Lanthanide biosensors and methods of use

Abstract

Disclosed herein are rare earth element (REE) sensors comprising an REE binding element and a fluorescent component, which can be expressed on a cell surface. Also disclosed are systems and methods of recovering rare earth elements from a sample, the method comprising exposing an aqueous solution comprising one or more rare earth elements. Also disclosed are methods of discovering new, useful REE binding elements.

Description

[0001] LANTHANIDE BIOSENSORS AND METHODS OF USE

[0002] GOVERNMENT SUPPORT CLAUSE

[0003] This invention was made with government support under Grant No. EFMA2223735 awarded by the National Science Foundation. The government has certain rights in the invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims benefit of U.S. Provisional Application No. 63 / 664,673, filed June 26, 2024, incorporated herein by reference in its entirety.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The sequence listing submitted on June 26, 2025, as an .XML file entitled “10046- 627W01_ST26.xmF’ created on June 23, 2025, and having a file size of 72,460 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0008] BACKGROUND

[0009] Rare-earth elements (REEs) are indispensable to industrial economies, yet despite their relative abundance, their purification remains challenging due to their chemical similarity and co-occurrence with each other and with radioactive actinides (Abaka-Wood et al. 2022; Jordens et al. 2013; Ji et al. 2022; Nassar et al. 2023; Gkika et al. 2024). These challenges render traditional REE processing both economically and environmentally costly, requiring multistage, energy-intensive solvent extractions that generate significant hazardous waste and limit the viability of large-scale recovery (Jordens et al. 2013; Gkika et al. 2024; Deblonde et al. 2020; Thompson et al. 2018; Neves et al. 2022). In contrast, protein-based metal refining holds promise as a highly specific, adaptable, and scalable approach for REE recovery and separation. For example, the protein Lanmodulin (LanM) has previously been applied to aqueous-phase extraction of REEs, owing to its picomolar affinity, selectivity over more abundant ions (e.g., Ca2+), and reversible binding mechanism (Cotruvo et al. 2018). These properties have been previously applied to create fluorescence-based REE biosensors such as LaMPl , which couples LanM’s conformational change upon binding to a Forster resonance energy transfer (FRET) signal (Mattocks et al. 2019). Further, LanM and its natural variants have been demonstrated to exhibit some selectivity between REEs, with wild-type Mex-LanM exhibiting a 5-fold preference for La3+over certain heavy REEs and the Elans-LanM dimer exhibiting 100-fold preference for Dy over Nd (Mattocks et al. 2023). Despite these intrinsic preferences, LanM lacks the single-metal specificity and tunable affinity needed for REE purification, and remains cost-prohibitive when used as purified protein.

[0010] What is needed in the art are cost-effective, rapid ways to identify rare earth elements as well as REE binding elements.

[0011] SUMMARY

[0012] Disclosed herein are rare earth element (REE) sensors comprising an REE binding element and a fluorescent component, wherein the REE binding element is not naturally occurring.

[0013] This REE sensor can comprise an REE binding element is lanmodulin (LanM, encoded by SEQ ID NO: 1), and further wherein LanM has been modified with at least one amino acid mutation within the EF hand region (encoded by nucleotides 40-75, 112-147, 187-222, 259- 294 of SEQ ID NO: 1).

[0014] Further disclosed is a construct compnsing an REE sensor, wherein the REE sensor comprises: a) an REE binding protein, wherein the REE binding protein comprises any of SEQ ID NOS: 1-20; and b) a fluorescent component comprising a donor and an acceptor, wherein the acceptor comprises any of SEQ ID NOS: 35-38, and wherein the donor comprises any of SEQ ID NOS: 21-34.

[0015] Also disclosed is a system for recovering rare earth elements, the system comprising a cell modified to express the REE sensors disclosed herein, and a means for exposing the cell to one or more rare earth elements.

[0016] Likewise, a method of using this system is disclosed, wherein the method is for recovering rare earth elements from a sample, and comprises the step of exposing an aqueous solution comprising one or more rare earth elements to the system.

