Cell-based materials for lanthanide recovery, methods of making and uses thereof

Abstract

Compositions for improved recovery of lanthanide are provided, which include cells genetically engineered to express one or more lanthanide binding proteins on their surface (herein-after, lanthanide-binding cells (LBCs)). The compositions include cells genetically engineered to express a lanthanide-binding protein extracellularly, with the lanthanide binding protein linked to the surface of the cell via a surface expression tag connected thereto. The compositions can be used to isolate lanthanide from a lanthanide-containing sample / material, by contacting the material / sample with the LBC to form a complex between the LBC and lanthanide, and isolating the complex from the sample material.

Description

[0001] ATTORNEY DOCKET NO. MIT 26099 PCT

[0002] CELL-BASED MATERIALS FOR LANTHANIDE RECOVERY, METHODS OF MAKING AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 753,780, filed February 4, 2025, the contents of which is specifically incorporated by reference herein in its entirety.

[0004] REFERENCE TO THE SEQUENCE LISTING

[0005] The Sequence Listing submitted as an XML file named “MIT_26099_PCT_ST26”, created February 4, 2026, and having a size of 19,959 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under Grant or Contract Number 2022-22072700001, awarded by USGA. The government has certain rights in the invention.

[0008] FIELD OF THE INVENTION

[0009] The disclosed invention is generally in the field of isolation of rare earth metals and specifically in the area of lanthanide recovery.

[0010] BACKGROUND OF THE INVENTION

[0011] Rare earth elements (REEs), including lanthanides, are essential components of technologies such as cell phones, electronic displays, and laser guidance systems.1,2Their importance, especially for clean energy technologies, has led the Department of Energy to classify REEs as critical, particularly because they are a limited resource.3Currently, REEs are extracted through destructive mining and harsh leaching methods, as most occur as insoluble phosphate or carbonate minerals. Further, these elements are often found at low levels (parts per million - ppm) along with more abundant ions such as calcium and magnesium, necessitating costly and energy-intensive extraction and separation.4Not only is recovery burdensome, but current approaches also cause ecosystem damage through acid runoff and heavy metal contamination.3,6Despite the drawbacks of mining, less than one percent of REEs are recycled because of current process limitations.7,8The energy intensity and environmental damage associated with REE recovery eliminate any climate benefits resulting from the clean technologies into which they are incorporated.5,6To enable a global energy transition and maintain technologies necessary for national security, a sustainable supply of REEs (such as lanthanides) is needed. This goal is only achievable through safe, efficient, and inexpensive extraction and recycling.3,6

[0012] 45821207.1 1ATTORNEY DOCKET NO. MIT 26099 PCT

[0013] The demand for REEs is expected to increase by more than 40% before 2030, leading to substantial work to improve extraction and recycling technologies. An important approach to decrease the need for caustic chemicals in these processes is the use of designed materials for REE adsorption.9 18Polymers, metal-organic frameworks, and biocomposites have all been reported for lanthanide recovery, but their high cost, synthetic intractability, and instability prevent their broad deployment. Microbes have also been investigated as adsorbents, reducing operational and environmental costs associated with REE recovery.9 12 19Microbes display high levels of metal adsorption due to the natural abundance of chelating groups present on their surface (e.g., carboxylates and phosphates).9Even without specific targeting,20microbes display partial selectivity for light over heavy REEs.21 2’ However, adsorption is hindered by competing metals, leading to the integration of metal-binding tags onto the surface of microbes.1 I l2‘1924 28Even with these chelators, the microbial capacity for REE biosorption remains low.19,24

[0014] To improve the specificity and capacity of microbial lanthanide capture, researchers have employed proteins with evolved specificity for these metals. These protein systems have been engineered to support lanthanide accumulation in the cells14 17. However, approaches that integrate microbial uptake of targets require subsequent cell lysis for recovery. Recently, the microbial protein lanmodulin (LanM) was identified and shown to have selective binding for lanthanides (100 million-fold higher affinity than for calcium).29,30However, recombinant LanM expression in E. coli yielded low recovery efficiency, likely due to high intracellular phosphate concentrations.

[0015] There remains a need for compositions and methods to improve lanthanide recovery. It is an object of the present invention to provide compositions for improved recovery of lanthanide.

[0016] It is also an object of the present invention to provide methods for recovering lanthanide from a lanthanide-containing sample / material.

