Gln Ala Leu 420 425 430

cag gtc cag cag cag ctc gcc acc cag gcg ctg leg ale gcc aac cag 1344 Gln Val Gln Gln Gln Leu Ala Thr Gln Ala Leu Ser Ile Ala Asn Gln 435 440 445

ggt ccg cag aac att ctc leg ctg ttc cgc tea 1377 Gly Pro Gln Asn Ile Leu Ser Leu Phe Arg = 450 455

c210> 20 c211> 458 c212> PRT c213> Artificial c220> c223> The sequence is a combination of elements from Azospirillum brasi lense and Trichoderma reesei <400> 20

Met Ala Ser Ile Met Thr Asn Thr Ser Ala Met Thr Ala Leu Gln Thr 1 5 10 15

! Val Arg Arg Val Thr Asp Asp Leu Ala Thr Thr Gln Asp Arg Ile Ser 20 25 30

Thr Gly Leu Lys Val Asn Asn Ala Lys Asp Asn Ala Ala Tyr Trp Ser 35 40 45

Ile Ala Thr Thr Met Arg Ala Asp Val Ala Gly Phe Lys Ala Val Lys 50 55 60

Glu Ser Leu Glu Leu Gly Ser Gly Thr Thr Asn Thr Ala Ser Val Ala 65 70 75 80

Ser Lys Asn Ile Val Glu Asn Leu Gln Thr Leu Lys Ala Arg Val Ile 85 90 95

Ala Gly Gln Thr Asn Gly Val Asp Lys Ser Leu Ile Gln Asn Asp Ile 100 105 110

Asp Gln Leu Val Lys Leu Val Lys Gly Ala Ala Ala Asp Ala Ser Phe 11S 120 125

Asn Gly Asp Asn Leu Leu Arg Ile Thr Tyr Ser Asn Asp Gly Thr Ala 130 135 140

Lys Asp Gln Asn Val Asp Ile Leu Ala Ser Leu Ser Arg Ser Ala Gly 145 150 155 160

Thr Val Asp Pro Ser Tyr Ile Ser Phe Gln Arg Gln Asp Met Gln Val 165 170 175

Thr Ser Ile Val Gly Lys Ala Thr Ile Glu Gln Gln Val Asp Ser Thr 180 185 190 -

Asn Asp Leu Lys Ala Ser Val Gly Ile Ala Ile Gly Ala Pro Asp Thr 195 200 Z05

Thr Phe Ile Asp Gly Gln Asn Leu Gly Leu Gly Asn Leu Thr Leu Asn 210 215 220

Val Thr Asn Glu Ala Gly Thr Lys Thr Ala Val Thr Val Asp Leu Ser 225 230 235 240

Gly Ile Asp Tyr Thr Thr Asp Leu Ala Thr Thr Gln Gly Leu Ile Glu 245 250 255:

i Thr Ala Val Asn Ala Ala Leu Thr Thr Ala Gly Gly Asp Phe Gln Val 260 265 270

Ala Phe Asn Gly Asp Lys Leu Glu Phe Thr Asp Gln Asp Leu Asn Thr 275 280 285

Asp Gly Arg Pro Leu Ile Asn Gly Ile Thr Gln Ser His Tyr Gly Gln 290 295 300

Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala Ser Gly Thr 305 310 315 320

Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu Gly Arg Pro 325 330 335

Thr Ala Lys Val Asp Ser Leu Trp Val Gly Ser Lys Glu Ser Asp Ala 340 345 350

Phe Gly Gly Leu Ala Asp Leu Thr Gln Ile Asp Val Thr Ser Asn Ala 355 360 365

Thr Lys Ser Leu Glu Val Ile Asn Gly Leu Leu Asp Lys Ala Ile Gly 370 375 380

Lys Ala Ala Val Ile Gly Ser Ile Glu Asn Arg Val Ser Val Gln Asn 385 390 395 400

Asp Phe Val Ser Lys Leu Thr Asp Ser Met Asn Lys Gly Ile Gly Ser 405 410 415

Leu Val Asp Ala Asp Met Asn Glu Glu Ser Ser Arg Leu Gln Ala Leu 420 425 430

Gln Val Gln Gln Gln Leu Ala Thr Gln Ala Leu Ser Ile Ala Asn Gln 435 440 445

Gly Pro Gln Asn Ile Leu Ser Leu Phe Arg 450 455

c210> 21 c211> 1113 <212> DNA

c213> Artificial c220> c223> The sequence is a combination of elements from Pseudomonas fluore scens and Trichoderma reesei