[0017] Disclosed herein is a method of determining effectiveness of a test REE binding element to bind a rare earth element, the method comprising: a) integrating one or more test REE binding elements with a fluorescent component, thereby forming a test REE sensor; b) expressing the test REE sensors one or more cell surfaces; c) exposing the cells to one or more rare earth elements under conditions sufficient to elicit a fluorescent response if said test REE binding element is effective; d) sorting cells by fluorescence activated cell sorting (FACS); and e) determining if a test REE sensor underwent a change in fluorescence when exposed to the rare earth element, thereby determining the effectiveness of the test REE binding element.

[0018] Additional aspects and advantages of the disclosure will be set forth, in part, in the detailed description and any claims which follow, and in part will be derived from the detailed description or can be learned by practice of the various aspects of the disclosure. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and together with the description, serve to explain, without limitation, the principles of the disclosure. Like numbers represent the same elements throughout the figures.

[0021] Figure 1A-B shows screened multiple REE-binding proteins against a panel of REEs using chelator-buffered titrations. (A) shows fold change of FRET ratio for free La3+. (B) shows REE selectivity for LanM wildtype and two variants.

[0022] Figure 2 shows a schematic for selection and counterselection using FACS. 1. A library7of REE-binding protein variants within a FRET biosensor is transformed into yeast. 2. Surfaceexpressing yeast are enriched based on their positive response to a target metal. 3. Yeast are enriched based on their negative response against non-target metals or in the absence of metals. 4. Sequencing of extracted plasmid.

[0023] Figure 3A-B shows 2D histograms of fluorescence intensity for a mixed population before (A) and after (B) addition of REE. The FRET response (B) corresponds to a decrease in donor intensity (x axis) and an increase in acceptor intensify (y axis).

[0024] Figure 4A-B shows Figure 4. (A) S. cerevisiae encapsulation in 1% (w / v) sodium alginate and (B) test of alginate resistance to acid. Alginate hydrogel was exposed to HN03 at pH 1. representative of conditions used for REE elution. The beads remain intact after 30 days. (C) Removal of Dy3+ from solution by phosphorylated and untreated S. cerevisiae cells. S. cerevisiae cells (20 mg / mL, wet weight) were incubated in solutions containing Dy3+ before being removed by centrifugation. Supernatant ion concentration was measured by ICP-OES. At 100 ppm, phosphory lation increases Dy 3+ binding capacity 4-fold.

[0025] Figure 5 shows the process of yeast functionalization, encapsulation, and element analysis.

[0026] Figure 6 shows LaMPl inducible expression.

[0027] DETAILED DESCRIPTION

[0028] Definitions

[0029] Reference is made herein to nucleic acid and nucleic acid sequences. The terms ‘"nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

[0030] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999. Brooks / Cole, 110).

[0031] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity' to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and / or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereof).

[0032] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C). aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue’’ also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxy lysine, 0-alanine, P-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutync acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid. N-Methylglycine, sarcosine, 3 -Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid. 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0033] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4- dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g.. the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g.. of thyroid hormones), and phosphorylation (e g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0034] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion’7refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3. 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5'-terminal or 3'-terminal truncation or both of a reference polynucleotide).

[0035] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide / amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and / or the C-terminal region of a polypeptide or the 5'-terminal region and / or the 3' terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.

[0036] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0037] Fusion proteins and fusion polynucleotides also are contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N- terminus, the C-terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a "portion", “region” or “moiety” of the fusion protein.

[0038] A fusion polynucleotide refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3' end of a first polynucleotide to a 5' end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0039] “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences or polynucleotide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.

[0040] The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity7for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.

[0041] Percent identity may be measured over the length of an entire defined polypeptide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 1 , at least 20, at least 30, at least 40, at least 50. at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity’ may be measured.

[0042] A “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity7to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999). “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%. or at least 99% or greater sequence identity over a certain defined length relative to a reference polypeptide.

[0043] A variant polypeptide may have substantially the same functional activity as a reference polypeptide. For example, a variant polypeptide may exhibit or more biological activities associated with binding a ligand and / or binding DNA at a specific binding site.