[0017] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0018] BRIEF SUMMARY OF THE INVENTION

[0019] Compositions for improved recovery of lanthanide are provided. The compositions include a population of cells genetically engineered to express one or more lanthanide binding proteins on their surface (herein-after, lanthanide-binding cells (LBCs)). The compositions can be in wet form, including live cells or in dry form (lyophilized) and can include viable and / or 45821207.1 2ATTORNEY DOCKET NO. MIT 26099 PCT

[0020] non-viable LBC. The compositions include in some forms, one or more cells genetically engineered to express a lanthanide -binding protein extracellularly, with the lanthanide binding protein linked to the surface of the cell via a surface expression tag connected thereto. The compositions include in some forms, one or more cells expressing a lanthanide-binding protein extracellularly, with the lanthanide binding protein linked to the surface of the cell via a surface expression tag connected thereto, wherein the cells have been freeze-dried or lyophilized.

[0021] In some forms, the genetically engineered cell is a microbe such as a bacterium or bacteriophage. In some forms, the bacterium is a Grain-negative bacterium. In other embodiments, the bacterium is a Gram-positive bacterium.

[0022] In some forms, the genetically engineered cell is a eukaryotic cell such as a yeast or mammalian cell.

[0023] In some forms, the LBC can be generally represented by the formula:

[0024] B L1 (Set L2LBP L PT)

[0025] Formula II, where,

[0026] B represents a cell,

[0027] Set represents a surface expression tag,

[0028] LBP represents a lanthanide-binding protein, and

[0029] PT represents a purification tag. The Set, LBP and PT are preferably expressed on the surface of the cell B, as a fusion protein.

[0030] Thus, the LBC of formula II is an embodiment of the LBC of formula I.

[0031] The surface-expressed portion can also be in the order PT-Set-LBP; PT-LBP-Set; Set-PT-LBP, etc., optionally separated by a linker as shown in formula I herein.

[0032] Optional linkers Li, L2, L3 can be introduced between B and Set, between Set and LBP, and / or between LBP and PT. Thus, in some forms, Li, L2, and L3 are absent.

[0033] In some forms, the cells include SEQ ID Nos: 19, 20 and 21, and express a protein represented by SEQ ID NO: 22 or a lanthanide binding portion thereof.

[0034] Thus, Nucleic acids and polypeptides can have at least 50% sequence identity (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a given nucleic acid (SEQ ID NO: 19, 20, 21) or polypeptide sequence (SEQ ID NO:22) so long as the lanthanide binding portion of the protein is retained.

[0035] Methods for extracting rare earth elements (REE) such as lanthanide from a material containing the REE, are also provided. The methods include the steps of: (a) providing engineered cells engineered to express an exogenous nucleic acid sequence encoding at least one 45821207.1 3ATTORNEY DOCKET NO. MIT 26099 PCT

[0036] lanthanide-binding protein and expressing the lanthanide-binding protein on the cell surface; (b) contacting the genetically engineered cells with a lanthanide containing material, whereupon the lanthanide-binding protein specifically binds at least a portion of the lanthanide to form a cell-lanthanide complex; and (c) separating the cell-lanthanide complex from at least a portion of the material.

[0037] In some aspects, the present disclosure provides methods for preferentially separating lanthanide from a lanthanide containing material including the steps of; (a) contacting one or more LBCs, for example the LBCs of Formula I with the lanthanide containing material at an effective pH to form a LBC-Lanthanide complex; (b) contacting the LBC-Lanthanide complex with a composition containing a binding partner to the purification tag (PT) on the LBC; and (c) separating the LBC-Lanthanide complex based on affinity of the PT for its binding partner. The method further comprises separating the lanthanide from the LBC-Lanthanide complex by contacting the LBC-Lanthanide complex with a washing buffer with an acidic pH for example, pH less than 3, for example, PH 1, 2, 3 or 4.

[0038] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions 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 invention as claimed.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0041] Histograms of the flow cytometric analysis indicating the FITC fluorescent and not fluorescent populations of FIG. 1A) induced and La3+bound FIG. IB) induced and FIG. 1C) uninduced cells.

[0042] FIGS. 2A-2B show determination of engineered E. coli binding capacity for lanthanides.

