Tardigrade secretory proteins stabilize biological structures

Abstract

Described herein are methods and compositions relating to tardigrade secreted-abundant heat soluble (SAHS) proteins.

Description

Attorney Docket No.002806-000126WOPT TARDIGRADE SECRETORY PROTEINS STABILIZE BIOLOGICAL STRUCTURES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 605,229 filed December 1, 2023, the contents of which are incorporated herein by reference in their entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under 1752172 awarded by National Science Foundation (NSF) and under N000141912317 awarded by U.S. Office of Naval Research (NAVY / ONR) and under W911NF1920017 awarded by U.S. Department of Defense / Defense Advanced Research Projects Agency (DOD / DARPA). The government has certain rights in this invention. TECHNICAL FIELD

[0003] The technology disclosed herein relates to proteins that stabilize biological structures. BACKGROUND

[0004] Water is the universal solvent essential for survival. However, several organisms demonstrate remarkable abilities to withstand almost complete loss of water for a prolonged time and recover upon rehydration. [1,2] This phenomenon of “anhydrobiosis”, or “life without water”, has attracted a great deal of attention because it may reveal Nature’s strategies to endure adverse environments and open up ways to achieve efficient preservation of living matter. However, the precise mechanisms behind anhydrobiosis remain elusive, as they may involve a complex combination of protective proteins, solutes and cellular machineries working in a concert. [3]

[0005] Several molecules have been identified as mediators of desiccation tolerance in various organisms. One is the disaccharide trehalose, which accumulates in a large quantity in animals such as brine shrimps or nematodes. [4] Trehalose uptake enhances survival of yeast and human cells under drying conditions, and may protect dehydrated cells by vitrification (turning the cell interior into a glass- like state). [4-6] Several “intrinsically disordered proteins (IDPs)”, which lack fixed tertiary structure, have also been associated with desiccation tolerance. [2] For instance, late embryogenesis-abundant (LEA) proteins are tolerance molecules found in plant seeds and anhydrobiotic species. [7] LEA proteins are mostly unstructured in aqueous environment, and assume helical structure upon desiccation to alleviate damage due to drying. [7, 8]

[0006] Recent studies on the survival mode of tardigrades revealed novel classes of stress proteins. Tardigrades (water bears) are microscopic animals that can survive under a broad range of environmental stressors including desiccation, high and low temperatures, radiation, and even an exposure to outerAttorney Docket No.002806-000126WOPT space. [9] Tardigrades express LEA proteins, and also three different families termed “tardigrade disordered proteins” (TDPs) unique to the phylum Tardigrada. [10-12] Some of these TDPs are highly disordered and less likely to undergo aggregation at high temperatures. (The inference that TDPs are in fact disordered is indirect and not established for every such protein.) Three different groups of TDPs were termed cytoplasmic-, mitochondrial- and secreted-abundant heat soluble (CAHS, MAHS, SAHS) proteins according to their putative subcellular localization. In each family the proteins show sequence similarity. [13, 14] TDPs are either constitutively expressed at high levels or enriched upon desiccation, and knocking out even a certain CAHS or SAHS gene can cause diminished desiccation tolerance.

[0015]

[0007] SAHS proteins are classified as secreted as they are generally encoded with signal sequences and are exported to the extracellular space upon expression in eukaryotes.

[0013] Fukuda et al. solved the structure of Ramazzottius varieornatus SAHS1 and SAHS4 [16, 17], indicating that, at least under hydrated conditions, the SAHS proteins have defined structures and their description as “intrinsically disordered proteins” is not accurate. RvSAHS1 and 4 proteins share sequence and structural similarity to fatty acid binding proteins (FABPs): their structures include β-barrels with large internal cavities that correspond to the fatty acid binding pockets in FABPs. [16, 17]. The function of these large cavities in the SAHS proteins is unclear, as is their biochemical mechanism in protecting against desiccation.

[0008] References 1. Moberg, N. et al. Survival in extreme environments – on the current knowledge of adaptations in tardigrades. Acta Physiol.202, 409-420 (2011). 2. Boothby, T. C. & Pielak, G. J. Intrinsically disordered proteins and desiccation tolerance: elucidating functional and mechanistic underpinnings of anhydrobiosis. BioEssays 39, 1700119 (2017). 3. Belott, C., Janis, B. & Menze, M. A. Liquid-liquid phase separation promotes animal desiccation tolerance. Proc. Natl. Acad. Sci. U. S. A.117, 27676-27684 (2020). 4. Tanaka, A. et al. Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel. PLoS Biol. 20, e3001780; 10.1371 / journal.pbio.3001780 (2022). 5. Tapia, H., Young, L., Fox, D., Bertozzi, C. R. & Koshland, D. Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. A.112, 6122-6127 (2015). 6. Li, S. et al. Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation. Proc. Natl. Acad. Sci. U. S. A.109, 20859-20864 (2012). 7. Hand, S. C., Menze, M. A., Toner, M., Boswell, L. & Moore, D. LEA proteins during water stress: not just for plants anymore. Annu. Rev. Physiol.73, 115-134 (2011).Attorney Docket No.002806-000126WOPT 8. Veling, M. T. et al. Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells. ACS Synth. Biol.11, 1292-1302 (2022). 9. Persson, D. et al. Extreme stress tolerance in tardigrades: surviving space conditions in low earth orbit. J. Zool. Syst. Evol. Res.49, 90-97 (2011). 10. Hengherr, S., Heyer, A. G., Köhler, H. R. & Schill, R. O. Trehalose and anhydrobiosis in tardigrades–evidence for divergence in responses to dehydration. FEBS J.275, 281-288 (2008). 11. Ingemar Jonsson, K. & Persson, O. Trehalose in three species of desiccation tolerant tardigrades. Open Zool. J.3, 1-5 (2010). 12. Hesgrove, C. & Boothby, T. C. The biology of tardigrade disordered proteins in extreme stress tolerance. Cell Commun. Signal.18, 178; 10.1186 / s12964-020-00670-2 (2020). 13. Yamaguchi, A. et al. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade. PLoS One 7, e44209; 10.1371 / journal.pone.0044209 (2012). 14. Tanaka, S. et al. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic tardigrade improve osmotic tolerance of human cells. PLoS One 10, e0118272; 10.1371 / journal.pone.0118272 (2015). 15. Boothby, T. C. et al. Tardigrades use intrinsically disordered proteins to survive desiccation. Mol. Cell.65, 975-984 (2017). 16. Fukuda, Y., Miura, Y., Mizohata, E. & Inoue, T. Structural insights into a secretory abundant heat‐ soluble protein from an anhydrobiotic tardigrade, Ramazzottius varieornatus. FEBS Lett. 591, 2458-2469 (2017). 17. Fukuda, Y. & Inoue, T. Crystal structure of secretory abundant heat soluble protein 4 from one of the toughest “water bears” micro‐animals Ramazzottius varieornatus. Protein Sci. 27, 993-999 (2018). SUMMARY

[0009] Here, investigations focused on the proteins belonging to the secretory family of tardigrade proteins termed secreted-abundant heat soluble (“SAHS”) proteins, and investigated their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevented desiccated liposomes from fusion, and enhanced desiccation tolerance of bacteria, exemplified by E. coli and Rhizobium tropici, upon extracellular application. Molecular dynamics simulation and comparative structural analysis indicate a model by which SAHS proteins undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the applications of SAHS proteins as stabilizing molecules for lipid membrane containing structures, and uses including but not limited toAttorney Docket No.002806-000126WOPT stabilization or preservation of liposome formulations or liposome-containing preparations, and, for example, cells, such as bacterial cells.

[0010] In one aspect, described herein is a dried composition comprising a tardigrade secreted- abundant heat-soluble (SAHS) polypeptide in admixture with a viable microbe. It is specifically contemplated that any species of tardigrade SAHS can be used. However, in one embodiment of this or any other aspect described herein, the SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

[0011] In another embodiment of this or any other aspect described herein, the SAHS polypeptide is selected from those set out in Table 1.

[0012] In another aspect, described herein is a composition comprising a tardigrade SAHS polypeptide in admixture with a liposome preparation. It is specifically contemplated that any species of tardigrade SAHS can be used. However, in one embodiment of this or any other aspect described herein, the SAHS polypeptide comprises a R. varieornatus or H. exemplaris SAHS polypeptide.

[0013] In another embodiment of this or any other aspect described herein, the SAHS polypeptide is selected from those set out in Table 1.

[0014] In another embodiment of this or any other aspect described herein, the composition is a dried composition.

[0015] In another embodiment of this or any other aspect described herein, the liposomes retain liposome structure upon rehydration. For example, the liposomes do not exhibit substantial aggregation upon drying and re-constitution.

[0016] In another aspect, described herein is a method of stabilizing a liposomal membrane, the method comprising contacting the liposomal membrane with a tardigrade SAHS polypeptide. It is specifically contemplated that any species of tardigrade SAHS can be used. However, in one embodiment of this or any other aspect described herein, the SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

[0017] In another embodiment of this or any other aspect describe herein, the SAHS polypeptide comprises a R. varieornatus or H. exemplaris SAHS polypeptide.

[0018] In another embodiment of this or any other aspect describe herein, the SAHS polypeptide is selected from those set out in Table 1.

[0019] In another aspect, described herein is a method of stabilizing a cosmetic formulation, the method comprising contacting the cosmetic formulation with a tardigrade SAHS polypeptide. It is specifically contemplated that any species of tardigrade SAHS can be used. However, in one embodiment of this or any other aspect described herein, the SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.Attorney Docket No.002806-000126WOPT

[0020] In another embodiment of this or any other aspect describe herein, the SAHS polypeptide comprises a R. varieornatus or H. exemplaris SAHS polypeptide.

[0021] In another embodiment of this or any other aspect describe herein, the SAHS polypeptide is selected from those set out in Table 1.

[0022] In another embodiment of this or any other aspect described herein, the method further comprises contacting the cosmetic formulation with trehalose.

[0023] In another embodiment of this or any other aspect described herein, the cosmetic formulation comprises liposomes.

[0024] In another aspect, described herein is a composition comprising a cosmetic formulation in admixture with a tardigrade SAHS polypeptide. It is specifically contemplated that any species of tardigrade SAHS can be used. However, in one embodiment of this or any other aspect described herein, the SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

[0025] In another embodiment of this or any other aspect described herein, the composition further comprises trehalose.

[0026] In another embodiment of this or any other aspect described herein, the cosmetic formulation comprises liposomes.

[0027] In another embodiment of this or any other aspect described herein, the SAHS polypeptide is selected from those set out in Table 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figs.1A-1B. Fig.1A depicts classification of tardigrade-specific desiccation-protective proteins. Tardigrades express unique sets of proteins termed as tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are further classified into three different subgroups, Cytosolic-, Mitochondrial- and Secretory-abundant heat soluble (C / M / SAHS) proteins based on their subcellular localization. Fig. 1B depicts an alignment of SAHS1-13 of R. varieornatus, SAHS4 of H. exemplaris, and two FABP (fatty acid binding protein) sequences, with amino acids whose side chains point inward toward the open cavity, and those with side chains in the hydrophobic core highlighted. Underlined amino acid sequences at the N-termini of SAHS9, 10 and 12 are newly identified, based on analysis of the R. varieornatus genome sequence. Underlined and bold sequences are putative N-linked glycosylation sites based on the consensus sequence Asn-X-Ser / Thr (X not Pro).

[0029] Figs.2A-2H demonstrate that tardigrade SAHS proteins stabilize liposomes from desiccation- induced damages. POPC liposomes at 1.4 mg / mL were dried with and without the addition of SAHS proteins and BSA at varying concentrations of 0.1 – 10 mg / mL, and their size distributions wereAttorney Docket No.002806-000126WOPT measured by DLS. (Fig.2A) Size distribution of non-dried POPC liposomes (Fig.2BB) Size distribution of POPC liposomes dried and rehydrated without additives. (Figs.2C-2G) Size distributions of the liposomes dried with (Fig.2C) RvSAHS1, (Fig.2D) RvSAHS4, (Fig.2E) HySAHS4, (Fig.2F) RvSAHS6, (Fig.2G) BSA and (Fig.2H) trehalose.

[0030] Figs.3A-3B demonstrate that tardigrade SAHS proteins enhance bacterial desiccation survival. (Fig.3A) E. Coli cells were dried for 48 hours with and without addition of 0.5 mg / mL concentration of SAHS proteins, BSA and trehalose. Survival percentage was calculated by comparing the number of viable colonies after plating the rehydrated cells to the initial number of cells added. (Fig. 3B) R. tropici cells were dried for 48 hours with and without addition of 0.5 mg / mL concentration of SAHS proteins, BSA and trehalose. Survival percentage was calculated the same way as E. coli experiment. Individual data points represent independent replicates and lines represent the mean survival. Student’s t-test was used to determine the statistical significance between the negative control (no excipient) and each group, which is indicated as asterisks. * p < 0.05; ** p < 0.01; *** p < 0.001.

[0031] Figs.4A-4B demonstrate that tardigrade SAHS proteins undergo structural changes upon dehydration-mimicking conditions. (Fig.4A) CD spectra of SAHS proteins upon addition of increasing amounts of TFE from 0 – 75%. (Fig.4B) Secondary structure compositions of SAHS proteins under different TFE level, calculated from the CD spectra.

[0032] Figs.5A-5D depict MD simulation of SAHS protein structural change upon desolvation. (Fig. 5A) RMSD over time during the first 100 ns MD simulation of RvSAHS1 homotrimer. Results during the first 100 ns are magnified. Different shading indicates each monomer chain in a trimer. Representative images of the RvSAHS1 protein structures reconstituted from the MD simulation at 0, 50 and 100 ns time points are provided at the top. (Fig.5B) Changes in secondary structure components of RvSAHS1 during the MD simulation. (Fig.5C) Representative image showing how conserved beta-sheet motifs of RvSAHS1 becomes closely aligned upon structural shift into helices. Only the conserved C3 segments of RvSAHS1 of each monomer are highlighted to show the changes in their alignments as the simulation progresses. (Fig.5D) Detailed image of C3 motif sheet-to-helix structural change (left). Highlighted are two highly conserved regions found within the beta sheet region that directly interact through ionic bonds. Structures of these regions at 0 and 180 ns are indicated (right).

[0033] Figs.6A-6B demonstrate that glycosylation sites in the SAHS proteins of R. varieornatus cluster near the cavity opening. (Fig.6A) Structure of SAHS1 from Fukuda et al.

[0016] , showing an alpha- carbon trace of all amino acids, with the beta-hairpins surrounding the cavity. Amino acids with highlighted side chains shown define the hydrophobic core. Large spheres show the alpha carbons of asparagines in an Asn-X-Ser / Thr sequence in at least one SAHS protein. The putative opening of the cavity is on the left. (Fig.6B) Structure of SAHS1, rotated 90oC relative to panel A with the left sideAttorney Docket No.002806-000126WOPT coming forward. All the side chains are shown. This view illustrates the opening into the cavity of the protein, such that the highlighted hydrophobic core side chains at the back of the protein can be seen through this hole.

[0034] Fig.7 depicts the sequence design and purification of the SAHS proteins.

[0035] Fig.8 depicts expression and purification of the SAHS proteins from E. Coli host. Left gel shows the SDS-PAGE of E. coli whole cell expressing each SUMO-SAHS construct, and the right gel shows the final protein products after lysis, purification and SUMO cleavage. Each number indicates corresponding RvSAHS protein, and “4H” indicates HeSAHS4. RvSAHS1, 4, 6 and HeSAHS4 showed high soluble expression, whereas RvSAHS2, 7, 8 showed low soluble expression and the rest were expressed as insoluble fraction.

[0036] Figs.9A-9H depict data from a repeat experiment showing that SAHS proteins stabilize liposomes from desiccation-induced damages. POPC liposomes at 1.4 mg / mL were dried with and without the addition of SAHS proteins and BSA at varying concentrations of 0.1 – 10 mg / mL, and their size distributions were measured by DLS. (Fig.9A) Size distribution of non-dried POPC liposomes (Fig. 9B) Size distribution of POPC liposomes dried and rehydrated without additives. (Figs.9C-9G) Size distributions of the liposomes dried with (Fig.9C) RvSAHS1, (Fig.9D) RvSAHS4, (Fig.9E) HySAHS4, (Fig.9F) RvSAHS6, (Fig.9G) BSA and (Fig.9H) trehalose.

[0037] Figs.10A-10B. (Fig.10A) Tardigrade SAHS proteins protect dried enzyme activity. LDH enzymes at 0.01 mg / mL were desiccated and rehydrated with and without the addition of SAHS proteins and BSA. Percent activity was determined using non-desiccated control samples stored at 4ºC as the reference to compare the activity. (Fig.10B) The data from the same experiment as (Fig.10A), depicted using molar ratio between excipient and LDH enzyme as the x-axis. Error bars represent the standard deviations from triplicate measurements.