[0044] The terms “percent identity ” and “% identity ,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity7for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety ). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety7of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. ’‘BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

[0045] Percent identity7may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30. at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0046] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0047] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0048] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0049] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably7linked DNA sequences may be in close proximity or contiguous and, where necessary' to join two protein coding regions, in the same reading frame.

[0050] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by7chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3. Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0051] “Transformation"’ describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as w ell as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0052] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

[0053] By "fluorescent detection" is meant any method by which the presence or concentration of a target molecule is detected or quantified using a fluorescent signal. Such methods may involve direct labeling of the target, fluorescent reporter proteins, dye-based systems, or sensor constructs that undergo conformational or environmental changes resulting in a measurable change in fluorescence.

[0054] By "FRET" is meant Forster Resonance Energy Transfer, a distance-dependent interaction between two fluorophores — a donor and an acceptor — where energy transfer occurs from the excited donor to the acceptor if the fluorophores are within approximately 1-10 nm of each other. FRET is commonly used in biosensing applications to report molecular interactions, conformational changes, or binding events based on changes in energy transfer efficiency.

[0055] By "donor fluorophore" is meant a fluorescent molecule that, upon excitation by an external light source (such as a laser), can transfer its excitation energy to a nearby acceptor fluorophore via Forster Resonance Energy Transfer (FRET). The donor's emission spectrum must overlap with the excitation spectrum of the acceptor, and FRET efficiency depends on their spatial proximity, typically within 1-10 nanometers.

[0056] By "acceptor fluorophore" is meant a fluorescent molecule capable of receiving excitation energy from a donor fluorophore via FRET. Upon energy transfer, the acceptor emits light at a longer wavelength than the donor. The presence or intensity of acceptor emission can be used to infer molecular interactions, conformational changes, or proximity -dependent events in biosensor systems.

[0057] By "FACS" or "fluorescence-activated cell sorting" is meant a specialized type of flow cytometry used to sort and isolate individual cells from a heterogeneous population based on the presence, absence, or intensity of fluorescent markers. In FACS, cells are passed in single file through a laser beam that excites fluorescent tags on or within the cells. The emitted fluorescence is measured, and based on pre-set gating criteria, the instrument applies an electric charge to droplets containing individual cells. These charged droplets are then deflected by an electric field into separate collection vessels, enabling physical separation of cells with desired properties. FACS enables high-throughput screening and selection of cells expressing specific proteins, binding specific targets, or undergoing particular biological responses.

[0058] By "flow cytometry" is meant a technique used to analyze the physical and chemical characteristics of cells or particles as they pass individually through a fluidic system intersected by one or more laser beams. Properties such as size, granularity, and fluorescence intensity can be simultaneously measured at high speed, enabling multiparametric analysis of heterogeneous populations.

[0059] By "gating" is meant the process of defining a subset of events (e.g., cells) within a flow cytometry dataset based on user-defined parameters, typically using fluorescence intensity or scatter characteristics. Gates are used to isolate populations of interest — such as live cells, fluorescent protein-expressing cells, or cells with specific binding activity — for further analysis or sorting.

[0060] By "rare earth element" or "REE" is meant any of the elements in the lanthanide series of the periodic table, specifically the fifteen elements with atomic numbers 57 through 71 : lanthanum (La), cerium (Ce), praseodymium (Pr). neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu). The term may also optionally include scandium (Sc) and yttrium (Y) due to their chemical similarity and common co-occurrence with lanthanides in geological deposits. REEs are typically trivalent cations and exhibit unique spectroscopic, magnetic, and catalytic properties.

[0061] By "lanmodulin" or "LanM" is meant a naturally occurring lanthanide-binding protein identified in certain methylotrophic bacteria, such as Methylorubrum extorquens. Lanmodulin contains EF-hand motifs and exhibits extremely high affinity (in the picomolar range) and selectivity7for trivalent lanthanides over biologically relevant metal ions, including calcium and magnesium. Upon binding REEs, LanM undergoes a conformational change, which can be exploited for metal sensing, separation, or bio-based purification systems.