<22u <221> CDS

*<222, (1)..(1113)

<223> <400> 21

atg aaa ctg aaa aac acc ttg ggc tet gcc att ggt tct att att gcc 48 Met Lys Leu Lys Asn Thr Leu Gly Phe Ala Ile Gly Ser Ile Ile Ala 1 5 10 15

gct act tct ttc ggc gca ctg goa caa ggc caa ggc gca gtt gaa ggc 96 Ala Thr Ser Phe Gly Ala Leu Ala Gln Gly Gln Gly Ala Val Glu Gly 20 25 30

gag ctg ttc tac aag aag cag tac aac gat agc get aag cac atc gaa 144 Glu Leu Phe Tyr Lys Lys Gln Tyr Asn Asp Ser Val Lys His Ile Glu 35 40 45

gac ggc ttc aac cca ggc gct cgc att ggt tac ttc ctg acc gac gac 192 Asp Gly Phe Asn Pro Gly Ala Arg Ile Gly Tyr Phe Leu Thr Asp Asp 50 55 60

ctg tog ttg aac chg tcc tac gac aaa acc aac cac acc cgt tcg aac 240 Leu Ser Leu Asn Leu Ser Tyr Asp Lys Thr Asn His Thr Arg Ser Asn 65 70 75 80

gac ggt act ggc cgg cca tta att aac ggg atc acc cag tcg cac tac 288 Asp Gly Thr Gly Arg Pro Leu Ile Asn Gly Ile Thr Gln Ser His Tyr 85 90 95

ggc cag tgc ggc ggc atc ggc tac agc ggc ccc acc gtg tgc gac teg 336 Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val Cys Ala Ser 100 105 110

ggc acc acg tgc caa gtc ctg aac ccg tac tac tag cag tgc ctg ggc 384 Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cys Leu Gly 115 120 125

cgg ccg aag atc ggt ggc gac act agc agc ctg act gct cag tac cac 432 Arg Pro Lys Ile Gly Gly Asp Thr Ser Ser Leu Thr Ala Gln Tyr His 130 135 140

ttc ggc cag gct ggc gtt gac agc ctg cgt cca tac gta gaa ggc ggt 480 Phe Gly Gln Ala Gly Val Asp Ser Leu Arg Pro Tyr Val Glu Gly Gly 145 150 155 160

ttc ggc cac caa agc cgt ggc aac gtc aaa gct gac ggc cac agc ggt 528 Phe Gly His Gln Ser Arg Gly Asn Val Lys Ala Asp Gly His Ser Gly 165 170 175

cgc gac cag tcc acc ctg gct atc gcc ggc gat gOc gtg aag tac tac 576 Arg Asp Gln Ser Thr Leu Ala Ile Ala Gly Ala Gly Val Lys Tyr Tyr 180 185 190

ttc acc aac aac gtg tac gat cgt gac ggt gtt gaa gct gac tac gca 624 Phe Thr Asn Asn Val Tyr Ala Arg Ala Gly Val Glu Ala Asp Tyr Ala 195 200 205

chg gac aac ggc aeg tgg gac tac tcc goa ctg gec gOc ctg gOt gtg 672 Leu Asp Asn Gly LYB Trp Asp Tyr Ser Ala Leu Val Gly Leu Gly Val 210 215 220

aac ttc gOc gOt aac get ggc gca gct gct cca gct cct acc cca gca 720 Asn Phe Gly Gly Asn Ala Gly Ala Ala Ala Pro Ala Pro Thr Pro Ala 225 230 235 240

cca get cca gag cca act cca gag cca gaa gct cca gtt gct cag gtt 768 Pro Ala Pro Glu Pro Thr Pro Glu Pro Glu Ala Pro Val Ala Gln Val 245 250 255

gtt cgt ght gag ctg gac gtt aag ttc gac ttc gac aag tog gtt gtt 816 Val Arg Val Glu Leu Asp Val Lys Phe Asp Phe Asp Lys Ser Val Val 260 265 270

aag cct aac agc tac ggc gac gtg aaa aac ctg gcc gac ttc atg gct 864 Lys Pro Asn Ser Tyr Gly Asp Val Lys Asn Leu Ala Asp Phe Met Ala 275 2B0 285

cag tac cca gct acc aac gta gaa gtt get ggt cac act gac tcc ale 912 Gln Tyr Pro Ala Thr Asn Val Glu Val Ala Gly His Thr Asp Ser Ile 290 295 300

ggt cca gac gcc tac aac cag aag ctg tcc cag cgt cOt get gac cgt 960 Gly Pro Asp Ala Tyr Asn Gln Lys Leu Ser Gln Arg Arg Ala Asp Arg -