[0038] Figs.11A-11D depict the desiccation protection of bacterial cells by SAHS proteins. (Fig. 11A) Desiccation survival percentage of E. coli cells dried for 48 hours with 0.5 mg / mL concentrations of control proteins including BSA, lactate dehydrogenase (LDH) and citrate synthase (CS). (Fig.11B) Desiccation survival percentage of E. coli cells dried with 0.1 mg / mL concentration of extracellularly added SAHS proteins and control excipients. (Fig.11C) SDS-PAGE of the whole E. Coli cells intracellularly overexpressing heterologous RvSAHS1, HySAHS4 and GFP. (Fig.11D) Comparison of desiccation survival of the cells intracellularly expressing each protein. Individual data points represent independent replicates and lines represent the mean survival. Student’s t-test was used to determine the statistical significance between the negative control (no excipient) and each group, which is indicated as asterisks. * p < 0.05; ** p < 0.01; *** p < 0.001.Attorney Docket No.002806-000126WOPT

[0039] Figs.12A-12B depict the effect of molecular crowding on SAHS protein structure. SAHS protein secondary structures upon glycerol addition were determined using circular dichroism. (Fig.12A) CD spectra of SAHS proteins upon addition of increasing amounts of glycerol from 0 – 75%. (Fig.12B) Secondary structure compositions of SAHS proteins under different glycerol level, calculated from the CD spectra.

[0040] Fig.13 depicts TEM images of the fibrous network structure formed by RvSAHS1 proteins dried at 1 mg / mL (left) and 10 mg / mL (right) concentrations. Scale bar = 200 nm.

[0041] Figs.14A-14G depict the MD simulation of RvSAHS1 protein. (Fig.14A) Changes in the structures of RvSAHS1 trimer during the first 100 ns of simulation. (Fig.14B) RMSD over time during the entire 1 microsecond of MD simulation. Each series indicates each monomer chain in a trimer. (Fig. 14C) Conserved motifs C1-C3 of RvSAHS1 protein sequence in a single monomer. Amino acid sequence of RvSAHS1 is indicated. (Figs.14D-14F) The distribution of sheet, helix and coil conformations of amino acid backbones for each of the three SAHS1 proteins in the simulation. The sheet / helix / coil classification is based on the psi / phi dihedral angles of each amino acid, and not on hydrogen-bonding patterns that may or may not be present. (Fig.14G) State of the simulation at 100 nsec (left), and a segment from each of the three SAHS1 proteins represented to illustrate the formation of short helical segments. During the course of the high-temperature simulation, helical segments throughout the proteins are unstable.

[0042] Figs.15A-15C depict the MD simulation showing structural changes in the conserved C3 motif of RvSAHS1. (Fig.15A) Representative structural changes in C3 motif during the first 170 ns of simulation. This shows that this motif is highly conserved among the SAHS family. (Fig.15B) Close-up representation of the C3 motif sheet-to-helix structural change. Highlighted are two highly conserved regions found within the beta sheet region that directly interact through ionic bonds. Structures of these regions at 0 and 180 ns are indicated, along with an additional depiction of the same regions in which positive residues (Lys, Arg) and negative residues (Asp, Glu) are highlighted. (Fig.15C) Evolutionary conservation analysis of the RvSAHS1 sequence. Boxes indicate the same sequences depicted in (Fig. 15B).

[0043] Figs.16A-16B depicts AlphaFold-predicted insertion of the SAHS1 N-terminal tail into the SAHS1 cavity. The AlphaFold-predicted structure of three SAHS1 proteins indicated that the N-terminal ~9 amino acids of mature SAHS1, which are predicted to be unstructured in a monomer [4], might be able to occupy the cavity of a second SAHS1 protein. The T=0 nanosecond structure in Fig.14A shows the trimeric structure predicted by AlphaFold, which contains three different insertions of an N-terminus into the cavity of an adjacent SAHS1 protein. These differ in detail, and we regard the complex in this figure as the most structurally plausible. (Fig.16A) A SAHS1 protein (left) with an N-terminal tail located in theAttorney Docket No.002806-000126WOPT cavity of a second SAHS1 protein (right); this is a close-up view of two of the three proteins depicted in Fig.14A, T=0. (Fig.16B) An expanded view of the region in the box of Fig.16A in which the first 14 amino acids of the left subunit are shown (Ala20-Trp32, with interacting amino acids lettered) and select interacting amino acids from the right SAHS1 protein shown. Also shown are the distances, in Angstroms, between oxygens in one side chain and protonated nitrogens in another, in cases of possible hydrogen bonding. Amino acid numbering is relative to the first amino acid in the translated sequence (as in Fig.1), such that after removal of the 19-amino acid signal sequence, Ala20 is the first amino acid in the mature SAHS1. We note that when Fukuda et al. solved the structure of SAHS1 [4], they removed the N-terminal 10 amino acids of the protein because this segment might interfere with crystallization, and the cavity was filled with solvent molecules. The crystallized form of SAHS4 included an extra serine at its N-terminus, which may have disrupted insertion of the N-terminus into the SAHS4 cavity, given the tight packing and specific contacts made by the N-terminal amino group in the SAHS1 model of Fig.16B [5]. The human liver and heart muscle fatty acid binding proteins, which are otherwise similar to the SAHS proteins and possess a large cavity, lack these N-terminal tails (see Fig.1B). DETAILED DESCRIPTION

[0044] As described herein, the inventors have found that tardigrade SAHS polypeptides protect microbes and lipid formulations (e.g., liposomes) from the harmful effects of degradation, providing a surprisingly strong protective effect as compared to state-of-the art chemical protectants. Accordingly, described herein are compositions comprising at least one tardigrade secreted-abundant heat-soluble (SAHS) polypeptide and at least one viable microbe, lipid, or liposome and methods of making and using the same. Such compositions have improved shelf-life, stability, and performance.

[0045] In one aspect of any of the embodiments, described herein is a composition comprising at least one tardigrade secreted-abundant heat-soluble (SAHS) polypeptide and at least one viable microbe. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide and the at least one viable microbe are in admixture.

[0046] In one aspect of any of the embodiments, described herein is an agricultural composition comprising at least one tardigrade secreted-abundant heat-soluble (SAHS) polypeptide in admixture with at least one viable microbe. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide and the at least one viable microbe are in admixture. An agricultural composition can comprise a fertilizer, a biofertilizer, a seed coating composition, a pesticide, a foliar spray, tabs, pellets, and a solution. In some embodiments of any of the aspects, the agricultural composition is applied above the soil, e.g., to the leaves and / or stems of a plant. In some embodiments of any of the aspects, theAttorney Docket No.002806-000126WOPT agricultural composition is applied below the soil, e.g., by irrigation of the soil or spraying onto the soil surface. In some embodiments of any of the aspects, the agricultural composition is applied below the soil, e.g., to the roots of a plant. In some embodiments of any of the aspects, the agricultural composition is applied directly to the roots of a plant, e.g., before planting / transplanting. In some embodiments of any of the aspects, the agricultural composition is applied to the seeds of a plant, e.g., before or during planting. In some embodiments of any of the aspects, the agricultural composition is applied to the soil, e.g., before or during the planting process. In some embodiments of any of the aspects, the agricultural composition is hydrated, dissolved, or suspended prior to application. In some embodiments of any of the aspects, the agricultural composition is dried or dessciated at the time of application.

[0047] In some embodiments of any of the aspects, the agricultural composition further comprises an insecticide, a fungicide, a nematicide, and / or a biological agent.

[0048] A plant referred to herein can be any plant, e.g., a plant of commercial or agricultural interest of value. In some embodiments of any of the aspects, the plant is a plant that can form root nodules. For example, leguminous plants are generally known to form root nodules. Examples of leguminous plants include, but are not limited to, soybean, adzuki bean (Phaseolus angularis), broad bean (Vicia faba), garden pea (Pisum sativum), peanut, cowpea (Vigna sinensis), lupin bean, clover, and alfalfa.

[0049] As used herein, “admixture” refers to refers to a composition comprising two or more elements, wherein at least a portion of a first element is in physical contact with at least a portion of the second element.

[0050] In one aspect of any of the embodiments, described herein is a composition comprising at least one tardigrade SAHS polypeptide and a liposome preparation. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide and the liposome preparation are in admixture.

[0051] In some embodiments of any of the aspects, the liposomes retain liposome structure upon rehydration.

[0052] In one aspect of any of the embodiments, described herein is a composition comprising a cosmetic composition and at least one tardigrade SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide and the cosmetic composition are in admixture. As used herein, “cosmetic” refers to a substance of beautification or preparation that preserves, restores, grants, simulates, or improves the appearance of body beauty or appears to improve beauty or youth, specifically as which refers to the appearance of tissue or skin.

[0053] In some embodiments of any of the aspects, a composition described herein comprises a liposome. In some embodiments of any of the aspects, a cosmetic composition described herein comprises a liposome.Attorney Docket No.002806-000126WOPT

[0054] In some embodiments of any of the aspects, the composition described herein further comprises trehalose.

[0055] As used herein “SAHS” or “secreted-abundant heat-soluble” refers to a family of proteins which are intrinsically disordered in a hydrated state, assume stable structures on desiccation, and comprise secretory signal sequences. SAHS proteins promote desiccation survival of tardigrades.

[0056] As used herein, “tardigrade” refers to an organism of the phylum Tardigrada, a group of eight-legged, segmented micro-animals. A tardigrade SAHS is a polypeptide obtained from, derived from, or engineered from a SAHS found in a naturally-occurring tardigrade.

[0057] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

[0058] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a naturally-occurring Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide or naturally-occurring Hypsibius exemplaris (H. exemplaris) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a naturally-occurring Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a naturally-occurring Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

[0059] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to a naturally-occurring Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide or naturally-occurring Hypsibius exemplaris (H. exemplaris) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to a naturally-occurring Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to a naturally-occurring Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

[0060] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to a naturally-occurring Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide or naturally-occurring Hypsibius exemplaris (H. exemplaris) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to a naturally-occurringAttorney Docket No.002806-000126WOPT Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to a naturally-occurring Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

[0061] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to a naturally-occurring Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide or naturally-occurring Hypsibius exemplaris (H. exemplaris) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to a naturally-occurring Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to a naturally-occurring Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

[0062] Exemplary tardigrade SAHS polypeptides include SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least one of SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, or SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, or SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, or SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, or SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, or SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, or SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, or SAHS12.

[0063] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptideAttorney Docket No.002806-000126WOPT comprises SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS1. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS1.

[0064] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS2. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS2.

[0065] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS3. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS3. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS3. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS3. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS3. InAttorney Docket No.002806-000126WOPT some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS3. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS3. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS3. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS3. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS3. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS3.

[0066] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS4. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS4.

[0067] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptideAttorney Docket No.002806-000126WOPT comprises a sequence with at least 95% sequence identity to SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS6.

[0068] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS7.

[0069] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHSAttorney Docket No.002806-000126WOPT polypeptide comprises a sequence with at least 99% sequence identity to SAHS8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS8.

[0070] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS9.

[0071] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS10.Attorney Docket No.002806-000126WOPT

[0072] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS11.

[0073] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to SAHS12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of SAHS12.

[0074] Exemplary sequences of tardigrade SAHS polypeptides are provided in Table 4.

[0075] Table 4Attorney Docket No.002806-000126WOPT Protein Sequence SEQ ID NO: With Secretory SequenceAttorney Docket No.002806-000126WOPT YKNDVVFKLGQESAGSYNGSSFTVKYEDKDGALVGTIHYTGTKEQSL DKTINNEYKVEGNQLVKTSTLEGVTHKRYYNKRNAttorney Docket No.002806-000126WOPT SEDGEKLVADVSIPTKGKTIRSEYEVQGDQLIKTYKTGDIVAKKWFKK VANPTEAPAQAA

[0076] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a polypeptide comprising the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of a polypeptide comprising theAttorney Docket No.002806-000126WOPT sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of a polypeptide comprising the sequence of one of SEQ ID NOs: 5-28.

[0077] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a polypeptide consisting essentially of the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of a polypeptide consisting essentially of the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of a polypeptide consisting essentially of the sequence of one of SEQ ID NOs: 5-28.

[0078] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a polypeptide consisting of the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of a polypeptide consisting of the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of a polypeptide consisting of the sequence of one of SEQ ID NOs: 5-28.

[0079] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a polypeptide comprising the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of a polypeptide comprising the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of a polypeptide comprising the sequence of SEQ ID NO: 5.

[0080] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a polypeptide consisting essentially of the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of a polypeptide consisting essentially of the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of a polypeptide consisting essentially of the sequence of SEQ ID NO: 5.

[0081] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a polypeptide consisting of the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of a polypeptide consisting of the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of a polypeptide consisting of the sequence of SEQ ID NO: 5.

[0082] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a polypeptide comprising the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of a polypeptide comprising the sequenceAttorney Docket No.002806-000126WOPT of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of a polypeptide comprising the sequence of SEQ ID NO: 9.

[0083] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a polypeptide consisting essentially of the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of a polypeptide consisting essentially of the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of a polypeptide consisting essentially of the sequence of SEQ ID NO: 9.

[0084] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a polypeptide consisting of the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists essentially of a polypeptide consisting of the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide consists of a polypeptide consisting of the sequence of SEQ ID NO: 9.

[0085] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 5-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of one of SEQ ID NOs: 5-28.

[0086] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 5-8 and 21-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 5-8 and 21-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 5-8 and 21-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 5-8 andAttorney Docket No.002806-000126WOPT 21-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of one of SEQ ID NOs: 5-8 and 21-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 5-8 and 21-28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of one of SEQ ID NOs: 5-8 and 21-28.

[0087] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 5.

[0088] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 6.

[0089] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 7. In someAttorney Docket No.002806-000126WOPT embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 7.

[0090] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 8.

[0091] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises aAttorney Docket No.002806-000126WOPT sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 9.

[0092] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 10.

[0093] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 11.

[0094] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90%Attorney Docket No.002806-000126WOPT sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 12.

[0095] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 13.

[0096] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 14.Attorney Docket No.002806-000126WOPT

[0097] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 15.

[0098] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 16.

[0099] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 17 In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one tardigradeAttorney Docket No.002806-000126WOPT SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 17.

[0100] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 18.

[0101] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 19.

[0102] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequenceAttorney Docket No.002806-000126WOPT with at least 85% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 20.

[0103] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 21.

[0104] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 22. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 22. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 22. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 22. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 22. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 22. In someAttorney Docket No.002806-000126WOPT embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 22.

[0105] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 23. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 23. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 23. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 23. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 23. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 23. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 23.

[0106] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 24. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 24.

[0107] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, the atAttorney Docket No.002806-000126WOPT least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 25. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 25.

[0108] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 26. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 26. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 26. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 26. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 26. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 26. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 26.

[0109] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 27. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 27. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 27. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 27. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 27. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 27. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 27.Attorney Docket No.002806-000126WOPT

[0110] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 28. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises the sequence of the sequence of SEQ ID NO: 28.

[0111] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is an isolated tardigrade SAHS polypeptide. As used herein with respect to proteins or peptides, the term "isolated" refers to a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

[0112] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 90% of all tardigrade proteins present in the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 95% of all tardigrade proteins present in the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 98% of all tardigrade proteins present in the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 99% of all tardigrade proteins present in the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 100% of all tardigrade proteins present in the composition.

[0113] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is at least 90% of all tardigrade proteins present in the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is at least 95% of all tardigrade proteins present in the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is at least 98% of all tardigrade proteins present in the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is at least 99% of all tardigrade proteins present in the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is at least 100% of all tardigrade proteins present in the composition.Attorney Docket No.002806-000126WOPT

[0114] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is the only tardigrade protein present in the composition.

[0115] In some embodiments of any of the aspects, the composition does not comprise a tardigrade. In some embodiments of any of the aspects, the composition does not comprise a viable tardigrade.

[0116] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 0.1% wt / wt of the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 1% wt / wt of the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 2% wt / wt of the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 3% wt / wt of the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 5% wt / wt of the composition. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide comprises at least 10% wt / wt of the composition.