[0043] FIG. 2A) Titration of metal mixture (YC13, LaC13, GdC13, and TbC13; 0.2 to 2 mM) to a constant number of cells (9.6 x 109cells), with relative La3+ binding as determined by ICP-MS. FIG. 2B) ICP-MS -determined relative binding of surface expressed LanM (dark) as compared to uninduced (light) and BL21 (striped) cells for four REE cations (1.2 x 109cells; 62.5 nmol total metal

[0044] 45821207.1 4ATTORNEY DOCKET NO. MIT 26099 PCT

[0045] mixture). Statistical significance indicated with * P < 0.05, ** P< 0.01, *** P < 0.001, and n.s. (not significant) > 0.05 based on n = 3 technical replicates.

[0046] FIGS. 3A-3B show scaffolded LanM binding to REE mixtures in the presence of competing ions and pH variations. Relative binding to La3+(from an equimolar mixture of YC13, LaC13, GdC13, and TbC13: 62.5 nmol) to LanM-expressing (dark) or uninduced (light) cells (1.2 x 109cells) at a constant concentration of M3+FIG. 3A) in the presence of increasing concentrations of Mg2+and Ca2+(0, 20, and 200-fold excess relative to M3+) and FIG. 3B) with decreasing pH (pH 5, 4, 3.5, and 2.5). Statistically significant changes are indicated with * P < 0.05, ** P < 0.01, *** P < 0.001, and n.s. (not significant) > 0.05 based on n = 3 technical replicates.

[0047] FIGS.4A-4D show colorimetric lanthanide quantification. FIG. 4A) Absorption spectra for La3+titration to a constant alizarin-complcxonc concentration. Lanthanides arc quantified through the ratio of absorbance at 490 nm and 405 nm, indicated by dashed vertical lines. FIG.

[0048] 4B) Standard curves of A490nm / 405nm show a linear dynamic range between 0 - 0.5 mM for Tb3+and Y3+(violet and orange) and 0 - 1 mM for Gd3+, La3+, and an equimolar mixture of the four metals (green, blue, and brown). No signal increase was observed for Ca2+(grey) or Mg2+(black) complexation. Relative binding of induced and uninduced cells to four lanthanide cations based on FIG. 4C) the colorimetric assay and FIG.4D) ICP-MS readout. Statistically significant changes are indicated with * P < 0.05, ** P < 0.01, *** P < 0.001, and n.s. (not significant) > 0.05 based on n - 3 biological replicates.

[0049] FIGS. 5A-5B. Bio-scaffolded proteins as packed-bed filter materials. FIG. 5A) Freeze- dried cells are packed onto a 0.22 pm filter, enabling flow-through capture and subsequent recovery of REEs. FIG.5B) Percent of initially loaded M3+in effluent, as calculated from relative absorbance at A490 / 405 nm after filtration with LanM- expressing cells (binding at pH 5, orange). Metal release was achieved using 0.25 mM HC1 in acetate buffer (pH = 1.7; purple). Measurements were acquired on subsequent days with the same filter and

[0050] cells. FIG.5C shows the plasmid map of the INPC-LamN-His6 constmct. A previously described plasmid that enables Gibson assembly of a target gene block with A-terminally-integrated INPNC was used.2,3The sequences were cloned into a pSKBb3 vector using standard NEB’s Gibson Assembly Cloning Kit (E5510S, Ipswitch, MA). Completed, sequenced plasmids were purified using a Qiagen Plasmid Kit (12143, Germantown, MD).

[0051] FIG.6 shows SDS-page gel of protein extract from LanM expressing cells. Lane 1: ladder; lanes 2 -4 uninduced cells; lanes 5 - 7 induced cells showing a strongly amplified band at ~ 39 kDa indicating successful INPNC-LanM expression.

[0052] 45821207.1 5ATTORNEY DOCKET NO. MIT 26099 PCT

[0053] FIGS. 7A-7C show electron microscopy imaging of lanthanide -binding cells.

[0054] SEM images (left) and EDX analysis (right) of LanM expressing cells incubated with FIG. 7A) LaC13, FIG.7B) GdC13 and FIG.7C) control cells without metal, indicating the presence of lanthanum or gadolinium in the respective samples but not in the control.