[0062] By "LaMPl" is meant a genetically encoded, protein-based biosensor composed of lanmodulin fused between a donor and acceptor fluorescent protein pair. Binding of a rare earth element to LanM induces a conformational rearrangement that alters the spatial relationship between the fluorophores. thereby modulating FRET efficiency and providing a ratiometric signal indicative of metal binding. LaMPl enables sensitive, reversible, and selective detection of REEs in complex biological or environmental matrices.

[0063] By "EF-hand" is meant a helix-loop-helix structural motif commonly found in calcium- binding proteins and adapted in LanM to coordinate trivalent lanthanide ions. The loop region of the EF-hand contains conserved residues that coordinate metal ions through oxygen atoms, ty pically from side chains or backbone carbonyl groups. In LanM, multiple EF-hands cooperate to confer high-affinity, multi-ligand coordination suitable for lanthanide selectivity.

[0064] General Description

[0065] Lanthanide-binding cell surfaces offer an efficient, modular, and sustainable approach for rare earth element (REE) recovery. This approach is made scalable by the incorporation of cells into solid support matrices, which enhance cellular robustness and facilitate efficient metal recovery7from dilute aqueous media. Disclosed herein are surface-functionalized Saccharomyces cerevisiae cells for enhanced lanthanide binding and incorporation of these cells into hydrogel beads. S. cerevisiae was functionalized by two distinct methods: 1) chemical phosphory lation and 2) cell surface expression of lanmodulin-based protein sensor 1 (LaMPl). The first method, chemical phosphorylation, introduces a high density of phosphate groups onto the cell surface, providing a high metal binding capacity. Surface expression of LaMPl provides high-affinity, lanthanide-specific binding that can be tuned. LaMPl additionally enables fluorescent detection of lanthanide binding by fluorescence resonance energy transfer (FRET). Using elemental analysis and fluorescent assays, it is shown herein that these cell surface modifications can significantly7enhance REE extraction from dilute aqueous media. While metal-binding proteins such as lanmodulin exhibit high affinity for REEs, their high purification costs hinder use in bio-based metal separation processes. To overcome this, the direct use of 5. cerevisiae biomass was investigated, which we modified either with low- cost phosphorylating reagents or by inducible expression of LaMPl. It was found that surface phosphory lation increased REE binding capacity up to four times over untreated cells. Further, it was demonstrated that these phosphorylated cells could be incorporated into hydrogel beads for enhanced scalability, and that these beads could withstand the harsh conditions (e.g. low pH) required for REE elution. These findings indicate that surface expression, chemical functionalization, and hydrogel encapsulation of S. cerevisiae can enable scalable, bio-based recovery of rare-earth elements.

[0066] The present disclosure provides compositions and methods for detecting, sorting, and recovering rare earth elements (REEs) using engineered REE sensors. The disclosed REE sensors comprise a rare earth element binding element and a fluorescent component. The REE binding element includes lanmodulin (LanM), variants thereof, or other REE-binding proteins. The fluorescent component may include a Forster Resonance Energy Transfer (FRET) pair or any other fluorescent detection modality. These sensors are particularly useful when genetically encoded and expressed on the surface of host cells, enabling applications in high- throughput screening and rare earth element recovery'.

[0067] Compositions

[0068] As mentioned above, the REE sensor comprises two components: the REE binding element and a fluorescent component, which allows for detection of binding of REE to the binding element. This REE sensor can be thought of as a modular component, in which various REE binding elements can be combined with various fluorescent components.

[0069] The REE binding element can comprise lanmodulin (LanM), or a variant thereof. LanM is represented by SEQ ID NO: 40. Variants of lanmodulin are encoded by SEQ ID NOS: 2-20, and represent one or more nucleic acid variations which lead to a change in amino acid sequence. These variants have properties which allow them to have unexpected benefits or desirable characteristics compared to LanM. These may include rationally designed or randomly mutagenized variants of LanM that retain the ability to bind one or more REEs with an affinity of at least 10"9M, 112M, or lower.

