Enzymes and methods of using same

Abstract

The present invention provides a cell including a recombinant DNA molecule including a first nucleic acid sequence encoding at least one first polypeptide being an enzyme selected from: inosine monophosphate dehydrogenase (Impdh), guanosine monophosphate synthetase (gmps), nucleotidase 5C (nt5c), polynucleotide phosphorylase (Pnp), or any combination thereof. Further provided is a method of using the cell of the invention, such as for synthesizing guanine.

Description

ENZYMES AND METHODS OF USING SAMEREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (YEDA-P-037-PCT.xml; size: 52,652 bytes; and date of creation: January 13, 2025) is herein incorporated by reference in its entirety.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of IL patent application no. 310520, titled “ENZYMES AND METHODS OF USING SAME”, filed 29 January 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0003] The present invention relates to enzymes, and methods of using the same, such as for producing guanine crystals.BACKGROUND

[0004] Across the animal kingdom, many organisms harness intracellular crystals made from organic materials, particularly guanine, for a wide variety of purposes. These range from enhancing vision to creating the vibrant colors observed in fish and chameleons. Recent studies have revealed that molecular crystals are much more widespread than initially believed. For instance, evidence indicates that unicellular organisms from all eukaryotic supergroups utilize molecular crystals for nitrogen storage. Yet, while extensive research has elucidated the cellular machinery and biosynthetic pathways of non-crystalline, pigmentbased systems, such as melanin production, the mechanisms governing molecular crystal formation remain largely enigmatic. Thus, these underexplored mechanisms carry profound implications for both cell biology and ecology.

[0005] The cellular processes driving crystal formation are intricate, relying on the synthesis of substantial quantities of guanine, an essential nucleobase in purine metabolism. Crystalforming cells execute this process with remarkable precision and control. Notably, each crystal is composed of billions of nucleobases and there may be hundreds of crystals in asingle cell. Yet, remarkably, these specialized cells manage to produce vast quantities of crystal-forming nucleobases while delicately maintaining cellular homeostasis, including DNA and RNA synthesis and overall cell function. Despite extensive research of these cells, which have long been recognized, the mechanisms regulating these intricately balanced biological processes have remained elusive.

[0006] It is generally believed that in both unicellular and multicellular crystals-forming organisms, nucleobases are synthesized within the cytoplasm. Then, they are transported by dedicated machinery into membrane-bound organelles, where the meticulously regulated process of crystal formation takes place. This process results in the generation of crystals with precise morphologies, which far exceed the capabilities of state-of-the-art materials science and solid-state chemistry. These crystals are specifically designed to serve a range of functions, including light scattering by prism-shaped crystals, light reflection by elongated plates, and space-filling squares. Recent studies have shown that thin crystal plates are formed by templated nucleation on protein scaffolds. However, the molecular mechanisms governing the morphology of these plates remain elusive.

[0007] Biogenic molecular crystals seldom consist of a single nucleobase; rather, they comprise a blend of nucleobases, primarily guanine and hypoxanthine. Previous research has identified several, often conflicting, biosynthetic pathways for guanine. Consequently, the exact route of crystal-forming guanine biosynthesis is uncertain.

[0008] There is still a great need for a deeper understanding of the precise mechanisms governing nucleobase biosynthesis and their contribution to crystal morphogenesis, which currently remains an underexplored avenue in the fields of cell biology and biomineralization.SUMMARY

[0009] To unravel the intricate molecular mechanisms and catalytic networks underpinning these unique biosynthetic pathways, the inventors embarked on a multifaceted investigation encompassing proteomics and spatial metabolomics, in vitro enzymatic activity assays, cellular localization studies, and genetic manipulations using the zebrafish (Danio rerio) iridophores as a model system (Fig. 2A). Zebrafish have harnessed the remarkable properties of guanine crystals for a multitude of purposes. These crystals, located in their skin and eyes, play a pivotal role in color and pattern formation, serve for camouflage, act as a light barrier, and enhance visual sensitivity, especially in low light conditions. Although the exactmorphologies of the guanine crystals in zebrafish vary between tissues, they all exhibit an elongated, semi-hexagonal thin plate structure.

[0010] The present invention, in some embodiments, is based, at least in part, on the findings of the entire purine biosynthetic pathway in iridophores. The inventors show that these cells exhibit a remarkable ability to boost guanine production while effectively suppressing the formation of undesirable byproducts. This regulatory process involves the upregulation of a distinct subset of paralogous enzymes, which are exclusive to crystal-forming iridophores. Moreover, some of these enzymes display remarkable substrate selectivity in comparison to their paralogues. Through genetic manipulation of essential enzymatic regulators associated with guanine synthesis, the inventors have uncovered their influence not only on cellular crystal quantities, but also on crystal composition and, surprisingly, crystal morphology. These variations occur by the incorporation of elevated levels of hypoxanthine within the crystals. Additionally, the current research has revealed that biogenic crystals of different morphologies, locations and functions also differ in purine composition. Significantly, the current 10 in vitro experiments demonstrate that alterations of crystal composition directly correlate with the resulting crystal morphology. This implies the intriguing possibility that over the course of evolution, organisms may have fine-tuned crystal morphology by precisely adjusting crystal composition.[Oi l] According to the first aspect, there is provided a cell comprising a recombinant DNA molecule comprising a nucleic acid sequence encoding a first enzyme selected from the group consisting of: inosine monophosphate dehydrogenase (Impdh), guanosine monophosphate synthetase (gmps), nucleotidase 5C (nt5c), polynucleotide phosphorylase (Pnp), and any combination thereof.

[0012] According to another aspect, there is provided an extract derived from the cell of the invention.

[0013] According to another aspect, there is provided a composition comprising any one of: (a) the cell of the invention; and (b) the extract of the cell of the invention, and an acceptable carrier.

[0014] According to another aspect, there is provided a method for synthesizing a purine- based compound, the method comprising: (a) providing the cell of the invention; and culturing the cell from step (a) such that the first enzyme is expressed, thereby synthesizing the purine- based compound.

[0015] According to another aspect, there is provided an extract obtained according to the method of the invention.

[0016] According to another aspect, there is provided a method for controlling morphogenesis of a crystal comprising a purine -based compound in a cell producing the crystal, the method comprises modulating gene expression ratio between a first gene encoding the enzyme Pnp4a, a functional analog thereof, or both, and a second gene encoding the enzyme Pnp5a / b, a functional analog thereof, or both, in the cell, thereby controlling the morphogenesis of a crystal produced in a cell.

[0017] In some embodiments, the Pnp is selected from the group consisting of: pnp4a, pnp4b, pnp5a, pnp5b, pnp6, and any combination thereof.

[0018] In some embodiments, the Pnp is pnp4a.

[0019] In some embodiments, the Impdh is selected from the group consisting of: Impdhlb, Impdhla, Impdh2a, and any combination thereof.

[0020] In some embodiments, the Impdh is Impdh lb.

[0021] In some embodiments, the nt5c is selected from the group consisting of: nt5clbb, nt5claa, nt5clba, nt5c2a, nt5c2b, and any combination thereof.

[0022] In some embodiments, the nt5c is nt5clbb.

[0023] In some embodiments, the recombinant DNA molecule further encodes a second enzyme being selected from the group consisting of: guanylate kinase 1 (gukl), ribonucleotide reductase catalytic subunit Ml (rrml), NME / NM23 nucleoside diphosphate kinase 4 (nme4), Nudix Hydrolase 5 (nudt5), and any combination thereof.

[0024] In some embodiments, the gukl is gukla, guklb, or both.

[0025] In some embodiments, the gukl is gukla.

[0026] In some embodiments, the first enzyme comprises an amino acid sequence as set forth in SEQ ID Nos: 1-4, or a functional analog thereof having at least 80% homology thereto.

[0027] In some embodiments, the second enzyme comprises an amino acid sequence as set forth in SEQ ID Nos: 5-10, or a functional analog thereof having at least 80% homology thereto.

[0028] In some embodiments, the recombinant DNA molecule further encodes a third enzyme being selected from the group consisting of: phosphoribosyl pyrophosphate amidotransferase (ppat), phosphoribosylglycinamide formyltransferase (gart), adenylosuccinate lyase (adsl), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (atic), phosphoribosylformylglycinamidine synthase (pfas), phosphoribosylaminoimidazole carboxylase (paics), and any combination thereof.

[0029] In some embodiments, the third enzyme comprises an amino acid sequence as set forth in SEQ ID Nos: 36-41, or a functional analog thereof having at least 80% homology thereto.

[0030] In some embodiments, the recombinant DNA molecule is codon optimized for expression in the cell.

[0031] In some embodiments, the cell comprises an inactive guanine deaminase (gda) gene, an inactive urate oxidase (uox) gene, or both.

[0032] In some embodiments, the inactive gene is knocked out, knocked down, mutated, chemically inhibited, or any combination thereof.

[0033] In some embodiments, the cell is a transgenic cell, a transformed cell, a transfected cell, a transduced cell, or any combination thereof.

[0034] In some embodiments, the cell is any one of: a unicellular organism, a cell of a multicellular organism, and a cell in a culture.

[0035] In some embodiments, the extract comprises guanine.

[0036] In some embodiments, the guanine is in a form of a crystal comprising guanine.

[0037] In some embodiments, the culturing is in a cell culture medium.

[0038] In some embodiments, the purine-based compound is selected from the group consisting of: guanine, xanthine, hypoxanthine, any enantiomer, isomer, or tautomer thereof, and any combination thereof.

[0039] In some embodiments, the culturing comprises supplementing the cell with an effective amount of a purine-based precursor.

[0040] In some embodiments, the purine-based precursor is selected from the group consisting of: inosine monophosphate (IMP), xanthosine monophosphate (XMP), guanosine monophosphate (GMP), guanosine, guanosine diphosphate (GDP), deoxyguanosinediphosphate (dGDP), deoxyguanosine monophosphate (dGMP), deoxyguanosine, guanosine triphosphate (GTP), deoxyguanosine triphosphate (dGTP), and any combination thereof.

[0041] In some embodiments, the method further comprises a step after the step (b) comprising extracting the cell or the cell culture medium wherein the cell is cultured.

[0042] In some embodiments, the extracting is from the culture medium wherein the cell is cultured, and wherein the cell comprises the recombinant DNA molecule encoding: (i) the first enzyme being selected from Impdh, gmps, and both; (ii) the third enzyme being ppat; or both (i) and (ii).

[0043] In some embodiments, the guanine is in a form of a crystal comprising guanine.

[0044] In some embodiments, the crystal further comprises hypoxanthine.

[0045] In some embodiments, the purine-based compound is selected from the group consisting of: guanine, xanthine, hypoxanthine, adenine, any enantiomer, isomer, or tautomer thereof, and any combination thereof.

[0046] In some embodiments, increasing the gene expression ratio above a predetermined threshold results in an elongated semi -hexagonal crystal comprising a combination of guanine and hypoxanthine in a weight per weight ratio ranging 1:2 (w / w) and 1,000:1 (w / w).

[0047] In some embodiments, reducing the gene expression ratio below the predetermined threshold results in semi-hexagonal crystal, a square-like crystal, or a combination thereof, comprising a combination of guanine and hypoxanthine in a w / w ranging between 1 : 1 (w / w) and 100:1 (w / w).

[0048] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0049] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0050] Figs. 1A-1D include an illustration, a volcano plot, and graphs showing that iridophores utilize unique molecular machinery to facilitate guanine production and crystal formation. (1A) Illustration of an iridophore packed with iridosomes, i.e., membrane-bound crystals. Insets in grayscale show high resolution reconstructed cryo-electron tomography (cryoET) images of iridosomes. Different organelles are color-coded. (IB) Volcano plot showing differentially expressed proteins between iridophores and a control heterogeneous cell population in zebrafish larvae at 5 dpf (p = 0.01 and FC > 2 or FC > 0.5). (1C) Bar graph showing the elevated expression of different iridophore marker proteins. (ID) GO enrichment analysis revealing upregulated (purple) and downregulated biological processes (pink).

[0051] Figs. 2A-2D include non-limiting schemes, fluorescent micrographs, and a vertical bar graph showing comprehensive upregulation of the purine metabolic network in iridophores. (2A) Schematic representation of the guanine biosynthetic pathway, highlighting differential protein regulation. The selective upregulation of specific enzymes within iridophores emphasizes their distinct roles in guanine biosynthesis and crystal formation. (2B) Schematic representation of the preparation of thin (70-100 pm) zebrafish larval eye slices for spatial metabolomics using matrix-assisted laser desorption / ionization (MAEDI) imaging. (2C) MAEDI images depict the spatial distribution of guanine ([M+K+], m / z 190.013, pink), guanosine ([M+K+], m / z 332.254, cyan), and deoxyguanosine ([M+K+], m / z306.060, green) during early development. Scale bar: 50pm. (2D) Quantification of metabolite ratios from (2C) at 2 days post-fertilization (dpf), 3 dpf and 10 dpf. Note the changes in precursor levels relative to guanine across the different developmental stages of eye iridophore.

[0052] Figs. 3A-3D include graphs and a ribbon diagram showing that Pnp4a has specialized activity and selectivity compared to other members of the PNPase family. (3 A) Phylogenetic analysis of the vertebrates PNP protein family. Clades are highlighted in the background, with their phylogenetic classes color-coded: blue for fish, green for amphibians, light pink for birds, yellow for reptiles, and pink for mammals. (3B) Enzymatic activity assays of the zebrafish PNPase family using different nucleosides as substrates. Mean ± SD are shown. (3C) Substrate competition experiments, in which residual nucleoside concentrations weredetermined for each enzyme using LC-MS following a 30 min incubation with the specified nucleoside mixture. Mean ± SD are shown. (3D) An overlay of the different folded states of Danio rerio PNPases using AlphaFold-2.

[0053] Figs. 4A-4D include photographs, fluorescent micrographs, micrographs, and vertical bar graphs showing functional role of Pnp4a in crystal formation and compensatory responses. Phenotypes observed in wild-type (4A-4A’”) and pnp4a' / ' mutant (4B-4B’”) larvae. (4A-4B) Gross morphology at 72 hours post fertilization (hpf). (4A’-4B”) Larval eyes at 72 hpf with iridophores shown in green (4 A’ and 4B’), transgenic TDL358:GFP) and crystal reflection in grey (4A”and 4B”). (4A’” and 4B’”) Zoom in of crystal morphology at 72 hpf. Note the reduced crystal formation and altered morphology in the mutant compared to the WT control. Scale bars: 50 pm in (4A’-4B”), and 5 pm in 4A’” and B’”. (4C) Comparison of mean iridophore reflection per 50 pm in wild-type, mutant, and rescue conditions. Wild-type control animals were subjected to the same rescue treatment as pnp4a / _mutants (see Materials and Methods). Wild type, n=7; wild-type control, n=12; pnp4 / n=16; pnp4a / ~ rescue, n=20 larvae; ****, p < 0.0001; **, p < 0.01, *, p < 0.05; two-tailed, unpaired, non-parametric Mann-Whitney tests. (4D) qPCR analysis of pnp5a and pnp5b mRNA expression levels in iridophores of wild-type or pnp4a' / ' mutants. ****, p < 0.0001; two-tailed, non-parametric paired Wilcoxon test. RNA was extracted at 5 dpf. Mean ± SEM are shown in all graphs.

[0054] Figs. 5A-5C include micrographs, and a graph showing that the incorporation of hypoxanthine into guanine crystals alters crystal morphology by inhibiting the (012) facet. (5A) TEM images of biogenic guanine crystals isolated from WT zebrafish operculum (left panel) and body (middle panel) and from pnp4 / ' mutant zebrafish larval eye, showing increasingly more pronounced (012) facets and under-expressed (001) facets. Insets show electron diffraction of individual crystals. (5B) SEM images of in vitro-formed crystals mirror the trend observed in the isolated biogenic crystals, with more pronounced (012) facets as hypoxanthine levels increase. (5C) A schematic illustration of the in vitro and in vivo observations.

[0055] Figs. 6A-6H include an illustration, graphs, and schemes showing cell isolation and enrichment of iridophores via FACs. (6A) Illustration depicting FACs isolation of iridophore cells from zebrafish larvae at 5 days post-fertilization (dpf). The sorting process was guided by the mCherry signal originating from the transgene Tg(pnp4a:PALM-mCherry) and sidescatter from the crystals facilitating the precise separation and enrichment of iridophores. (6B) A graph showing the pre-sorted cell population. (6C) A graph showing the post-sorted population, in which iridophores were enriched over 100-fold compared to the initial cell population. The isolation process effectively targeted and retained these specialized cells, exhibiting their distinct characteristics which can be observed in (6D). (6E) Principal component analysis (PC A) score plot showcasing the proteomic profiles of iridophores. (6F) KEGG pathways analysis reveals the vital pathways enriched in iridophores, including the de novo inosine monophosphate (IMP) biosynthesis, purine nucleobase biosynthesis, and purine metabolism pathways, unveiling their significance in the cellular processes of iridophores. (6G) STRING analyses of protein -protein interaction network within the purine metabolism pathway, based on upregulated and downregulated proteins. (6H) Metascape analysis of GO terms enriched within iridophores.

[0056] Figs. 7A-7X include micrographs showing that temporal dynamics of metabolites in iridophores revealed a decrease in precursor materials and an increase in guanine accumulation by MALDI imaging. A detailed exploration of the temporal dynamics of metabolites within iridophores, spanning multiple developmental time points: 2 (7A-7D), 3 (7E-7H), 4 (7I-7L), 5 (7M-7P), 7 (7Q-7T), and 10 (7U-7X) dpf). The MALDI images visually depict the changing concentrations of key metabolites — such as guanine ([M+K]+, m / z 190.013; 7B, 7F, 7J, 7N, 7R, and 7V), guanosine ([M+K]+, m / z 332.254; 7C, 7G, 7K, 70, 7S, and 7W), and deoxyguanosine ([M+K], m / z 306.060; 7D, 7H, 7L, 7P, 7T, and 7X) — within iridophores at distinct developmental stages. These images were generated with a spatial resolution of 10 pm and a narrow mass-to-charge ratio (m / z) bin width of Am / z = ± 0.001 Da. Importantly, the same metabolite is visualized under identical scale intensity to allow for their semi-quantitative comparison. (7A, 7E, 71, 7M, 7Q, and 7U) Optical images.