305 310 315 320

gtt aag caa gtc ctg gUt aaa gac gOc gtt gct cct agc cgc ale acc 1008 Val Lys Gln Val Leu Val Lys Asp Gly Val Ala Pro Ser Arg Ile Thr 325 330 335

gcg gta ggt tac ggc gaa tcc cgc cca gtt gct gac aac gca act gaa 1056 Ala Val Gly Tyr Gly Glu Ser Arg Pro Val Ala Asp Asn Ala Thr Glu: 340 345 350 -

gct ggt cgc gct gtt aac cgt cgc gta gaa gcg tog gtt gee got caa 1104 Ala Gly Arg Ala Val Asn Arg Arg Val Glu Ala Ser Val Glu Ala Gln 355 360 365

get cag tea 1113 Ala Gln -

c210> 22 <211> 370

c212> PRT <213> Artificial <220> c223> The sequence is a combination of elements from Pseudomonas fluore scens and Trichoderma reesei c400> 22 Met Lys Leu Lys Asn Thr Leu Gly Phe Ala Ile Gly Ser Ile Ile Ala 1 S 10 15

Ala Thr Ser Phe Gly Ala Leu Ala Gln Gly Gln Gly Ala Val Glu Gly 20 25 30 -

Glu Leu Phe Tyr Lys Lys Gln Tyr Asn Asp Ser Val Lys His Ile Glu 35 40 45

Asp Gly Phe Asn Pro Gly Ala Arg Ile Gly Tyr Phe Leu Thr Asp Asp 50 55 60

Leu Ser Leu Asn Leu Ser Tyr Asp Lys Thr Asn His Thr Arg Ser Asn 65 70 75 80

Asp Gly Thr Gly Arg Pro Leu Ile Asn Gly Ile Thr Gln Ser His Tyr 85 90 95

Gly Gln Cys Gly Gly 1le Gly Tyr Ser Gly Pro Thr Val Cy5 Ala Ser 100 105 110

Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln Cy6 Leu Gly 115 120 125

Arg Pro Lys Ile Gly Gly Asp Thr Ser Ser Leu Thr Ala Gln Tyr His 130 135 140

Phe Gly Gln Ala Gly Val Asp Ser Leu Arg Pro Tyr Val Glu Gly Gly 145 lSO 155 160 Phe Gly Xis Gln Ser Arg Gly Asn Val Lys Ala Asp Gly His Ser Gly 165 170 175

Arg Asp Gln Ser Thr Leu Ala Ile Ala Gly Ala Gly Val Lys Tyr Tyr 180 185 190

Phe Thr Asn Asn Val Tyr Ala Arg Ala Gly Val Glu Ala Asp Tyr Ala 195 200 205

Leu Asp Asn Gly Lys Trp Asp Tyr Ser Ala Leu Val Gly Leu Gly Val 210 215 220

Asn Phe Gly Gly Asn Ala Gly Ala Ala Ala Pro Ala Pro Thr Pro Ala 225 230 235 240

Pro Ala Pro Glu Pro Thr Pro Glu Pro Glu Ala Pro Val Ala Gln Val 245 250 265

Val Arg Val Glu Leu Asp Val Lys Phe Asp Phe Asp Lys Ser Val Val 260 265 270

Lys Pro Asn Ser Tyr Gly Asp Val Lys Asn Leu Ala Asp Phe Met Ala

/ ( 275 280 285

Gln Tyr Pro Ala Thr Asn Val Glu Val Ala Gly His Thr Asp Ser Ile 290 295 300

Gly Pro Asp Ala Tyr Asn Gln Lys Leu Ser Gln Arg Arg Ala Asp Arg 305 310 315 320

Val Lys Gln Val Leu Val Lys Asp Gly Val Ala Pro Ser Arg Ile Thr 325 330 335

Ala Val Gly Tyr Gly Glu Ser Arg Pro Val Ala Asp Asn Ala Thr Glu 340 345 350

Ala Gly Arg Ala Val Asn Arg Arg Val Glu Ala Ser Val Glu Ala Gln 355 360 365

Ala Gln <210> 23

<211> 1113

<212> DNA

<213> Artificial <220> c223> The sequence is a combination of elements from Pseudomonas fluore scens and Trichoderma reesei <220> c221> CDS <222> (1)..(1113)