[0070] The REE sensor also comprises a fluorescent detection component. These include, but are not limited to, a donor fluorophore and an acceptor fluorophore suitable for use in FRET detection. In particular, the donor may be selected from SEQ ID NOS: 21-34. and the acceptor from SEQ ID NOS: 35-38. These sequences can vary by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides, as long as they encode a fluorescent protein which is capable of detection when an REE binds the REE binding element. Put another way, SEQ ID NOS: 21-28 can vary by 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or any amount above, below, or in-between these values. Furthermore, any fluorescent molecule can be used which allows for the detection of binding of an REE to the REE binding element.

[0071] In one embodiment, the REE sensor is a construct comprising a fusion protein in which the REE binding element is positioned between the donor and acceptor fluorophores, forming a ratiometric biosensor that exhibits a FRET signal upon binding to one or more REEs. Therefore, in a specific aspect, the disclosure provides an REE sensor comprising (i) a non- naturally occurring REE binding element and (ii) a fluorescent component. The REE binding element may be a protein having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1, optionally including amino acid mutations encoded by nucleotides 40-75, 112- 147, 187-222, and / or 259-294 of SEQ ID NO: 1. Such mutations may alter EF-hand regions involved in metal binding. An example construct can be found in SEQ ID NO: 39.

[0072] Host Cells and Display Systems

[0073] In certain embodiments, the REE sensor is genetically encoded and expressed in a host cell. The sensor may be localized to the surface of the host cell using one of several cell surface display systems, allowing direct interaction with extracellular rare earth elements (REEs). Displaying the sensor on the cell surface enables real-time detection of REEs. simplifies downstream processing, and facilitates use of techniques such as fluorescence-activated cell sorting (FACS). Cell-based expression systems also offer the potential for rapid screening, live sensing, and environmentally responsive biosensing platforms.

[0074] A wide range of host cells may be used for expression and display of REE sensors. These include, but are not limited to, yeast cells such as Saccharomyces cerevisiae or Pichia pastoris,' bacterial cells such as Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicunr, mammalian cells including HEK293 or CHO cells; insect cells such as Sf9; or even microalgal cells. The choice of host cell may depend on the desired expression level, post- translational modification compatibility, environmental tolerance, or application-specific factors such as scalability or sortability.

[0075] The REE sensor, or a portion thereof (e.g., the REE binding element or the full FRET construct), may be anchored to the cell surface using a variety of well-established protein display systems. In S. cerevisiae. a preferred system is the Agalp-Aga2p yeast display platform, in which the REE sensor is fused to Aga2p, which is covalently tethered to cell-wall- anchored Agalp via disulfide bonds. This system supports proper folding, secretion, and surface localization of the sensor protein in a functional conformation. Alternatively, a glycosylphosphatidylinositol (GPI) anchor can be employed to tether the REE sensor to the outer membrane in yeast or mammalian cells.

[0076] In gram-negative bacteria such as E. coll, surface display may be achieved using autotransporter proteins of the Type V secretion system, such as the IgA protease or AIDA-I. which allow the passenger domain (e g., REE sensor) to be translocated and exposed on the cell surface. Other bacterial display systems include the Lpp-OmpA fusion, which links the REE sensor to a hybrid outer membrane protein scaffold, and the ice nucleation protein (INP), which enables surface expression of large fusion proteins. Covalent anchoring strategies such as SpyTag / SpyCatcher or sortase-mediated ligation may also be used, allowing the REE sensor to be post-translationally tethered to the cell surface or to synthetic scaffolds.

[0077] In mammalian systems, the REE sensor can be fused to a transmembrane domain such as PDGFR. CD8a, or MHC class I for stable insertion into the plasma membrane. Orientation of the sensor — whether N-terminaL C-terminal, or within a sandwich fusion — may be controlled to optimize exposure of the REE binding site and enhance detection sensitivity. Flexible or rigid linkers, such as glycine-serine repeats or alpha-helical spacers, may be inserted between sensor domains to reduce steric hindrance and maintain proper fluorophore alignment for FRET signaling. Epitope tags such as FLAG, HA. or Hiss may also be included for detection, purification, or surface quantification purposes.