[0057] Figs. 8A-8D include phylogenic analysis showing selective upregulation of paralogous enzymes in the guanine biosynthetic pathway. The exploration of enzymes participating in the guanine biosynthetic pathway revealed a subset of paralogues that exhibited significant upregulation in iridophores. Notably, in the inosine monophosphate dehydrogenase family (8A) impdhlb, in the nucleotidase 5C family (8B) nt5clbb, and in the guanylate kinase family (8C) gukla were among the selectively upregulated paralogues. Additionally, the enzyme guanosine monophosphate synthetase (gm .s) (8D) was identified as an example of a gene that appears to be universally expressed without the presence of specific paralogues evolved.

[0058] Figs. 9A-9B include ribbon diagrams showing structural predictions of Danio rerio PNPase enzymes. (9A) Predicted three-dimensional structures of the five PNPase enzymes: Pnp4a, Pnp4b, Pnp5a, and Pnp6, acquired through the application of the AlphaFold-2 algorithm. (9B) Structural consensus analysis for the predicted PNPase enzyme structures. Five independent structural predictions were conducted for each enzyme using different computational methods (only Pnp4a is shown). Notably, all five predictions for each enzyme exhibit remarkable agreement.

[0059] Fig. 10 includes structural based multiple sequence alignment of D. rerio PNPases. Amino-acid sequence alignment of the five homologous PNPases from D. rerio (4a (SEQ ID NO: 4), 4b (SEQ ID NO: 42), 5a (SEQ ID NO: 43), 5b (SEQ ID NO: 44) and 6 (SEQ ID NO: 45)). Secondary structure elements are labeled above the corresponding sequences according to the structure of PNPase 4a, predicted using Alphafold v2.01. Sequence numbering is based on the sequence of PNPase 4b, the longest sequence, and sequences are colored according to residue conservation from white (low score) to red (identity). The alignment was done using MultAlin2 and the figure was created using ESPript3.

[0060] Figs. 11A-11F include micrographs and graphs showing that Pnp4a is located in the iridophore cytoplasm. (11A-11D) Eye iridophores of double transgenic TDL358:GFP (green, 11A) and hsp70:Pnp4a-mKate2 (magenta, 11B), with guanine crystals (cyan, 11C), at 56 hpf. Note the Pnp4a-mKate2 subcellular expression pattern at 4 h post-heat shock induction (hpHS) of the transgene hsp70:Pnp4a-mKate2 is mainly cytoplasmic (11D). (HE) Average intensities of normalized TDL358:GFP, Pnp4a-mKate2, and reflection signals versus relative position (ROI line in HD, 50 pixels width average). Signals were normalized to respective maxima. Note the discernible lack of correlation between the reflection (guanine crystals) and mKate2 signals (Pnp4a-mKate2 fusion protein), while the latter overlaps ROI positions with cytoplasmic GFP signal (TDL358:GFP). (HF) Rate activity profiles of different zebrafish PNPases across different pH ranges. Enzymatic rates of guanosine phosphorolysis were assessed at varying pH values (2-9) for PNPases (0.25 pM) at saturating substrate concentrations (50 mM phosphate; 390 pM guanosine). The graph displays optimal activity at pH 7 for most PNPases, while PNPase 4a exhibited maximal activity at pH 7.5.

[0061] Fig 12 includes schemes and sequences showing generation of pnp4a' / ' mutant zebrafish using CRISPR / Cas9 technology. Non-limiting schematic representation of theCRISPR / Cas9-mediated knockout strategy targeting the pnp4a gene is provided. Illustration created with Biorender.

[0062] Figs. 13A-13B include photographs showing long-term resilience and phenotypic adaptation in adult zebrafish depleted of Pnp4a. Long-term observation of adult pnp4a' / ' mutant zebrafish reveals intriguing viability, eventual development of almost normal stripe patterns, and eye iridescence despite the absence of functional Pnp4a. This unexpected outcome underscores the complexity of compensatory mechanisms in the absence of Pnp4a. Scale bar: 2 mm. (13A) wild-type 3 mpf (months post fertilization); (13B) pnp4a' / ' 3 mpf.

[0063] Figs. 14 includes a vertical bar graph showing compensatory gene expression in pnp4a' / ' mutant zebrafish reveals insights into guanine crystal formation and regulation. Depicted is mRNA expression levels of PNPase family members in pnp4a' / ' mutants compared to wild-type using real-time PCR.

[0064] Figs. 15A-15F includes photographs, micrographs, and fluorescent micrographs showing the selective influence of pnp4a' / ' knockout on crystal morphology. Morpholino (MO) knockdown experiments targeting various members of the PNPase family, including pnp4a (15B); pnp4b (15C); pnp5a (15D); pnp5b (15E); and pnp6 (15F), were conducted to assess their influence on crystal quantities and morphology. The knockdown of these family members did not alter crystal quantities or morphology, which retained their typical elongated semi-hexagonal shape, similar to wild-type fish. Iridophores are marked in green (TDL358:GFP) and crystals in white (reflectance). (15A) Control MO.

[0065] Figs. 16A-16J include a graph, and micrographs showing the impact of Pnp4a mutation on crystal morphology. (16A) Micro-Raman spectroscopy confirmed that despite altered crystal morphology, the mutant crystals were still composed of beta-anhydrous guanine. (16B-16C) Conventional TEM imaging of the mutant larvae displayed multiple small, underdeveloped iridosomes. (16D-16E) Cryo-scanning electron microscopy (Cryo- SEM) imaging showed thin, plate-like crystals similar to those found in the wild type. (16F- 16J) Transmission electron microscopy (TEM) imaging revealed the Pnp4a mutant crystals exhibited overdeveloped (012) facets while the (010) facets were underdeveloped.

[0066] Fig. 17 includes images and a graph showing the effect of hypoxanthine on crystal morphology. OrbiTrap secondary ion mass spectrometry (Orbi SIMS) imaging indicated that the hypoxanthine-to-guanine ratios in the mutant crystals were almost two times higher thanthat in the wild type, suggesting that the inclusion of hypoxanthine significantly modifies crystal morphology.

[0067] Fig. 18 includes a graph showing fine-tuning of crystal morphology through composition. Stripes crystals, with higher hypoxanthine levels, exhibit a lower aspect ratio, suggesting that organisms adjust guanine-to-hypoxanthine composition to fine-tune crystal morphology.

[0068] Fig. 19 includes a non-limiting scheme of the regulatory mechanisms governing tissue-specific crystal morphogenesis provided as an illustration showing the coupling of the expression levels and selectivity of PNPases to crystal composition and subsequent morphology. Morphogenesis relies on the development of paralogous enzymes with exceptional substrate selectivity. The tissue- specific expression patterns of these enzymes yield varied substrate compositions that are instrumental in forming crystals with distinct compositions, directly influencing their morphology and, consequently, their optical characteristics.

[0069] Figs. 20A-20B include vertical bar graphs showing production levels of guanine (20A) or hypoxanthine (20B) in recombinant bacteria expressing pnp4a, pnp5a, or pnp4b.

[0070] Figs. 21A-21B includes vertical bar graphs showing guanine levels in pellet (21A) or supernatant (21B) of recombinant bacterial cultures expressing gmps, impdhlb, or ppat.DETAILED DESCRIPTIONCells, DNA, and polypeptide

[0071] According to the first aspect, there is provided a cell comprising a DNA molecule comprising a first nucleic acid sequence encoding at least one first polypeptide being an enzyme selected from: inosine-5’-monophosphate dehydrogenase (Impdh), guanosine monophosphate synthase (gmps), cytosolic 5 ’-nucleotidase (nt5c), purine nucleoside phosphorylase (Pnp), or any combination thereof.

[0072] In some embodiments, the DNA molecule comprising a first nucleic acid sequence encoding at least one first polypeptide being an enzyme is a DNA molecule comprising a nucleic acid sequence encoding a first enzyme. In some embodiments, the recombinant DNA molecule comprises a nucleic acid sequence encoding a first enzyme. In some embodiments, the at least one first polypeptide is an enzyme or a first enzyme. The expressions “a firstnucleic acid sequence encoding at least one first polypeptide being an enzyme” and “a nucleic acid sequence encoding a first enzyme” are used herein interchangeably.

[0073] In some embodiments, the at least one first polypeptide has an enzymatic activity of: Impdh, gmps, nt5c, Pnp, or any combination thereof.

[0074] In some embodiments, the DNA molecule comprises a first nucleic acid sequence encoding Impdh, gmps, nt5c, and Pnp.

[0075] In some embodiments, the DNA molecule is a recombinant DNA molecule. In some embodiments, the DNA molecule is an exogenous DNA molecule. In some embodiments, the DNA molecule is integrated into the genome of the cell. In some embodiments, the DNA molecule is not integrated into the genome of the cell. In some embodiments, the DNA molecule is capable of being replicated by the cell. In some embodiments, the DNA molecule replicated in synchronization with the division of the cell, DNA replication of the cell, or both. In some embodiments the DNA molecule comprises a nucleic acid sequence that is non- endogenous, non-native, or both, to the cell. In some embodiments, the DNA molecule comprises a nucleic acid sequence that is derived or originates from a different species of the cell. In some embodiments, the DNA molecule comprises a nucleic acid sequence that is derived or originates from fish. In some embodiments, a fish comprises or consists of Zebrafish (Danio rerio). The terms “DNA molecule” and “Recombinant DNA molecule” are used herein interchangeably.

[0076] As used herein, the term “recombinant protein” refers to a protein which is coded for by a recombinant DNA and is thus not naturally occurring. The term “recombinant DNA” refers to DNA molecules formed by laboratory methods of genetic recombination. Generally, this recombinant DNA is in the form of a vector, plasmid or virus used to express the recombinant protein in a cell.

[0077] As used herein, the terms “peptide”, “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms “peptide”, “polypeptide” and “protein” as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides, polypeptides and proteins described have modifications rendering them more stable while in the body or more capable of penetrating into cells. In one embodiment, the terms “peptide”, “polypeptide” and “protein” apply to naturally occurringamino acid polymers. In another embodiment, the terms “peptide”, “polypeptide” and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0078] The term “analog” as used herein, refers to a polypeptide that is similar, but not identical, to the polypeptide of the invention that still is capable of binding succinate or still comprises the succinate binding pocket. An analog may have deletions or mutations that result in an amino acids sequence that is different than the amino acid sequence of the polypeptide of the invention. It should be understood that all analogs of the polypeptide of the invention would still be capable of binding succinate or still comprise the succinate binding pocket. Further, an analog may be analogous to a fragment of the polypeptide of the invention, however, in such a case the fragment must comprise at least 50 consecutive amino acids of the polypeptide of the invention.

[0079] In some embodiments, a polypeptide of the invention is an enzyme.

[0080] In some embodiments, an analog to the polypeptide disclosed herein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology or identity to the amino acid sequence of the polypeptide, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0081] In some embodiments, an analog comprises a functional analog. In some embodiments, a functional analog comprises any compound, e.g., a polypeptide (such an enzyme as disclosed herein), having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% the activity of a polypeptide, e.g., an enzyme, as disclosed herein, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0082] As used herein, the term "analog" includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or thesubstitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention.

[0083] In some embodiments, Pnp is selected from: pnp4a, pnp4b, pnp5a, pnp5b, pnp6, and or combination thereof. In some embodiments, Pnp is or comprises pnp4a.

[0084] In some embodiments, Impdh is selected from: Impdhlb, Impdhla, Impdh2a, or any combination thereof. In some embodiments, Impdh is or comprises Impdhlb.

[0085] In some embodiments, nt5c is selected from: nt5clbb, nt5claa, nt5clba, nt5c2a, nt5c2b, or any combination thereof. In some embodiments, nt5c is nt5clbb.

[0086] In some embodiments, the DNA molecule further comprises a second nucleic acid sequence. In some embodiments, the second nucleic acid sequence encodes at least one second polypeptide. In some embodiments, the at least one second polypeptide comprises or is an enzyme selected from: guanylate kinase 1 (gukl), ribonucleoside-diphosphate reductase large subunit (rrml), nucleoside diphosphate kinase 4, ADP-sugar (nudt5), or any combination thereof. In some embodiments, the recombinant DNA molecule further encodes a second enzyme. In some embodiments, the second enzyme is selected from: guanylate kinase 1 (gukl), ribonucleoside-diphosphate reductase large subunit (rrml), nucleoside diphosphate kinase 4, ADP-sugar (nudt5), or any combination thereof.

[0087] In some embodiments, the DNA molecule further comprises a second nucleic acid sequence encoding at least one second polypeptide being an enzyme, is a DNA molecule comprising a nucleic acid sequence encoding a second enzyme. In some embodiments, the recombinant DNA molecule further comprises a nucleic acid sequence encoding a second enzyme. In some embodiments, the at least one second polypeptide is an enzyme or a second enzyme. The expressions “a second nucleic acid sequence encoding at least one second polypeptide being an enzyme” and “a nucleic acid sequence encoding a second enzyme” are used herein interchangeably.

[0088] In some embodiments, the DNA molecule further comprises a third nucleic acid sequence. In some embodiments, the third nucleic acid sequence encodes at least one third polypeptide. In some embodiments, the at least one third polypeptide comprises or is an enzyme selected from: phosphoribosyl pyrophosphate amidotransferase (ppat), phosphoribosylglycinamide formyltransferase (gart), adenylosuccinate lyase^a sY), 5- aminoimidazole-4-carboxamide ribonucleotide formyltransferase (atic),phosphoribosylformylglycinamidine synthase (pfas), phosphoribosylaminoimidazole carboxylase (paics), a functional analog thereof, and any combination thereof. In some embodiments, the DNA molecule further encodes a third enzyme. In some embodiments, the third enzyme comprises or is an enzyme selected from: phosphoribosyl pyrophosphate amidotransferase (ppat), phosphoribosylglycinamide formyltransferase (gart), adenylosuccinate lyase (adsl), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (atic), phosphoribosylformylglycinamidine synthase (pfas), phosphoribosylaminoimidazole carboxylase (paics), a functional analog thereof, and any combination thereof.

[0089] In some embodiments, the recombinant DNA molecule further comprises a third nucleic acid sequence encoding at least one third polypeptide being an enzyme, is a recombinant DNA molecule comprising a nucleic acid sequence encoding a third enzyme. In some embodiments, the recombinant DNA molecule further comprises a nucleic acid sequence encoding a third enzyme. In some embodiments, the at least one third polypeptide is an enzyme or a third enzyme. The expressions “a third nucleic acid sequence encoding at least one third polypeptide being an enzyme” and “a nucleic acid sequence encoding a third enzyme” are used herein interchangeably.

[0090] In some embodiments, gukl comprises or is gukla, guklb, or both. In some embodiments, gukl comprises or is gukla.

[0091] In some embodiments, the cell comprises a plurality of DNA molecules. In some embodiments, the plurality of DNA molecules comprises a plurality of different nucleic acid sequences. In some embodiments, each different nucleic acid sequence encodes one of the at least one first polypeptide and / or the at least one second polypeptide and / or the at least one third polypeptide.

[0092] As used herein, the term “plurality” refers to any integer being equal to or greater than 2.

[0093] In some embodiments, any one of the at least one first polypeptide, the at least one second polypeptide, the at least one third polypeptide, or any combination thereof, is encoded by a different nucleic acid sequence of a different DNA molecule.

[0094] In some embodiments, the DNA molecule is an artificial DNA molecule. In some embodiments, the DNA molecule comprises a plasmid and / or an expression vector. In someembodiments, the DNA molecule comprises the genome of the cell. In some embodiments, the DNA molecule is contained or integrated into a plasmid and / or an expression vector. In some embodiments, the DNA molecule is contained or integrated into the genome of the cell.

[0095] In some embodiments, the cell comprises a DNA molecule comprising nucleic acid sequences encoding enzymes: Impdh, gmps, nt5c, and Pnp. In some embodiments, the cell comprises a DNA molecule comprising nucleic acid sequences encoding the enzymes: Impdhlb, gmps, nt5clbb, and Pnp4a. In some embodiments, the cell comprises recombinant enzymes of: Impdh, gmps, nt5c, and Pnp. In some embodiments, the cell comprises recombinant enzymes of: Impdhlb, gmps, nt5clbb, and Pnp4a.

[0096] In some embodiments, the cell comprises a DNA molecule comprising nucleic acid sequences encoding enzymes: Impdh, gmps, gukl, nme4, rrml, nudt5, and Pnp. In some embodiments, the cell comprises a DNA molecule comprising nucleic acid sequences encoding the enzymes: Impdhlb, gmps, gukla, nme4, rrml, nudt5, and Pnp4a. In some embodiments, the cell comprises recombinant enzymes of: Impdh, gmps, gukl, nme4, rrml, nudt5, and Pnp. In some embodiments, the cell comprises recombinant enzymes of: Impdhlb, gmps, gukla, nme4, rrml, nudt5, and Pnp4a.

[0097] In some embodiments, the cell comprises a DNA molecule comprising nucleic acid sequences encoding enzymes: Impdh, gmps, gukl, rrml, nt5c, and Pnp. In some embodiments, the cell comprises a DNA molecule comprising nucleic acid sequences encoding the enzymes: Impdhlb, gmps, gukla, rrml, nt5clbb, and Pnp4a. In some embodiments, the cell comprises recombinant enzymes of: Impdh, gmps, gukl, rrml, nt5c, and Pnp. In some embodiments, the cell comprises recombinant enzymes of: Impdhlb, gmps, gukla, rrml, nt5clbb, and Pnp4a.