[0055] FIGS. 8A-8D show determination of engineered E. coli binding capacity for lanthanides, b) Titration of metal mixture (YC13, LaC13, GdC13, and TbC13; 0.2 to 2 mM, 0.5 mL, 0.1 to 1 pmol) versus a constant number of cells (9.6 x 109 cells) Relative binding (% bound) of FIG. 8A-) Y3+, FIG. SAB) La3+, FIG.8AC) Gd3+, and FIG. 8D) Tb3+was determined by ICP-MS. LanM expressing cells show a linear binding with respect concentration. Uninduced cell binding follows an exponential one phase decay. Statistically significant changes arc displayed with * P < 0.05, ** P < 0.01, *** P < 0.001, and n.s. (not significant) > 0.05 based on n = 3 technical replicates.

[0056] FIGS. 9A-9D show scaffolded LanM binding to REE mixtures under varying competing Mg2+and Ca2+concentrations. Relative binding to FIG. 9A) Y3+, FIG.9B) La3+, FIG.9C) Gd3+, and FIG. 9D) Tb3+(equimolar mixture of YCh, LaCh, GdCh, and TbCh; 62.5 nmol) to LanM expressing (dark) or uninduced (light) cells (1.2 x 109cells) at a constant concentration of M3+in the presence of increasing concentrations of Mg2+and Ca2+(0, 20, and 200-fold excess relative to M3+). Statistically significant changes in absorbance ratio are displayed with * P < 0.05, ** P < 0.01, *** P < 0.001, and n.s. (not significant) > 0.05 based on n - 3 technical replicates.

[0057] FIG. 10 shows Scaffolded LanM binding to REE mixtures under varying competing Mg2+and Ca2+concentrations. A490 / 405 nm for the supernatant after incubating LanM-expressing cells (2.4 x 109cells; orange) with an equimolar mixture of (YCh, LaCh, GdCh, and TbCh; 62.5 nmol) in the presence of increasing concentrations of Mg2+and Ca2+(0, to 200-fold excess relative to M3+). Statistically significant changes in absorbance ratio are displayed with * P < 0.05, ** P < 0.01, *** P < 0.001, and n.s. (not significant) > 0.05 based on n = 3 technical replicates.

[0058] FIGS. 11A-11D show scaffolded LanM binding to REE mixtures under varying competing salt concentrations and pH. Relative binding to FIG. 11A) Y3+, FIG. 11B) La3+, FIG. 11C) Gd3+, and FIG. 11D) Tb3+(from and equimolar mixture of (YCh. LaCh, GdCh, and TbCh; 0.1 to 1 pmol) to LanM expressing (dark) oruninduced (light) cells (1.2 x 109cells) with decreasing pH (pH 5, 4, 3.5, and 2.5). Statistically significant changes in absorbance ratio are

[0059] 45821207.1 6ATTORNEY DOCKET NO. MIT 26099 PCT

[0060] displayed with * P < 0.05, ** P < 0.01, *** P < 0.001, and n.s. (not significant) > 0.05 based on n = 3 technical replicates.

[0061] FIGS. 12A-12D show absorbance spectra and lanthanide titration for colorimetric quantification. FIGS. 12A-12C) Full absorption spectra of the titration curve for constant alizarin-3-methyliminodiacetic acid concentration (TCI A3227, 0.5 mM, 150 mM NaOAc, pH 5.0) and varying GdCh (FIG. 12A), YCh (FIG. 12B), and TbCh (FIG. 12C) concentrations.

[0062] FIG. 12D) Standard curves of relative absorption measured at 405 nm and 490 nm with a linear dynamic range between 0-0.5 mM for Tb3+and Y3+(violet and orange) and 0-1 mM for Gd3+, La+, and an equimolar mixture of the four metals (green, blue, and brown). No increase in signal was observed for Ca2+(grey) and Mg2+(black) complexation.

[0063] FIGS. 13A-13B show quantification of metal bound to LanM expressing cells. FIG. 13A) Relative absorbance at A490 / 405 nm of supernatant mixed in a 1:2 ratio with alizarin-3-methyliminodiacetic acid. FIG. 13B) Concentrations of metals determined by ICP-MS. Varying amounts of induced (dark) or uninduced (light) cells (9.6, 4.8, 2.4 x 109cells) and a control sample (no cells added, striped) were incubated with a constant amount of metal solution (250 nmol; GdCh, green; LaCL, blue; TbCh, purple; or YCh, orange).