[0078] Methods of Use

[0079] Cells displaying REE sensors on their surface may be used for a variety of applications. For example, they may serve as biosensors for detecting REEs, such as in aqueous samples, or as sorting targets in high-throughput screening workflows to evaluate libraries of REE binding elements. Cells may be immobilized on hydrogel beads, membranes, or within microfluidic systems to create deployable sensing platforms or metal recovery' interfaces. In some embodiments, different sensor variants may be expressed on different cells, enabling multiplexed REE detection with a common fluorescent output system. In other embodiments. REE-binding cells may serve as bioadsorbents for environmental or industrial REE recovery, including from mining effluent, wastewater, or electronic recycling streams.

[0080] In one embodiment, the REE sensor is used to detect REEs in an aqueous solution. The system may comprise one or more cells displaying the REE sensor, optionally immobilized, and exposed to a fluid containing one or more REEs. The fluorescent output may be measured to determine the presence or concentration of the REE.

[0081] Specifically, disclosed is a method of sensing a rare earth element (REE), the method comprising exposing a sample which may contain an REE to the REE sensor as described herein, wherein the REE sensor can detect an REE or distinguish between different REEs. The REE sensor can be displayed on a cell, for example. This method can be carried out in multiplex so 2, 3. 4, 5, 6, or more different rare earth elements can be identified simultaneously. This can be done by using different fluorescent molecules for each different REE to be detected. Each REE would then cause it’s fluorescent component to fluoresce in a different way.

[0082] In another embodiment, the REE sensor is used in a high-throughput method to evaluate candidate REE binding elements. A test REE sensor is created by fusing a candidate REE binding element to a fluorescent component. Cells expressing these test sensors are exposed to one or more REEs under conditions sufficient to elicit a fluorescent response. The cells are sorted using fluorescence-activated cell sorting (FACS) based on changes in fluorescence, and the REE binding effectiveness of each candidate is determined.

[0083] In certain embodiments, multiple REE binding elements are screened simultaneously, with each variant expressed on a separate cell. In some embodiments, REE binding elements are generated by random mutagenesis of one or more EF-hand domains, allowing for directed evolution of high-affinity, REE-selective sensors.

[0084] In still other embodiments, cells expressing the REE sensors are used in systems or methods for recovering REEs from environmental, industrial, or waste samples.

[0085] FACS-based sorting of variants by affinity is shown in Figure 1. In addition to REE- affinity characterizations of a single variant, the surface-displayed REE sensor can be used to screen millions of REE-binding proteins via FACS, enabling the screening of protein mutant libraries. One example schematic for this screening is given in Figure 2.

[0086] FACS was also used to separate REE-binding proteins by affinity, for which example data is shown in Figure 3. Here, yeast populations expressing sensors were combined with either high-affinity or no-affinity REE-binding proteins. Upon exposure to REEs, the subpopulation expressing high-affinity REE-binding proteins exhibited a FRET response and was separated by gating. Upon outgrowth of sorted cells, it was found that the ratio of high-affinity to no-affinity expressing cells had increased by up to 1000-fold after a single round of enrichment. EXAMPLES

[0087] To further illustrate the principles of the present disclosure, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compositions, articles, and methods claimed herein are made and evaluated. They are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc ); however, some errors and deviations should be accounted for. Unless indicated otherwise, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of process conditions that can be used to optimize product quality and performance. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0088] EXAMPLE 1

[0089] Disclosed herein is a platform to rapidly test, evolve, and scalably produce REE- specific LanM variants, and have applied this platform to identify monomeric LanM variants with enhanced binding specificity7. Using yeast surface-display on Saccharomyces cerevisiae, we (1) designed a yeast surface-displayed, FRET-based REE biosensor to create a whole-cell sensor which uses LanM as the REE binding domain; (2) characterized REE binding to this sensor where we substituted rationally-designed REE-binding proteins for LanM; and (3) coupled this sensor with fluorescence-activated cell sorting (FACS) to facilitate high- throughput screening approaches such as random mutagenesis. We additionally demonstrated methods for REE pre-concentration via capture by phosphorylated S. cerevisiae cells.