[0117] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide present in a composition, or combination, of the disclosure exhibits an increased utility that is not exhibited when said at least one tardigrade SAHS polypeptide occurs alone or when said at least one tardigrade SAHS polypeptide is present at a naturally occurring concentration. In some embodiments of any of the aspects, compositions of the disclosure, comprising at least one tardigrade SAHS polypeptide as taught herein, exhibit a synergistic effect on imparting at least one improved trait in a microbe or cosmetic composition. In some embodiments of any of the aspects, the compositions of the disclosure-- comprising at least one tardigrade SAHS polypeptide as taught herein--exhibit markedly different characteristics / properties compared to their closest naturally occurring counterpart. That is, the compositions of the disclosure exhibit markedly different functional and / or structural characteristics / properties, as compared to their closest naturally occurring counterpart. For instance, the at least one tardigrade SAHS polypeptide of the disclosure are structurally different from a tardigrade SAHS polypeptide as it naturally exists in a tardigrade, for at least the following reasons: said at least one tardigrade SAHS polypeptide can be isolated and purified, such that it is not found in the milieu of the tardigrade, said at least one tardigrade SAHS polypeptide can be present at concentrations that do not occur in the tardigrade, said at least one tardigrade SAHS polypeptide can be associated with acceptable carriers that do not occur in the tardigrade, said at least one tardigrade SAHS polypeptide can be formulated to be shelf-stable and exist outside the tardigrade’s environment, and said at least one tardigrade SAHS polypeptide can be combined with microbes or other compositions at concentrations that do not exist in the tardigrade. Further, the at least one tardigrade SAHS polypeptide of the disclosure are functionally different from a tardigrade SAHS polypeptide as it naturally exists in a tardigrade, for atAttorney Docket No.002806-000126WOPT least the following reasons: said at least one tardigrade SAHS polypeptide when applied in an isolated and purified form can lead to stabilization of microbes and / or liposomes, said at least one tardigrade SAHS polypeptide can be formulated to be shelf-stable and able to exist outside the tardigrade environment, such that the at least one tardigrade SAHS polypeptide now has a new utility as a supplement capable of use in agricultural or cosmetic compositions wherein the at least one tardigrade SAHS polypeptide could not have such a utility in its natural state in the tardigrade, as the at least one tardigrade SAHS polypeptide would not be accessible to the agricultural or cosmetic composition components in effective amounts without the intervention of the hand of man to formulate the at least one tardigrade SAHS polypeptide into an accessible state and impart this new utility that has the aforementioned functional characteristics not possessed by the at least one tardigrade SAHS polypeptide in its natural state of existence in the tardigrade.

[0118] In some embodiments of any of the aspects, the composition is dried. As used herein “dried” refers to a composition from which water or another solvent has been removed, e.g., by heating, air- drying, or freeze-drying. In some embodiments, a dried composition is therefore a composition which has less water present in the composition than is normally present in such a composition.

[0119] In some embodiments of any of the aspects, a dried composition comprises less than 50% water by weight. In some embodiments of any of the aspects, a dried composition comprises less than 40% water by weight. In some embodiments of any of the aspects, a dried composition comprises less than 30% water by weight. In some embodiments of any of the aspects, a dried composition comprises less than 20% water by weight. In some embodiments of any of the aspects, a dried composition comprises less than 10% water by weight.

[0120] In some embodiments of any of the aspects, the composition is desiccated. As used herein, “desiccated” refers to a composition from which substantially all water has been removed, e.g., by heating, air-drying, or freeze-drying. In some embodiments, a desiccated composition is therefore a composition which has substantially no water present.

[0121] In some embodiments of any of the aspects, a dried or desiccated composition comprises less than 5% water by weight. In some embodiments of any of the aspects, a dried or desiccated composition comprises less than 4% water by weight. In some embodiments of any of the aspects, a dried or desiccated composition comprises less than 3% water by weight. In some embodiments of any of the aspects, a dried or desiccated composition comprises less than 2% water by weight. In some embodiments of any of the aspects, a dried or desiccated composition comprises less than 1% water by weight.

[0122] In some embodiments of any of the aspects, a composition described herein can further comprise a carrier. In some embodiments of any of the aspects, a carrier can be water. In some embodiments of any of the aspects, a carrier can be a carrier other than water. In some embodiments ofAttorney Docket No.002806-000126WOPT any of the aspects, a carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[0123] As used herein, “microbe” or "microorganism" refers to cellular microorganisms including bacteria, fungi, and archaea, and encompasses both individual organisms and populations comprising any number of the organisms. The term is generally used to refer to any prokaryotic or eukaryotic microscopic organism capable of growing and propagating in a suitable medium, including but not limited to bacteria and fungi. Microorganisms encompassed by the scope of the present invention include prokaryotes, i.e., bacteria and archaea; and various forms of eukaryotes, including protozoa, fungi, yeasts (e.g., anaerobic yeasts), algae, and the like.

[0124] As used herein, “viable” refers to refers to the ability of a cell to complete at least one cell cycle and, ultimately proliferate.

[0125] In some embodiments of any of the aspects, the at least one viable microbe comprises a microbe that is symbiotic with at least one plant. In some embodiments of any of the aspects, the at least one viable microbe comprises a microbe that is beneficial to at least one plant. As used herein, the term "beneficial microbe" refers to a microbe (e.g., bacterium) strain or species that inhibits the growth of pathogens cells and / or facilitates the host’s growth and / or survival. In some embodiments of any of the aspects, the at least one viable microbe comprises a microbe that is commensal with at least one plant. As used herein, the term "commensal microbe" refers to a microorganism that is nonpathogenic to a host and is part of the normal microflora of the host.

[0126] In some embodiments of any of the aspects, the at least one viable microbe is a nitrogen- fixing microbe. In some embodiments of any of the aspects, the at least one viable microbe is a diazotroph. Diazotrophs are bacteria and archaea that fix atmospheric nitrogen gas into a more usable form such as ammonia. Examples of diazotrophs include bacteria from the genera Rhizobium spp. (e.g., R. cellulosilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R. huautlense, R. indigoferae, R. leguminosarum, R.loessense, R. lupini, R. lusitanum, R. meliloti, R. mongolense, R.miluonense, R. sullae, R. tropici, R. undicola, and / or R. yanglingense), Bradyrhizobium spp. (e.g., B. bete, B. canariense, B. elkanii, B.iriomotense, B. japonicum, B. jicamae, B. liaoningense, B. pachyrhizi, and / or B. yuanmingense), Azorhizobium spp. (e.g., A. caulinodans and / or A. doebereinerae), Sinorhizobium spp. (e.g., S. abri, S. adhaerens, S. americanum, S. aborts, S. fredii, S. indiaense, S. kostiense, S.kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. morelense, S. saheli, S. terangae, and / or S. xinjiangense), Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M. pluifarium, M. septentrionale, M. temperatum, and / or M. tianshanense), and combinations thereof. In some embodiments of any of the aspects, the diazotroph isAttorney Docket No.002806-000126WOPT selected from the group consisting of B. japonicum, R leguminosarum, R meliloti, S. meliloti, and combinations thereof.

[0127] In some embodiments of any of the aspects, the at least one viable microbe is a mycorrhizal fungi. Mycorrhizal fungi form symbiotic associations with the roots of a vascular plant, and provide, e.g., absorptive capacity for water and mineral nutrients due to the comparatively large surface area of mycelium. Mycorrhizal fungi include endomycorrhizal fungi (also called vesicular arbuscular mycorrhizae, VAMs, arbuscular mycorrhizae, or AMs), an ectomycorrhizal fungi, or a combination thereof. In one embodiment, the mycorrhizal fungi is an endomycorrhizae of the phylum Glomeromycota and genera Glomus and Gigaspora. In still a further embodiment, the endomycorrhizae is a strain of Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus fasciculatum, Glomus intraradices, Glomus monosporum, or Glomus mosseae, Gigaspora margarita, or a combination thereof. Examples of mycorrhizal fungi include ectomycorrhizae of the phylum Basidiomycota, Ascomycota, and Zygomycota. Other examples include a strain of Laccaria bicolor, Laccaria laccata, Pisolithus tinctorius, Rhizopogon amylopogon, Rhizopogon fulvigleba, Rhizopogon luteolus, Rhizopogon villosuli, Scleroderma cepa, Scleroderma citrinum, or a combination thereof. The mycorrhizal fungi include ecroid mycorrhizae, arbutoid mycorrhizae, or monotropoid mycorrhizae. Arbuscular and ectomycorrhizae form ericoid mycorrhiza with many plants belonging to the order Ericales, while some Ericales form arbutoid and monotropoid mycorrhizae. In some embodiments, the mycorrhiza can be an ericoid mycorrhiza, for example, of the phylum Ascomycota, such as Hymenoscyphous ericae or Oidiodendron sp. In another embodiment, the mycorrhiza also can be an arbutoid mycorrhiza, for example, of the phylum Basidiomycota. In yet another embodiment, the mycorrhiza can be a monotripoid mycorrhiza, for example, of the phylum Basidiomycota. In still yet another embodiment, the mycorrhiza can be an orchid mycorrhiza, for example, of the genus Rhizoctonia.

[0128] In some embodiments of any of the aspects, the microbe can be a Bacillus or Paenibacillus, e.g., Bacillus amyloliquifaciens (Khan, et al., in Bioresource Technology, 99(8): 3016-3023 (2008); Plant Growth and Health Promoting Bacteria (Microbiology Monographs), D. K. Maheshwari, Ed., Springer- Verlag, Berlin, 2010. In some embodiments of any of the aspects, the microbe can be a species within the fungal genera Cladosporum, Corvularia, Fusarium, Gliocladium, Metarhizium, Penicilliunn or Trichoderma (Kim, et al. in BMC Microbiology, 8:231 (2008); Khan, et al., in World Journal of Microbiology and Biotechnology, 28(4): 1483-1494 (2012), Biotechnology of Microbes and Sustainable Utilization, R. C. Rajak, Ed., Scientific Publishers, Jodhpur, India, 2002, pp.116-120. Each of the foregoing references is incorporated by reference herein in its entirety. In some embodiments of any of the aspects, the microbe can be a species within the genera Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter, Eupenicillium,Attorney Docket No.002806-000126WOPT Exiguobacterium, Klebsiella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacter, and Xanthomonas, e.g., Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans,Bacillus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, Eupenicillium parvum, Exiguobacterium sp., Klebsiella sp., Kluyvera cryocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marquandii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea aglomerans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, Vibrio proteolytics, Xanthobacter agilis, and Xanthomonas campestris. One of skill in the art can select a suitable microbe depending on the plant to be treated and the stresses or challenges facing the plant. See e.g., U.S. Patent 11,118,159, e.g., at Table 1; which is incorporated by reference herein in its entirety.

[0129] In some embodiments of any of the aspects, the at least one viable microbe comprises a Rhizobium, a Bradyrhizobium, an Azorhizobium, a Sinorhizobium, a Mesorhizobium, or a mycorrhizal fungi. In some embodiments of any of the aspects, the at least one viable microbe comprises R. tropici, B. elkani, B. japonicum, M. loti, M. huakuii, R. leguminosarum, R. meliloti, S. fredii, or S. meliloti. In some embodiments of any of the aspects, the at least one viable microbe comprises R. tropici. In some embodiments of any of the aspects, the at least one viable microbe consists of R. tropici.

[0130] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 10 mg / mL or greater. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1 mg / mL or greater. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5 mg / mL or greater. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.1 mg / mL or greater. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.1-10 mg / mL. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5-10 mg / mL. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1-10 mg / mL. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in theAttorney Docket No.002806-000126WOPT composition at 0.01-100 mg / mL. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5-100 mg / mL. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1-100 mg / mL.

[0131] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 10 mg / mL or greater and the liposome is present in the composition at 1.4 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1 mg / mL or greater and the liposome is present in the composition at 1.4 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5 mg / mL or greater and the liposome is present in the composition at 1.4 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.1 mg / mL or greater and the liposome is present in the composition at 1.4 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.1-10 mg / mL and the liposome is present in the composition at 1.4 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5-10 mg / mL and the liposome is present in the composition at 1.4 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1-10 mg / mL and the liposome is present in the composition at 1.4 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.01-100 mg / mL and the liposome is present in the composition at 1.4 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5-10 mg / mL and the liposome is present in the composition at 1.4 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1-100 mg / mL and the liposome is present in the composition at 1.4 mg / mL lipid concentration.

[0132] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 10 mg / mL or greater and the liposome is present in the composition at 0.1- 10 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1 mg / mL or greater and the liposome is present in the composition at 0.1-10 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5 mg / mL or greater and the liposome is present in the composition at 0.1-10 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.1 mg / mL or greater and the liposome is present in the composition at 0.1-10 mg / mL lipid concentration. In someAttorney Docket No.002806-000126WOPT embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.1-10 mg / mL and the liposome is present in the composition at 0.1-10 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5-10 mg / mL and the liposome is present in the composition at 0.1-10 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1-10 mg / mL and the liposome is present in the composition at 0.1-10 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.01-100 mg / mL and the liposome is present in the composition at 0.1-10 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5-10 mg / mL and the liposome is present in the composition at 0.1-10 mg / mL lipid concentration. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1-100 mg / mL and the liposome is present in the composition at 0.1-10 mg / mL lipid concentration.

[0133] In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 10 mg / mL or greater and the microbe is present in the composition at 1x108– 1x1010cells. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1 mg / mL or greater and the liposome is present in the composition at1x108– 1x1010cells. In some embodiments of any of the aspects, the at least one tardigrade SAHSpolypeptide is present in the composition at 0.5 mg / mL or greater and the liposome is present in the composition at 1x108– 1x1010cells. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.1 mg / mL or greater and the liposome is present in the composition at 1x108– 1x1010cells. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.1-10 mg / mL and the liposome is present in the composition at 1x108– 1x1010cells. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5-10 mg / mL and the liposome is present in the composition at 1x108– 1x1010cells. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1-10 mg / mL and the liposome is present in the composition at 1x108– 1x1010cells. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.01-100 mg / mL and the liposome is present in the composition at 1x108– 1x1010cells. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 0.5-10 mg / mL and the liposome is present in the composition at 1x108– 1x1010cells. In some embodiments of any of the aspects, the at least one tardigrade SAHS polypeptide is present in the composition at 1-100 mg / mL and the liposome is present in the composition at 1x108– 1x1010cells.Attorney Docket No.002806-000126WOPT

[0134] Solutions, including emulsifiable concentrates, can be prepared by simply mixing the ingredients. Dusts and powders can be prepared by blending and, usually, grinding as in a hammer mill or fluid-energy mill. Suspensions are usually prepared by wet-milling; see, for example, U.S.3,060,084. Granules and pellets can be prepared by spraying the active material upon preformed granular carriers or by agglomeration techniques. See Browning, "Agglomeration", Chemical Engineering, December 4, 1967, pp.147-48, Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963, pp.8- 57 and following, and PCT Publication WO 91 / 13546. Pellets can be prepared as described in U.S. 4,172,714. Water-dispersible and water-soluble granules can be prepared as taught in U.S.4,144,050, U.S.3,920,442 and DE 3,246,493. Tablets can be prepared as taught in U.S.5,180,587, U.S.5,232,701 and U.S.5,208,030. Films can be prepared as taught in GB 2,095,558 and U.S.3,299,566. For further information regarding the art of formulation, see T. S. Woods, "The Formulator's Toolbox - Product Forms for Modern Agriculture" in Pesticide Chemistry and Bioscience, The Food- Environment Challenge, T. Brooks and T. R. Roberts, Eds., Proceedings of the 9th International Congress on Pesticide Chemistry, The Royal Society of Chemistry, Cambridge, 1999, pp.120-133. See also U.S.3,235,361 , Col.6, line 16 through Col.7, line 19 and Examples 10- 41 ; U.S.3,309,192, Col.5, line 43 through Col. 7, line 62 and Examples 8, 12, 15, 39, 41 , 52, 53, 58, 132, 138-140, 162-164, 166, 167 and 169-182; U.S. 2,891 ,855, Col.3, line 66 through Col.5, line 17 and Examples 1 -4; Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961 , pp.81 -96; and Hance et al., Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989. Each of the foregoing references is incorporated by reference herein in its entirety.

[0135] In one aspect of any of the embodiments, described herein is a method of stabilizing at least one viable microbe, the method comprising contacting the at least one viable microbe with at least one tardigrade SAHS polypeptide. In one aspect of any of the embodiments, described herein is a method comprising contacting the agricultural composition with at least one tardigrade SAHS polypeptide.

[0136] In one aspect of any of the embodiments, described herein is a method of stabilizing a liposomal membrane, the method comprising contacting the liposomal membrane with at least one tardigrade SAHS polypeptide. In one aspect of any of the embodiments, described herein is a method comprising contacting the cosmetic composition with at least one tardigrade SAHS polypeptide.

[0137] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of” the elementsAttorney Docket No.002806-000126WOPT described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of” the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.

[0138] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0139] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level.

[0140] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.Attorney Docket No.002806-000126WOPT

[0141] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha- amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, e.g. microbe stabilizing activity, of the full length polypeptide, e.g., a tardigrade SAHS polypeptide of any one of SEQ ID NOs: 5-28, e.g. as measured according to the assays described herein. Conservative substitution variants that maintain the activity of a wildtype tardigrade SAHS polypeptide described herein will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with SAHS homologs or paralogs from other species. Amino acids that are identical between SAHS homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and / or fusion proteins can be tested for activity, for example, by drying or desiccating a microbe as described herein. Further discussion of the structure of tardigrade SAHS polypeptides can be found, e.g. in Boothby, T. C. et al. Tardigrades use intrinsically disordered proteins to survive desiccation. Mol. Cell.65, 975-984 (2017); and Fukuda, Y., et al. Structural insights into a secretory abundant heat‐soluble protein from an anhydrobiotic tardigrade, Ramazzottius varieornatus. FEBS Lett.591, 2458-2469 (2017); and Fukuda, Y. & Inoue, T. Crystal structure of secretory abundant heat soluble protein 4 from one of the toughest “water bears” micro‐animals Ramazzottius varieornatus. Protein Sci.27, 993-999 (2018); each of which is incorporated by reference herein in its entirety.