[0064] FIG. 14 shows Percent of initially loaded Ln3+in effluent calculated from relative absorbance at A490 / 405 nm after filtration with induced, LanM-expressing (dark circles) and uninduced (light squares) cells. Metal release (purple) was achieved using 0.25 mM HC1 in acetate buffer (pH = 1.7). Measurements were taken on subsequent days with the same filter and cells.

[0065] Binding capacity in mg g1dry cell weight (dew) was calculated using well defined conversions for E. coli:

[0066] ODCM = 1 ~ 8 x 108cells ml'1(Equation S3) and

[0067] 1 cell = C = 3 x 10'13g (Equation S4)

[0068] Binding capacity of cells loaded on a filter were determined using Equation S5. binding capacity mggldcw = Mavx V x c x 1000

[0069] 24 x C x Vceiis x ODeoo

[0070] (Equation S5)

[0071] With Mavbeing the average molecular weight of the equimolar lanthanide mixture (136 g mol1), V and c are the volume (L) and concentration (mol L1) of the added lanthanide solution and Vceiis is the volume (mL) of cell suspension added.

[0072] 45821207.1 7ATTORNEY DOCKET NO. MIT 26099 PCT

[0073] DETAILED DESCRIPTION OF THE INVENTION

[0074] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0075] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0076] I. DEFINITIONS

[0077] The term "cell" refers to a membrane-bound biological unit capable of replication or division.

[0078] The term “construct” refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences. Genetic constructs used for transgene expression in a host organism include a series of cassettes including units with (in the 5’-3’ direction), a promoter sequence; a sequence encoding a gene of interest; and a termination sequence. The construct may also include selectable marker gene(s) and other regulatory elements for expression.

[0079] “Cryoprotectant” is any agent that prevents the formation of ice crystals, which can rupture cell membranes.

[0080] The term “expression” as used herein refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0081] The term “expression vector” refers to a vector that includes one or more expression control sequences.

[0082] The term “expression control sequence” refers to a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. In eukaryotes the term “expression vector” refers to a vector that includes one or more expression control sequences regardless of the origin of the sequence (prokaryote or eukaryote).

[0083] 45821207.1 8ATTORNEY DOCKET NO. MIT 26099 PCT

[0084] The term “gene” refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein. The term “gene” also refers to a DNA sequence that encodes an RNA product. The term gene as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5’ and 3’ ends.

[0085] As used herein the term “heterologous” means from another host. The other host can be the same or different species.

[0086] As used herein the term “heterologous” means from another host. The other host can be the same or different species.

[0087] The term “promoter” refers to a regulatory nucleic acid sequence, typically located upstream (5’) of a gene or protein coding sequence that, in conjunction with various elements, is responsible for regulating the expression of the gene or protein coding sequence.

[0088] A “transgene” as used herein refers to an artificial gene, manipulated in the molecular biology lab that incorporate all appropriate elements critical for gene expression generally derived from a different species.

[0089] The terms “transformed,” “transgenic,” “transfected” and “recombinant” refer to a host organism such as a bacterium or a plant into which an exogenous nucleic acid molecule has been introduced.

[0090] The term “vector” refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors can be expression vectors.

[0091] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0092] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0093] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the description and does not pose a limitation on the scope of the description unless otherwise claimed.

[0094] 45821207.1 9ATTORNEY DOCKET NO. MIT 26099 PCT

[0095] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the embodiments unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the

[0096] II. COMPOSITIONS

[0097] The compositions include in some forms include lanthanide-binding cells. The lanthanide binding cells include one or more cells expressing a lanthanide-binding protein extracellularly, with the lanthanide binding protein linked to the surface of the cell via a surface expression tag connected thereto. The compositions include in some forms, include cell-based lanthanide binding scaffolds. The cell-based lanthanide binding scaffolds include one or more cells expressing a lanthanide-binding protein extracellularly, with the lanthanide binding protein linked to the surface of the cell via a surface expression tag connected thereto, wherein the cells have been lyophilized.

[0098] A. Lanthanide-binding cells

[0099] Lanthanide binding cells include one or more cells genetically engineered to express a lanthanide-binding protein extracellularly, with the lanthanide binding protein linked to the surface of the cell via a surface expression tag connected thereto. The lanthanide binding cell can be generally represented by the formula:

[0100] B L1 (Set L2LBP-L3 PT) / (LBP L2-Set L3PT) / (PT L2-LBP L3Set)

[0101] Formula I, where,

[0102] B represents a cell.