<223> <400> 23

atg aaa ctg aaa aac acc ttg ggc ttt gcc att ggt let att att gcc 48 Met Lys Leu Lys Asn Thr Leu Gly Phe Ala Ile Gly Ser Ile Ile Ala 1 5 10 15

gct act let tcc ggc gca ctg gca caa ggc caa ggc gca gtt gaa ggc 96 Ala Thr Ser Phe Gly Ala Leu Ala Gln Gly Gln Gly Ala Val Glu Gly 20 25 30

gag ctg ttc tac aag aag cag tac aac get agc gtt aag cac ale gaa 144 Glu Leu Phe Tyr Lys Lys Gln Tyr Asn Asp Ser Val Lys His Ile Glu 35 40 45

gac ggc ttc aac cca ggc gct cgc att ggt tac ttc ctg acc gac gac 192 Asp Gly Phe Asn Pro Gly Ala Arg Ile Gly Tyr Phe Leu Thr Asp Asp 50 55 60

ctg leg ttg aac ctg tcc tac gac aaa acc aac cac acc cgt leg aac 240 Leu Ser Leu Asn Leu Ser Tyr Asp Lys Thr Asn His Thr Arg Ser Asn 65 70 75 80

gac ggt act ggc agc cag aag ale ggt ggc gac act agc agc ctg act 288 Asp Gly Thr Gly Ser Gln Lys Ile Gly Gly Asp Thr Ser Ser Leu Thr 85 90 95

got cag tac cac ttc ggc cag gct ggc gtt gac agc ctg cgt cca tac 336 Ala Gln Tyr His Phe Gly Gln Ala Gly Val Asp Ser Leu Arg Pro Tyr 100 105 110

gta gaa ggc ggt ttc ggc cac caa agc cgt ggc aac gtc aaa gct gac 384 Val Glu Gly Gly Phe Gly His Gln Ser Arg Gly Asn Val Lys Ala Asp 115 120 125

ggc cac agc ggt cgc gac cag tcc acc ctg gct ale gcc ggc gct ggc 432 Gly His Ser Gly Arg Asp Gln Ser Thr Leu Ala Ile Ala Gly Ala Gly 130 135 140 gtg aag tac tac ttc acc aac aac gtg tac get cgt gcc ggt gtt gaa 480 Val

Lys Tyr Tyr Phe Thr Asn Asn Val Tyr Ala Arg Ala Gly Val Glu 145 150 155 160

gct gac tac gca ctg gac aac ggc aag tgg gac tac tcc gca ctg gtc 528 Ala Asp Tyr Ala Leu Asp Asn Gly Lys Trp Asp Tyr Ser Ala Leu Val 165 170 175

gOc ctg ggt gUg aac ttc ggc ggt aac gct gOc gca gct gct cca gct 576 Gly Leu Gly Val Asn Phe Gly Gly Asn Ala Gly Ala Ala Ala Pro Ala 180 185 190

cat acc cca gca cca gct cca ggc cgg cca tta att aac ggg ale acc G24 Pro Thr Pro Ala Pro Ala Pro Gly Arg Pro Leu Ile Asn Gly Ile Thr 195 200 205

cag leg cac tac ggc cag tgc ggc ggc ale ggc tac agc ggc ccc acc 672 Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr 210 215 220

gtg tgc gac leg ggc acc acg tgc caa gtc ctg aac ccg tac tac tag 720 Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser 225 230 235 240

cag tgc ckg ggc cgg cct cca gag cca gaa gct cca gtt gct cag gtt 768 Gln Cys Leu Gly Arg Pro Pro Glu Pro Glu Ala Pro Val Ala Gln Val 245 250 255

gtt cgt gtt gag ctg gac gtt aag ttc gac ttc gac aeg tog gtt gtt 816 Val Arg Val Glu Leu Asp Val Lys Phe Asp Phe Asp Lys Ser Val Val 260 265 270

aag cct aac agc tac ggc gac gtg aaa aac ctg gcc gac ttc aLg gct 864 Lys Pro Asn Ser Tyr Gly Asp Val Lys Asn Leu Ala Asp Phe Met Ala 275 280 285