[0142] In some embodiments, a polypeptide, e.g., a tardigrade SAHS polypeptide described herein, can be a variant of a sequence described herein, e.g. a variant of a tardigrade SAHS polypeptide comprising the amino acid sequence of one of SEQ ID NOs: 5-28. In some embodiments, the variant is a conservative substitution variant. Variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native orAttorney Docket No.002806-000126WOPT reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., can stabilize microbes at least 50% as well as a wildtype tardigrade SAHS polypeptide described herein. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e.5% or fewer, e.g.4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, has more than 100% of the activity of wildtype, e.g.110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.

[0143] One method of identifying amino acid residues which can be substituted is to align, for example, a tardigrade SAHS polypeptide described herein to a homolog from one or more other species. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. The variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence, e.g. a tardigrade SAHS polypeptide described herein or a nucleic acid encoding one of those amino acid sequences. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp or BLASTn (available on the world wide web at blast.ncbi.nlm.nih.gov), with default parameters set.Attorney Docket No.002806-000126WOPT

[0144] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0145] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. stabilizing activity and specificity of a native or reference polypeptide is retained.

[0146] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0147] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)): (1) non- polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, forAttorney Docket No.002806-000126WOPT example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Val, into Ile or into Leu. Typically conservative substitutions for one another also include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0148] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0149] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0150] In some embodiments, a polypeptide, e.g., a tardigrade SAHS polypeptide can comprise one or more amino acid substitutions or modifications. In some embodiments, the substitutions and / or modifications can prevent or reduce proteolytic degradation and / or prolong half-life of the polypeptide in a subject. In some embodiments, a polypeptide can be modified by conjugating or fusing it to other polypeptide or polypeptide domains such as, by way of non-limiting example, transferrin (WO06096515A2), albumin (Yeh et al., 1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov, 2002); and / or Fc fragments (Ashkenazi and Chamow, 1997). The references in the foregoing paragraph are incorporated by reference herein in their entireties.

[0151] In some embodiments, a polypeptide, e.g., a tardigrade SAHS polypeptide, as described herein can comprise at least one peptide bond replacement. A tardigrade SAHS polypeptide as describedAttorney Docket No.002806-000126WOPT herein can comprise one type of peptide bond replacement or multiple types of peptide bond replacements, e.g.2 types, 3 types, 4 types, 5 types, or more types of peptide bond replacements. Non- limiting examples of peptide bond replacements include urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylacetic acid, meta- (aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

[0152] In some embodiments, a polypeptide, e.g., a tardigrade SAHS polypeptide, as described herein can comprise naturally occurring amino acids commonly found in polypeptides and / or proteins produced by living organisms, e.g. Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, a tardigrade SAHS polypeptide as described herein can comprise alternative amino acids. Non-limiting examples of alternative amino acids include, D-amino acids; beta- amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma- carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,- tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citruline, alpha- methyl-alanine, para-benzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine), diaminobutyric acid, 7-hydroxy- tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, amino- isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-amino-l- cyclopentanecarboxylic acid, 1-amino-l-cyclohexanecarboxylic acid, amino-benzoic acid, amino- naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine, nipecotic acid, alpha-amino butyric acid, thienyl-alanine, t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs; azide- modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof.

[0153] In some embodiments, a polypeptide, e.g. a tardigrade SAHS polypeptide, can be modified, e.g. by addition of a moiety to one or more of the amino acids that together comprise the peptide. In some embodiments, a polypeptide as described herein can comprise one or more moiety molecules, e.g.1 or more moiety molecules per polypeptide, 2 or more moiety molecules per polypeptide, 5 or more moiety molecules per polypeptide, 10 or more moiety molecules per polypeptide or more moiety molecules per polypeptide. In some embodiments, a polypeptide as described herein can comprise one more types of modifications and / or moieties, e.g.1 type of modification, 2 types of modifications, 3 types of modifications or more types of modifications. Non-limiting examples of modifications and / or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; end-Attorney Docket No.002806-000126WOPT capping modifications; cyano groups; phosphorylation; albumin, and cyclization. In some embodiments, an end-capping modification can comprise acetylation at the N-terminus, N-terminal acylation, and N- terminal formylation. In some embodiments, an end-capping modification can comprise amidation at the C-terminus, introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties. The half-life of a polypeptide can be increased by the addition of moieties, e.g. PEG, albumin, or other fusion partners (e.g. Fc fragment of an immunoglobin).

[0154] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0155] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide- directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established. Alterations of the original amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Khudyakov et al. “Artificial DNA: Methods and Applications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols” Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook” Springer 2000; which are herein incorporated by reference in their entireties. In some embodiments, a polypeptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[0156] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can beAttorney Docket No.002806-000126WOPT a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0157] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0158] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the term "detecting" or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.

[0159] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0160] In some embodiments of any of the aspects, the tardigrade SAHS polypeptide described herein is exogenous. In some embodiments of any of the aspects, the tardigrade SAHS polypeptide described herein is ectopic. In some embodiments of any of the aspects, the tardigrade SAHS polypeptide described herein is not endogenous.

[0161] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a composition or biological system such as a cell or organism in which it is not normally found. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a composition or biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid orAttorney Docket No.002806-000126WOPT polypeptide in the composition or cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the composition or biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and / or amount. An ectopic substance can be one that is normally found in a given location or environment, but at a much lower amount and / or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given location or cell in its natural environment.

[0162] As used herein, the term “nanoparticle” refers to particles that are on the order of about 1 to 1,000 nanometers in diameter or width. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; these nanoparticles may be part of a nanonetwork. The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. Exemplary nanoparticles include lipid nanoparticles or ferritin nanoparticles. Lipid nanoparticles can comprise multiple components, including, e.g., ionizable lipids (such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102), pegylated lipids (such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), and cholesterol.

[0163] Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof.

[0164] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one protein. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine. In some embodiments, the contacting provides direct physical contact of the protein and the at least one microbe, e.g., the protein and the at least one microbe are touching, e.g., at least one surface of the protein is touching or forming a junction with at least one aspect of the at least one microbe, or is capable of contact with at least one aspect of the of the at least one microbe (e.g., stable binding is not required for “contact”).

[0165] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0166] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.Attorney Docket No.002806-000126WOPT

[0167] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0168] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0169] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0170] As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.

[0171] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0172] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0173] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in cell and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Bruce Alberts et al., Molecular Biology of the Cell, published by W.W. Norton & Company, 2022 (ISBN 0393884821, 978-0393884821); John M. Lackie eat al. (eds.), The Dictionary of Cell and MolecularAttorney Docket No.002806-000126WOPT Biology, 5thEdition, published by Academic Press, 2013 (ISBN 0123849314, 978-0123849311); Nalini Chandar et al., Lippincott Illustrated Reviews: Cell and Molecular Biology, 3rdEdition, published by LWW, 2023 (ISBN 1975180895, 978-1975180898); Teresa Atwood et al., Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); Johnathan Law et al., (eds.), A Dictionary of Chemistry, 8thEdition, published by Oxford University Press, 2020 (ISBN 9780198841227, 9780191876783); Robert C. King et al. (eds.), A Dictionary of Genetics, 8thEdition, published by Oxford University Press, 2013 (ISBN 9780199766444, 9780199376865); Richard Cammack et al. (eds.), Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006 (ISBN 9780198529170, 9780191727641); John Lackie et al. (eds.), A Dictionary of Biomedicine, 2ndEdition, published by Oxford University Press, 2019 (ISBN 9780191829116); Lodish et al., Molecular Cell Biology, 8thEdition, published by W.H. Freeman, 2016 (ISBN 1464183392, 978-1464183393); Abul K. Abbas et al., Cellular and Molecular Immunology, 10thEdition, published by Elsevier, 2021 (ISBN 0323757480, 978-0323757485); Kenneth M. Murphy et al., Janeway's Immunobiology, 10thEdition, published by W. W. Norton & Company, 2022 (ISBN 0393884899, 978-0393884890); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 0444569464); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, 1987-2010 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0174] Other terms are defined herein within the description of the various aspects of the invention.

[0175] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are notAttorney Docket No.002806-000126WOPT entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0176] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0177] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0178] In some embodiments, the present technology may be defined in any of the following numbered paragraphs: 1. A dried composition comprising a tardigrade secreted-abundant heat-soluble (SAHS) polypeptide in admixture with a viable microbe. 2. The composition of paragraph 1, wherein the SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide. 3. The composition of paragraph 1 or paragraph 2, wherein the SAHS polypeptide is selected from those set out in Table 1. 4. A composition comprising a tardigrade SAHS polypeptide in admixture with a liposome preparation. 5. The composition of paragraph 4, wherein the SAHS polypeptide comprises a R. varieornatus or H. exemplaris SAHS polypeptide.Attorney Docket No.002806-000126WOPT 6. The composition of paragraph 4 or paragraph 5, wherein the SAHS polypeptide is selected from those set out in Table 1. 7. The composition of any one of paragraphs 4-6, which is a dried composition. 8. The composition of paragraph 7, wherein the liposomes retain liposome structure upon rehydration. 9. A method of stabilizing a liposomal membrane, the method comprising contacting the liposomal membrane with a tardigrade SAHS polypeptide. 10. The method of paragraph 9, wherein the SAHS polypeptide comprises a R. varieornatus or H. exemplaris SAHS polypeptide. 11. The method of paragraph 9 or paragraph 10, wherein the SAHS polypeptide is selected from those set out in Table 1. 12. A method of stabilizing a cosmetic formulation, the method comprising contacting the cosmetic formulation with a tardigrade SAHS polypeptide. 13. The method of paragraph 12, further comprising contacting the cosmetic formulation with trehalose. 14. The method of paragraph 12 or 13, wherein the cosmetic formulation comprises liposomes. 15. The method of any one of paragraphs 12-143, wherein the SAHS polypeptide comprises a R. varieornatus or H. exemplaris SAHS polypeptide. 16. The method of any one of paragraphs 12-15, wherein the SAHS polypeptide is selected from those set out in Table 1. 17. A composition comprising a cosmetic formulation in admixture with a tardigrade SAHS polypeptide. 18. The composition of paragraph 17, further comprising trehalose. 19. The composition of paragraph 17 or 18, wherein the cosmetic formulation comprises liposomes. 20. The composition of any one of paragraphs 17-19, wherein the SAHS polypeptide comprises a R. varieornatus or H. exemplaris SAHS polypeptide. 21. The composition of any one of paragraphs 17-20, wherein the SAHS polypeptide is selected from those set out in Table 1.

[0179] In some embodiments, the present technology may be defined in any of the following numbered paragraphs: 1. A composition comprising at least one tardigrade secreted-abundant heat-soluble (SAHS) polypeptide and at least one viable microbe. 2. An agricultural composition comprising at least one tardigrade secreted-abundant heat-soluble (SAHS) polypeptide and at least one viable microbe. 3. A composition comprising at least one tardigrade SAHS polypeptide and a liposome preparation. 4. A composition comprising a cosmetic composition and at least one tardigrade SAHS polypeptide.Attorney Docket No.002806-000126WOPT 5. The composition of any one of the preceding paragraphs, wherein the composition is an admixture of the at least one tardigrade SAHS polypeptide and at least one of a viable microbe, a liposome preparation, and a cosmetic composition. 6. The composition of any one of the preceding paragraphs, further comprising trehalose. 7. The composition of any one of the preceding paragraphs, wherein the composition comprises liposomes. 8. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is an isolated tardigrade SAHS polypeptide. 9. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide comprises at least 90% of all tardigrade proteins present in the composition. 10. The composition of any one of the preceding paragraphs, wherein the composition does not comprise a viable tardigrade. 11. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide. 12. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is selected from SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, SAHS12. 13. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is SAHS1. 14. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is selected from RvSAHS1, RvSAHS2, RvSAHS3, RvSAHS4, HeSAHS4, RvSAHS6, RvSAHS7, RvSAHS8, RvSAHS9, RvSAHS10, RvSAHS11, and RvSAHS12. 15. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is RvSAHS1. 16. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to one of SEQ ID NOs: 5-28. 17. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 5 or 9. 18. The composition of any one of the preceding paragraphs, wherein the composition is dried. 19. The composition of any one of the preceding paragraphs, wherein the composition is desiccated. 20. The composition of any one of paragraphs 3-19, wherein the liposomes retain liposome structure upon rehydration.Attorney Docket No.002806-000126WOPT 21. The composition of any one of the preceding paragraphs, wherein the at least one viable microbe comprises a Rhizobium, a Bradyrhizobium, an Azorhizobium, a Sinorhizobium, a Mesorhizobium, or a mycorrhizal fungi. 22. The composition of any one of the preceding paragraphs, wherein the at least one viable microbe comprises R. tropici, B. elkani, B. japonicum, M. loti, M. huakuii, R. leguminosarum, R. meliloti, S. fredii, or S. meliloti. 23. The composition of any one of the preceding paragraphs, wherein the viable microbe is R. tropici. 24. A method of stabilizing at least one viable microbe, the method comprising contacting the at least one viable microbe with at least one tardigrade SAHS polypeptide. 25. A method of stabilizing an agricultural composition, the method comprising contacting the agricultural composition with at least one tardigrade SAHS polypeptide. 26. A method of stabilizing a liposomal membrane, the method comprising contacting the liposomal membrane with at least one tardigrade SAHS polypeptide. 27. A method of stabilizing a cosmetic composition, the method comprising contacting the cosmetic composition with at least one tardigrade SAHS polypeptide. 28. The method of any one of the preceding paragraphs, further comprising contacting the composition with trehalose. 29. The method of any one of the preceding paragraphs, wherein the composition comprises liposomes. 30. The method of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is an isolated tardigrade SAHS polypeptide. 31. The method of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is provided in a composition and the at least one tardigrade SAHS polypeptide comprises at least 90% of all tardigrade proteins present in the composition. 32. The method of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is provided in a composition that does not comprise a viable tardigrade. 33. The method of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide. 34. The method of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is SAHS1. 35. The method of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is selected from RvSAHS1, RvSAHS2, RvSAHS3, RvSAHS4, HeSAHS4, RvSAHS6, RvSAHS7, RvSAHS8, RvSAHS9, RvSAHS10, RvSAHS11, and RvSAHS12.Attorney Docket No.002806-000126WOPT 36. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide is RvSAHS1. 37. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to one of SEQ ID NOs: 5-28. 38. The composition of any one of the preceding paragraphs, wherein the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 5 or 9. 39. The composition of any one of the preceding paragraphs, wherein the at least one viable microbe comprises a Rhizobium, a Bradyrhizobium, an Azorhizobium, a Sinorhizobium, a Mesorhizobium, or a mycorrhizal fungi. 40. The composition of any one of the preceding paragraphs, wherein the at least one viable microbe comprises R. tropici, B. elkani, B. japonicum, M. loti, M. huakuii, R. leguminosarum, R. meliloti, S. fredii, or S. meliloti. 41. The composition of any one of the preceding paragraphs, wherein the at least one viable microbe comprises R. tropici..

[0180] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. EXAMPLES Example 1

[0181] In this study, SAHS proteins from the tardigrades R. varieornatus and Hypsibius exemplaris were investigated for their potential to protect various biological structures from desiccation-induced damages. Four different SAHS proteins were expressed, and it was found that they can specifically protect liposomes and bacterial cells but not enzymes upon desiccation. SAHS proteins undergo structural changes under dehydration with trifluoroethanol. Further, molecular dynamic and bioinformatic analyses were performed to begin to identify structural features of the SAHS proteins that may contribute to desiccation protection. These studies lead to a proposed model for SAHS protein action, and also illustrate that SAHS proteins can be practical preservatives for cells in commercial applications.

[0182] Results

[0183] Protein sequence analysis and expression

[0184] 12 SAHS proteins from R. varieornatus and one from H. exemplaris were selected to test for expression in the E. coli cytoplasm (Table 1). R. varieornatus is the most desiccation-resistant tardigrade and is well-characterized; a draft genome sequence is available, and crystal structures of two SAHS proteins from this organism have been solved [16, 17]. The TargetP program

[0018] that computationally predicts protein subcellular localization based on the N-terminal signal peptide was used, and it predictedAttorney Docket No.002806-000126WOPT that only 8 out of 12 SAHS proteins would be secreted (Table 2). However, the surprising implication that some of the SAHS proteins might not be secreted also prompted the investigation of genomic regions encoding these genes.