[0103] Set represents a surface expression tag,

[0104] LBP represent a lanthanide-binding protein, and

[0105] PT represents a purification tag, preferably, where the portion including Set, LBP and PT is expressed on the surface of the cell B, as a fusion protein.

[0106] -(Set-L2-LBP-L3-PT) / (LBP-L2-Set-L3-PT) / (PT-L2-LBP-L3-Set) as set forth in Formula I shows exemplary alternative arrangements of, LBP, PT and Seton the surface of B. The surface- expressed portion can also be in the order PT-Set-LBP; Set-PT-LBP; etc., optionally separated by a linker as shown in the formula above.

[0107] A LBP as used herein is distinct and different from lanthanum binding tags as described for example, in US 2003 / 0228622, which are short peptide sequences (fewer than 20 amino

[0108] 45821207.1 10ATTORNEY DOCKET NO. MIT 26099 PCT

[0109] acids) that are optimized to bind trivalent lanthanide (Ln 3+) ions. Preferably, the disclosed LBC does not express lanthanide binding tags.

[0110] Optional linkers Li, L2, L3 can be introduced between B and Set, between SetandLBP, and / or between LBP and PT, as shown above in Formula I. Thus, in some forms, Li. L2, and L3 are absent.

[0111] In some forms, the cells include SEQ ID Nos: 19, 20 and 21, and express a protein represented by SEQ ID NO: 22 or a lanthanide binding portion thereof.

[0112] Thus, Nucleic acids and polypeptides can have at least 50% sequence identity (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a given nucleic acid (SEQ ID NO: 19, 20, 21) or polypeptide sequence (SEQ ID NO:22) so long as the lanthanide binding portion of the protein is retained.

[0113] In calculating percent sequence identity, two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It also will be appreciated that a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over each aligned region.

[0114] The alignment of two or more sequences to determine percent sequence identity can be performed using the computer program ClustalW and default parameters, which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500. ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the default parameters can be used (i.e., word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5); for an alignment of multiple nucleic acid sequences, the following parameters can be used: gap opening penalty: 10.0: gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of polypeptide sequences, the following parameters can be used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; and gap penalty: 3. For multiple alignment of polypeptide sequences, the following parameters can be used: weight 45821207.1 11ATTORNEY DOCKET NO. MIT 26099 PCT

[0115] matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, and Lys; and residue-specific gap penalties: on. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website or at the European Bioinformatics Institute website on the World Wide Web.

[0116] Changes can be introduced into a nucleic acid molecule, thereby leading to changes in the amino acid sequence of the encoded polypeptide. For example, changes can be introduced into nucleic acid coding sequences using mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis) or by chemically synthesizing a nucleic acid molecule having such changes. Such nucleic acid changes can lead to conservative and / or non-conservative amino acid substitutions at one or more amino acid residues. A “conservative amino acid substitution” is one in which one amino acid residue is replaced with a different amino acid residue having a similar side chain (sec, for example, Dayhoff ct al. (1978, in Atlas of Protein Sequence and Structure, 5(Suppl. 3):345-352), which provides frequency tables for amino acid substitutions), and a nonconservative substitution is one in which an amino acid residue is replaced with an amino acid residue that does not have a similar side chain. Nucleic acid and / or polypeptide sequences may be modified as described herein to improve one or more properties including, without limitation, increased expression (e.g., transcription and / or translation), tighter regulation, deregulation, loss of catabolite repression, modified specificity, secretion, thermostability, solvent stability, oxidative stability, protease resistance, etc.

[0117] The genetically engineered LBC are viable or non-viable.

[0118] 1. Cells

[0119] Lanthanide-binding cells (LBCs) can be any cells which can be genetically modified as exemplified herein using E. coli, to express on its surface, a surface expression tag linked to a lanthanide binding protein. In some forms the cells are prokaryotic cells. In some forms, the cells are eukaryotic cells. Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art include, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.

[0120] Lanmodulin (LanM) surface-displayed on cells such as E. coli through modification with an INPNC sequence serves as a capture agent for rare earth elements (REEs). The freeze-dried (lyophilized) cell serves as an inert scaffold for the protein to bind metals (pink spheres) and release them upon a change in pH.