[0090] Design of biosensor and identification of REE-selective binding proteins. S. cerevisiae was surface-expressed using a fusion protein consisting of a cell surface-tethering domain (e.g., Aga2p), a green donor fluorescent protein (FP), an REE-binding protein, and a red acceptor FP. A green / red pair was selected for efficient excitation at 488 nm, a common laser wavelength available for FACS, which is incompatible with the cyan / yellow pair used in LaMPl due to weak excitation of the donor cyan FP (Kremers et al. 2006). Using this sensor, multiple REE-binding proteins were screened against a panel of REEs using chelator-buffered titrations (Mattocks et al. 2021), as shown in Figure 1. New REE binding proteins were created via rational design, modifying the wildtype LanM sequence by altering the binding loops (EF- hands) to prefer heavier REEs. One sequence (LanM-Var2) exhibited up to a 100-fold binding preference for a heavy REE over La3+, out-performing the wildtype variant while not requiring dimerization for selectivity, unlike Hans-LanM (Mattocks et al. 2023).

[0091] Lastly, it should be understood that while the present disclosure has been provided in detail with respect to certain illustrative and specific aspects thereof, it should not be considered limited to such, as numerous modifications are possible without departing from the broad spirit and scope of the present disclosure as defined in the appended claims.

[0092] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0093] SEQUENCES

[0094] REFERENCES

[0095] 1. Abaka-Wood, G. B., Ehrig, K., Addai-Mensah, J. & Skinner. W. Recovery’ of Rare Earth Elements Minerals from Iron-Oxide-Silicate-Rich Tailings: Research Review. Eng 3, 259-275 (2022).

[0096] 2. Jordens, A., Cheng, Y. P. & Waters, K. E. A review of the beneficiation of rare earth element bearing minerals. Minerals Engineering 41, 97-114 (2013).

[0097] 3. Ji, B.. Li. Q. & Zhang. W. Leaching recovery of rare earth elements from the calcination product of a coal coarse refuse using organic acids. Journal of Rare Earths 40, 318— 327 (2022).

[0098] 4. Nassar, N. T. et al. Rock-to-metal ratios of the rare earth elements. Journal of Cleaner Production 405, 136958 (2023).

[0099] 5. Gkika, D. A., Chalaris, M. & Kyzas, G. Z. Review of Methods for Obtaining Rare Earth Elements from Recycling and Their Impact on the Environment and Human Health. Processes 12, (2024).

[0100] 6. Deblonde, G. J.-P. et al. Selective and Efficient Biomacromolecular Extraction of Rare-Earth Elements using Lanmodulin. Inorg. Chem. 59, 11855-11867 (2020).

[0101] 7. Thompson, V. S. et al. Techno-economic and Life Cycle Analysis for Bioleaching Rare-Earth Elements from Waste Materials. ACS Sustainable Chem. Eng. 6, 1602-1609 (2018).

[0102] 8. Neves, H. P. et al. Liquid-liquid extraction of rare earth elements using systems that are more environmentally friendly: Advances, challenges and perspectives. Separation and Purification Technology 282, 120064 (2022).

[0103] 9. Cotruvo, J. A., Featherston, E. R., Mattocks, J. A., Ho. J. V. & Laremore, T. N. Lanmodulin: A Highly Selective Lanthanide-Binding Protein from a Lanthanide-Utilizing Bacterium. J. Am. Chem. Soc. 140, 15056-15061 (2018).

[0104] 10. Mattocks, J. A., Ho, J. V. & Cotruvo, J. A. A Selective, Protein-Based Fluorescent Sensor with Picomolar Affinity for Rare Earth Elements. J. Am. Chem. Soc. 141, 2857-2861 (2019).

[0105] 11. Mattocks. J. A. et al. Enhanced rare-earth separation with a metal-sensitive lanmodulin dimer. Nature 618, 87-93 (2023).