[0185] It was found that the “short” SAHS protein sequences in Uniprot that lack amino acids corresponding to the first ~40 amino acids of mature SAHS1 and SAHS4 may be incorrectly annotated; an alignment shown in Figure 5 of Fukuda et al.

[0016] shows three such proteins of the 13 SAHS proteins encoded by R. varieornatus. For example, the DNA encoding RvSAHS9 contains a splice acceptor immediately upstream of the putative start codon, further preceded by a splice donor and an in-frame coding sequence that encodes an amino acid sequence that aligns well with the N-terminus of other SAHS proteins; RvSAHS10 and RvSAHS12 have a similar organization (Figure 1B; Example 2). In each case, the new putative intron has a consensus splice donor but the polypyrimidine tract of the splice acceptor has rather few pyrimidines, which may have misled the automated systems that analyzed the genomic sequences. When spliced out, the new N-terminal amino acid sequences align well with the other SAHS mature protein N-termini. This analysis has further identified other candidate splice sites in these genes (Supplementary Discussion).

[0186] The Uniprot-predicted mature proteins RvSAHS1-4, and RvSAHS6-12 as well as HeSAHS4 were recombinantly expressed in the E. Coli cytoplasm. The target proteins were expressed with a N- terminal SUMO tag, whose cleavage with a highly specific protease leaves the protein “scarless” without any extraneous amino acids remaining (Figure 7A).

[0019] Since the N-terminal signal sequences are expected to be cleaved during secretion, certain proteins were expressed certain proteins without these sequences, based on the cleavage sites predicted by TargetP (Table 3). As an initial study, four proteins were successfully expressed and purified (RvSAHS1, 4, 6, and HeSAHS4) with high soluble expression and purity, and subsequent studies were conducted using them (Figs.7-8).

[0187] SAHS proteins stabilize liposomes from dehydration-induced damage

[0188] As a first step of assessing the protective potential of SAHS proteins, is was tested if they can stabilize lipid membranes upon dehydration-induced damages, using POPC (1-palmitoyl-2-oleoyl- glycero-3-phosphocholine) liposomes as a model membrane. Prior to drying, the diameter of the liposomes as measured by dynamic light scattering (DLS) was in the range of 50-100 nm (Figure 2A). Upon drying and rehydration at 1.4 mg / mL lipid concentration, less than 10% of the particles remained in this range, while larger particles appeared around 360 nm and 4000 nm (Figure 2B). This marked increase in particle diameter indicated that dehydration damaged liposome membranes and promoted their fusion and / or aggregation

[0020] .

[0189] Liposomes dried in the presence of SAHS proteins or other excipients at 10, 1 and 0.1 mg / mL concentrations were partially protected, with higher levels of excipient providing more protection. For allAttorney Docket No.002806-000126WOPT four SAHS proteins tested, particle sizes after a dehydration-rehydration cycle were notably smaller compared to those dried without an excipient (Figure 2C-2F; Figure 93). While the diameters of the major peaks were slightly increased from ~60 nm to ~100 nm, none of the bigger particles having diameters larger than 200 nm were observed, which indicated that addition of SAHS proteins prevented fusion and aggregation of dried liposomes. Bovine serum albumin (BSA), a commonly used excipient for protein preservation, was less effective in protecting liposomes than SAHS proteins, as indicated by the signal in ~500 nm range (Figure 2G). Liposomes dried in 10 mg / mL trehalose showed no particles over 150 nm, suggesting that trehalose also prevented membrane fusion of dried liposomes (Figure 2H). These observations indicated that SAHS proteins have comparable effects to trehalose in terms of their ability to stabilize membranes. SAHS proteins at lower concentrations of 1 mg / mL and were also able to protect liposomes, whereas at 0.1 mg / mL the extent of protection was diminished and larger aggregates ~1000 nm were formed (Figure 2C-2F, dotted lines); also, the repeat experiments showed consistent results (Figure 9).

[0190] SAHS proteins enhance survival of desiccated bacterial cells

[0191] It was then tested whether extracellularly added SAHS proteins can enhance the desiccation tolerance of bacterial cells (Figure 3, Figure 11). E. Coli cells were dried for 48 hours at 108 cells / mL with 0.5 mg / mL of SAHS proteins. As measured by colony-forming units, E. coli showed enhanced survival compared to those dried without added excipients or with BSA, lactate dehydrogenase, or citrate synthase proteins (Figure 11), indicating that this was not a general effect caused by adding high concentration of protein. RvSAHS1 provided the most reproducible protection of E. coli, with all replicates in each experiment showing >10-fold enhancement of survival. At 0.1 mg / mL RvSAHS1, the protective effect decreased but was still statistically significant (Figure 11B). Trehalose, a well-known cryoprotectant that is expressed intracellularly in many organisms during desiccation, also protected E. coli against drying when added extracellularly, but not as strongly as RvSAHS1. Intracellular expression of SAHS proteins showed no clear-cut enhancement of E. coli survival upon desiccation (Figure 5C, 5D).

[0192] It was also tested if the SAHS proteins could enhance desiccation survival of the plant- symbiotic bacterium Rhizobium tropici, which is a model organism for nitrogen-fixing biofertilizers. When R. tropici cells that were dried at concentration of 7.2 x 109 cells / mL with and without 0.5 mg / mL excipients, we observed ~40-fold enhancement of cell survival upon addition of SAHS proteins (Figure 3B). Cells dried with trehalose at 0.5 mg / mL concentration showed 16-fold increased survival, while 0.5 mg / mL BSA caused a 4.2-fold increased survival.

[0193] SAHS proteins and BSA protect enzymes from dehydration-induced inactivation

[0194] Tardigrade proteins such as CAHS have been shown to protect desiccated enzyme activities

[0021] . It was tested if the SAHS proteins can preserve an enzyme, specifically lactate dehydrogenaseAttorney Docket No.002806-000126WOPT (LDH) from desiccation-induced inactivation in vitro. When dried, LDH (0.01 mg / mL) alone lost >90% of its activity compared to the non-dried control. The four SAHS proteins helped preserve LDH activity as a function of increasing concentration from 0.001 to 5 mg / mL, but BSA’s protective effect was comparable at each concentration (Figure 10). This observation contrasts with the specific effect of SAHS proteins vs BSA on protection of membranous structures such as liposomes and cells.

[0195] Structural changes of SAHS proteins under desolvated conditions

[0196] To investigate the behaviors of SAHS proteins upon water loss, their secondary structures were examined using circular dichroism (CD). CD spectra of all four SAHS proteins showed minima at ~215 nm, indicating that they adopt β-structures under aqueous condition (Figure 4A). This result was consistent with previous literature reporting β-sheet structures in RvSAHS1 and SAHS4. [13, 16-17] To investigate if these proteins can change their structures upon environmental stress, increasing concentrations of trifluoroethanol (TFE) and glycerol were used to mimic water-deficit conditions. All four proteins remained mostly β-stranded up to 25% TFE added, but displayed notable increase in helical structures at 50% TFE. At 50-75% TFE, all proteins showed spectra with two minima around 208 and 222 nm, which are characteristic of α-helices. Calculating relative secondary structure content confirmed that the helical contents in these proteins increased from 10% up to 40% with addition of TFE (Figure 4B). In contrast, increasing concentrations of glycerol had no such effect, even though glycerol is a ‘crowding’ agent (Figure 12).

[0197] A filamentous, mesh-like structure was observed from dried RvSAHS1 proteins upon transmission electron microscopy (TEM) imaging (Figure 13). RvSAHS1 proteins dried at concentration of 1 mg / mL revealed entangled network of fibrous structures, while those dried at higher concentration of 10 mg / mL showed denser network that covered most of the grid. Such observation suggested that RvSAHS1 may undergo structural changes upon loss of water to allow for the higher-order structure formation.

[0198] To further investigate the pathway for structural change of SAHS proteins, a molecular dynamics (MD) simulation of the RvSAHS1 protein was conducted. To account for the possible inter- protein interactions, a water bath was first generated with three copies of a RvSAHS1 monomer, and the simulation was conducted using explicit solvent, openMM, and the Amber force field. The simulation was performed for a microsecond at 550K to induce breaking of hydrogen bonds in a time frame amenable to simulation. It was found that the root mean squared deviation (RMSD) of alpha carbons in the ensemble relative to the starting structures increased within the first 100 ns and then plateaued for the remainder of the 1 µs (Figure 5A, 14). As the simulation progressed, RvSAHS1 proteins showed the structural unfolding of beta-sheet structures and formation of alpha-helices: the beta-sheet componentAttorney Docket No.002806-000126WOPT decreased from ~50% to ~3%, and the alpha-helical component increased from ~7 to ~20% as the simulation progressed from 0 ns to 100 ns (Figure 5B).

[0199] Figure 5C shows the C-terminal segments of each monomer that are conserved among SAHS proteins, which contribute significantly to the initial beta-barrel structure (identified as C3 motif in

[0013] ). As the simulation progressed, this region highlighted how, as helices form, the motifs between subunits may become more closely aligned during the 0 ns to 100 ns simulation period. Figure 5D indicates how the beta sheet region within C3 motif, which is maintained through interactions between positively and negatively charged amino acid residues, gets separated to form a helix as the simulation proceeds. These sequences, highlighted in blue and red, were found to be highly conserved among SAHS family (Figure 15) based on an evolutionary conservation analysis using ConSurf

[0022] , indicating that such structural shift may be a shared trait among SAHS proteins.

[0200] Structural bioinformatic analysis of SAHS proteins and related binding proteins

[0201] To understand how SAHS proteins might undergo a dramatic transition from a beta sheet conformation to a predominantly alpha-helical state and protect vesicles and cells during drying, the solved structures of RvSAHS1 and RvSAHS4 were examined, compared with solved structures of fatty acid binding proteins from higher organisms, and patterns of sequence conservation were analyzed.

[0202] The structures of RvSAHS1 and RvSAHS4 (PDB IDs 5xna and 5z4g), fatty acid binding protein from human muscle (1hmr)

[0023] , the intracellular lipid binding protein FABP1 (6do6)

[0024] were examined, along with alignments of related sequences.

[0203] RvSAHS1 and RvSAHS4 are highly structured and similar to FABPs and related proteins [16, 17]. Thus, the description of these proteins as “intrinsically disordered proteins” is not accurate, at least in the hydrated state. All of these proteins are unusual in that they have a large internal cavity that may be filled with ligands, solvent molecules, or both. The sides of the cavity are defined by beta sheets that, for the most part, do not pack against a typical hydrophobic core. In the RvSAHS1 and RvSAHS4 structures solved by Fukuda and colleagues, this volume is occupied by solvent molecules.

[0204] In RvSAHS1, there is a small hydrophobic core, while the cavity is bordered by beta-hairpin loops from Phe50-His72, His83-Val96, Thr106-Ile113, Ala126-Asp139, and Lys150-Tyr163. (The Phe50-His72 loop also contains two short alpha helices.) In the absence of a ligand in the cavity, both faces of these loops are simply beta-sheets that face water on both sides. Thus, these structures would not be as stabilized as in a typical protein. Without wishing to be bound by theory, it is hypothesized that during a desiccation process, water would equilibrate out of the cavity, and as the environment becomes more dehydrated, other more stable proteins would pack against a SAHS protein, essentially crushing it and disrupting the beta sheet structure that surrounds the cavity. In this way, the SAHS proteins could actAttorney Docket No.002806-000126WOPT as deformable packing material to prevent other biological structures from deforming or rupturing during desiccation.

[0205] Among the 13 SAHS proteins of R. varieornatus, the amino acids pointing into the small hydrophobic center are generally conserved, while amino acids pointing into the cavity are not. The “hydrophobic core” is located to one side of the much larger interior and includes the amino acids Trp 32, Trp37, Met38, Trp41, Tyr64, Ile74, Trp75, Tyr81, Phe98, Leu100, Leu124, Tyr141, Val143, Leu148, and Ala159. Of these, Trp 32, Trp37, Met38, Trp41, Phe98, Leu100, Leu124, Tyr / Phe141, Val143, and Leu148 are rather well conserved, while the others are not. It is noted that the highly conserved tryptophans at positions 32, 37 and 41 are present in the newly proposed sequences for RvSAHS 9, 10, and 12, supporting the idea that these sequences were previously misannotated.

[0206] The amino acids pointing inward towards the cavity include Phe50, Phe53, Ile54, Leu57, Leu59, His83, Ile85, Val87, Lys90, Tyr92, Asp94, Thr106, His108, Leu113, Tyr115, Tyr117, Ala126, Val128, Val130, Ile137, Asp139, Lys150, Tyr152, Arg151, and Tyr165. Most of these are within 4 Angstroms of an ethylene glycol or the putative fatty acid in the RvSAHS1 structure

[0016] . Of these, Phe53, Val / Ile87, Tyr117, Arg / Lys161 and Tyr163 are largely conserved, and the hydrophobic residues Phe50, Ile54, Ile85, Ile118, Val123, Val125, and Ile137 are generally interchangeable with other hydrophobic residues. Arg / Lys161 is notable because it is conserved in fatty acid binding proteins and interacts with the carboxyl group in the fatty acid in the same way that this amino acid interacts with a carboxyl group on a putative fatty acid in the SAHS1 structure.

[0207] Another feature of the SAHS proteins and fatty acid binding proteins is the presence of an entry / exit point. In RvSAHS1, four turns define this area – three beta-hairpins with tips at Lys90, Asn109 and Ser132, and a one-turn alpha helix with Gln63 pointing towards the hole. The amino acid sequences around this region are extremely variable among the R. varieornatus SAHS proteins, with Gln63 and Ser132 near the edge of a gap in alignment. Moreover, since the SAHS proteins are secreted eukaryotic proteins, they will undergo N-linked glycosylation at Asn-X-Ser / Thr sites (which is not captured in proteins made in E. coli). The distribution of putative N-linked glycosylation sites in RvSAHS1-13 is strongly biased toward the amino acids surrounding the entry / exit site: Asn109 is likely N-glycosylated in RvSAHS1, 2, 8, 10, and 11 (and Asn107 in RvSAHS5); in RvSAHS3, 8 and 9 sites near Gln63 are likely glycosylated; and in RvSAHS5, 9 and 11 sites near Ser132 are glycosylated. RvSAHS4, 6, 12 and 13 do not have N-linked glycosylation sites. Of the 14 putative N-linked glycosylation sites in RvSAHS1- 13, twelve are near the opening into the cavity. The presence / absence and positioning of these sites may have a significant effect on movement of water and other small molecules in and out of the cavity.

[0208] Fukuda et al. discussed the possibility that RvSAHS1 may bind a ligand with a carboxyl group (e.g. acetate), but noted that not all of the SAHS proteins have these carboxyl-binding residuesAttorney Docket No.002806-000126WOPT

[0017] . Our analysis of alignments of the R. varieornatus SAHS proteins indicates a great deal of variation in the cavity-forming residues, implying that these proteins do not bind a common ligand, if they bind one at all.

[0209] Based on this analysis and data presented here, a model is proposed by which the thirteen SAHS proteins encoded by R. varieornatus undergo a transition upon dehydration in which water molecules equilibrate out of the cavity and crowding and pressure of other proteins then disrupts the structure of the SAHS proteins, which then reconfigure into a completely different conformation dominated by alpha-helices. The thirteen different SAHS proteins would have different characteristics for stability and water retention, such that they would collapse under different conditions of dehydration and pressure, allowing for a gradual process rather than a sudden phase transition. This idea is explored further in the Discussion.

[0210] Discussion

[0211] In this study, proteins belonging to the SAHS family of TDPs were tested for their potential to protect diverse biological structures. In addition, it was observed that the SAHS proteins undergo structural shift upon desiccation, and a diverse set of experimental approaches was used to suggest models of how the structural transition of SAHS proteins might occur to protect biological structures. Specifically, it was found that the SAHS proteins protect membrane-containing structures, specifically liposomes and living bacteria, against desiccation-induced disruption, but are no better than BSA at protecting the enzyme lactate dehydrogenase against desiccation. Based on CD analysis, the SAHS proteins appear to undergo a shift from a primarily beta-sheet structure to an alpha-helical structure under desiccation-mimicking conditions, and molecular dynamic simulation showed that alpha-helices can transiently form upon denaturation of proteins. Lastly, a comparative structural analysis based on the available putative sequences of SAHS proteins was performed. During this, it was found that sequences in Uniprot database corresponding to short (N-terminal truncated) SAHS proteins may have been incorrectly annotated, and that all SAHS proteins are likely secreted and share a common structure. As noted previously, SAHS proteins are distantly related to fatty acid binding proteins and consist of a large cavity and a small hydrophobic core off to one side. The structural analysis described herein indicated that amino acid side chains pointing into the cavity are variable, and putative N-linked glycosylation sites are also variable in position but tend to cluster around the opening from which solvent molecules might enter and exit.