cag tac cca get acc aac gta gaa gtt gct ggt cac act gac tcc ale 912 Gln Tyr Pro Ala Thr Asn Val Glu Val Ala Gly His Thr Asp Ser Ile 290 295 300

gOt cca gac gac tac aac cag aag ctg tcc cag cOt cgt gct gac cOt 960 Gly Pro Asp Ala Tyr Asn Gln Lys Leu Ser Gln Arg Arg Ala Asp Arg 305 310 315 320

q (, gtt aag caa gtc ctg gEt aaa gac ggc ght gct cct agc cOc ale acc 1008 Val Lys Gln val Leu Val Lys Asp Gly Val Ala Pro Ser Arg Ile Thr 325 330 335

gcg gta ggt tac ggc gaa tcc cgc cca gtt gct gac aac gca act gaa 1056 Ala Val Gly Tyr Gly Glu Ser Arg Pro Val Ala Asp Asn Ala Thr Glu 340 345 350

gct ggt cOc gct gtt aac cgt cgc gta gaa gcg leg gtt gaa got caa 1104 Ala Gly Arg Ala Val Asn Arg Arg val Glu Ala Ser Val Glu Ala Gln 355 360 365

get cag tea 1113 Ala Gln c210> 24 c211> 370 <212> PRT

<213> Artificial c220> c223> The sequence is a combination of elements from Pseudomonas fluore scens and Trichoderma reesei c400> 24 Met Lys Leu Lys Asn Thr Leu Gly Phe Ala Ile Gly Ser Ile Ile Ala 1 5 10 15

Ala Thr Ser Phe Gly Ala Leu Ala Gln Gly Gln Gly Ala Val Glu Gly 20 25 30

Glu Leu Phe Tyr Lys Lys Gln Tyr Asn Asp Ser Val Lys HiB Ile Glu 35 40 45

Asp Gly Phe Asn Pro Gly Ala Arg Ile Gly Tyr Phe Leu Thr Asp Asp 50 55 60

Leu Ser Leu Asn Leu Ser Tyr Asp Lys Thr Asn His Thr Arg Ser Asn 65 70 75 80

Asp Gly Thr Gly Ser Gln Lys Ile Gly Gly Asp Thr Ser Ser Leu Thr 85 90 95

Ala Gln Tyr His Phe Gly Gln Ala Gly Val Asp Ser Leu Arg Pro Tyr 100 105 110

Val Glu Gly Gly Phe Gly His Gln Ser Arg Gly Asn Val Lys Ala Asp 115 120 125

Gly His Ser Gly Arg Asp Gln Ser Thr Leu Ala Ile Ala Gly Ala Gly

7o ( 130 135 140

Val Lys Tyr Tyr Phe Thr Asn Asn Val Tyr Ala Arg Ala Gly Val Glu 145 150 155 160

Ala Asp Tyr Ala Leu Asp Asn Gly Lys Trp Asp Tyr Ser Ala Leu Val 165 170 175

Gly Leu Gly Val Asn Phe Gly Gly Asn Ala Gly Ala Ala Ala Pro Ala 180 185 190

Pro Thr Pro Ala Pro Ala Pro Gly Arg Pro Leu Ile Asn Gly Ile Thr 195 200 205

Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr 210 215 220

Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser 225 230 235 240

Gln Cys Leu Gly Arg Pro Pro Glu Pro Glu Ala Pro Val Ala Gln Val 245 250 255

Val Arg Val Glu Leu Asp Val Lys Phe Asp Phe Asp Lys Ser Val Val 260 265 270

Lys Pro Asn Ser Tyr Gly Asp Val Lys Asn Leu Ala Asp Phe Met Ala 275 280 285

Gln Tyr Pro Ala Thr Asn Val Glu Val Ala Gly His Thr Asp Ser Ile 290 295 300

Gly Pro Asp Ala Tyr Asn Gln Lys Leu Ser Gln Arg Arg Ala Asp Arg 305 310 315 320

Val Lys Gln Val Leu Val Lys Asp Gly Val Ala Pro Ser Arg Ile Thr 325 330 335

Ala Val Gly Tyr Gly Glu Ser Arg Pro Val Ala Asp Asn Ala Thr Glu 340 345 350

Ala Gly Arg Ala Val Asn Arg Arg Val Glu Ala Ser Val Glu Ala Gln 355 360 365

Ala Gln

Claims (1)

1\ CLAIMS

1. A fusion protein comprising: a cellulose binding domain peptide; and a cell surface polypeptide capable of causing the cellulose binding domain peptide to be exposed on the surface of a cell.