[0098] In some embodiments, the cell comprises an inactive guanine deaminase (gda) gene, an inactive urate oxidase (uox) gene, or both.

[0099] In some embodiments, an inactive gene is knocked out, knocked down, mutated, chemically inhibited, or any combination thereof.

[0100] In some embodiments, impdhlb comprises the amino acid sequence: MADYLISGGTGYIPDDGLSAQQLFAVGDGLTYNDFLILPGFIDFTSDEVDLTSALTK KITLKTPLISSPMDTVTESSMAIAMALMGGIGIIHHNCTPEFQANEVRKVKRFEQGFI TDPVVLSPHHTVGDVLEAKVRHGFSGIPITETGKMGSKLVGIVTSRDIDFLSEKDNNKYLEEAMTKREDLVVAPAGVTLKEANDILQRSKKGKLPIVNDKDELVAIIARTDLK KNRDYPLASKDSRKQLLCGAAIGTREDDKYRLDLLTQSGVDMVVLDSSQGNSVYQ INMIHYIKQKYPELQVVGGNVVTAAQAKNLIDAGVDALRVGMGCGSICITQEVMA CGRPQGTSVYKVAEYARRFGVPVIADGGIQTVGHVVKALSLGASTVMMGSLLAAT TEAPGEYFFSDGVRLKKYRGMGSLDAMEKNTSSQKRYFSEGDKVKVAQGVSGSV QDKGSIHKFVPYLIAGIQHGCQDIGAKSLSVLRSMMYSGELKFEKRTMSAQVEGGV HGLHSFEKRLY (SEQ ID NO: 1), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0101] In some embodiments, impdhlb is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number NM_001014369.2), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number NM_001014369.2), or a fragment thereof.

[0102] In some embodiments, gmps comprises the amino acid sequence: MALCNGDSKLDIVGEPKDGLCSYEGAVVILDAGAQYGKVIDRRVREMFVQSEILPL ETPAFAIREQGFRAIIISGGPNSVYAEDAPWFDPAIFTIGKPVLGICYGMQMMNKVF GGTVHRKSVREDGVFNIALDNSCSLFRGLQKEEHVLLTHGDSVDKVADGFKIVAQS GNIVAGIANEQKKLYGTQFHPEVDLTERGMDMLRNFLFEIAGCSSNFTVQNRQMSC IREIREKVDKSKVLVLLSGGVDSTVCTALLNKALNQEQVIAVHIDNGFMRKRESQS VEEALTKLGIKLKVVNAAHTFYNGTTTLPISEEDRTPRKRISKTLNMTTNPEEKRKIIGDTFVKVANEVIGEMNLKPEDVYLAQGTLRPDLIESASHLASGKAEVIKTHHNDTE LIRKLRDEGKVIEPLKDFHKDEVRALGRELGLPEEIVSRHPFPGPGLAIRVICADEPY VCKDFAETNNILKIITDFSASVKKPHTLLQRVKSCISDEEEEKLMQITSLHSLNAFLLP IKTVGVQGDSRSYSYVCGVSSKEAPHWDSLMFLARLIPRICHSINRVVYVFGSHVKE PPTDITPTFLTTGVLSTLRQADFVAHSILRESGYSSKISQMPVILTPLHFDRDPLQKQP SCRRSVVIRTFITSDFMTGIAATPDNQIPEEVVLKMVNEIKKIPGISRVMYDLTSKPPG TTEWE (SEQ ID NO: 2), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0103] In some embodiments, gmps is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number NM_200587.2), or a fragment thereof. In some embodiments,the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number NM_200587.2), or a fragment thereof.

[0104] In some embodiments, nt5clbb comprises the amino acid sequence: MSEIKTTEVASADIKAAPSEEKDWAAAKAHYENLKSKKPRPPKPKNAVTIAVSSRT LFDLTAERKIFEEEGVEKYVQHQQDNENQPLNPGPAFPFVKALMTVNERLRELYPE SEELFDIVLMTNNHAQVGVRLMNSINHYDLTIERFCMTGGQSPIGYLKAYMTNLYL SRDSKKVGEAIEEGIAAATMFKSDVETQLSETQLRVAFDGDAVLFSDESEIIVKQHG LDTFFEHEKQHENKPLAQGPLKCFLEALGKLQKKFYAKNERLNCPIRTFLVTARSA ASSGARVLKTLRSWGLEIDEALFLAGAPKGPLLQKIRPHIFFDDQMFHIEGAKEMGT IAAHVPYGIGQKYNKGKLIEPEKQQK (SEQ ID NO: 3), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0105] In some embodiments, nt5clbb is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number XM_685092.9), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number XM_685092.9), or a fragment thereof.

[0106] In some embodiments, pnp4a comprises the amino acid sequence: MHSKDQICHEDYQRAADWLLSQTQHRPKVAIICGSGLGMLADGLKCQDSFKYSDI PGFPQSTVKGHAGRLVFGELKGKTCVCMQGRFHMYEGHSLSKVTFPVRVFKLLGV DTLIVTNAAGSLADSYNCGDIMIIRDHINFPGLAGLNPLNGPNDEKFGPRFPPMSGV YDRGLRKMALDICKGMGVSQYVQEGVYCMVGGPNFESIAEARLLHRLGVDAVGM STAPEVLVASHCGIRVFGLSLITNKVVKSYEDNETVNHEAVLEVSKMRSETLQALV TELISRMDINNNTA (SEQ ID NO: 4), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0107] In some embodiments, pnp4a is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number NM_001002102.1), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number NM_001002102.1), or a fragment thereof.

[0108] In some embodiments, rrml comprises the amino acid sequence: MHVIKRDGRQERVMFDKITSRIQKLCYGLNSDFVDPTQITMKVIQGLYSGVTTVELDTLAAEIAATLTTKHPDYAILAARIAVSNLHKETKKVFSEVMEDLYNYVNPLNSRH SPMISKETLDIVLANKDRLNSAIIYDRDFSYNFFGFKTLERSYLLKINGKVAERPQHM LMRVSVGIHKEDIAAAIETYNLLSEKWFTHASPTLFNAGTNRPQLSSCFLLAMKDDS IEGIYDTLKQCALISKSAGGIGVAVSCIRATGSYIAGTNGNSNGLVPMLRVYNNTAR YVDQGGNKRPGAFAMYLEPWHFDIFDFLELKKNTGKEEQRARDLFYALWIPDLFM KRVETNGDWSLMCPNDCPGLDECWGEEFEKLYAKYEQEGRAKRVVKAQQLWYA IIESQTETGTPYMLYKDACNRKSNQQNLGTIKCSNLCTEIVEYTSADEVAVCNLASI ALNMYVTSERTFDFQKLASVTKVIVKNLNKIIDINYYPVKEAENSNKRHRPIGIGVQ GLADAFILMRFPFESAEAQLLNTQIFETIYYAALESSCELAAEYGPYQTYAGCPVSK GILQYDMWEKTPTDLWDWAALKEKIAKHGVRNSLLLAPMPTASTAQILGNNESIEP YTSNIYTRRVLSGEFQIVNPHLLKDLTERGLWNEEMKNQIIAQNGSIQTIPAIPDDLK ELYKTVWEISQKTILKMAADRGAYIDQSQSLNIHIAEPNYGKLTSMHFYGWKQGLK TGMYYLRTKPAANPIQFTLNKEKLKETQKTTSSEDEETKERNKAAMVCSLENRDEC LMCGS (SEQ ID NO: 5), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0109] In some embodiments, rrml is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number NM_131455.2), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number NM_131455.2), or a fragment thereof.

[0110] In some embodiments, nme4 comprises the amino acid sequence: MAVRFGLRVHAARAVTRNPSRALGIGAARSLSSDSDFSGVNERTLVAVKPDGVQR RLIGEVIKRFEQRGFRLVGLKMLQAPDKLLAQHYVSLQKKPFYSSLLYYMTSGPIV AMVWEGHNVVKTSRMMVGDTDPAAAAPGTIRGDFSVHISRNVVHASDSVEGAQR EISLWFHRSELVDWEGCDHKNIYHL (SEQ ID NO: 6), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0111] In some embodiments, nme4 is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number NM_201195.1), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number NM_201195.1), or a fragment thereof.

[0112] In some embodiments, nudt5 comprises the amino acid sequence: MSSPTKATTTEPHVIKEELVASGNWVKLEKTTYVDPSGSTRTWETVKRTTRVANT AADGVGIIALLKRTLHKDCVVMVKQFRPPMGCNTLEFPAGLIDENESAETAALREL KEETGYKGEVVGVTPVTCLDPGLSNCSTKIVMVHINGDDIENINPTQQLGDGEFVE VILLPLDEFQQKIDELLQKEKIVVDCKVYIYAMGMSQAFFKPRELPVLKQ (SEQ ID NO: 7), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0113] In some embodiments, nudt5 is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number NM_001002086.1), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number NM_001002086.1), or a fragment thereof.

[0114] In some embodiments, gukla comprises the amino acid sequence: MRAHTTDKAMAGPRPVVMSGPSGAGKSTLLKKLLKEFNGVFGFSVSHTTRNPRPG EENGKDYHFVSREVMQTSIAKGEFIESAEFSGNMYGTSKAAVQAVQAQNLICILDID MQGVKNIKKTDLNPIYVSVQAPSMDILEKRLRDRKTESEESLQKRLHAAKVDVEIS KEPGLFDVVIINDDLEAAYGKLKDVLLEEIQKVRDVNKSS (SEQ ID NO: 8);MYVKFISRLFSAMAGPRPVVMSGPSGAGKSTLLKKLLKEFNGVFGFSVSHTTRNPR PGEENGKDYHFVSREVMQTSIAKGEFIESAEFSGNMYGTSKAAVQAVQAQNLICIL DIDMQGVKNIKKTDLNPIYVSVQAPSMDILEKRLRDRKTESEESLQKRLHAAKVDV EISKEPGLFDVVIINDDLEAAYGKLKDVLLEEIQKVRDVNKSS (SEQ ID NO: 9); or MAGPRPVVMSGPSGAGKSTLLKKLLKEFNGVFGFSVSHTTRNPRPGEENGKDYHF VSREVMQTSIAKGEFIESAEFSGNMYGTSKAAVQAVQAQNLICILDIDMQGVKNIK KTDLNPIYVSVQAPSMDILEKRLRDRKTESEESLQKRLHAAKVDVEISKEPGLFDVV IINDDLEAAYGKLKDVLLEEIQKVRDVNKSS (SEQ ID NO: 10), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0115] In some embodiments, gukla is encoded by a nucleic acid sequence as disclosed in the Genbank (any one of: accession numbers NM_001002126.2, NM_001316896.1, NM_001316899.1, and a combination thereof), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (any oneof: accession numbers NM_001002126.2, NM_001316896.1, NM.001316899.1, and a combination thereof), or a fragment thereof.

[0116] In some embodiments, the at least one first polypeptide comprises an amino acid sequence as set forth in SEQ ID Nos: 1-4, or a functional analog thereof having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0117] In some embodiments, the at least one second polypeptide comprises an amino acids sequence as set forth in SEQ ID Nos: 5-10, or a functional analog thereof having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0118] In some embodiments, phosphoribosyl pyrophosphate amidotransferase (ppat) comprises the amino acid sequence:MEFEESGIGEECGVFGCVAAGEWPTQLEVAQILTLGLVALQHRGQESAGIVTSTGT NPPTFSTLKGMGLVNTAFKPEDLLKLRYGNLGIGHTRYSTTGISELHNCQPFVVDTL HGKIAVAHNGELVNASALRKKVMRHGVGLSTCSDSELITQLLALTPPMEELDNPD WVARIKNLMTETPTSYSLLVMYKDVIYAVRDPYGNRPLCIGRLVPISKLHSSGAGE ADTEGWVVSSESCSFQSIGAKYYREVKPGEIVQISKNGVESLSVVPRPEGDLPAFCIF EYVYFARPDSMFEGQMVYTVRQRCGRQLAIEAPTDADVVSTVPESATPAALGYAQ QSGLPYVEVLCKNRYVGRTFIQPNTRLRQLGVAKKFGALTDNLAGKRVVLIDDSIV RGNTISPIIKLLKEAGATEVHIRVASPPIRFPCYMGINIPTKEELIANKPEFKDIAGYIG ATSVRYLSVEGLLSAVQGGIESHGKDERISSTTKTTRIGHCTACLTGKYPVELEW (SEQ ID NO: 36), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0119] In some embodiments, ppat is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number BC 146730.1), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number BC146730.1, or a fragment thereof.

[0120] In some embodiments, phosphoribosylglycinamide formyltransferase (gart) comprises the amino acid sequence:MSERVLVIGSGGREHALAWKFAQSPHVQQVLVAPGNAGTANCGKISNSEVSVNNH SILAQYCKDHKVGLVVVGPEVPLAAGMVDDLTAAGVLCFGPSARAAQLEASKSFSKAFMDRHNIPTARWSSFTDPQQACAYIRDADFPALVVKASGLAAGKGVIVAQDKD EACQAVLDIMKDKAFGSAGETVVVEELLDGQEVSCLCFSDGVTVAPMPPAQDHKR LLDGDMGPNTGGMGAYCPTPQVSDELLQEISRSVLQKTVDGMREEGAPYVGVLY AGLMLTAQGPRVLEFNCRFGDPECQVLLPLLQSDLYEVCMQTLRCELDSASVQWL QDCAAVTVVMASGGYPNAYRKGLEISGLSQASEMGVQVFHAGTALKEGGGVITSG GRVLTVTAVRPTLESALQSANEGVGAISFPDAVYRRDIGHRAITYLTHTRGLTYKDS GVDIAAGNRLVDIIKPLAKATSRPGCNADLGGFAGLFDLKAAGFTDPILVSGTDGV GTKLKIAQECGVHSTLGQDLVAMCVNDVLAQGAEPLFFLDYFSCGRLDVNVAASV IGGIADACQMAGC ALLGGETAEMPGVYPPGEYDLAGFCVGA VERS ALLPRLKD ISE GDLLLGVSSSGIHSNGFSLVRTILERSGLNISSPAPFGRPGQTIGDVLLTPTKIYSRVL QPVLRSGAVKAFAHITGGGLLENIPRVLPADLTADLDACRWRIPPVFSWLQQQGGV CEQEFCRTFNCGLGAVLVVSKADAQRVLRLLQAHEESWIIGSLTHRHPGAESVVVR NLERSLRAGPDCSPDTGVLRNGSAAADQNSRRRTRVAVLISGSGTNLQALMDQAR KPSSSAEIVLVISNRPGVMGLKRAALAGIQTRVVDHKLYGSRAEFDGTIDKVLEEFS VELVCLAGFMRILTGPFVRKWSGKMLNIHPSLLPSFKGVNAQKQALQAGVRVTGC SVHFVAEDVDAGAIVVQEAVPVLVTDSEESLSERIREAEHRAFPAALELVSSGAVKL RDDGHIVWSTRETH (SEQ ID NO: 37), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0121] In some embodiments, gart is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number AF257743.1), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number AF257743.1, or a fragment thereof.

[0122] In some embodiments, adenylosuccinate lyase (adsl) comprises the amino acid sequence:MEGSGEEFLKYRSPLVSRYASKEMAYNFSDRKKFTTWRKLWIYLAKAEKSLGLPIS DAQVSEMESHSEDIDFVMAAEEERKLRHDVMAHVHTFAQCCPTAAPIIHLGATSCY VGDNTDLIMLRDGFDILLPKLARVIDRLANFAEKYADLPTLGFTHYQPAQLTTVGK RSCLWLQDLLMDMRNLQRARDDLRFRGVKGTTGTQASFLQLFQGDHDKVEELDK MVTEMAGFKKSYLVTGQTYSRKVDIDSLCVLSSLAATIHKICTDIRLLANLKEIEEPF EKEQIGSSAMPYKRNPMRAERCCSLARHLMALVSNPLQTAAVQWLERTLDDSANR RISLPEAFLTADIILSTLQNITEGLVVYPKVIERHIRHELPFMATENIIMAMVKAGGNR QDCHEKIRVLSQQAAAVVKQEGGDNDLLARVQADPYFAPILGELDALLDPKTFIGRAPQQVTRFLSEEVRPVLEPYKSKMDVKIELEL (SEQ ID NO: 38), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0123] In some embodiments, adsl is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number BC154295.1), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number BC 154295.1, or a fragment thereof.

[0124] In some embodiments, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (atic) comprises the amino acid sequence: MASEIALLSVSDKTGLVEFARRLVSVGLSLVASGGTAKTLRDAGLAVRDVSEITGFP EMLGGRVKTLHPAVHGGILARKTPSDNADMEKLGFSLVRVAVCNLYPFVKTVSSP GVTVEDAVEQIDIGGVTLLRAAAKNHARVTVVCDPSDYNVVAKEMETSEIHDTTM ETRKTLALKAFTHTAQYDEAISDYFRREYSRGVSQLPLRYGMNPHQAPAQLYTTRP ALPLAVLNGSPGFINLCDALNAWQLVRELKKALGLPAATSFKHVSPAGAAVGVPLS EDEAKVCMVNDMLQDLTPLATAYARARGSDRMSSFGDFIALSDVCDVPTAKIISRE VSDGIVAPGYEEDALRILSKKKNGNYCVLKMDPEYEPDEEEVRVLFGLHLKQKRN GAVIDKELFSNIVSKGKLSESALRDLIVASIAVKYTQSNSVCYAKDGQVIGIGAGQQ SRIHCTRLAGDKADNWWLRHHPGVLSMRFRSGVKRAEMANAIDQYVSGTVGEGP DKEVWKGLFEEVPEPLSEVEKKNWISSLQAVALSSDAFFPFRDNVDRAKQSGVEYI AAPSGSTADEVVVKACNELGITLVHTNIRLFHH (SEQ ID NO: 39), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0125] In some embodiments, atic is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number NM_001082796.2), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number NM_001082796.2, or a fragment thereof.