[0212] SAHS proteins from the tardigrades R. varieornatus and H. exemplaris are thought to contribute to the desiccation resistance of these organisms. R. varieornatus is of particular interest because this organism can tolerate rapid desiccation and encodes 13 different SAHS proteins, at least some of which are constitutively expressed. It is not clear why so many members of the SAHS family are encodedAttorney Docket No.002806-000126WOPT in this organism, or whether their diversity is important in desiccation protection. Several different SAHS proteins were tested for their ability to be expressed in the E. coli cytoplasm without signal sequences. From R. varieornatus, SAHS1, 2, 4, 6, 7, and 8 were expressed while the Uniprot-derived sequences of SAHS3, 9-12 were not. SAHS1, SAHS4 and SAHS6 from R. varieornatus and SAHS4 from H. exemplaris were expressed at the highest levels (Figure 8) and were characterized further.

[0213] The four tested SAHS proteins reduced the extent of liposome fusion and bacterial loss of viability during desiccation. When a commercial POPC-based liposome preparation was dried in the absence of excipients and then rehydrated, the average diameter of the liposomes increased from about 70 nm to about 350 nm, indicating that many liposomes fuse during this process to make much larger aggregates. In the presence of 10 mg / ml of any of the SAHS proteins, the extent of liposome fusion was much reduced and even at lower concentrations there was a protective effect. The excipient disaccharide trehalose showed a comparable protective effect, while BSA was less protective (Figure 2, 9). When either E. coli or R. tropici was desiccated and rehydrated, the SAHS proteins had a strong survival- promoting effect, generally improving viability by about 50-fold and performing at least 10-fold better than BSA and several other proteins, and about 3-fold better than trehalose (Figure 3, 11). R. tropici is a nitrogen-fixing plant symbiont that represents a working model for the preservation of nitrogen fixing bacteria such as rhizobia and could itself be deployed as biofertilizer.

[0214] In contrast, the SAHS proteins did not specifically protect the enzyme lactate dehydrogenase against loss of activity upon desiccation and rehydration – the SAHS proteins had a slight effect, comparable to the protective effect of BSA (Figure 12). The ability to stabilize proteins / enzymes and prevent their aggregation, which is often referred to as “molecular chaperoning”, has been associated with intracellular stress protection [25, 26]. For instance, heterologous expressions of tardigrade small heat shock proteins (sHSPs) improved bacterial desiccation survival, and their protective mechanism has been attributed to their ability to reduce protein aggregation during desiccation

[0027] . Similarly, CAHS proteins, whose heterologous expressions in bacteria and yeast enhanced their desiccation survival, showed higher ability to protect enzymes from desiccation-induced inactivation compared to BSA in vitro [15, 21]. Conversely, while not wishing to be bound by theory, where the SAHS proteins tested in this study are presumed to function extracellularly, it is not surprising that their main role is not to protect the proteins, which is also consistent with our observations that heterologous intracellular expression of SAHS proteins does not significantly improve bacterial desiccation survival (Figure 13).

[0215] SAHS proteins, similar to those reported for CAHS proteins

[0013] , appear to undergo a shift to a primarily alpha-helical conformation in a desiccation-mimicking condition. MD simulation of three SAHS proteins revealed that, as the beta barrel structure of the protein unraveled, the conserved beta- sheet regions shifted into helix upon breakage of hydrogen bonds between protein and solvent.Attorney Docket No.002806-000126WOPT Importantly, where the simulation was performed using multiple RvSAHS1 proteins, it was observed that helices formed upon structural shifts were aligned closely, indicating that loss of hydrogen bonds may lead to increased inter-protein interactions between the conserved motifs of protein molecules and potentially form oligomeric higher-order structures. Based on this simulation, while not wishing to be bound by theory, the structural shift was attributed to the disruptions of hydrogen bonds and ionic interactions, which are both expected to happen during dehydration. Furthermore, where structural shifts in evolutionarily conserved motifs have been observed, it is contemplated that such behaviors may be shared among the SAHS protein family.

[0216] A combined analysis of the structure of two solved SAHS proteins and the pattern of amino acid conservation in the SAHS proteins (Figure 6) indicates that the SAHS proteins all have a large cavity that may simply be full of water. The SAHS proteins are similar in sequence to fatty acid binding proteins (FABPs), and the SAHS and FABP structures are superimposable. However, the amino acid side chains that point inward in the SAHS protein cavity are not strongly conserved, suggesting that these proteins are not binding to a particular ligand but may simply be filled with water or with variable solutes. Moreover, in FABPs the cavity is significantly larger than can be occupied by a fatty acid, and since fatty acids are moving in and out during intracellular transport, they are presumably exchanged with water in this process.

[0217] A model is envisioned for the protective mechanism of SAHS proteins in which the SAHS protein cavity is filled with water and possibly other solutes in the hydrated state, but during dehydration the water is extruded. Thus, the SAHS protein would collapse as a result of Brownian motion of macromolecules and pressure on the outside of the protein without a corresponding pressure from the inside. This would lead to the denaturation of the protein and transition to an alpha-helical state. It is further envisioned that the different SAHS proteins have different kinetics and equilibria for this transition, such that when a tardigrade is being dehydrated, there is a gradual conversion of the different SAHS proteins to a protective alpha-helical state. The presence or absence and positioning of N-linked oligosaccharides in the different SAHS proteins near the entry / exit point may give them distinct dehydration-transition characteristics. After the conversion to an alpha-helical state, the SAHS proteins may form higher-order, gel-like structures that stabilize membrane-bound elements of cells, which may constitute rampart-like “special extracellular structure (SES)” observed outside of the secretory cell membranes of dried tardigrade

[0028] .

[0218] SAHS proteins can have a practical application in protecting non-tardigrade cells, such as agricultural or other microbes, against desiccation. It was found that two model bacteria, E. coli and R. tropici, could be stabilized against desiccation by the presence of a single SAHS protein. R. tropici is a “plant-growth promoting rhizobacterium” and PGPR use is often limited by their low desiccationAttorney Docket No.002806-000126WOPT tolerance [29, 30], highlighting the potential applicability of SAHS proteins to improve the durability of microbial biofertilizers. Also, desiccation would permit easier and lower-cost transportation and safer handling of these microbes in the field. In summary, this work provides the evaluation of tardigrade SAHS proteins in terms of their protective abilities, which supports their practical applications as stabilizing molecules.

[0219] Methods

[0220] Sequence analysis

[0221] SAHS protein sequences used for expression in E. coli (Figure 8) were obtained from Uniprot database (available on the world wide web at uniport.org). Protein subcellular localization and signal peptides were predicted using TargetP 2.0 server available at services.healthtech.dtu.dk.

[0018] Genomic sequences encoding R. varieornatus SAHS proteins were from the Ramazzottius varieornatus contig Scaffold001, strain YOKOZUNA-1, whole genome shotgun sequence, GenBank accession number BDGG01000001, which encodes SAHS1-12, and Ramazzottius varieornatus contig Scaffold002, GenBank accession number BDGG01000002, which encodes SAHS 13.

[0222] Protein expression and purification

[0223] Expression vectors encoding His6 / SUMO-tagged SAHS proteins were obtained from Twist Bioscience and transformed into LEMO21(DE3) competent cells (New England Biolabs) according to the provider’s protocol. Transformed cells were grown in TBM-5052 autoinduction media containing 100 µg / mL Kanamycin, 25 µg / mL Chloramphenicol and 2 mM L-rhamnose at 37°C overnight. The cells were harvested by centrifugation at 4,000 g for 10 min, resuspended in lysis buffer (20 mM NaH2PO4, 500 mM NaCl, 20 mM Imidazole, pH 7.5) supplemented with 0.25 mg / mL lysozyme (Milipore Sigma), turbonuclease (Accelagen) and 1% n-Nonyl-Beta-D-Glucopyranoside (Cube Biotech) and shaken vigorously for 1 hour. The total lysate was then centrifuged at 4,000 g for 30 min and supernatant was obtained. This clarified protein lysate was then shaken with Ni-charged IMAC Magbeads (Genscript) for 1 hour to bind tagged proteins. Beads were then washed 3 times with wash buffer (20 mM NaH2PO4, 500 mM NaCl, 20 mM Imidazole, pH 7.5), washed once more with PBS buffer, and incubated with PBS buffer supplemented with Cth SUMO protease

[0019] at 0.02 mg / mL shaking overnight to achieve the cleavage of SAHS proteins from N-terminal SUMO tag. Cleaved proteins were recovered from the supernatant, filtered through 0.2 µm filter, concentrated using Amicon ultra 0.5 centrifugal columns (Millipore, 10 kDa MWCO), and stored at -20°C. Purified proteins were inspected using SDS-PAGE and SimplyBlue staining (Invitrogen).

[0224] Circular Dichroism

[0225] Proteins were buffer exchanged by overnight dialysis to 20 mM NaH2PO4, pH 7.5 and diluted to a final concentration of 0.25 mg / mL. Protein secondary structure was examined using far-UVAttorney Docket No.002806-000126WOPT circular dichroism (CD) using a Jasco J-815 CD spectropolarimeter equipped with a Peltier temperature controller and single cuvette holder. The CD spectra were obtained by averaging three wavelength scans from 200 to 260 nm in 0.5 nm steps in a cuvette with a pathlength of 1 mm. To examine protein secondary structures in water loss-mimicking conditions and molecular crowding conditions, trifluoroethanol (TFE) or glycerol was added to the sample at final concentrations ranging from 25% to 75%.

[0226] Lactate dehydrogenase inactivation assay

[0227] L-Lactate dehydrogenase (LDH) enzyme (Roche) was diluted to 0.01 mg / mL concentration in 100 µL of 25 mM Tris / HCl buffer containing various concentrations of SAHS proteins or BSA (Sigma-Aldrich).50 µL of each sample was stored at 4°C while the other 50 µL was dried using a Savant SPD131DDA SpeedVac (Thermo Scientific) for 2 hrs without heating. Dried samples were rehydrated and activity was measured at 50 x dilution in a buffer containing 100 mM sodium phosphate (pH 6), 100 µM NADH and 2 mM pyruvate. Absorbance at 340 nm was measured using Synergy H1 plate reader (BioTek). Relative activity after dehydration was calculated by comparing the initial linear reaction rate of dried sample to that of non-dried control sample.

[0228] Liposome drying assay

[0229] POPC liposomes (100 nm, Tribio science) dialyzed against PBS buffer were diluted to 1.4 mg / mL concentration in 50 µL PBS buffer containing various concentrations of proteins and excipients. Samples were then dried in a microcentrifuge tube with an opened lid placed inside a sealed chamber filled with Drierite dessicant for 48 hrs at room temperature. Samples were rehydrated, diluted 10 times and their sizes were measured using Wyatt DynaPro Plate Reader III dynamic light scattering (DLS) device.

[0230] Bacterial cell drying assay

[0231] E. Coli vectors expressing SAHS proteins without SUMO tags were ordered and synthesized from Twist Bioscience and transformed into LEMO21(DE3) competent cells (NEB). To test for the effects of intracellular SAHS expression, cells transformed with each SAHS protein as well as mEGFP expression vector were grown in LB medium containing 100 µg / mL Kanamycin and 25 µg / mL Chloramphenicol at 37°C until OD600 of 0.6, added with 0.1 mM IPTG and 2 mM L-rhamnose and further grown at room temperature overnight. Cells were harvested and resuspended in PBS buffer, and diluted to contain 5x106 cells in 50 µL PBS buffer. Cells were then dried in a microcentrifuge tube with an opened lid placed inside a sealed chamber filled with Drierite dessicant for 48 hrs at room temperature. Subsequently, cells were rehydrated and immediately plated onto LB-agar Kan / Cm selection plates with a serial dilution to count the colony forming units the next day. To test for protection by extracellular SAHS proteins, LEMO21(DE3) cells transformed with mEGFP expression vector were grown in LBAttorney Docket No.002806-000126WOPT medium containing 100 µg / mL Kanamycin, 25 µg / mL Chloramphenicol at 37°C overnight, without any rhamnose and inducer added. Subsequently, cells were harvested and resuspended in PBS buffer, and diluted to contain 5x106 cells in 50 µL PBS buffer supplemented with varying concentrations of SAHS proteins or other control excipients, dried and rehydrated following the above protocol.

[0232] For R. tropici drying assay, R. tropici CIAT 899 cells were grown in PY media overnight in round-bottom culture tubes. The culture tubes were placed in an incubator set to 28°C and shaken at 200 rpm, then transferred to larger volume flasks the following day. Following incubation, the bacterial culture was centrifuged at 4,000 × g for 15 minutes. The resulting pellets were then resuspended in phosphate-buffered saline (PBS) and diluted to reach an OD600 of 0.4. The cultures were centrifuged again, and the supernatant was replaced with PBS buffer containing excipients. The solutions were pipetted into 250 µl tubes in 100 µl aliquots to contain total 7.2 x 108 cells and placed inside a sealed container with Drierite desiccant (10-20 mesh, Thermo Scientific) to allow the samples to dry for 48 hours. Once the samples were dried, three samples of each condition were taken out at relevant time points and resuspended in PBS. Subsequently, the samples were diluted and spread onto PY agar plates for colony counting.

[0233] Transmission Electron Microscopy (TEM)

[0234] SAHS proteins in PBS buffer were imaged by transmission electron microscopy using a Tecnai G2 Spirit BioTWIN TEM equipped with an AMT 2k CCD camera. TEM samples were prepared by depositing proteins onto carbon / formvar coated copper grids, staining with 2% uranyl acetate solution.

[0235] Computational modelling and conservation analysis of RvSAHS1

[0236] An initial structure for RvSAHS1 was generated using alphaFold using the sequence acquired from UniProt with the accession code J7MFT5. Ambertools generated amber input files with the ff19SB forcefield and explicit TIP3 solvent conditions. OpenMM was used to simulate 1000 ns at a temperature of 550 K after an equilibration at 310 K for 1 ns. Trajectories were generated with conformations captured every 10 ps. RMSD analysis of the simulation trajectory was performed using the Python package mdtraj. Visualizations of protein structures were produced using Houdini (SideFX software) and the Python packages mdtraj, biopython, and prody.

[0237] Evolutionary conservation analysis was performed on the RvSAHS1 sequence (J7MFT5) using ConSurf and the default parameters, where 60 homologs were collected from the UNIREF90 database using HMMER. Of these, 24 homologs passed the default thresholds (min / max similarity, coverage, etc.), and 25 were CD-HIT unique. The conservation calculations were conducted on the 25 unique hits, including the initial query.