[0106] 12. Kremers, G.-J., Goedhart, J., van Munster, E. B. & Gadella, T. W. J. Cyan and Yellow Super Fluorescent Proteins with Improved Brightness, Protein Folding, and FRET Forster Radius.. Biochemistry 45, 6570-6580 (2006). 13. Matocks, J. A., Tirsch, J. L. & Cotruvo, J. A. Chapter Two - Determination of affinities of lanthanide-binding proteins using chelator-buffered titrations, in Methods in Enzymology (ed. Cotruvo, J. A.) vol. 651 23-61 (Academic Press, 2021).

Claims

CLAIMSWhat is claimed is:

1. A rare earth element (REE) sensor comprising an REE binding element and a fluorescent component, wherein the REE binding element is not naturally occurring.

2. The REE sensor of claim 1, wherein the REE binding element is lanmodulin (LanM, encoded by SEQ ID NO: 1), and further wherein LanM has been modified with at least one amino acid mutation encoded by nucleotides 40-75, 112-147, 187-222, and 259- 294 of SEQ ID NO: 1.

3. The REE sensor of claim 1 or 2, wherein the REE binding element is encoded by a sequence comprising any of SEQ ID NOS: 2-20.

4. The REE sensor of any of claims 1-3. wherein the fluorescent component is a fluorescent resonant energy transfer (FRET) pair (a donor and an acceptor).

5. The REE sensor of claim 4, wherein the FRET donor is encoded by a sequence comprising any of SEQ ID NOS: 21-34.

6. The REE sensor of claim 4 or 5, wherein the FRET acceptor is encoded by a sequence comprising any of SEQ ID NOS: 35-38.

7. A construct comprising an REE sensor, wherein the REE sensor comprises: a. an REE binding protein, wherein the REE binding protein comprises any of SEQ ID NOS: 1-20; b. a fluorescent component comprising a donor and an acceptor, wherein the acceptor comprises any of SEQ ID NOS: 35-38, and wherein the donor comprises any of SEQ ID NOS: 21-34.

8. A construct of claim 7, wherein the construct comprises SEQ ID NO: 39.

9. A cell comprising the REE sensor of any one of claims 1-6.

10. The cell of claim 9, wherein the cell is modified to express an REE sensor on its surface.

11. A cell modified to express lanmodulin-based protein sensor 1 (LaMPl) on its surface.

12. The cell of any one of claims 9-11, wherein the cell is Saccharomyces cerevisiae.

13. The cell of any one of claims 9-12, wherein the cell is incorporated onto a hydrogel bead.

14. A method of sensing a rare earth element (REE), the method comprising exposing a sample which may contain an REE to the REE sensor of any one of claims 1-6 or to a cell comprising any one of claims 9-13, wherein the REE sensor can detect an REE or distinguish between different REEs.

15. The method of claim 14, wherein the method can be carried out in multiplex.

16. The method of claim 15, wherein two or more REEEs are distinguished from one another.

17. A system for recovering rare earth elements, the system comprising a cell of any one of claims 9-13 and a means for exposing the cell to one or more rare earth elements.

18. A method of recovering rare earth elements from a sample, the method comprising exposing an aqueous solution comprising one or more rare earth elements to the system of claim 17.

19. A method of determining effectiveness of a test REE binding element to bind a rare earth element, the method comprising: a) integrating one or more test REE binding elements with a fluorescent component, thereby forming a test REE sensor; b) expressing the test REE sensors one or more cell surfaces; c) exposing the cells to one or more rare earth elements under conditions sufficient to elicit a fluorescent response if said test REE binding element is effective; d) sorting cells by fluorescence activated cell sorting (FACS); and e) determining if a test REE sensor underwent a change in fluorescence when exposed to the rare earth element, thereby determining the effectiveness of the test REE binding element.

20. The method of claim 19, wherein the method is high throughput.

21. The method of claim 20, wherein multiple REE binding elements are tested simultaneously.

22. The method of claim 21 , wherein the fluorescent component for the REE sensor is identical but the REE binding elements differ from each other.

23. The method of any one of claims 19-22, wherein each REE sensor is on a different cell, and further wherein multiple cells are tested simultaneously.

24. The method of any one of claims 19-23, wherein each REE binding element is generated by random mutagenesis.

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