[0126] In some embodiments, phosphoribosylformylglycinamidine synthase (pfas) comprises the amino acid sequence:MPVVRFYRTEETGEARAIRRIAQLYPDVIITTELCYNVELDGPDSLSVAQKDILRWL FSPPYSVSLLEEPTLKAEHGARLVEIGPRLNFSTAWSTNAVSICQSAGLSQVTRVELSRRHLIKPQEGCKVGMKDGEMESLISCLYDSMTECIYAQPITSFAVDIRPQDVFEVDIL GKGRAALEKANDELGLAFDSWDLDYYTALFQKVKRNPTSVECFDLAQSNSEHSRH WFFRGRMVIDGKEQKETLFSLIMGTQQHSNQNNVIKFCDNSSGIKGMELRCMYPTN PAQASPYESRDTTRHVIFTAETHNFPTGVAPFSGATTGTGGRIRDVQSAGKGGHVIA GTAGYCFGNLHIPGFVLPWEEEGWEYPSSFAPPLQVAIEASDGASDYGNKFGEPVL AGFARSFGMRLANGERREWIKPIMFSGGLGSIEDPHVRKDQAEPGMEVVKIGGPVY RIGVGGGAASSVQVQGDNSSARDLGAVQRGDAEMEQKMNRALRACLERVEGNPI CSIHDQGAGGNGNVLKELSEPAGAVIYTEKFKRGDPTLSVLELWGAEYQESNALLL RPSDRSFLERVCQREKCPVDFVGKITGDGKIVLVDGLRKQNDVLEGARNPVDLELD WVLGKMPQKEFILEHRSVSLQPLTLPAGLSVLPALERVLRLPAVASKRYLTNKVDR SVTGLVAQQQCVGPLHTPLADVAVVALSPFSLQGAATAIGEQPIKGLLSPAAGARM AVGEALTNLVFARVSALKDVKCSGNWMWAAKLPGEGACLWDACQAMCEVMGQ LGVAVDGGKDSLSMAARVSGETVKAPGSLVISVYAVCPDITATVTPDLDNPEGKG VLLYVPVSAGKYRLGGSALAQCFGQLGDCSPDMDQPDKLSACFNTTQTLIQDRLLT AGHDVSDGGLISCLLEMAFAGNYGIEVDLPLEGVDVMEALFSEELSLVLEVCERNA SSVCQRYTDAGLLCHRIGTTSGFGPDAKVRVSLCGREVLNERLPTLRAIWESTSFEL ERLQANPLCVQEEEQGLASRTQPYLKLTFDPSQTPIIKELATGKARVAVVREEGSNG DREMSASLFMAGFEVWDVTMQDLCSGSTTLDPFRAVVFVGGFSYADVLGSAKGW AATVTFNNRAREEFERFRKREDTLSLGVCNGCQLLALLGWVGEREDGGSDVTLTH NKSGRFESRFVSVGILPSPAIMLKGMEGSALGVWVAHGEGLMQFRSPEAQQKLIGS SLAPLRYVDDSGNPTEIYPINPNGSAQGVAGICSADGRHLAMMPHPERAVLSWQW AWAPQHLRGSLEPSPWLSMFRNAAAWCQNS (SEQ ID NO: 40), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0127] In some embodiments, pfas is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number NM_001045202.1), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number NM_001045202.1, or a fragment thereof.

[0128] In some embodiments, phosphoribosylaminoimidazole carboxylase (paics) comprises the amino acid sequence:MSTATELKLGQKLNEGKTKQIFEILDEPGHVLVQSKDQITAGNAVRKDQMEGKAAIANKTTSCVFKLLQDAGLKTAFVRQHSDTAFVASRCEMIPIEWVCRRIATGSFLKRNPGVKEGYRFTPLKMEMFFKDDANNDPQWSEEQLLAAGFDLAGLTIGRCEVDIMSK STVAIFEVLEKAWATQDCTLVDMKIEFGVNVTTKEIVLADVIDNDSWRLWPAGDR SQQKDKQVYRDLKEVTPEAMQMVKRNFEWVAERVKLLLESQARGRVVVMMGST SDVAHCEKIRKACASYGIPCHLRVNSAHKGPDETLRIKAEYEGDGEPTIFVAVAGRS NGLGPVMSGNTAYPVINCPPVTPDWGAQDIWSSLRMPSGLGCSTVLSPEAAAQFA AQILGLNNHLIWAKLRASMLNTWVSLKQADKKMQDCSL (SEQ ID NO: 41), or a functional analog thereof, having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0129] In some embodiments, paics is encoded by a nucleic acid sequence as disclosed in the Genbank (accession number BC071527.1), or a fragment thereof. In some embodiments, the DNA molecule comprises a nucleic acid sequence as disclosed in the Genbank (accession number BC071527.1, or a fragment thereof.

[0130] In some embodiments, the at least one third polypeptide comprises an amino acids sequence as set forth in SEQ ID Nos 36-41, or a functional analog thereof having at least 60%, 70%, 80%, 90%, 95%, or 99% homology or sequence identity thereto, or any value and range therebetween. Each possibility represents a separate embodiment thereto.

[0131] The terms “homology” or “identity”, as used interchangeably herein, refer to sequence identity between two amino acid sequences or two nucleic acid sequences, with identity being a stricter comparison. The phrases “percent identity or homology” and “% identity or homology” refer to the percentage of sequence identity found in a comparison of two or more amino acid sequences or nucleic acid sequences. Two or more sequences can be anywhere from 0-100% identical, or any value there between. Identity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison to a reference sequence. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of homology of amino acid sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

[0132] The following is a non-limiting example for calculating homology or sequence identity between two sequences (the terms are used interchangeably herein). The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non- homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences.

[0133] In some embodiments, % homology or identity as described herein are calculated or determined using the basic local alignment search tool (BLAST). In some embodiments, % homology or identity as described herein are calculated or determined using Blossum 62 scoring matrix.

[0134] In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, or any combination thereof, is codon optimized for expression in the cell.

[0135] In some embodiments, a cell is or comprises a transgenic cell, a transformed cell, a transfected cell, a transduced cell, or any combination thereof.

[0136] In some embodiments, a cell is or comprises: a unicellular organism, a cell of a multicellular organism, or a cell in a culture (such as of a cell line, a stable line, a primary cell line).

[0137] As used herein, the term "transgenic cell" refers to any cell that has undergone human manipulation on the genomic or gene level. In some embodiments, the transgenic cell has had exogenous polynucleotide, such as an isolated DNA molecule as disclosed herein, introduced into it. In some embodiments, a transgenic cell comprises a cell that has an artificial vector introduced into it. In some embodiments, a transgenic cell is a cell which has undergone genome mutation or modification. In some embodiments, a transgenic cell is a cell that has undergone CRISPR genome editing. In some embodiments, a transgenic cell is a cell that hasundergone targeted mutation of at least one base pair of its genome. In some embodiments, the exogenous polynucleotide (e.g., the isolated DNA molecule disclosed herein) or vector is stably integrated into the cell. In some embodiments, the transgenic cell expresses a polynucleotide of the invention. In some embodiments, the transgenic cell expresses a vector of the invention. In some embodiments, the transgenic cell expresses a protein of the invention. In some embodiments, the transgenic cell, is a cell that is devoid of a polynucleotide of the invention that has been transformed or genetically modified to include the polynucleotide of the invention. In some embodiments, CRISPR technology is used to modify the genome of the cell, as described herein.

[0138] In some embodiments, the cell comprises: a unicellular organism, a cell of a multicellular organism, or a cell in a culture.

[0139] In some embodiments, a unicellular organism comprises a fungus or a bacterium. In some embodiments, the fungus is a yeast cell.

[0140] In some embodiments, any one of the inactive genes is knocked out, knocked down, mutated, or chemically inhibited. As used herein, the terms “knocked out” and “knockout” are used interchangeably and refer to a gene that is inactive. In some embodiments, the inactivation is by a genetic technique. In some embodiments, the genetic technique comprises a method in which the gene, or at least part of it (e.g., the operon or the protein coding sequence), is taken out from the organism’s genome.

[0141] Methods for generating knocked out genes are well known in the art. Non-limiting examples comprise homologous recombination, site-specific nucleases, zinc finger nuclease, transcription activator-like effector nuclease (TALENTS), and clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9.

[0142] In some embodiments, any one of the inactive genes is knocked down. As used herein, the term “knocked down”, or “knockdown”, refers to a condition in which the expression of the gene is reduced. In some embodiments, the transcription of the gene is reduced. In some embodiments, the translation of the gene is reduced. In some embodiments, the expression of the gene is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or by at least 99%. Each embodiment refers to a separate embodiment of the invention. In some embodiments, the reduction can occur either through a genetic modification or by treatment with a reagent suchas a short DNA or RNA oligonucleotide that has a nucleic acid sequence complementary to either gene or a mRNA transcript.

[0143] In some embodiments, the knocked down gene is achieved by a genetic modification or mutation, leading to gene silencing. In some embodiments, the knocked down gene is by a stable knockdown, referring to constituent reduced gene expression. In some embodiments, the gene reduced expression is caused by an oligonucleotide binding to mRNA or temporarily binding to a gene. In some embodiments, the knocked down gene is a transient knockdown referring to a temporary change in gene expression that does not modify the chromosomal DNA. In some embodiments, the transient knockdown, is achieved by at least one of: blocking or reducing transcription, degradation of the mRNA transcript, blocking or reducing mRNA translation, blocking, or reducing the binding of pre-mRNA splicing sites, or nuclease cleavage sites used for maturation of other functional RNAs, including miRNA. In some embodiments, blocking or reducing transcription can be achieved by small interfering RNA (siRNA) or RNase-H dependent antisense. Methods for generation of knockdown genes are known in the art, comprising RNA interference, CRISPRs, and TALENs.

[0144] According to some embodiments, there is provided a homogenate, lysate, extract, derived from a transgenic cell disclosed herein, any combination thereof, or any fraction thereof.

[0145] According to another aspect, there is provided an extract derived from the cell of the invention, or any fraction thereof.

[0146] In some embodiments, the extract comprises a purine -based compound.

[0147] In some embodiments, the extract comprises guanine. In some embodiments, guanine is in a form of a crystal comprising guanine. In some embodiments, the crystal further comprises hypoxanthine.

[0148] In some embodiments, the extract comprises the DNA molecule as disclosed herein, the polypeptide(s) disclosed herein, or both.

[0149] Methods and / or means for extracting, lysing, homogenizing, fractionating, or any combination thereof, a cell or a culture of same, are common and would be apparent to one of ordinary skill in the art of cell biology and biochemistry. Non-limiting examples include, but are not limited to, pressure lysis (e.g., such as using a French press), enzymatic lysis, soluble-insoluble phase separation (such for obtaining a supernatant and a pellet), detergent-based lysis, solvent (e.g., polar, or nonpolar solvent), liquid chromatography mass spectrometry, or others.Compositions

[0150] According to another aspect, there is provided a composition comprising: (a) the cell of the invention; and / or the extract as disclosed herein, and an acceptable carrier.

[0151] In some embodiments, the carrier is or comprises a pharmaceutical carrier. In some embodiments, the carrier is or comprises a cosmeceutical carrier.

[0152] As used herein, the term “carrier”, “excipient”, or “adjuvant” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds.,Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0153] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.Methods of synthesis

[0154] According to another aspect, there is provided a method for synthesizing guanine.

[0155] According to another aspect, there is provided a method for controlling morphogenesis of a crystal comprising a purine -based compound in a cell producing the crystal.

[0156] In some embodiments, the method comprises: (a) providing the cell of the invention; and (b) culturing the cell from step (a) such that the at least one first polypeptide is expressed.

[0157] In some embodiments, the culturing comprises supplementing the cell with an effective amount of a guanine precursor. In some embodiments, a guanine precursor comprises a nitrogen source. In some embodiments, a nitrogen source is selected from: nitrate, ammonium sulfate, urea, ammonia, or any combination thereof. In some embodiments, a guanine precursor and / or a nitrogen source is selected from: soybean, peanut, cottonseed meal, corn pulp, corn steep, meat extract, peptone, fish meal, yeast extract, yeast peptone, or any combination thereof. In some embodiments, a guanine precursor and / or a nitrogen source is in the form of: soybean, peanut, cottonseed meal, corn pulp, com steep, meat extract, peptone, fish meal, yeast extract, yeast peptone, or any combination thereof.

[0158] In some embodiments, a guanine precursor is selected from: inosine monophosphate (IMP), xanthosine monophosphate (XMP), guanosine monophosphate (GMP), guanosine, guanosine diphosphate (GDP), deoxygu ano sine diphosphate (dGDP), deoxyguanosine monophosphate (dGMP), deoxyguanosine, guanosine triphosphate (GTP), deoxyguanosine triphosphate (dGTP), or any combination thereof.

[0159] In some embodiments, culturing is in a cell culture medium.

[0160] In some embodiments, the method further comprises a step after step (b), comprising extracting the cell (e.g., step (c)).

[0161] In some embodiments, the method further comprises a step after step (b), comprising extracting the cell culture medium cell (e.g., step (c)). In some embodiments, the method further comprises a step after step (b), comprising extracting the cell culture medium, wherein the cell is, was, or both, being cultured.

[0162] In some embodiments, extracting is from a cell comprising a recombinant DNA molecule comprising a first nucleic acid sequence encoding at least one first polypeptide being an enzyme selected from Impdh, gmps, and both.

[0163] In some embodiments, extracting is from a cell comprising a recombinant DNA molecule comprising a third nucleic acid sequence encoding at least one third polypeptide being the enzyme ppat; or both (i) and (ii).In some embodiments, extracting is from a cell comprising a recombinant DNA molecule comprising: (i) a first nucleic acid sequence encoding at least one first polypeptide being an enzyme selected from Impdh, gmps, and both;and (ii) a third nucleic acid sequence encoding at least one third polypeptide being the enzyme ppat.

[0164] In some embodiments, extracting is from the culture medium wherein the cell is, was, or both, being cultured, and wherein the cell comprises a recombinant DNA molecule comprising a first nucleic acid sequence encoding at least one first polypeptide being an enzyme selected from Impdh, gmps, and both.

[0165] In some embodiments, extracting is from the culture medium wherein the cell is, was, or both, being cultured, and wherein the cell comprises a recombinant DNA molecule comprising a third nucleic acid sequence encoding at least one third polypeptide being the enzyme ppat.

[0166] In some embodiments, extracting is from the culture medium wherein the cell is, was, or both, being cultured, and wherein the cell comprises a recombinant DNA molecule comprising: (i) a first nucleic acid sequence encoding at least one first polypeptide being an enzyme selected from Impdh, gmps, and both; and (ii) a third nucleic acid sequence encoding at least one third polypeptide being the enzyme ppat.

[0167] In some embodiments, the method comprises culturing the cell in a medium and extracting the cell.

[0168] In some embodiments, extracting is from the cell, and wherein the cell comprises a recombinant DNA molecule comprising the first nucleic acid sequence encoding at least one first polypeptide being the enzyme pnp4a.

[0169] In some embodiments, the method comprises the steps: (a) culturing the cell in a medium; and (b) extracting the cell, thereby obtaining an extract from the cell.

[0170] In some embodiments, the medium comprises cell culture medium or growth medium.

[0171] As used herein the term “purine-based compound” encompasses a compound represented by Formula 1 :, wherein represents a single or a double bond; wherein each X is independently N or NH, as allowed by valency; wherein each of R1 and R is independently H or represents one or more substituents selected from alkyl, oxo, amino, halo,cyano, nitro, -COOR’, mercapto and hydroxy; and wherein R’ is H or alkyl, including any salt, any hydrate, and tautomer and any stereoisomer of the compound. In some embodiments, each of R1 and R is independently H or represents one or more substituents selected from alkyl, oxo and amino. In some embodiments, alkyl is methyl. In some embodiments, amino is -NH2.

[0172] In some embodiments, the purine-based compound is represented by Formula 2:, wherein X is as described above; wherein each XI is independently absent, or is selected from O and NH2; and wherein each R2 is independently methyl, or is absent.

[0173] In some embodiments, the purine -based compound is a natural compound of Formula 1 or of Formula 2.

[0174] In some embodiments, a purine-based compound is selected from: guanine, xanthine, adenine, theobromine, caffeine, uric acid, isoguanine, adenine, theophylline, or hypoxanthine, including any salt, isomer, or tautomer thereof, and any combination thereof.

[0175] In some embodiments, a purine-based compound is selected from: guanine, xanthine, hypoxanthine, adenine, including any salt, isomer, or tautomer thereof, and any combination thereof.

[0176] In some embodiments, a method for producing guanine, comprises: (a) providing a comprising a cell comprising: (i) a recombinant DNA molecule comprising nucleic acid sequence encoding the enzymes: ppat, gar, pfas, paics, adsl, atic, impdh, gmps, nt5c, pnp, nme, and / or functional analog(s) thereof; (ii) the inactive genes: gda, uox, xdh, urah, and / or functional analog(s) thereof ; and (b) culturing the cell from step (a) such that the enzymes are expressed, thereby producing guanine.

[0177] In some embodiments, a method for producing xanthine, comprises: (a) providing a comprising a cell comprising: (i) a recombinant DNA molecule comprising nucleic acid sequence encoding the enzymes: ppat, gar, pfas, paics, adsl, atic, impdh, gmps, nt5c, pnp,gda, and / or functional analog(s) thereof; and (ii) inactive genes: uox, xdh, urah, and / or functional analog(s) thereof ; and (b) culturing the cell from step (a) such that the enzymes are expressed, thereby producing xanthine.