[0238] ReferencesAttorney Docket No.002806-000126WOPT Moberg, N. et al. Survival in extreme environments – on the current knowledge of adaptations in tardigrades. Acta Physiol.202, 409-420 (2011). Boothby, T. C. & Pielak, G. J. Intrinsically disordered proteins and desiccation tolerance: elucidating functional and mechanistic underpinnings of anhydrobiosis. BioEssays 39, 1700119 (2017). Belott, C., Janis, B. & Menze, M. A. Liquid-liquid phase separation promotes animal desiccation tolerance. Proc. Natl. Acad. Sci. U. S. A.117, 27676-27684 (2020). Tanaka, A. et al. Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel. PLoS Biol. 20, e3001780; 10.1371 / journal.pbio.3001780 (2022). Tapia, H., Young, L., Fox, D., Bertozzi, C. R. & Koshland, D. Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. A.112, 6122-6127 (2015). Li, S. et al. Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation. Proc. Natl. Acad. Sci. U. S. A.109, 20859-20864 (2012). Hand, S. C., Menze, M. A., Toner, M., Boswell, L. & Moore, D. LEA proteins during water stress: not just for plants anymore. Annu. Rev. Physiol.73, 115-134 (2011). Veling, M. T. et al. Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells. ACS Synth. Biol.11, 1292-1302 (2022). Persson, D. et al. Extreme stress tolerance in tardigrades: surviving space conditions in low earth orbit. J. Zool. Syst. Evol. Res.49, 90-97 (2011). Hengherr, S., Heyer, A. G., Köhler, H. R. & Schill, R. O. Trehalose and anhydrobiosis in tardigrades–evidence for divergence in responses to dehydration. FEBS J.275, 281-288 (2008). Ingemar Jonsson, K. & Persson, O. Trehalose in three species of desiccation tolerant tardigrades. Open Zool. J.3, 1-5 (2010). Hesgrove, C. & Boothby, T. C. The biology of tardigrade disordered proteins in extreme stress tolerance. Cell Commun. Signal.18, 178; 10.1186 / s12964-020-00670-2 (2020). Yamaguchi, A. et al. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade. PLoS One 7, e44209; 10.1371 / journal.pone.0044209 (2012). Tanaka, S. et al. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic tardigrade improve osmotic tolerance of human cells. PLoS One 10, e0118272; 10.1371 / journal.pone.0118272 (2015). Boothby, T. C. et al. Tardigrades use intrinsically disordered proteins to survive desiccation. Mol. Cell.65, 975-984 (2017).Attorney Docket No.002806-000126WOPT 16. Fukuda, Y., Miura, Y., Mizohata, E. & Inoue, T. Structural insights into a secretory abundant heat‐ soluble protein from an anhydrobiotic tardigrade, Ramazzottius varieornatus. FEBS Lett. 591, 2458-2469 (2017). 17. Fukuda, Y. & Inoue, T. Crystal structure of secretory abundant heat soluble protein 4 from one of the toughest “water bears” micro‐animals Ramazzottius varieornatus. Protein Sci. 27, 993-999 (2018). 18. Emanuelsson, O., Nielsen, H., Brunak, S. & Von Heijne, G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol.300, 1005-1016 (2000). 19. Lau, Y. T. et al. Discovery and engineering of enhanced SUMO protease enzymes. J. Biol. Chem. 293, 13224-13233 (2018). 20. Furuki, T. & Sakurai, M. Group 3 LEA protein model peptides protect liposomes during desiccation. Biochim. Biophys. Acta Biomembr.1838, 2757-2766 (2014). 21. Piszkiewicz, S. et al. Protecting activity of desiccated enzymes. Protein Sci.28, 941-951 (2019). 22. Ben Chorin, A. et al. ConSurf-DB: An accessible repository for the evolutionary conservation patterns of the majority of PDB proteins. Protein Sci.29, 258-267 (2020). 23. Patil, R. et al. A ligand-induced structural change in fatty acid-binding protein 1 is associated with potentiation of peroxisome proliferator-activated receptor α agonists. J. Biol. Chem. 294, 3720- 3734 (2019). 24. Young, A. C. et al. Structural studies on human muscle fatty acid binding protein at 1.4 Å resolution: binding interactions with three C18 fatty acids. Structure 2, 523-534 (1994). 25. Balchin, D., Hayer-Hartl, M. & Hartl, F. U. In vivo aspects of protein folding and quality control. Science 353, aac4354; 10.1126 / science.aac43 (2016). 26. Lim, S. Glover, D. J. & Clark, D. S. Prefoldins in archaea. in Prefoldins: the new chaperones, Adv. Exp. Med. Biol.1106 (eds. Djouder, N.) (Springer, 2018). 27. Hibshman, J. D., Carra, S. & Goldstein, B. Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation. Commun. Biol.6, 121 (2023). 28. Richaud, M. et al. Ultrastructural analysis of the dehydrated tardigrade Hypsibius exemplaris unveils an anhydrobiotic-specific architecture. Sci. Rep.10, 4324 (2020). 29. Zvinavashe, A. T. et al., Programmable design of seed coating function induces water-stress tolerance in semi-arid regions. Nat. Food 2, 485-493 (2021). 30. Heveran, C. M. et al. , Biomineralization and successive regeneration of engineered living building materials. Matter 2, 481-494 (2020).

[0239] Table 1. Information of SAHS proteins used in this study.Attorney Docket No.002806-000126WOPT Name Uniprot ID Host organism Amino acid lengths (including secretion

[0240] Table 2. Estimation of protein localization using TargetP software. Protein name Other Secretory MitochondrialAttorney Docket No.002806-000126WOPT RvSAHS2 .0001 .9999 0

[0241] Table 3. Amino acid sequences of the SAHS proteins (after SUMO cleavage). Protein name Amino acid sequence Mw(kDa) SEQ IDAttorney Docket No.002806-000126WOPT KMGQEGTLSHDGVDLKYVCTEDGEQLITKINIPSKNQETIV TYTATGDDLEQTFTSNGVTGKRWYKKIHA

[0242] The “short” SAHS proteins, SAHS9, 10 and 12 from R. varieornatus are truncated at their N- termini relative to other SAHS proteins. The Uniprot versions of SAHS9, 10 and 12 lack signal sequences, could not be expressed in a soluble form, lack residues that appear to be important in forming the hydrophobic core in SAHS1 and SAHS4, and for SAHS9 and SAHS12 began with a methionine corresponding to a methionine also found in SAHS3, 7 and 8. We therefore explored whether these might be incorrectly annotated and examined the genome sequence of R. varieornatus for upstream coding sequences that might have been overlooked. The results of this analysis are that the SAHS9, 10 and 12 genes encode proteins that may extend in the N-terminal direction further than the annotation would indicate, and that the new predicted protein sequences align well with N-termini of other SAHS proteins. However, we have not found clear-cut start codons for SAHS9 and 10, and SAHS12 may be much larger than the other SAHS proteins. The annotation below is intended to be a work in progress.

[0243] The SAHS9, 10 and 12 genomic regions were obtained from Genbank accession # BDGG01000001 (contig 1), a large contig from the R. varieornatus genome. [1] Relevant genome sequence fragments were obtained as reverse complements, then copy-pasted into Microsoft Word, and visually scanned for the presence of splice acceptors upstream of putative start codons for ORFs that translate into the Uniprot sequences. Because all of the SAHS genes in BDGG01000001 are on the anti- sense strand, we generated the reverse complement of this ~ 9Mb sequence for presentation purposes. The numbering used below is according to this reverse complement. Upstream regions of SAHS9, 10 and 12 were also copy-pasted into the Expasy Translate Tool (web.expasy.org / translate), translated, and checked for long ORFs. A visual scan of the translated sequences revealed a pattern of tryptophans and other amino acids that align well with the N-terminus of other mature SAHS proteins (Fig.1B), but which are not found in the annotated sequences of these genes.Attorney Docket No.002806-000126WOPT

[0244] We also examined the intron / exon boundaries of these SAHS proteins and compared them to regions encoding other SAHS proteins to further validate the proposed mature amino acid sequences.

[0245] SAHS9 analysis

[0246] The SAHS9 protein sequence is most similar to SAHS10, 2 and 8. SAHS9 is also adjacent to SAHS2 and SAHS8 in the genome and is in a ~30 kilobase region that codes for SAHS11 plus SAHS10 (also adjacent) and SAHS7 plus SAHS1 (also adjacent).

[0247] When the SAHS9 genomic sequence is aligned with, for example that of SAHS2, there is a putative splice acceptor in SAHS9 DNA upstream of the annotated start codon at the same position as a splice acceptor annotated in SAHS2. Moving upstream, there is a putative splice donor in SAHS9 such that an intron of 79 bp is predicted, similar in size to the 82 bp intron in SAHS2. Moving further upstream, there is a predicted exon encoding a protein segment that aligns well with other full-length SAHS proteins, including Trp residues that help define the hydrophobic core. However, the predicted splice acceptor upstream of this exon has the sequence ... TCCTTCTTACCG, lacking the canonical AG, and yet further upstream it is difficult to identify a splice donor and start codon that would align with other SAHS genomic sequences. It is possible that SAHS9 is a pseudogene, that this region of the sequence contains one or more errors, or that some other mechanism operates to express this gene.

[0248] Below is the genomic region encoding SAHS 9, 2 and 8.

[0249] SEQ ID NO: 1. Bold text is the TATA box. Italicized text is the splice donor. Plain underlined text is the end of the signal. Bold underlined text are the Trp codons. Captilized codons are, in order from 5’ to 3’, the SAHS9 start and the SAHS8 start. 2575501 atttttcgcc tgtacagaca taagctgtta gagctgatgg agacacacct agatctcggg 2575561 ttgacaccca atgacacggt aatgatcctt agaccattgt ccgaaaaatt gctcatcacc 2575621 gttccctgta tggaaaaaaa ctggcttggc gggtaccttc gtgaacaata gtatgtcagt 2575681 tgagttttcg gtgagtcata aggggcagat agccgcgtgg cggaagcagc tgtttcggcc 2575741 tgtactgcaa accatgcata ggaaattggt ttaaaaaacg acttttccag ttttcttccc 2575801 tacatggata tttccgccca aatttagcca tccattcgtt tgtagaatgt ttccagcattAttorney Docket No.002806-000126WOPT 2575861 cgatacctta aggcatgctc ttgataacta cataagcggt ttcaagagtt cgactaacgc 2575921 ccctccagaa aacgtcgttt cctgaacagt ttagaaaata cctcggaacg gccaattaga 2575981 aaaaaacagg cgtcagttat gctgtacatt gctgcaacgg tgtacaggtc gaccgagcca 2576041 acggaatccg aataacatcc gaataatatc cttcttaccg gcgctgccat cgccattctc 2576101 atcgaagacc ctgctgacga aaaaggagca gaatggaccg gaaaaccgtg gctgggcgaa 2576161 tgggtctctg tacccgagca ggacaaaaac ctggcacagt tcaagaggaa gcttcgtaag 2576221 ttttacgtct cctgtgtgac tttgatcttt ctgtacgatt gatgacgttc ttcccgaata 2576281 tatatcgttc acagagctgc ctATGagcca tccggaagtc aatctcaact ctactgtctt 2576341 ggtcaaccac ctcaagaagg gagatgaata ccatcacaag attatcatca aagaatatta exon 3 2576401 caccaatcac gtaagtttat aaccgttagc gcgctcttaa aaaaagcaaa atggtcacgc 2576461 gaaaacgttt ttcaggtcgt ttacaagctg ggcgagcagt cacccggctc gtacgacggt 2576521 ttgtcctata gtgtaaagta tggagagaaa gatggcgcgt tggttggaac ggcccattac 2576581 acaggcacca aagaccagcg tctcaacata accatgcaca acgtctacaa gctcgaaggg 2576641 gatcgtcttc tcaagagctc caccatcgac ggagtaacac tgaattgcca tcacaagagg 2576701 cgcatctgaa gctgtgcacg tcgtttcagt tcttcgactt ttatcgatat tcttatagtc 2576761 ttgatcaacg cttgagtaaa ggtgtttcaa gatgcataaa gactttcttc gtccttggcg 2576821 aagcttatca gtcatcttct cgaatcaaga tatccattcc atcggcagaa ggagagattcAttorney Docket No.002806-000126WOPT 2576881 tccgctttcc acacttgcgt gggtagactc gtgtagagcc gcgcgtatcg cggtgaacat 2576941 cgagaaaggt tcgaagcttg gcttagagtt ctcagacggt cacgggacaa gatcagatac 2577001 ggcgcaaggg tgttttcccc agcatgcaga ccgttatgac acatagttga ctttgatgcg 2577061 actggtggta ctgggaagaa tgtaacgctc caatattcgc ctaaggtgta atcctaaggt 2577121 atacgtacag tagacgcaaa cagtacagac tcctccagca acggtacttc ctggccgtat 2577181 ataaaatcaa ggttcgggtt ccttccaacc tgtacctctt tacttcttgt aactttctca 2577241 cggagaatac tcgcagaacc atgcatcgat ttgtccttgc tctcgtcgtt tttgccggta 2577301 aacttgaaag cttcttaaga gatttaccgt cttgtgtgct atgaaagagg acgagcatga Intron 1 2577361 gctaaaaggt catcgtaaga aacgtgcgtg gctatatatg tacattttct cctggctttg 2577421 caggtgctgc catcgtctgg gccgctgatg acgctgctca cgaagaaggc gtagaatgga 2577481 ctgggaaacc gtggatgggc aaatgggaat ccgacccatc gaaggacgag aacgttgagg 2577541 aattcaaaaa gaagctccgt aagttacttt gcatttgcat ctcctcgtta gttagttttt 2577601 ttagtgttca gtcgacgctg gttgtctggt ggattcgtcg aattccccgg cctgatattt 2577661 tttgcatagc tttatagatt tacccgttca gatccgtaca agcgagaaac caagacacct 2577721 atgtttgttt gcagagcttc cgatgagcca ctcggaaatg aacaaaaact ccaaagtttg 2577781 gatccatcac tacaagaagg gagacgagta ccatcacaaa atcatcatca acgacgccca 2577841 ttacaaaaac gatgtaagtc cgcaaacttt cccggttaca ttgtttctta cgtctttgctAttorney Docket No.002806-000126WOPT 2577901 gcacagatca aagaaaatta ttttcggctt tgtagatcgt cttcaagctg ggtcaagagt 2577961 ccgccggttc gtataacggc tcatctttca gcgtgaagta cgaggacaaa gacggcgctc 2578021 tagtcggaag cgtccactac actggcacca aagaacagtc tcttgacaag accatcaaca 2578081 acgtcttcaa gctcgaaggt gaccatctgg ttaagacttc caccatcgag ggagtgacca 2578141 tgaagcgcca ctacaacaaa cgccagtgaa gttgtcgttg cggctaaatt ttttcctttc 2578201 tgcaaattca tgcccgtttt gtcgagtctc tcctgcttcc catcgttcta aagatttttg 2578261 cagtactgag ttatcagggc tttgtttctg ttctcgttct atcctcgtat tttcttttcg 2578321 ttcaccggat acagtaaagc tgcgtttcaa agccaggttt tttatctgcc tgttggtcgg 2578381 acggattgtc ggaccaactc agatatcgat cgggctgatt gtaaacagat actacgtatt 2578441 ttctcgtact cttcgcactg gctaacgtta ggttatacgc tctaaacggt gatcgagaaa 2578501 gttttaaaag caggcacgac atactttaaa ttctcaaacg aacagtactt ttcccatcag 2578561 atgaagcata aagcgggttt tcccagcaaa gacgcttcag cccatggcca attgaccttt 2578621 ggccgaaggc tgctggctac gaaagcgctg agtaatgata agtactcgta ctctattgtt 2578681 caactaagaa acctccgaat gtaacccaaa ggagaacgac ttcgtgccaa cttgatctta 2578741 gactgcagaa taaaccagta taaaatcaaa ccttcgccat ttcacagtac aaagcacgtt 2578801 ttaaactttt ccagccactt tcttcacgta gaagtcgagc cgcaagcATG aagtgtatcc 2578861 tagctctcgc tctgtttgcc ggtacgtcga cagacaatta aaattttggt gctttttagtAttorney Docket No.002806-000126WOPT 2578921 atcatagggt aatgcaagac gttgactcga cttgtgtgga tgatcacatg agcatgtgat 2578981 tcatagttga tttcattggt ctgttgcagg tgttgccgtc gtgtgggctg gcgatgatgc 2579041 cgctcatgaa gaaggagtcg actggacctc caagccttgg ttgggcaagt gggaatctat 2579101 cccggagaag gatgaaaacc tcgtggagtt tctcaagaag ctcagtacgg tgtatagctt 2579161 tctcttcctt cagtattttc tgttctggac acatatcata aacagggtcc tcgctccagt 2579221 ccccgtccac cttagtctta gaaaatttag agttttcgag ctttacttct gttcacagat 2579281 gttcccatgg accactctaa aatgaacgcc accgtcaagg tccacctcaa ccactacaag 2579341 aaaggagacg attaccacca caagatcatc gtcaaggagg ctgagtacaa gaacgatgta 2579401 agtttgaccg ctttcgatga gttgacccgt cagctagatg acccctttcg gaagtctatc 2579461 ctatggggtt tccgactgac actagacacg taatatctgt ttcgattttt aggttgtctt 2579521 caagttaggc caagagtccg ctggttcgta caacggttcg tccttcaccg tgaagtacga 2579581 agataaggat ggcgcactgg tcggaaccat tcactacacc ggtacgaagg aacagagcct 2579641 cgacaagacc atcaacaacg agtacaaggt tgaaggcaat caactggtca agacctcaac 2579701 cctcgaagga gtgacacaca agagatacta caacaaacgc aactgaggtt gttcttgccg 2579761 ctatatggtt gttagttcgg ccaagttttt cctattttcg catcttttgg ctttttctca 2579821 tcattcttcc agtctttatg ttcgcctgtt ctcactgtac tttgctcaag cccatttcca 2579881 gcaacaagtg ctttattcac gtcccagaac cagctttccg ctcgtcgttt acctattcgcAttorney Docket No.002806-000126WOPT 2579941 ggaagaaatc atccaacatg accaatactg cttctgagcc gataaagtca acgattctcg 2580001 tgctggattc gatttttcga tgtagatact ggacttgact ttgacttgta cggagcgaca 2580061 ccgagcgcag caggaacgca agaagaaaac caagcagata ctttaacccg cttcaaattt 2580121 agagtgggtc tttccaggct gccactgcat tgacccgtgt tttgtttcct gaggtagact 2580181 gatccctctc ctcgaggttt caataggttc gagaacggga aagcgttgta gattacggga

[0250] SAHS10 analysis

[0251] SAHS11 and 10, shown below, are adjacent in the R. varieornatus genome and about 5 kilobases from the SAHS, 2, 8 cluster. The SAHS10 annotated start codon lies within a putative intron corresponding to the second intron in SAHS11 and other SAHS genes. SAHS10 encodes two of the three conserved tryptophans near the N-terminus, while the first tryptophan is replaced with a structurally plausible arginine. Upstream of this coding region, at the position of the splice acceptor found in most SAHS genes, a potential splice site is mutated, reminiscent of this site in SAHS9.