5 2. A fusion protein according to claim 1, wherein the cellulose binding domain peptide comprises the sequence BXQCGGX--XB-XXXX-CX)O<-XCXXXXX-XXXQC

wherein "B is an amino acid having an aromatic group; "X" is any amino acid and is any or no amino acid.

10 3. A fusion protein according to claim 2, wherein the cellulose binding domain peptide has a sequence comprising BXQCGGX--XXXXX-CXXX-XCX) O(NX-BYXQC

wherein BIB" is an amino acid having an aromatic group; hX" is any amino acid and "-"

is any or no amino acid.

15 4. A fusion protein according to claim 2, wherein the cellulose binding domain peptide comprises the following sequence YXXXXXX--XX-XXXXXXXX-XXXXXNX--WXQ

wherein "X" is any amino acid and a is any or no amino acid.

20 5. A fusion protein according to claim 3 or 4, wherein the cellulose binding domain peptide comprises the sequence TQSHYGQCGGIGYSGPTVCASGTTCQVLNPWSQCL

6. A fusion protein according to any one of the preceding claims, wherein the cell surface polypeptide comprises the sequence of SEQ ID NO.:4, SEQ ID NO.: 6 or 25 SEQ ID NO.: 8.

7. A fusion protein according to claim 6 comprising the sequence of SEQ ID NO.: 12, SEQ ID NO.: 14, SEQ ID NO.: 16, SEQ ID BO.:18, SEQ ID NO.:20, SEQ

ID NO.: 22 or SEQ ID NO.: 24.

8. A fusion protein according to any one of the preceding claims wherein the cell 5 is a microorganism, preferably a bacterium or fungus.

9. A nucleic acid encoding a fusion protein according to any one of the preceding claims.

10. A vector comprising the nucleic acid of claim 9.

11. A vector according to claim 10, wherein the vector is a plasmid.

10 12. A cell transformed with a nucleic acid encoding a cellulose binding domain peptide such that, when the nucleic acid is expressed, the cellulose binding domain peptide is exposed on the surface of the cell.

13. A cell according to claim 12 wherein the cellulose binding domain peptide comprises the sequence 15 BXQCGGX-XB-XXXX-CXXX-XCXX)(XX--X)O(QC

wherein "B" is an amino acid having an aromatic group; UX" is any amino acid and a w is any or no amino acid.

14. A cell according to claim 13 wherein the cellulose binding domain peptide has a sequence comprising 20 BXQCGGX--XB-XX)(X-CXXX-XCXXXNX--BYXQC

wherein "B" is an amino acid having an aromatic group; "X" is any amino acid and "_n is any or no amino acid.

15. A cell according to claim 13, wherein the cellulose binding domain peptide comprises the following sequence 25 YXXXXXX-XX-X)(XX-XXX)(XXYV(XNX-WXQ

( wherein AXE is any amino acid and _r iS any or no amino acid.

16. A cell according to claim 14 or 15, wherein the cellulose binding domain peptide comprises the sequence TQSHYGQCGGIGYSGPIVCASGTTCQVLNPWSQCL

5 17. A cell according to any one of claims 12 to 16, wherein the nucleic acid is a nucleic acid according to claim 10.

18. A cell according to any one of claims 12 to 17, wherein the cell is a bacterium or a fungal cell.

19. A cell according to claim 18, wherein the bacterium is a Plant-Growth 10 Promoting Rhizobacterium.

20. A cell according to claim 19, wherein the bacterium is Azosp/nilum brasilense.

21. A plant seed coated with one or more cells according to any one of claims 12 to20. 22. A method of increasing cellular adhesion comprising transforming a cell with a 15 cellulose binding domain peptide such that the cellulose binding domain peptide is exposed on the surface of the cell.

23. A method according to claim 22 comprising transforming the cell with a nucleic acid according to claim 9.

24. Use of a cellulose binding domain peptide to improve cellular adhesion.

20 25. Use according to claim 24, wherein the cellulose binding domain peptide is part of a fusion protein according to any one of claims 1 to 8.

26. A method of improving plant growth or health, prophylactically or otherwise comprising applying a cell according to any one of claims 12 to 20 to the roots of a plant.