[0178] In some embodiments, a method for producing hypoxanthine, comprises: (a) providing a comprising a cell comprising a recombinant DNA molecule comprising nucleic acid sequence encoding the enzymes: ppat, gart, pfas,paics, adsl, atic,nt5, pnp and / or functional analog(s) thereof; and (b) culturing the cell from step (a) such that the enzymes are expressed, thereby producing hypoxanthine.

[0179] In some embodiments, a method for producing uric acid, comprises: (a) providing a comprising a cell comprising: (i) a recombinant DNA molecule comprising nucleic acid sequence encoding the enzymes: ppat, gar, pfas, paics, adsl, atic, impdh, gmps, nt5c, pnp, gda, xdh, and / or functional analog(s) thereof; and (ii) inactive uox gene and / or functional analog(s) thereof ; and (b) culturing the cell from step (a) such that the enzymes are expressed, thereby producing uric acid.

[0180] In some embodiments, a method for producing IMP, comprises: (a) providing a comprising a cell comprising: (i) a recombinant DNA molecule comprising nucleic acid sequence encoding the enzymes: ppat, gar, pfas, paics, adsl, atic, and / or functional analog(s) thereof; and (ii) inactive genes: Impdh, nt5, and / or functional analog(s) thereof ; and (b) culturing the cell from step (a) such that the enzymes are expressed, thereby producing IMP.

[0181] In some embodiments, a method for producing XMP, comprises: (a) providing a comprising a cell comprising: (i) a recombinant DNA molecule comprising nucleic acid sequence encoding the enzymes: ppat, gar, pfas, paics, adsl, atic, impdh, and / or functional analog(s) thereof; and (ii) inactive nt5 gene, and / or functional analog(s) thereof ; and (b) culturing the cell from step (a) such that the enzymes are expressed, thereby producing XMP.

[0182] In some embodiments, a method for producing GMP, comprises: (a) providing a comprising a cell comprising: (i) a recombinant DNA molecule comprising nucleic acid sequence encoding the enzymes: ppat, gar, pfas, paics, adsl, atic, impdh, gmps, and / or functional analog(s) thereof; and (ii) inactive nt5 gene, and / or functional analog(s) thereof ; and (b) culturing the cell from step (a) such that the enzymes are expressed, thereby producing GMP.

[0183] In some embodiments, a method for producing GDP, comprises: (a) providing a comprising a cell comprising: (i) a recombinant DNA molecule comprising nucleic acid sequence encoding the enzymes: ppat, gar, pfas, paics, adsl, atic, impdh, gmps, gukla, and / or functional analog(s) thereof; and (ii) inactive rrm gene, and / or functional analog(s) thereof ; and (b) culturing the cell from step (a) such that the enzymes are expressed, thereby producing GDP.

[0184] In some embodiments, a method for producing GTP, comprises: (a) providing a comprising a cell comprising: (i) a recombinant DNA molecule comprising nucleic acid sequence encoding the enzymes: ppat, gar, pfas, paics, adsl, atic, impdh, gmps, gukla, nme, and / or functional analog(s) thereof; and (ii) inactive rrm gene, and / or functional analog(s) thereof ; and (b) culturing the cell from step (a) such that the enzymes are expressed, thereby producing GTP.

[0185] In some embodiments, "producing" comprises increasing the production of, increasing the amount, abundance, concentration of, or any combination thereof, of a purine- based compound, as disclosed herein.

[0186] In some embodiments, the method comprises modulating gene expression ratio between a first gene encoding the enzyme Pnp4a, a functional analog thereof, or both, and a second gene encoding the enzyme Pnp5a / b, a functional analog thereof, or both, in the cell, thereby controlling the morphogenesis of a crystal produced in a cell.

[0187] In some embodiments, the gene expression ratio is between a first gene encoding the enzyme Pnp4a and a second gene encoding the enzyme Pnp5a, Pnp5b, or both.

[0188] In some embodiments, modulating comprises increasing or reducing.

[0189] In some embodiments, increasing the gene expression ratio above a predetermined threshold results in an elongated semi -hexagonal crystal comprising a combination of guanine and hypoxanthine in a weight per weight ratio ranging 1:2 (w / w) and 1,000:1 (w / w).

[0190] In some embodiments, reducing the gene expression ratio below the predetermined results in semi-hexagonal crystal, a square-like crystal, or a combination thereof, comprising a combination of guanine and hypoxanthine in a w / w ranging between 1:1 (w / w) and 100:1 (w / w).

[0011] In some embodiments, an elongated semi-hexagonal crystal is characterized by or comprises a length / width ratio ranging between 1: 1 (length / width) and 1:50 (length / width).

[0192] In some embodiments, a semi-hexagonal crystal or a square-like crystal is characterized by or comprises a length / width ratio length / width ratio ranging between 1:1 (length / width) and 1:20 (length / width).

[0193] Methods and means for determining length, width, compound concentration, or any combination thereof, of crystals such as disclosed herein, are common and would be apparent to one of ordinary skill in the art, such as exemplified herein (e.g., in the example section).

[0194] According to some embodiments, there is provided a medium or a portion thereof separated from a cultured cell, obtained according to the herein disclosed method.

[0195] According to some embodiments, there is provided a composition comprising: (a) the extract disclosed herein; (b) the medium disclosed herein or a portion thereof; or (c) any combination of (a) and (b), and an acceptable carrier, as described herein.

[0196] In some embodiments, a portion comprises a fraction or a plurality thereof.

[0197] In some embodiments, the method further comprises a step preceding or before step (a), comprising introducing or transfecting the cell with the DNA molecule or an artificial nucleic acid molecule or vector comprising same.

[0198] Methods for introducing or transfecting a cell with an artificial nucleic acid molecule or vector are common and would be apparent to one of ordinary skill in the art.

[0199] In some embodiments, introducing or transfecting comprises transferring an artificial nucleic acid molecule or vector comprising the DNA molecule as disclosed herein into a cell; or modifying the genome of a cell to include the DNA molecule as disclosed herein. In some embodiments, the transferring comprises transfection. In some embodiments, the transferring comprises transformation. In some embodiments, the transferring comprises lipofection. In some embodiments, the transferring comprises nucleofection. In some embodiments, the transferring comprises viral infection.

[0200] As used herein, the terms “transfecting” and “introducing” are interchangeable.

[0201] Types of plasmids and / or vector as well as protocols for using same are common and would be apparent to one of ordinary skill in the art.

[0202] Expressing polynucleotide within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell's genome. In some embodiments, the polynucleotide is in an expression vector suchas plasmid or viral vector. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly- Adenine sequence.

[0203] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno- associated viral vector, a virgaviridae viral vector, or a poxviral vector. The barley stripe mosaic virus (BSMV), the tobacco rattle virus and the cabbage leaf curl geminivirus (CbLCV) may also be used. The promoters may be active in plant cells. The promoters may be a viral promoter.

[0204] In some embodiments, the DNA molecule as disclosed herein is operably linked to a promoter. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). In some embodiments, the promoter is operably linked to the polynucleotide of the invention. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is the endogenous promoter.

[0205] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), such as biolistic use of coated particles, and needle-like particles, Agrobacterium Ti plasmids and / or the like.

[0096] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7- 20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. The promoter may extend upstream or downstream of the transcriptional start site and may be any size ranging from a few base pairs to several kilo-bases.

[0206] In some embodiments, the DNA molecule is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells, known to catalyze the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.

[0207] In some embodiments, a plant expression vector is used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3: 1671-1680 (1984); and Brogli et al., Science 224:838- 843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation, and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0208] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0209] In some embodiments, recombinant viral vectors, which offer advantages such as systemic infection and targeting specificity, are used for in vivo expression. In one embodiment, systemic infection is inherent in the life cycle of, for example, the retrovirus and is the process by which a single infected cell produces many progeny virions that infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread systemically. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0210] In some embodiments, plant viral vectors are used. In some embodiments, a wild-type virus is used. In some embodiments, a deconstructed virus such as are known in the art is used. In some embodiments, Agrobacterium is used to introduce the vector of the invention into a virus.

[0211] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, agrobacterium Ti plasmids and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0212] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield, or activity of the expressed polypeptide.

[0213] The term "nucleic acid" is well known in the art of molecular biology. A "nucleic acid" as used herein will generally refer to any molecule (e.g., a strand) of DNA, RNA or a derivative or analog thereof, comprising nucleotides. Nucleotides are comprised of nucleosides and phosphate groups. The nitrogenous bases of nucleosides include, for example, naturally occurring purine or pyrimidine nucleosides as found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C).General

[0214] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed withinthe invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0215] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ± 100 nm.

[0216] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0217] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0218] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embracedby the present invention and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0219] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0220] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0221] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Materials and MethodsEthics and safety statement

[0222] This study followed European Union directives (2010 / 63 / EU) and the German law, with project license #25-5131 / 522 / 54 (TVV52 / 2021) and according to the guidelines of the Weizmann Institute of Science Animal Care and Use Committee (Rehovot, Israel). Genetic engineering work was carried out in a SI area, following regulations from the German Genetic Engineering Act and German Genetic Engineering Safety Ordinance (GenTSV), with license #Az.: 54-8451 / 103.Zebrafish husbandry and handling

[0223] Zebrafish (Danio rerio) were housed at ~28 °C, 14 hours light: 10 hours dark and fed with Artemia and flake food. Fish were maintained and fed following the standard protocols. Crosses were performed with 3- to 12-months old wild-type AB strain adults. Embryos were kept in E3 zebrafish embryo medium at 28.5 °C until reaching the desired developmental stage. Published lines used were: Et(01a.Edar:GAL4,14xUAS:GFP)TDL358Et(RRID: ZDB- FISH- 150901-2380) for simplicity denominated TDL358 throughout manuscript; Tg(pnp4a:PALM-mCherry)wprtl0Tg(RRID: ZDB-FISH-210414-18).Cell dissociation and isolation

[0224] Fish were anesthetized with Tricaine and immersed in TrypLE Express (Invitrogen, 12604039) with 200 pg / ml Liberase™ TL Research Grade (Roche, 05401020001). Fish were incubated at 37 °C and shaken at 200 rpm for 40 min, followed by mechanical disruption with a Pasteur pipette to further dissociate the cells. Cells were then strained through a 40 pM cell strainer with HL- 15 buffer and centrifuged (HL- 15 Buffer contains: Hank’s Balanced salt solution 40% (Sigma H8264) and 60% Leibovitz's L-15 Medium (Gibco 21083-027)). Dissociated cells were then pelleted at 1000 ref for 10 min at 4°C, then resuspended 5 ml of fresh HL- 15 for FACs.Fluorescence-activated cell sorting (FACS)

[0225] Cells were isolated from Tg(pnp4a:PALM-mCherry) positive fish cells and sorted via FACS. Following resuspension in 5 ml cold HL-15, the isolated cells were incubated with Hoechst to mark the nuclei for 30 min prior to FACS. Cells were analyzed and sorted using a BD FACSAria™ III Cell Sorter with a 100 pM nozzle. Cells were illuminated using both 405 and 561 nm lasers. Cells were gated based on attributes to separate cells from each other as well as from cellular debris. Cellular debris were detected using forward, side scatter and Hoechst signals to select against the smallest particles (1 mm or less). Cells were additionallysorted and enriched based on detection using 561 nm filters, corresponding to the pnp4a:PALM-mCherry signal. Cells were collected into ice-cold PBS medium and kept at 4°C. Cells were then spun down at 1,000 ref for 5 min and cell pellets were flash frozen using liquid nitrogen and stored in -80 °C until downstream proteomics analysis.Proteomics sample preparation

[0226] Cell pellets were lysed with 5% SDS in 50 mM Tris-HCl. Lysates were incubated at 96 °C for 5 min, followed by six cycles of 30 s of sonication (Bioruptor Pico, Diagenode, USA). Protein concentration was measured using the BCA assay (Thermo Scientific, USA) and a total of 7.3 pg protein was reduced with 5 mM dithiothreitol and alkylated with 10 mM iodoacetamide in the dark. Each sample was loaded onto S-Trap microcolumns (Protifi, USA) according to the manufacturer’s instructions. Briefly, after loading, samples were washed with 90:10% methanol / 50 mM ammonium bicarbonate. Samples were then digested with trypsin (1:50 trypsin / protein) for 1.5 hours at 47 °C. The digested peptides were eluted using 50 mM ammonium bicarbonate; trypsin was added to this fraction and incubated overnight at 37 °C. Two more elutions were made using 0.2% formic acid and 0.2% formic acid in 50% acetonitrile. The three elutions were pooled together and vacuum-centrifuged to dry. Samples were kept at -20 °C until analysis.Liquid chromatography

[0227] ULC / MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nano-Ultra Performance Liquid Chromatography (10 kpsi nanoAcquity; Waters, Milford, MA, USA). The mobile phase was: A) H2O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed-phase Symmetry C18 trapping column (180 pm internal diameter, 20 mm length, 5 pm particle size; Waters). The peptides were then separated using a T3 HSS nanocolumn (75 pm internal diameter, 250 mm length, 1.8 pm particle size; Waters) at 0.35 pL / min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 30% B in 105 min, 30% to 90% B in 10 min, maintained at 90% for 7 min and then back to initial conditions.Mass spectrometry

[0228] The nanoUPLC was coupled online through a nanoESI emitter (10 pm tip; New Objective; Woburn, MA, USA) to a quadrupole orbitrap mass spectrometer (Exploris 480,Thermo Scientific) using a Flexion nanospray apparatus (Proxeon). Data was acquired in data dependent acquisition (DDA) mode, using 2 s cycle time. MSI resolution was set to 120,000 (at 200 m / z), mass range of 380-1,500 m / z, 200% AGC and maximum injection time was set to 50 ms. MS2 resolution was set to 15,000, quadrupole isolation 1.4 m / z, 75% AGC, dynamic exclusion of 30 s and maximum injection time was set to auto.High-Performance Liquid ChromatographySample preparation

[0229] Ammonium formate buffer (10 mM, pH 3.7) (70221-25G-F, Sigma- Aldrich) was prepared by dissolving 630 mg of ammonium formate in 1 L of Milli-Q® water and adjusting the pH with formic acid (27001-1L-R, Sigma- Aldrich).

[0230] For the pellet samples, bacteria pellets expressing different genes were dissolved in Perchloric acid (PCA) (244252- IL, Sigma- Aldrich) to a total volume of 200 pL. The solution was sonicated for 20 min. Subsequently, 50 pL of the sonicated solution was added to 1,950 pL of Ammonium formate buffer (10 mM, pH 3.7). The resulting solution was filtered with a PVDF filter (0.22 pm; SLGVO33RS, Merck Millipore) into HPLC vials. Chromatograms were recorded by injecting 10 pL of the filtered solution into the HPLC system.

[0231] For the supernatant (sup) samples, 50 pL of the sup were added to 150 pL of PCA. The solution was sonicated for 20 min. Then, 50 pL of the sonicated solution were added to 1,950 pL of Ammonium formate buffer (10 mM, pH 3.7). The solution was filtered with a PVDF filter (0.22 pm) into HPLC vials. Chromatograms were recorded by injecting 10 pL of the filtered solution into the HPLC system.HPLC analysis

[0232] HPLC data were recorded using an Agilent 1260 Infinity HPLC system (Agilent Technologies) equipped with an Agilent Zorbax Eclipse plus column (250 mm x 4.5 mm, 5 pm). The mobile phase consisted of an elution gradient containing phase A (Ammonium formate buffer, 10 mM, pH 3.7) and phase B (methanol; 1368350100, BioLab). The gradient elution was programmed as: 0-20 min: 1% B; 20-23 min: linear gradient to 80% B; 23-29 min: hold at 80% B; 29-31 min: linear gradient to 1% B; 31-35 min: hold at 1% B. The total run time was 35 min with a flow rate of 1 ml min-1. Throughout the experiment, the column temperature was maintained at 30 °C and the UV detection wavelength was set to 260 nm. The position and quantity of Guanine, Hypoxanthine and Adenine in the pellet and sup of thebacterial samples were determined using pre-recorded calibration curves from standard samples.Data processing for mass spectrometry

[0233] Raw data was processed with MetaMorpheus v0.0.320. The data was searched against the Danio rerio Uniprot proteome database (appended with common lab protein contaminants and the following modifications: Carbamidomethylation of C as a fixed modification and oxidation of M and protein N-terminal acetylation as variable ones. Quantification was performed using the embedded FlashLFQ and protein inference algorithms. The LFQ intensities were imported into Perseus v 1.6.2.3. Decoy hits were removed and the LFQ intensities were log2 transformed. The data were filtered to include only proteins that had at least 2 valid values in at least one experimental group. The remaining missing values were randomly imputed, and a student’s / -test was used to detect proteins that were significantly differentially expressed.Phylogenetic analysis of the vertebrates PNP protein family

[0234] The identification of orthologous genes and their associated protein sequences was extracted with Ensembl, using the canonical transcript for each gene. Multiple alignment was done with the clustal method in MEGA11. The tree was constructed using the Maximum Likelihood method and JTT matrix-based model with MEGAI L The numbers indicate the percentage of replicate trees in which the associated clades clustered together in a bootstrap test (100 replicates). The phylogenetic clades are highlighted in the background, with each clade's phylogenetic class indicated by a specific color code: orange for fishes, green for amphibians, blue for birds, grey for reptiles, and pink for mammals. Species abbreviations: Salmon, Atlantic Salmon; Cod, Atlantic cod; Lizard, Green Anole; Turtle, Painted Turtle; Snake, Eastern Brown Snake; Frog, African clawed Frog; Medaka, Japanese rice fish. The MTAP protein serves as an outgroup, and it is scaled outside of the figure. pnp4a cloning and transgenic generation

[0235] pnp4a (ID: ENSDARG00000057575) cDNA was amplified from 5 days post fertilization (dpf) zebrafish total cDNA together with primers containing Asci and Fsel digestion sites at their 5’ and 3’ ends, respectively (FWD: 5’- ATGGCGCGCCCATGCATAGTAAAG ACC-3’ (SEQ ID NO: 11); REV:5’ ATATGGCCGGCCTAATGCCGTGTTGTTGTTGATGTC-3’ (SEQ ID NO: 12)). The 876bp amplified fragment was purified using a gel extraction kit (QIAGEN 28706), digested with restriction enzymes Asci and Fsel (NEB) and cloned into pCS2+8CmCherry (Addgene #34935) - previously digested with the same restriction enzymes and treated with Calf intestinal alkaline phosphatase (NEB M0290S). For Tg(hsp70:Pnp4a-mkate2)cbgl9Tggeneration, Gateway Technology was used. A pnp4a middle entry clone was generated amplifying pnp4a from pCS2+pnp4amCherry with primers containing attB l and attB2 sites. (FWD : 5 ’ GGGGAC AAGTTTGT AC AAAAAAGC AGGCTT AATGC ATAGTAAAGACC A AATCTGCCAT-3' (SEQ ID NO: 13); REV:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTTAATGCCGTGTTG TTGTTGATGTCCATGC-3' (SEQ ID NO: 14)).