[0252] SEQ ID NO: 2. Bold text is the TATA box. Italicized text is the splice donor. Plain underlined text is the end of the signal. Bold underlined text are the Trp codons. Captilized codons are, in order from 5’ to 3’, the SAHS11 start and the SAHS10 start. 2568721 tcgtctgcca cgtgcacgat gccaggaacg aaaaagcaag ctaggttcag agccaacctg 2568781 ttcatacaca agtgctatcc agctatcctg ctctgcagac ttttaggcta ttctgcacag 2568841 tctccatttc tgttaactgc agttagaacg cctcaattgt cATGcggcga tttcttcgca 2568901 atcttctcat atttgccagt atgaaccttc ccacacctgt aacgttatgt gtttatgatt 2568961 tttctatttt cgtcctttga atcagaagcc tgactttcca aggataatat aagtaaacga 2569021 aatgtaatta ggtggaccga agggatctag tgttttcccc gtcgcattgg cgaatggctgAttorney Docket No.002806-000126WOPT 2569081 ctgttttgga taattatgat tttcgacgaa tcgataagag cttggctcaa gggactgtat 2569141 tatcccacag cttgatcctt tacaggtgcg gcagtctgcc tggcagagca cgatcctggc 2569201 catgaggaag gagccgaatg gactggaaaa ccatggctgg gcaaatgggt ttccgttccc 2569261 gagaaggacg taaacgtact aaacttcatc acagagatcg gtcagtgcca tgatgcatca 2569321 acttcagccc tcgtggtacg ctcagcggat ggcacgggac attcgaaatg gaatgaattt 2569381 tcacaggtgt cgctgcgagt catccggaac ttcctcccat cgttacggtc ctcgtcaacc 2569441 attacaaaaa gggcgacgag taccaccaga gactgcgcgt caaggaagta gctgatcttg 2569501 atgatcacga cgtaagtacg atacacggta ctcccttgac ccaggcttaa ggaaggcttc 2569561 ggaccgacgt taacgttaag gaacctgtac tgtagattgt ctacaaactg ggccaagaaa 2569621 ccaagaacgt ttttaacggt accaccttca gtgttaagta cgatgagaaa gatgacgctc 2569681 tcgtcggaca agtcatgcta ccctcgaaca acgcgactta caagaacgag ttcaaggtcg 2569741 aaggggactt ccttgtcaag gtatcgcagc ccactttgaa ttccgcctca gccattatgt 2569801 cactggtcac atcttttttc gcttgttgca gacctctgac gctcatggaa ttgtccacaa exon 5 2569861 acgatattac aagagacgga actaaatttc aagctggtgc cgaggttcag tctcagattt 2569921 tgtctttgga atcaagctcg cttgcgtgtt tgagttgtcc ccagagtaaa gtcacatgtc 2569981 gttcagttgg cgtgtcgata gaggttcttg tgcttagtct ttagtcgtac aagattttcc 2570041 cgagcagacg ggtggttcag tcggtttcca cttttttccc acattatccc gcatatctctAttorney Docket No.002806-000126WOPT 2570101 gtatcacaga cgaccactgc cagaaaccgc gccggggcgc ttgaccagat cagatggtga 2570161 gcaaagcagg caatcataat tcatcagcca acctttgaaa ccacgttttt ttcccccgag 2570221 tgtcctaatc ggcgaatgac aatgcttgag gtcgaaaaaa agtgactaca ggctactcac 2570281 aataagggca ggtcaacact atatatgcag gcctctcatc aaaccttcca ttagtttctc 2570341 tacccatcgt actttcttac atagccagct tgaacgatgc atcgatttat ccttcttctc 2570401 gcagtctttt ccggcaagat cgagctcata tcgggatatg gttccttata ctcgggagat 2570461 ggctctgtca tcggttggct ccagccgcta actttccgtg tgcgacgtca ctgcgagaag 2570521 catcggcagg cagaatccgc actgacgcca taagaaagct taagcaagag gagttcggtt 2570581 gtcaagatgc tggatgggaa gctgtctgcg tgttttgttc gttgtgcatg tactcgtact 2570641 gtcatgatgg tgtttcgcgg gtgtggcctt tatctgggcc gccgaagacg ctgttcacga 2570701 agaaggcgta gaacggactg gcaaaccgtg gatgggcaaa tgggtcgccg ttcctgagaa 2570761 ggacgaaaat cacgagggac tcaagaaaaa gctccgtgag tacattcgtg ttggcttctg 2570821 accttcattc gtcagttttc ctcgacaatc gtcgatgtga tgtatgagcc accATGggga 2570881 tgcgatctgc agatatcccc ttgagtcacc cgcatctgaa acacaacaac agagtgtggg 2570941 ttaacaccta caagaaggga gacgaatacc accacaagat tattatcaag gaagccggct 2571001 ataccaatga tgtacgtatg cgaagccatt atgattatgc aaactgccga acgctgcatg 2571061 gctttaatct ttccacacag tacgtgcgta ctgagatctc ttgcatacct gagactgattAttorney Docket No.002806-000126WOPT 2571121 ttcaggttgt cttcaagctg ggtcaagagt ccgccggctc gcataacggc tcatctttca 2571181 gcttgaagta cgaagacaag gatggcgcct tggtcggcac cgtccatcgc accggcacca 2571241 aggaacagcc cctggacaag acgatcaaca acgtcttcaa gctcgagggt gaccatttag 2571301 ttatgacctc caccatcgac ggagtaacca tgaaacgcta ctacaagaca cgaacgtgaa

[0253] SAHS12 analysis

[0254] In the annotation of Scaffold 1 of the R. varieornatus genome, the ORF upstream of SAHS12 (RvY_02619-1) encodes a protein whose C-terminal region aligns well with the N-terminus of the mature SAHS proteins. A splice donor is present near the end of this sequence that corresponds to intron 2 of SAHS2 and other well-annotated SAHS genes.

[0255] This region thus may encode a large protein with the sequence

[0256] SEQ ID NO: 3 MTQPMSFAQCSADRGKHSGTTIWTLLRIYLACQKAILRSKLRLRAPLFPTVEPNPAPIQNAAPAPS AAQRRRNFAASHAANVDLPGSVWHGETWGDQHDPPNRLAADVDNFDWRSKFWLGKWSSIPE KDQNLEAYLAVMgvdMNHPNMKKDQPVTLQTFKKGDKYHHKIVVEEAGYINDVIFRLGRETPGSYN GQQITVNYEEQGGALVGTVKYPAHNKVIHNTYEMDGQNLAKTSECEGVVHKRWYNKQQN, where the plain text amino acids are from RvY_02619-1, the “gvd” is glycine-valine-aspartate arising from the splice junction and segment upstream of the annotated SAHS12 start codon, and the italicized amino acids are from SAHS12 as annotated in the Scaffold 1 annotation.

[0257] SEQ ID NO: 4. Bold text is the TATA box. Italicized text is the splice donor. Plain underlined text is the end of the signal. Bold underlined text are the Trp codons. Captilized codons are, in order from 5’ to 3’, the RvY_02619 start and the SAHS12 start. 2002141 gatgagcaac aagtaatggc tcggtaccag ctaagacctg aggcaaaatc aacgcggatg 2002201 gatgcagatc tatttcagtg aacaaaggac ggaagatttc tgatgggagg aaaagtagta 2002261 cagtgtaata ataatccgag aaagaaaatc aaattctgac tttcagagtc atctgagATG 2002321 actcaaccga tgagtttcgc acagtgctcg gcggaccgcg gtaaacattc cggtaccacgAttorney Docket No.002806-000126WOPT 2002381 atctggacgc tgctgcgaat atacttggcc tgccaaaaag ccatccttcg atcaaaatta 2002441 aggctacgag gtaaacgtca caattcggac gggccaattt tctcgtcaca attcgggcgg 2002501 ctcgaaaagt tgatacgtta tttgctacgc tgtattgatt gatcaccgta gatcggttga 2002561 tacaacttta ggctttttaa cattgcgcat acccctcttc ctcccaccgc tgactccgca 2002621 actctttgac atctttcgat atctcaacct atcacaagag atagaagaga ggatgttcta 2002681 ccctgggaag caaaaactag gcagtttgga gcagaaacag ctgagataca gaccgcccaa 2002741 attgtgacga gaaaattggc ccgcccgaat tgggacgttt acctcgtaac gattatgtgt 2002801 tttgtcttct aatcagctaa tcaagtcatt ggttttcatg aggattatgt tgatgcatga 2002861 ttcctcgagc ttttgacagg ccgtaccgtg cttcacggag tgtttacccg ccgaatactg 2002921 ccaatgagtc attagaagca ccaggattgg tctgttactc tgtcctattc aaatcaggcg 2002981 aagcgggaga atgacgaagc ggatactgac gcttagcctt cgcatggtct gttgtttcgg 2003041 cttgcaccgt tccatccaga ttttagggtg agcaaccgtc agcaacgtgg ccgtcctggc 2003101 aaagtctttt cctgtcctca ccttcagctc ctttatttcc cactgtggag cctaatccgg 2003161 cacccatcca gaacgcagct cccgctccat cggctgccca acgtcgtcgg aactttgcag 2003221 ccagtcatgc cgccaatgtc gacctgccag gctccgtttg gcacggggaa acctggggag 2003281 atcaacatga tccgccgaac cgattagcag ctgatgtcga caacttcgac tggagatcga 2003341 aattctggct gggcaagtgg agctctatcc cagagaagga tcaaaatttg gaggcttaccAttorney Docket No.002806-000126WOPT 2003401 ttgctgtcat gggtaagccg gagacttacg cttgattgac tgtcattgat tgactggctt 2003461 ccaacttccg cttccgggtt caacaggtgt cgacATGaac catcccaaca tgaagaagga 2003521 tcaacccgtt acacttcaga ccttcaagaa gggtgacaag taccatcata agatcgtggt 2003581 cgaggaagcc ggctacatta acgatgtaag tttgatggac ctggcagctt tcttccagcc 2003641 ggatgtcatg ttgctttatc cgacgcgaat gtagtaacgc ctttcatatt actccatgtt 2003701 gtgccgtaat ggagaagcgt ggcgataaca tgcgtgtgat acgcatggta ttccggacag 2003761 ggaggcctat gtgtcctttt attgttattc caggttattt tccgcctcgg ccgagagact 2003821 cccggatctt ataacggtca acagatcact gtcaactatg aggaacaagg cggtgctttg 2003881 gtgggtaccg tcaagtatcc cgcccataac aaggtcatcc ataataccta cgagatggat 2003941 gggcagaatc tggccaaggt atcaaacctt acttcctctt ttgcagcttt tttcctggaa 2004001 acgccggtct gacaatttgc tgacagcggt gctcgtttgt tgcagacttc cgaatgtgag 2004061 ggtgtcgttc acaagcgctg gtataacaag cagcaaaact gaagcctgtc gcctccatta 2004121 attgtgatag ttttgccttc gagttacgat tcctcatgaa agtgcttttc atgtatgtct 2004181 gccattttaa ctaactgtac cagatgttga tttacggttt tggatagctg cagtattcct 2004241 tcagagaact ttgcgatgca acgaaccatg ttccttcttt gtccactgtg aatacgatgt 2004301 gctgcgatct actatggaag cactgcctac gtagagaaaa ccgaaaatgt cctgcctcag 2004361 aactagtttc cagtttccta gacatttcga caccctccag tatctttctc gcttagggtgAttorney Docket No.002806-000126WOPT 2004421 tcgcacgaac agaaactaca gttctaactg cgcctttcgg cggctgactt cgcattcgaa

[0258] Reference 1. Hashimoto T. et al. Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat. Commun.7, 12808; 10.1038 / ncomms12808 (2016). Example 3

[0259] Tardigrades, microscopic animals that survive a broad range of environmental stresses, express unique set of proteins termed as tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focused on the proteins belonging to the secretory family of tardigrade proteins termed secreted-abundant heat soluble (“SAHS”) proteins, and investigated their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevented desiccated liposomes from fusion, and enhanced desiccation tolerance of E. coli and Rhizobium tropici upon extracellular application. Molecular dynamics simulation and comparative structural analysis suggest a model by which SAHS proteins may undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the ability of SAHS proteins to act as stabilizing molecules for preservation of biological structures and cells.

Claims

Attorney Docket No.002806-000126WOPT What is claimed herein:

1. A composition comprising at least one tardigrade secreted-abundant heat-soluble (SAHS) polypeptide and at least one viable microbe.

2. An agricultural composition comprising at least one tardigrade secreted-abundant heat-soluble (SAHS) polypeptide and at least one viable microbe.

3. A composition comprising at least one tardigrade SAHS polypeptide and a liposome preparation.

4. A composition comprising a cosmetic composition and at least one tardigrade SAHS polypeptide.

5. The composition of any one of the preceding claims, wherein the composition is an admixture of the at least one tardigrade SAHS polypeptide and at least one of a viable microbe, a liposome preparation, and a cosmetic composition.

6. The composition of any one of the preceding claims, further comprising trehalose.

7. The composition of any one of the preceding claims, wherein the composition comprises liposomes.

8. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is an isolated tardigrade SAHS polypeptide.

9. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide comprises at least 90% of all tardigrade proteins present in the composition.

10. The composition of any one of the preceding claims, wherein the composition does not comprise a viable tardigrade.

11. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

12. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is selected from SAHS1, SAHS2, SAHS3, SAHS4, SAHS6, SAHS7, SAHS8, SAHS9, SAHS10, SAHS11, SAHS12.

13. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is SAHS1.

14. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is selected from RvSAHS1, RvSAHS2, RvSAHS3, RvSAHS4, HeSAHS4, RvSAHS6, RvSAHS7, RvSAHS8, RvSAHS9, RvSAHS10, RvSAHS11, and RvSAHS12.

15. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is RvSAHS1.Attorney Docket No.002806-000126WOPT 16. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to one of SEQ ID NOs: 5-28.

17. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 5 or 9.

18. The composition of any one of the preceding claims, wherein the composition is dried.

19. The composition of any one of the preceding claims, wherein the composition is desiccated.

20. The composition of any one of claims 3-19, wherein the liposomes retain liposome structure upon rehydration.

21. The composition of any one of the preceding claims, wherein the at least one viable microbe comprises a Rhizobium, a Bradyrhizobium, an Azorhizobium, a Sinorhizobium, a Mesorhizobium, or a mycorrhizal fungi.

22. The composition of any one of the preceding claims, wherein the at least one viable microbe comprises R. tropici, B. elkani, B. japonicum, M. loti, M. huakuii, R. leguminosarum, R. meliloti, S. fredii, or S. meliloti.

23. The composition of any one of the preceding claims, wherein the viable microbe is R. tropici.

24. A method of stabilizing at least one viable microbe, the method comprising contacting the at least one viable microbe with at least one tardigrade SAHS polypeptide.

25. A method of stabilizing an agricultural composition, the method comprising contacting the agricultural composition with at least one tardigrade SAHS polypeptide.

26. A method of stabilizing a liposomal membrane, the method comprising contacting the liposomal membrane with at least one tardigrade SAHS polypeptide.

27. A method of stabilizing a cosmetic composition, the method comprising contacting the cosmetic composition with at least one tardigrade SAHS polypeptide.

28. The method of any one of the preceding claims, further comprising contacting the composition with trehalose.

29. The method of any one of the preceding claims, wherein the composition comprises liposomes.

30. The method of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is an isolated tardigrade SAHS polypeptide.

31. The method of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is provided in a composition and the at least one tardigrade SAHS polypeptide comprises at least 90% of all tardigrade proteins present in the composition.

32. The method of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is provided in a composition that does not comprise a viable tardigrade.Attorney Docket No.002806-000126WOPT 33. The method of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide comprises a Ramazzottius varieornatus (R. varieornatus) SAHS polypeptide or Hypsibius exemplaris (H. exemplaris) SAHS polypeptide.

34. The method of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is SAHS1.

35. The method of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is selected from RvSAHS1, RvSAHS2, RvSAHS3, RvSAHS4, HeSAHS4, RvSAHS6, RvSAHS7, RvSAHS8, RvSAHS9, RvSAHS10, RvSAHS11, and RvSAHS12.

36. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide is RvSAHS1.

37. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to one of SEQ ID NOs: 5-28.

38. The composition of any one of the preceding claims, wherein the at least one tardigrade SAHS polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 5 or 9.

39. The composition of any one of the preceding claims, wherein the at least one viable microbe comprises a Rhizobium, a Bradyrhizobium, an Azorhizobium, a Sinorhizobium, a Mesorhizobium, or a mycorrhizal fungi.

40. The composition of any one of the preceding claims, wherein the at least one viable microbe comprises R. tropici, B. elkani, B. japonicum, M. loti, M. huakuii, R. leguminosarum, R. meliloti, S. fredii, or S. meliloti.

41. The composition of any one of the preceding claims, wherein the at least one viable microbe comprises R. tropici..

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