[0236] The 937 bp amplified fragment was cleaned using PCR clean up Kit (Promega A9281) and recombined with pDONOR221 (plasmid 208 from the Tol2 Kit) in a BP reaction (BP clonase II Enzyme-Mix Ivitrogen #11789020). The generated middle entry clone pME-pnp4a was then recombined in a LR reaction using pDEST-Tol2pA (plasmid 426 from the Tol2 Kit); p5E- hsp70 (plasmid 222 from the Tol2 kit) and p3E-mKate2 (gift from Oates Lab) (LR Clonase II Plus Enzyme from Invitrogen #12538120). One-cell stage TDL358 transgenic embryos were injected with 25 pg of transposase mRNA and 25 pg of the generated T2- hsp70:Pnp4a-mKate2 construct. The embryos were grown for 3 months. Afterwards, F0 fish have been crossed with wild-type AB. The obtained embryos have been heatshocked at 24 hours post fertilization (hpf) for 1 h at 37 °C and screened for red fluorescence with Olympus SZX16 fluorescence microscope. Positive Fl founder fish with TDL358 background have been selected and raised. pnp4acbg20and pnp4awz19CRISPR mutant and rescue

[0237] pnp4acbs20and pnp4awz19CRISPR mutants were generated as described (Kroll et al. 2021). Briefly, trans-activating Crispr RNAs (crRNA) were designed for specific loci of the pnp4a gene using the predesigned crRNAs dataset from IDT. Several crRNAs were tested for RNP mutagenesis and were chosen considering where the start codon is located, high on- target, and low off-target scores. The most efficient crRNA tested was located on exon 4 of the pnp4a canonical transcript, with target sequence: 5’-CGTGACCACATTAACTTCCC-3’ (SEQ ID NO: 15) (Dr.Cas9.PNP4A.l.AB, IDT). Each tested crRNA was separately annealed with an equal molar amount of tracrRNA (#1072533, IDT) and diluted to 57 pM in Duplex buffer (#11-01-03-01, IDT), generating the single guide RNA (sgRNA). The RNP mixes wereassembled using Cas9 protein (Alt-R S.p. Cas9 Nuclease V3 #1081058, IDT, 61 pM stock) and a sgRNA, in equal molar amounts, generating a 28.5 pM RNP solution. To improve mutagenesis efficiency, the mixes were kept at -20 °C overnight before being injected on the following day. One-cell stage WT AB or TDL358; hsp70:Pnp4a-mKate2 double transgenic embryos were injected with 1 nL of each of the RNP mixes. To screen for the efficiency of RNP mutagenesis and potential genotype to phenotype relationships, 10 embryos of each RNP-injected condition were individually genotyped, and reflection phenotypes were assessed using incident light from a stereoscope (Leica M165C) at 3 dpf and grown to adulthood. Founder fish containing frameshift mutations were identified by genotyping the resulting Fl progeny, resulting from outcrosses with wild-type AB fish. Two mutant lines were generated, the pnp4acbg20resulting in a 6 nt deletion, 1 nt insertion leading to an overall 5 nt frameshift and truncated protein resulting in 150 amino acids (AA) instead of the 291 AA on the background of TDL358; hsp70:Pnp4a-mKate2 as well as the pnp4awz19resulting in a 10 nt frameshift and truncated protein resulting in 142 AA on the background of WT AB. For rescue experiments, 400 pg of full-length pnp4a-mCherry mRNA were injected at onecell stage into incrossed heterozygous pnp4acbg20; TDL358; hsp70:Pnp4a-mKate2 mutant embryos. Then, injected embryos were heat activated at 36, 48, and 56 hpf in a water bath at 37C for 1 hour (in petri dishes containing 30mL embryo medium), subsequently kept at 28.5C, selected for fluorescence, and finally imaged at 72 hpf. After imaging, genotyping was performed in individual larvae, as described below. For mRNA synthesis details, see below.Genotyping

[0238] Genomic DNA from individual larvae was extracted using the Kapa Express Extract kit (Kapa Biosystems) according to the manufacturer’s protocol. This was followed by performing PCR with KAPA2G Robust HotStart ReadyMix (Kapa Biosystems) with primers surrounding the pnp4a mutation region (FWD: 5’-CAGAATTTGTGCTTGTGTTC3’ (SEQ ID NO: 16); REV: 5’-CCTTGTACTGGTGATTGTAATG-3’ (SEQ ID NO: 17)). As these are located in intron regions, amplicons were specific to the mutation and not the transgenes present. Then PCR products were sent for sequencing. Sequences were analyzed using Snapgene software.Microinjection of embryos with mRNAs and morpholinos

[0239] One-cell stage wild-type AB or TDL358; hsp70:Pnp4a-mkate2 embryos were injected using standard procedures with different pnp4a mRNA concentrations. This was generatedby linearization of pCS2-pnp4a-mCherry vector with Notl (NEB), and transcription using the SP6 mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit (#AM1340, Ambion), following the manufacturer’s protocol. The same procedure was applied for Tol2 Transposase mRNA synthesis, generated from a pCS2FA-transposase plasmid (Kwan et al; 2007). mRNAs were aliquoted and stored at -80 °C until use. Embryos were left to develop at 28.5 °C until the desired stage and then live imaged or grown to adulthood. A PV-820 Picoinjector (World Precision Instruments) and a Narashige micromanipulator were used for microinjection.

[0240] Gene knockdown experiments were performed using the following morpholinomodified antisense oligonucleotides (MO, Gene Tools, Philomath, OR): pnp4a'. 5’- GGCAGATTTGGTCTTTACTATGCAT-3’ (SEQ ID NO: 18); pnp4b 5’- CATGATCTCGGGTAATCACAGGCTG-3’ (SEQ ID NO:19); pnp5a 5’-GGGAAACATGATTCAAGTCGCTGCT-3’ (SEQ ID NO: 20); pnp5b 5’-ATAGAGGTGAAACCTGCCCTGCATG-3’ (SEQ ID NO: 21); pnp6 5’-GCTGGTCGAATTACTGAACGTGATA-3’ (SEQ ID NO: 22); and control morpholino 5'- CCTCTTACCTCAGTTACAATTT ATA-3' (SEQ ID NO: 23) (GENE TOOLS stock control). Each were diluted with ultra-pure (miliQ) water to 1 mM and 1-2 pmol of MO were injected into one-cell stage embryos.Live imaging and mounting

[0241] Confocal live imaging was performed using an upright Zeiss LSM88O confocal microscope, with a C-Apochromat 40x water dipping objective. Briefly, 72 hpf larvae were anesthetized with 0.1% MS-222 (Sigma) diluted in embryo medium, mounted in a concave slide (Sigma-Aldrich #BR475505) with 0.5% low-melting-point agarose (Sigma A9414) in E3 fish medium.Iridophore area and reflection quantification

[0242] After image acquisition, maximum intensity z-projections (MIP) of all acquired signals were obtained using Fiji. The images obtained from the GFP channel, corresponding to cytoplasmic signal from the TDL358 positive cells (iridophores), were segmented using Ilastik, using the pixel classification method. This provided a binary mask which labeled the contour of all iridophores in the samples. The mask was used to define the regions of interest (ROI) within the MIPs of other obtained channels, allowing to measure the corresponding reflectance within iridophores, as well as iridophore area, for each sample, using a custom-made Fiji macro. Individual sample reflection was normalized by dividing the reflectance signal per corresponding iridophore area, allowing statistical comparisons between samples.RT-PCR

[0243] The relative mRNA levels of pnp4a (NM_001002102.1), pnp4b (NM_205643.1), pnp5a (NM-213311.2), pnp5b (NM_001004628.1) and pnp6 (NM_205655.2) were determined using real-time PCR (RT-PCR). Total mRNA was extracted from three groups of WT and three groups of pnp4awz19mutant 6 dpf larvae (15 larvae per sample) or dissected tissue from the skin and gills of an adult zebrafish using the NucleoSpin RNA II Mini SpinKit (cat# 740955 Macherey-Nagel) according to the manufacturer instructions. A 0.5 pg sample of mRNA was reverse transcribed using qScript cDNA Synthesis Kit (cat# 95047- 100 Quanta BioSciences). Relative transcript levels were determined by the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Triplicates of each cDNA sample werePCR amplified using the Fast SYBR Green Master Mix (cat# 4385612 Thermo FisherScientific) and the following specific primers were used: pnp4cr. 5TCATTTGTGGCTCTGGACTG-3 ' (SEQ ID NO: 24) and 5TTTTCCCTTTGAGCTCCCC-3 ' (SEQ ID NO: 25); pnp4b 5TGAAGGGTGTCTGGTATTTGG-3 ' (SEQ ID NO: 26) and 5TGTCTCCCACTTTGAAATCCTG-3 ' (SEQ ID NO: 27); pnp5cr. 5ACATTCCTAACTTTCCCCAGAG-3 ' (SEQ ID NO: 28) and 5CCCAGCATCTTGAAAATCCG-3 ' (SEQ ID NO: 29); pnp5b 5CTAGAGATTGAGGC ATGTCAGG-3 ' (SEQ ID NO: 30) and 5GCCATTTCACTCTTCATTGTCC-3 ' (SEQ ID NO: 31); pnp6 5CAAAATCCCACGCTTTCCAC-3 ' (SEQ ID NO: 32) and 5GGTCACAATCAAAGTCTCAATTCC-3’ (SEQ ID NO: 33). The relative quantification of gene expression levels was normalized against rpll3 (NM_198143.1) in all assays with the rpl!3 primers 5'-AGCTCAAGATGGCAACAG-3' (SEQ ID NO: 34) and 5'- AAGTTCTTCTCGTCCTCC-3' (SEQ ID NO: 35), and AACT analysis. The rpl!3 gene was selected since its relative expression did not significantly change between pnp4awz19mutants and WT during the various developmental stages. Gene levels were normalized by dividing the absolute levels of each sample by the average of all WT samples.Matrix-assisted Laser Desorption / Ionization (MALDI) imaging

[0244] MALDI imaging analysis and data processing were performed as described previously. PICA: Pixel Intensity Correlation Analysis for Deconvolution and Metabolite Identification in Mass Spectrometry Imaging. Briefly, zebrafish at different developmental stages were embedded with Ml embedding matrix (Thermo Scientific, Waltham, MA) in Peel-A-Way disposable embedding molds (Peel-A-Way Scientific, South El Monte, California). The embedded tissues were transferred to a cryostat (Leica CM3050) and allowed to thermally equilibrate at -18 °C for at least 2 hours. The frozen tissues were cut into 70-100 pm-thick sections. The sections were then thaw mounted onto Superfrost Plus slides (Fisher Scientific, Pittsburg, PA) and vacuum dried in a desiccator. TM sprayer (HTX Technologies, LLC, NC, USA) was used to coat the slide with DHB matrix (40 mg mL1in water / methanol v / v 30 / 70 containing 0.2% trifluoroacetic acid). The nozzle temperature was set at 70 °C, and the DHB matrix solution was sprayed in 16 passes over the tissue sections at a linear velocity of 120 cm min1with a flow rate of 50 pl min1. MALDI imaging measurements were performed using a 7T Solarix FT-ICR (Fourier transform ion cyclotron resonance) mass spectrometer (Bruker Daltonics, Bremen, Germany). MSI datasets were collected with 10 pm spatial resolution in a positive ion mode using lock mass calibration (DHB matrix peak: [3DHB+H-3H2O]+, m / z 409.0554) at a frequency of 1 kHz and a laser power of 50%, with 100 laser shots per pixel. Unless stated otherwise, each mass spectrum was recorded in the range of 118-1000 m / z in broadband mode with a time domain for acquisition of 1 M, providing an estimated resolving power of 115,000 at 400 m / z. Raw data were converted into the imzML format using Fleximaging software (V 5.0, Bruker Daltonics, Germany). MALDI images were plotted with the R package Cardinal with mass bin width of ± 0.001 Da. Cardinal: an R package for statistical analysis of mass spectrometry-based imaging experiments. Images were optimized with Gaussian smoothing and contrast enhancement as described.Orbitrap-SIMS imaging

[0245] Cryosections, prepared as described above were analyzed using the M6 Hybrid SIMS instrument (IONTOF GmbH, Munster, Germany) using a top-mount sample holder. In order to detect, assign and determine the distribution of guanine and hypoxanthine, sample areas (field of view: 400x400 pm, raster size 200x200 pixels) were analyzed with a 20keV Anew gas cluster ion beam in positive ionization mode (3 replicates of WT 10 dpf, 3 replicates pnp4awz19mutants 10 dpf). Ion transfer was performed with a He collision cell pressure of 5xl0-2bar and RF amplitude of 70%. The primary ion current was 27 pA. For detection theOrbitrap™ HF high-resolution mass analyzer (mass range 50-750, mass resolution 120.000 @ m / z 200, injection time: 261 ms) was used. Since the sample-setup on glass slides resembles an insulator, all measurements were performed with charge compensation and Ar- gas flooding of the main chamber (Surface potential: -40 V, floodgun: on, pmain= 9xl0-7mbar). Data analysis was performed with Surfacelab 7.3 software (IONTOF GmbH, Munster, Germany). To determine the ratios of guanine and hypoxanthine, 5 regions of interest (ROIs) per imaging run were selected based on the highest relative intensity of guanine (M+H+). Mass peaks of Guanine ([M+H+], m / z 152.056 and [M+Na+], m / z 174.038) and Hypoxanthine ([M+H+], m / z 137.046, no sodium adduct observed) were integrated and intensity values used to calculate the ratio of the two molecules ((louanine + H+ + iGuanine +Na+) / lHypoxaiithiiie + H+) for each individual ROI.Orbitrap-SIMS spectrometry

[0246] Isolated crystals from operculum (gills) and skin tissues were fixed onto glass slides using double sides tape and analyzed using the M6 Hybrid SIMS instrument (IONTOF GmbH, Munster, Germany) using a top-mount sample holder. Five random areas per sample (480x480 pm) were analyzed with a 20 keV Anew gas cluster ion beam in positive ionization mode with a primary ion current of 26.5 pA and a spot size of 2 pm. Ion transfer was performed with a He collision cell pressure of 5xl0-2bar and RF amplitude of 70%. For detection the Orbitrap™ HF high-resolution mass analyzer (mass range 50-750, mass resolution 240.000 @ m / z 200, injection time: 512 ms) was used. For each measurement, 600 scans were summed up. Floodgun was on, surface potential set to -40V and the main chamber was flooded with Argon and regulated to pmain=9xl0’7mbar. Mass signals of Guanine ([M+H+], m / z 152.056 and [M+Na+], m / z 174.038) and Hypoxanthine ([M+H+], m / z 137.046, no sodium adduct observed) were integrated and intensity values used to calculate the ratio of the tWO molecules ((IGuanine + H+ + IGuanine +Na+) / lHypoxanthine + H+) for each individual measurement.CryoET

[0247] Sorted iridophores cells (3.5 pL) with 15 nm gold beads (1 pL) were applied to glow- discharged holey carbon R2 / 2 Cu 200 SiO2 mesh grids (Quantifoil) coated with collagen, Type I, Rat Tail (EMD Millipore 08-115) for cell adherence. The grids were blotted and vitrified by plunging into liquid ethane using a Leica EM GP automatic plunger, under 4 °C and 90% humidity conditions. Frozen grids were kept in liquid nitrogen until used. Data wascollected on a Titan Krios TEM G3i (Thermo Fisher Scientific) equipped with a BioQuantum energy filter with a K3 direct electron detector (Gatan Inc.). Data sets were collected at 300 kV with the K3 camera (counting mode) using SerialEM software. The TEM magnification corresponded to a camera pixel size of 1.6 A, and the target defocus was set to 3 pm. The total dose for a full tilt series was 120 electrons per A . Tomograms tilt series were collected using the dose-symmetric scheme, ±60° at 2°-degree steps. The tilt series images alignment and reconstruction were performed in IMOD.In vitro crystallization

[0248] Crystals were produced by customizing a protocol by Chen et al. Briefly, the following solutions were prepared: a) 1 mg of PVP-co-VA (Mw~50,000, 190845-250G, Sigma- Aldrich) in 10 ml of Formamide (F7503, Sigma- Aldrich), b) 4.5 mg of guanine powder (G6779, Sigma- Aldrich) in 1 ml of 0.4M NaOH solution (CAS 1310-73-2, BioLab), c) 1 mg of adenine powder (A8626, Sigma- Aldrich) in 1.5 ml of distilled water, and d). Different percentages of hypoxanthine powder (weight\weight) (0%-30%, H9377, Sigma- Aldrich) dissolved in 1.5 ml of distilled water. Guanine solution (b) was then filtered using PVDF filter (0.22 pm, SLGVO33RS, Merck Millipore), and adenine and hypoxanthine solutions (c & d) were filtered using Whatman filters (0.2 pm, 10462200, Cytiva). Guanine crystals were produced by adding (c) and (d) into (a) and mixing, followed by the addition of (b). The obtained suspensions were collected and filtered after 24 hours.Chemical fixation and conventional TEM

[0249] Samples (whole larvae) were fixed with 4% paraformaldehyde, 2% glutaraldehyde in 0.1 M cacodylate buffer containing 5 mM CaCh (pH 7.4) for 1 hour, postfixed in 1% osmium tetroxide supplemented with 0.5% potassium hexacyanoferrate tryhidrate and potassium dichromate in 0.1 M cacodylate for 1 hour, stained with 2% uranyl acetate in double distilled water for 1 hour, dehydrated in graded ethanol solutions and embedded in epoxy resin. Ultrathin sections (70-90 nm) were obtained with a Leica EMUC7 ultramicrotome and transferred to Formvar Support film slot grids (EMS). Grids were stained with lead citrate and examined with a Tecnai SPIRIT transmission electron microscope (Thermo Fisher Scientific). Digital electron micrographs were acquired with a bottom-mounted Gatan OneView camera.TEM imaging and electron diffractions of in vitro formed and isolated crystals

[0250] Crystals were placed on top of a copper-carbon coated TEM grid, allowing the crystals to dry. Samples were observed on a Thermo Fisher Scientific Tecnai T12 transmission electron microscope operated at 120 kV. Images and diffraction patterns were recorded on a bottom mounted TVIPS TemCam-XF416 4k x 4k CMOS using imaging and diffraction modes respectively.Micro-Raman spectroscopy

[0251] Micro-Raman spectroscopy (X = 532 nm excitation) was collected by a Horiba EabRAM HR Evolution (Horiba, France) spectrometer equipped with four laser lines (325 nm, 532 nm, 633 nm, and 785 nm). The system has an 800 mm focal length spectrograph for high resolution and low stray light, with several interchangeable gratings and mounted with an open electrode, front illuminated, cooled CCD detector. The sample is placed under a modular microscope (Olympus BX-FM) with a suitable objective. For this work, LUMPlanFL N x60 NA=1.0 WI, and MPlanFL N xl50 NA=0.9 BD (Olympus Japan) objectives with spatial resolution better than 1 pm are used. The light from the Raman scattered 532 nm laser was dispersed on 600 and 1,800 gr / mm gratings and the pixel resolution is better than 2 cm1, or 0.5 cm1respectively. Spectra is normally collected between 100 to 1,800 cm1, with a power of up to 2 mW, and an exposure of 20-60 s using 2-5 averages. The data underwent polynomial baseline subtraction, and spike removal. The spectra were smoothed using a Savitzky Golay algorithm (third order, 11 points), as is commonly done.Cloning, expression, and purification of PNPases

[0252] The sequences of PNPase variants 4a, 4b, 5a, 5b, and 6 were codon -optimized for expression in E. coli, synthesized and cloned into a pET28-His-bdSUMO expression vector (Twist Bioscience©). Plasmids were transformed into electrocompetent BL21 / DE3 cells harboring a chaperone-expressing plasmid pGJKE8 (Takara©) and plated on LB agar plates with 35 pg / ml chloramphenicol, 50 pg / ml kanamycin and 1% w / v glucose. Following O / N incubation at 37 °C, individual colonies from each transformation were randomly picked into 5ml culture tubes (Falcon®) containing growth media (2YT + Cap 35 pg / ml + Kanamycin 50 pg / ml; 1ml) and grown O / N (at 37 °C; shaking, 250 RPM). The resulting cultures were used to inoculate 100ml growth media cultures (1:100 dilution) and grown at 37 °C to ODeoo ~ 0.5. Protein expression was induced by IPTG (1 mM), and cultures were grown for 48 hours (16 °C; shaking 250 RPM). Subsequently, the cells were pelleted (4,000 RPM, 4 °C, 20 min), frozen at -80 °C, thawed, and resuspended in 3 mL cell lysis buffer (20 mM Tris, pH 7.5; 50mM NaCl; 0.6 mg / ml lysozyme; 10 U / ml benzonase nuclease®; bacterial protease inhibitor cocktail without EDTA 1:50). Cells were lysed by sonication and the lysate was clarified by centrifugation (30 min, 4 °C, 20,000 xg). The clarified lysates were loaded onto columns (BioRad©) containing Ni-NTA-resin (Merck©), washed with wash buffer (50 mM Tris, pH 7.5, 100 mM NaCl, imidazole 35 mM) and eluted using elution buffer (50 mM Tris, pH 7.5, 100 mM NaCl, imidazole 300 mM). Eluted proteins were concentrated following exchange to storage buffer (100 mM Tris, pH 7.5, 200 mM NaCl, Glycerol 10%). The His-SUMO tag was cleaved by incubation (18-20 hours, 4 °C) with 1 mM DTT and 0.1 mg / mL bdSumo protease and the untagged proteins were then purified by size exclusion chromatography using a Superdex 75-Increase 10 / 300 GL (Cytiva©) equilibrated with PBS buffer + 100 mM NaCl. Purified PNPase variants eluted using as a single peak corresponding to ~90 kDa trimer, and were pooled, concentrated and buffer exchanged to storage buffer, and frozen in aliquots at -80 °C.Kinetic analyses of PNPases

[0253] Freshly made stock solutions of guanosine or deoxyguanosine were diluted to varying final concentrations (0-500 pM) in potassium-phosphate buffer (50 mM, pH 7.5) containing purified PNPases (0.1 pM). The absorbance of the reaction mixtures was monitored at 258 nm in a quartz 96-well plate using an ELISA plate reader (BioTek©) for 10 min. Initial velocities of purified PNPases with other nucleoside substrates were similarly obtained using their respective maximal absorbance wavelengths (i.e., Inosine = 249 nm; adenosine, deoxyadenosine, or xanthosine = 258 nm; cytidine, deoxy cytidine, or thymidine = 270 nm). For substrate competition experiments, four nucleosides (guanosine, deoxyguanosine, inosine and adenosine) were mixed in equimolar concentrations (200 pM). The nucleoside mix was added to a potassium phosphate buffer solution (50 mM, pH 7.5) containing a purified PNPase (0.25 pM) and incubated for 30 min at RT. Following incubation, the reaction mixtures were inactivated (65 °C, 10 min), and frozen -80 °C. Residual nucleoside concentrations were determined using LC-MS. The pH rate profiles of individual PNPases were determined by mixing dilute enzyme solutions (20 ml, 2.5 mM) with potassium phosphate buffered guanosine solutions (430 mM, 180 mM) at different pH values (2-9) in a 96-well quartz ELISA plate, and monitoring solution absorbance at 258 nm for 10 min at RT. The latter were obtained by titrating a stock solution of guanosine in potassium phosphate buffer (100 mM) using either phosphoric acid, K2HPO4 (100 mM, pH 4.2) or KH2PO4 (100 mM, pH 9)solutions to the appropriate pH values.Structural predictions of PNPases

[0254] The 3D structures of PNPases variants 4a, 4b, 5a, 5b and 6 were predicted using: AlphaFold2, locally installed as of the 29thof May 2023 (github.com / deepmind / alphafold), a ColabFold version of AlphaFold2(colab.research.google.com / github / deepmind / alphafold / blob / main / notebooks / AlphaFold.ipy nb, with and without template -pdblOO), with relaxation in all cases., a ColabFold version of OmegaFold(colab .research .google . com / github / sokrypton / ColabFold / blob / main / beta / omegafold . ipy nb ) , an API-version of ESM (esmatlas.com / resources ?action=fold), Uni-Fold Colab Notebook (colab.research.google.com / github / dptech-corp / Uni-Fold / blob / main / notebooks / unifold.ipynb), and a Rosetta server implementation of RoseTTAFold (robetta.bakerlab.org / submit.php).Statistical analysis

[0255] All statistical analyses were performed using GraphPad Prism software by carrying out non-parametric, unpaired, two-tailed Mann Whitney and student t-tests between the different conditions. Statistical experimental details can be found in relevant figure legends.EXAMPLE 1

[0256] To investigate the proteomic landscape associated with crystal formation, the inventors isolated iridophores from zebrafish larvae at 5 days post-fertilization (dpf), a stage at which intensive crystal formation takes place, by fluorescence-activated cell sorting (FACS) and performed proteomic analysis using liquid chromatography mass spectrometry (LC-MS) (Figs. 1B-1C, and 6A-6E).

[0257] The inventors identified 190 significantly upregulated proteins specific to iridophores, including those encoded by the established iridophore marker genes pnp4a, slc2al5a, gmps, impdhlb, gpnmb, and alex4a (Figs. 1B-1C). Gene Ontology (GO) enrichment and KEGG pathways analyses unveiled key pathways enriched in iridophores, notably de novo inosine monophosphate (IMP) biosynthesis, and purine nucleobase biosynthesis (Figs. ID and 6F). To delve into the functional role of these upregulated iridophore-specific proteins, the inventors applied MetaScape and STRING analyses. As excepted, the purine metabolism pathway emerged as a prominently enriched and interconnected network, underscoring itspivotal role in iridophore biology (Figs. 6G-6H). Specifically, proteins involved in the conversion of phosphoribosyl pyrophosphate (PRPP) to IMP in the early steps of purine metabolism, such as ppat, gart, adsl, atic, paics, and pfas, were upregulated (Fig. 6G). Interestingly, the inventors also identified downregulated proteins, such as gda and uox, which are involved in the breakdown of guanine and its derivatives (Figs. ID and 6F-6G).

[0258] Subsequently, the inventors extended the current proteomic analysis to comprehensively map the intricate purine metabolic network within iridophores, starting from IMP. Strikingly, the inventors observed a comprehensive upregulation of the entire guanine metabolic network. This encompassed the enzymatic conversions of guanosine monophosphate (GMP), guanosine diphosphate (GDP), and deoxyguanosine diphosphate (dGDP), ultimately leading to the formation of guanine (Figs. IB, ID, and 7A). These results indicate that in addition to guanosine, deoxyguanosine is also converted into guanine. This finding underscores the importance of coordinated expression and activity of multiple proteins and enzymes in the intricate guanine biosynthesis pathway within iridophores (Figs. IB, ID, and 7A). To validate the current findings and gain insight into the metabolic regulation of these processes, the inventors performed spatial metabolomics on iridophores in the zebrafish eye at various developmental stages using matrix-assisted laser desorption / ionization (MALDI) imaging (Figs. 2B-2C, and 7A). The inventors observed a consistent rise in guanine, along with a consistent decrease in guanosine and deoxyguanosine levels throughout eye development (Figs. 2B-2C, and 7). These results strongly support the notion that both guanosine and deoxyguanosine are converted into guanine, and that the pathways leading to their production are upregulated.

[0259] In-depth examination of the enzymes within the guanine biosynthetic pathway unveiled the selective upregulation of specific paralogues in iridophores (Fig. 8A-8D). These encompassed inosine monophosphate dehydrogenase lb, but not impdhla or impdh2, nucleotidase 5Clbb, but not nt5clba, nt5claa, or nt5clab, and guanylate kinase la, rather than guklb. The targeted enhancement of a subset of paralogues underscores their specialized role in the guanine biosynthesis pathway and the subsequent crystal formation process (Fig. 7A, and 8A-8D). This raises the intriguing possibility that they may have evolved specialized catalytic activities and increased specificity for this purpose.

[0260] Of particular significance was the substantial upregulation of Pnp4a within iridophores, compared to other members of the PNPase family (Pnp4b, Pnp5a, Pnp5b, Pnp6)(Figs. 1B-1C). To elucidate the evolutionary relationships and functional distinctions among the PNPase enzymes involved in guanine biosynthesis, the inventors first performed phylogenetic analyses. A phylogenetic tree showed the distinct nature of Pnp4a compared to other PNPase members in zebrafish, suggesting its potential evolution towards acquiring a unique catalytic activity for the formation of guanine crystals within iridophores specifically (Fig. 3A).

[0261] To explore this hypothesis, the inventors conducted a comprehensive series of in vitro enzymatic activity assays and structural predictions. For this purpose, the inventors expressed and purified all zebrafish PNPase variants, including Pnp4a, from Escherichia coli (E. coll), and exposed them to a diverse range of nucleosides as substrates (Fig. 3B). The inventors found that Pnp4a displayed heightened enzymatic activity when using guanosine and deoxyguanosine as substrates, in contrast to some of its paralogues (Fig. 3B). Conversely, for nucleosides such as inosine, the precursor of hypoxanthine, and cytidine, the precursor of cytosine, Pnp4a activity was markedly lower compared to its highly active paralogues, namely Pnp5a or Pnp5b (Fig. 3B). This trend was also evident in substrate competition experiments (Fig. 3C). Collectively, these results offer compelling evidence for the selective and distinct functional role of Pnp4a in guanine biosynthesis, a vital process underlying crystal formation within iridophores.

[0262] To gain insights into the unique catalytic specificity of Pnp4a relative to its paralogs, the inventors compared their sequences and computationally predicted structures. For this purpose, the inventors utilized a number of structural prediction algorithms, including AlphaFold-2, to predict the three-dimensional structures of PNPases in D. rerio. Despite the fact that different prediction tools can yield different structural models, the algorithms the inventors used produced almost identical structures for each variant, and their AlphFold-2 models were assigned high confidence scores (Figs. 3D and 9). Furthermore, when overlayed, the structures of all PNPases were very similar with differences localized at their N- and C-termini (Fig. 3D), reflecting their substantial sequence similarities (Fig. 10). Based on their specificity towards 6-oxo-purine nucleosides (Fig. 3B) and their quaternary assemblies (data not shown), they belong to the trimeric PNP family. A comparison of the predicted structure of PNPase4a and the crystal structure of bovine PNPase (PDB ID IB 80) revealed significant sequence and structural similarities between them (60% seq id., 0.55A RMSD). The wealth of crystal structures of bovine PNPase with different ligands (e.g., 1 A9Q, 1A9T, 1A9R, 3FUC, 2QPL) enabled a comprehensive analysis of its active site and ligandbinding residues. The inventors compared the sequences of the corresponding positions in PNPases and found that most of them (Fig. 10) were highly conserved between the different paralogs and bovine PNPase. However, the divergence of some of these conserved active site residues in PNPase4b (e.g., Glu63Val, Alal22Ser, Asnl50His) and in PNPase6 (e.g., Asp56Lys, Glu62His, Phe204Tyr) may account for the variations in substrate specificities and reduced enzymatic activities the inventors observed relative to PNPase4a, 5a and 5b (Figs. 2B-2C, and 10). For example, Glu58 is involved in the active site of bovine PNPase, while the corresponding residue in PNPase4b, at position 63, is mutated to a valine and that of PNPase6 at position 62, is mutated to a histidine.

[0263] Similarly, the Asnl50His mutation in PNPase4b and the Phe204Tyr mutation in PNPase6 correspond to residues known to participate in inter-subunit interactions in bovine PNPase (Asnl45, Phe200) and their replacement results in trimer destabilization. Finally, the substitution of alanine to serine in position 122 of PNPase4b may affect phosphate binding in that variant, as it neighbors Alal21, who’s main chain oxygen in bovine PNPase position 116 helps coordinate phosphate binding.

[0264] To elucidate the structural basis of the unique catalytic specificity of Pnp4a, the inventors studied the three-dimensional structures of PNPases in D. rerio using a number of structural prediction algorithms, including AlphaFold-2. Although different prediction tools can yield different structural models, the algorithms the inventors used produced highly similar structures for each PNPase, and their AlphFold-2 models were assigned very high confidence values (Figs. 3D and 9). In addition, all PNPases seemed to share a common fold with differences localized to their N- and C-termini (Fig. 3D). Capitalizing on the significant sequence identity and structural homology shared between the PNPases (Figs. 9-10), and between Pnp4a and bovine PNPase (PDB ID 1A9O; 59% seq id., 0.61A RMSD), the inventors identified residues that are likely to play a role in the substrate binding and catalytic activity of Pnp4a. Thus, seven residues are likely to coordinate phosphate binding in Pnp4a (Ser35, His66, Arg86, His88, Asnl l7, Alai 18, Ser222) and eleven residues are expected to comprise the nucleoside binding site (Tyr90, Alai 19, Glyl20, Phe202, Glu203, Val219, Met221, Thr244, Asn245, His259, Asp261).

[0265] The divergence in conserved active site residues between Pnp4a, 5a and 5b versus Pnp4b (e.g., Gln60Val, Alal l9Ser, Asnl47) and Pnp6 (e.g., Asp56Lys, Gln60His,Phe202Tyr) may account for the variations in substrate preferences and enzymatic activities the inventors observed (Figs. 2B-2C, and 10).

[0266] Given the upregulation of the purine metabolic network and the role of Pnp4a in catalyzing directly guanosine and deoxyguanosine, the inventors investigated the subcellular localization of Pnp4a. To achieve this, the inventors cloned and engineered a transgenic zebrafish expressing a fluorescent tag fused to Pnp4a under the conditional expression of a heat-shock promoter (hsp70:Pnp4a-mKate2m; see Materials and Methods). The inventors then examined the fish following heat shock using confocal microscopy (Fig. 11A-11E). Live imaging revealed limited co-localization between Pnp4a-mKate2 and the crystal-forming iridosomes. This observation suggests a predominant localization of Pnp4a in the iridophore cytoplasm (Figs. 11A-11E). To validate this, the inventors examined the catalytic activity rate of the different purified zebrafish PNPases across a pH range, using guanosine as a substrate. For most PNPases, peak activity occurred at pH 7, with Pnp4a performing optimally at pH 7.5 (Fig. 11F). This pH value is consistent with cytoplasmic conditions, rather than with the presumably acidic iridosome.

[0267] Based on the current observations, the inventors hypothesized that inhibiting Pnp4a would significantly hinder crystal formation with minimal effects on the production of nucleobases for regular RNA and DNA synthesis or overall larval development. To test this hypothesis, the inventors employed CRISPR / Cas9 technology to create Pnp4a mutants (Fig. 12; see Materials and Methods). Homozygous pnp4a mutants exhibited a marked reduction in crystal quantity compared to the wild type (Figs.4A-4B). A similar phenotype was observed in knock-down experiments using morpholinos targeting the AUG region of pnp4a, thus confirming the specificity of the pnp4a mutations. Intriguingly, crystals produced by pnp4a' / ' mutants and morpholino -injected fish displayed a distinct square-like morphology, as compared to the typical elongated semi-hexagonal shape observed in wildtype fish (Figs. 4 and 13). Collectively, these results highlight the pivotal role of Pnp4a in facilitating crystal formation within iridophores.

[0268] To gain deeper insights into the specific contribution of Pnp4a to crystal formation, the inventors conducted rescue experiments and restored pnp4a expression in the null mutants (Fig. 4C). The rescued embryos exhibited elevated crystal quantities compared to the control group, confirming that the observed phenotype was specific to the perturbation of Pnp4a. Interestingly, the square-like crystal morphology remained unchanged in the rescued mutantlarvae (Fig. 4B). This underscores the intricate interplay between the chemical, physical, and biological factors influencing in vivo crystal formation (Figs. 4A-4B).

[0269] Intriguingly, despite the pivotal role of Pnp4a as the principal enzyme governing guanine biosynthesis, crystals were formed in the absence of functional Pnp4a, and almost normal stripe pattern and eye iridescence appeared to develop in adult pnp4a' / ' mutant fish (Fig. 14). Therefore, to further explore the mechanisms underlying guanine crystal formation and the compensatory processes that may be at play in the absence of functional Pnp4a, the inventors performed real-time PCR to assess the mRNA expression level of the other members of the PNPase family in pnp4a' / ' mutants and wild-type fish (Figs. 4C and 15). While pnp4a expression levels were reduced compared to the wild-type control, its paralogues pnp5a and pnp5b were significantly increased in the mutants (Figs. 4C and 15). This points towards a cell adaptive response or compensatory mechanism, wherein the absence of Pnp4a triggers the upregulation of these closely related family members.

[0270] To investigate whether other members of the PNPase family contribute to crystal morphology, the inventors performed morpholino knockdown experiments targeting each enzyme (Fig. 13). Interestingly, the knockdown of pnp4b, pnp5a, pnp5b, and pnp6 did not significantly affect crystal quantities or typical morphology (Fig. 13). These findings indicate that crystal morphology is largely determined by the dominant activity of Pnp4a, and that altered morphologies are apparent only when Pnp4a is suppressed.

[0271] To understand the effect of Pnp4a on crystal morphogenesis, the inventors conducted structural analyses on crystals from mutant and wild-type fish. Despite the differences in crystal morphology in the Pnp4a mutant, micro-Raman spectroscopy and electron diffraction confirmed that these crystals were still beta- anhydrous guanine (Figs. 5 and 16). However, transmission electron microscopy (TEM) imaging showed that the Pnp4a mutant crystals exhibited over developed (012) facets, while the (010) facets were underdeveloped (Figs. 5 and 16). TEM imaging of the mutant larvae revealed multiple small, underdeveloped iridosomes, and Cryo-SEM imaging of these larvae showed thin, plate-like crystals similar to those found in the wild type (Figs. 5A and 16).

[0272] Given the lower quantities of guanine crystals in the pnp4a' / ' mutant, the inventors suspected that the altered crystal morphology might be attributed to changes in crystal composition. Indeed, comparing crystal compositions in situ using OrbiTrap Secondary Ion Mass Spectrometry (Orbi SIMS) imaging indicated that the hypoxanthine to guanine ratios inthe mutant crystals were almost two times higher than in the wild type (Fig. 17), suggesting that the inclusion of hypoxanthine within the crystal lattice modifies its morphology (Figs. 5A and 16). To further investigate the effect of crystal composition on morphology, the inventors conducted a series of in vitro experiments producing crystals with increasing ratios of hypoxanthine to guanine (Fig. 5B). While the inventors observed variations in crystal morphologies during the crystallization process, a consistent pattern emerged. As the quantity of hypoxanthine increased, the crystals consistently exhibited greater width and more prominent (012) facets. This finding confirms the significant influence of hypoxanthine incorporation on crystal morphology in both in vivo and in vitro systems (Figs. 5, and 16- 17).

[0273] Organisms have long been known to produce guanine crystals with diverse morphologies; yet the mechanisms governing these precise morphogenetic processes have remained unknown. To gain a better understanding of these mechanisms and explore the possibility that organisms adjust guanine-to-hypoxanthine composition to regulate crystal morphology, the compared the composition of naturally occurring crystals with different morphologies in zebrafish. Using Orbitrap-SIMS, the inventors examined elongated crystals isolated from adult wild-type zebrafish operculum skin, where they are used to form silvery iridescence for camouflage, and shorter, wider crystals from the fish's body, where based on their assembly, they produce the alternating blue and yellow skin colors (Figs. 5, and 16-17). Indeed, the inventors found the expected correlation between crystal composition and morphology. Body crystals, characterized by a lower aspect ratio, exhibited significantly higher levels of hypoxanthine than operculum crystals, mirroring the current findings in both the pnp4a' / ' mutant fish and in vitro formed crystals (Fig. 18). This finding suggests that organisms control crystal morphology by meticulously fine-tuning its composition, which may explain how biogenic crystals are optimized to perform a large variety of functions (Fig. 19).Discussion

[0274] The formation of intracellular guanine crystals is a remarkable example of tightly regulated biosynthesis taken to its extreme. This intricate process involves the prodigious production of nucleobases, while simultaneously maintaining nucleotide homeostasis to support routine cellular functions. Although these cells have long been known and have been extensively studied, how this is achieved remains enigmatic. To elucidate this process andunravel the specific roles of enzymes and the broader enzymatic network, the inventors conducted an extensive investigation that included proteomics and spatial metabolomics, cryoET imaging, in vitro enzymatic activity assays, cellular localization studies, and genetic manipulations.

[0275] The current proteomic and spatial metabolomic analyses unveiled the intricate biosynthetic pathway of iridophores, showcasing their exceptional ability to selectively upregulate guanine production while simultaneously preventing the formation of other metabolites. This finely tuned regulatory mechanism, which shields the cell from toxicity while maintaining the delicate balance of nucleobases required for normal cellular function, involves a carefully orchestrated modulation of specific enzymes, some of which are upregulated while others are suppressed. Particularly noteworthy is the elevation of a distinct subset of paralogous enzymes uniquely expressed in crystal-forming cells.

[0276] The current study highlights the pivotal role of Pnp4a in guanine biosynthesis and crystal formation within iridophores. The inventors found that Pnp4a displayed remarkable substrate selectivity when compared to its paralogues. This selectivity provides an additional layer of control to enhance the specific production of guanine within iridophores. Furthermore, although in pnp4a' / ' mutant larvae, the differentiation and development of iridophores appeared minimally affected, they produced fewer crystals in their eyes and skin. Notably, the ability of pnp4a' / ' mutants to produce any crystals, along with the eventual formation of a nearly normal stripe pattern and iridescence in the eyes of adult fish (Fig. 14), suggests that other members of the PNPase family may partially compensate for the absence of Pnp4a. The observed variations in both sequence and structural aspects, along with the resulting differences in catalytic activity between Pnp4a and its paralogues, imply that Pnp4a has undergone specific evolutionary changes that optimize its activity in guanine synthesis for crystal formation.

[0277] The current investigations have also provided new insights into the evolutionary origins of guanine -hypoxanthine co-crystals, which are found in various aquatic and terrestrial organisms. Since the enzymes responsible for the synthesis of guanine and hypoxanthine from IMP, i.e., nucleotidase and PNPase, are shared (Figs. IB and 2A), the upregulation of guanine production also leads to an increase in hypoxanthine levels. The higher efficiency of Pnp4a in converting guanine precursors compared to hypoxanthine precursors such as inosine may account for the elevated guanine-to-hypoxanthine ratio theinventors observed. Likewise, differences in enzyme efficiency and substrate affinity could underline the substantial variations in the composition of guanine -hypoxanthine co-crystals observed among different organisms.

[0278] The most surprising phenotype observed in pnp4a / ' mutant fish is the altered crystal morphology. The inventors found that the hypoxanthine-to-guanine ratio in pnp4a' / ' mutant crystals was almost two times higher than that in wild-type crystals. The current findings challenge previous claims that hypoxanthine is merely an energetically "cheap" building block whose incorporation does not affect crystal morphology. The inventors demonstrated that the incorporation of hypoxanthine into guanine crystals drastically altered crystal morphology and resulted in the over-development of (012) facets and the under-development of (010) facets both in vitro and in vivo. Considering that the expression of a crystallographic facet is inversely proportional to its growth rate, this suggests that hypoxanthine selectively hinders growth along the (012) plane.

[0279] The inventors further found that crystals with lower aspect ratio isolated from the fish skin, where they are assembled into precise arrays to produce color, exhibited significantly higher levels of hypoxanthine when compared to the higher aspect ratio crystals isolated from the operculum, where their disorganized assembly results in silvery shimmer. Considering that arrays of shorter crystals are much easier to assemble and less sensitive to assembly imperfections, this may suggest that the zebrafish meticulously fine-tunes crystal composition and morphology to optimize function. Furthermore, elevated hypoxanthine levels in squareshaped guanine crystals of other organisms, such as the scallop, suggest that the phenomenon the inventors have uncovered is widespread in the animal kingdom. These findings may also inspire the rational design of molecular crystals and may pave the way for the efficient bioengineered synthesis of nucleobases in general, particularly of the highly commercially valuable guanine.EXAMPLE 2

[0280] Building on the elucidation of the biosynthetic pathway responsible for nucleobase formation in zebrafish and the identification of biosynthetic enzymes with distinct substrate specificities — validated through overexpression in bacteria, purification, and in vitro assays — the inventors further engineered various bacterial strains, including BL21, to express selected genes from the identified pathway. Subsequent analyses revealed elevated levels ofseveral nucleobases, including guanine and hypoxanthine (Figs. 20A-20B, respectively), in the engineered bacteria, thus confirming that the enzymes are active and functional in vivo.

[0281] Notably, the substrate selectivity observed in vitro was recapitulated in the bacterial system. For instance, the enzyme Pnp4a predominantly increased guanine levels (Fig. 20A) with somewhat lower effect on hypoxanthine (Fig. 20B), whereas Pnp5a elevated levels of both guanine and hypoxanthine (Figs. 20A-20B). Furthermore, the current study demonstrated that specific enzyme combinations and tailored bacterial growth conditions enhance the excretion of nucleobases into the medium, as is the exemplified case of guanine (Figs. 21A-21B).

[0282] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A cell comprising a recombinant DNA molecule comprising a nucleic acid sequence encoding a first enzyme selected from the group consisting of inosine monophosphate dehydrogenase (Impdh), guanosine monophosphate synthetase (gmps), nucleotidase 5C (nt5c), polynucleotide phosphorylase (Pnp), and any combination thereof.

2. The cell of claim 1, wherein said Pnp is selected from the group consisting of pnp4a, pnp4b, pnp5a, pnp5b, pnp6, and any combination thereof.

3. The cell of claim 1 or 2, wherein said Pnp is pnp4a.

4. The cell of any one of claims 1 to 3, wherein said Impdh is selected from the group consisting of Impdhlb, Impdhla, Impdh2a, and any combination thereof.

5. The cell of any one of claims 1 to 4, wherein said Impdh is Impdh lb.

6. The cell of any one of claims 1 to 5, wherein said nt5c is selected from the group consisting of nt5clbb, nt5claa, nt5clba, nt5c2a, nt5c2b, and any combination thereof.

7. The cell of any one of claims 1 to 6, wherein said nt5c is nt5clbb.

8. The cell of any one of claims 1 to 7, wherein said recombinant DNA molecule further encodes a second enzyme selected from the group consisting of guanylate kinase 1 (gukl), ribonucleotide reductase catalytic subunit Ml (rrml), NME / NM23 nucleoside diphosphate kinase 4 (nme4), Nudix Hydrolase 5 (nudt5), and any combination thereof.

9. The cell of claim 8, wherein said gukl is gukl a, guklb, or both.

10. The cell of claim 8 or 9, wherein said gukl is gukl a.

11. The cell of any one of claims 1 to 10, wherein said first enzyme comprises an amino acid sequence as set forth in SEQ ID Nos: 1-4, or a functional analog thereof having at least 80% homology thereto.

12. The cell of any one of claims 8 to 11, wherein said second enzyme comprises an amino acid sequence as set forth in SEQ ID Nos: 5-10, or a functional analog thereof having at least 80% homology thereto.

13. The cell of any one of claims 1 to 12, wherein said recombinant DNA molecule further encodes a third enzyme selected from the group consisting of: phosphoribosyl pyrophosphate amidotransferase (ppat), phosphoribosylglycinamide formyltransferase (gart), adenylosuccinate lyase (adsl), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (atic), phosphoribosylformylglycinamidine synthase (pfas), phosphoribosylaminoimidazole carboxylase (paics), and any combination thereof.

14. The cell of claim 13, wherein said third enzyme comprises an amino acid sequence as set forth in SEQ ID Nos: 36-41, or a functional analog thereof having at least 80% homology thereto.

15. The cell of any one of claims 1 to 14, wherein recombinant DNA molecule is codon optimized for expression in said cell.

16. The cell of any one of claims 1 to 15, comprising an inactive guanine deaminase (gda) gene, an inactive urate oxidase (uox) gene, or both.

17. The cell of claim 16, wherein said inactive gene is knocked out, knocked down, mutated, chemically inhibited, or any combination thereof.

18. The cell of any one of claims 1 to 17, wherein said cell is atransgenic cell, atransformed cell, a transfected cell, a transduced cell, or any combination thereof.

19. The cell of any one of claims 1 to 18, being any one of: a unicellular organism, a cell of a multicellular organism, and a cell in a culture.

20. An extract derived from the cell of any one of claims 1 to 19, or any fraction thereof.

21. The extract of claim 20, comprising guanine.

22. The extract of claim 21, wherein said guanine is in a form of a crystal comprising guanine.

23. A composition comprising any one of:a. the cell of any one of claims 1 to 11; and b. the extract of any one of claims 20 to 22, and an acceptable carrier.

24. A method for synthesizing a purine-based compound, the method comprising: a. providing the cell of any one of claims 1 to 19; and b. culturing said cell from step (a) such that said first enzyme is expressed, thereby synthesizing the purine-based compound.

25. The method of claim 24, wherein said culturing is in a cell culture medium.

26. The method of claim 24 or 25, wherein said purine-based compound is selected from the group consisting of: guanine, xanthine, hypoxanthine, any enantiomer, isomer, or tautomer thereof, and any combination thereof.

27. The method of any one of claims 24 to 26, wherein said culturing comprises supplementing said cell with an effective amount of a purine-based precursor.

28. The method of claim 27, wherein said purine-based precursor is selected from the group consisting of: inosine monophosphate (IMP), xanthosine monophosphate (XMP), guanosine monophosphate (GMP), guanosine, guanosine diphosphate (GDP), deoxyguanosine diphosphate (dGDP), deoxyguanosine monophosphate (dGMP), deoxyguanosine, guanosine triphosphate (GTP), deoxyguanosine triphosphate (dGTP), and any combination thereof.

29. The method of any of claims 25 to 27, further comprising a step after said step (b) comprising extracting said cell or said cell culture medium wherein said cell being cultured.

30. The method of claim 29, wherein said extracting is from said culture medium wherein said cell being cultured, and wherein said cell comprises said recombinant DNA molecule encoding: (i) said first enzyme being selected from Impdh, gmps, and both; (ii) said third enzyme being ppat; or both (i) and (ii).

31. An extract obtained according to the method of claim 29 or 30.

32. The extract of claim 31, comprising guanine.

33. The extract of claim 32, wherein said guanine is in a form of a crystal comprising guanine.

34. The extract of claim 33, wherein said crystal further comprises hypoxanthine.

35. A method for controlling morphogenesis of a crystal comprising a purine-based compound in a cell producing said crystal, the method comprises modulating gene expression ratio between a first gene encoding the enzyme Pnp4a, a functional analog thereof, or both, and a second gene encoding the enzyme Pnp5a / b, a functional analog thereof, or both, in said cell, thereby controlling the morphogenesis of a crystal produced in a cell.

36. The method of claim 35, wherein said purine-based compound is selected from the group consisting of: guanine, xanthine, hypoxanthine, adenine, any enantiomer, isomer, or tautomer thereof, and any combination thereof.

37. The method of claim 35 or 36, wherein increasing said gene expression ratio above a predetermined threshold results in an elongated semi-hexagonal crystal comprising a combination of guanine and hypoxanthine in a weight per weight ratio ranging 1 :2 (w / w) and 1,000: 1 (w / w).

38. The method of any one of claims 35 to 37, wherein reducing said gene expression ratio below said predetermined threshold results in semi -hexagonal crystal, a square-like crystal, or a combination thereof, comprising a combination of guanine and hypoxanthine in a w / w ranging between 1 : 1 (w / w) and 100: 1 (w / w).

39. The method of any one of claims 35 to 38, wherein said cell is a transgenic cell, a transduced cell, or a transformed cell.

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