[0121] 45821207.1 12ATTORNEY DOCKET NO. MIT 26099 PCT

[0122] 2. Cell Surface Expression tags and purification tags

[0123] The disclosed The LPC include a cell surface expression tag and are preferably purified by reliance on covalent interactions. Accordingly, LPC also includes a purification tag as shown in Formula I.

[0124] INPNC C-terminal extracellular domain encoding sequence is shown below.

[0125] AGACTCTGGGACGGGAAGAGGTACAGGCAACTGGTCGCCAGAACGGGTGAGAACG GTGTTGAGGCCGACATACCGTATTACGTGAACGAAGATGACGATATTGTCGATAAA CCCGACGAGGACGATGACTGGATAGAGGTAAAG (SEQ ID NO: 19).

[0126] INPNC N-terminal extracellular domain encoding sequence is shown below.

[0127] TCTCGACAAGGCGTTGGTGCTGCGTACCTGTGCAAATAACATGGCCGATCAC TGCGCTCCTTATATGGCCCGCCTTCCGGCACCTGTGGAATCCAGATACTGGCAGTCAAC CAGGCGGCATGAGAATGGTCTGGTCGGTTTACTGTGGGGCGCTGGAACCAGCGCTT TTCTAAGCGTGCATGCCGATGCTCGATGGATTGTCTGTGAAGTTGCCGTTGCAGACA TCATCAGTCTGGAAGAGCCGGGAATGGTCAAGTTTCCGCGGGCCGAGGTGGTTCAT GTCGGCGACAGGATCAGCGCGTCACACTTCATTTCGGCACGTCAGGCCGACCCTGC GTCAACGTCAACGTCAACGTCAACGTCAACGTTAACGCCAATGC CTACGGCATACCCACGCCCATGCCTGCGGTAGCAAGTGTCACGTTACCGGTGGCCG AACAGGCCCGTCATGAAGTGTTCGATGTCGCGTCGGTCAGCGCGGCTGCCGCCCCA GTAAACACCCTGCCGGTGACGACGCCGCAGAATTTGCAGACC (SEQ ID NO:20).

[0128] Lanmodulin (LanM) surface-displayed on cells such as E. coli through modification with an INPNC sequence serves as a capture agent for rare earth elements (REEs). The freeze-dried (lyophilized) cell serves as an inert scaffold for the protein to bind metals (pink spheres) and release them upon a change in pH.

[0129] Set LBP PT is preferably expressed as a cell membrane -bound as a recombinant fusion protein which includes component that binds to an affinity binding partner, the PT in Formula I. A preferred non-covalent linkage is provided by the affinity interactions involved receptorligand complex formation. Binding of a ligand to its binding partner can occur by intermolecular forces, such as ionic bonds, hydrogen bonds, hydrophobic interactions and Van der Waals forces. Thus, affinity interactions as used herein refers to the combination of non-covalent interactions between a ligand and its binding partner to form a complex.

[0130] Protein purification tags which are affinity tags are known in the art (reviewed in Kimple, et al., Curr Protoc Protein Sci. 2013; 73: Unit-9.9) Exemplary affinity tags include the polyhistidine affinity tag, also known as the His-tag, for example, 4 to 10 histidine residues, preferably, 6 histidine residues (His6), usually consists of six consecutive histidine residues, but 45821207.1 13ATTORNEY DOCKET NO. MIT 26099 PCT

[0131] can vary in length from two to ten histidine residues; FLAG tags, which typically include the sequence H6 (SEQ ID NO:1); DYKDDDDK (SEQ ID NO:2); haemagglutinin (HA) for example, YPYDVP (SEQ ID NO:3); MYC tag for example ILKKATAYIL (SEQ ID NO:4) or EQKLISEEDL (SEQ ID NO:5); Strep-tag WSHPQFEK (SEQ ID NO: 6), an aviTag (GLNDIFEAQKIEWHE (SEQ ID NO: 7), ( glutathione S-transferase (GST); Maltose binding protein (MBP), calmodulin binding peptide (CBP); the intein-chitin binding domain (intein-CBD), the streptavidin tag, etc. Methods of using purification tags to facilitate protein purification are known in the art and include, for example, a chromatography step wherein the tag reversibly binds to a chromatography resin.

[0132] Linkers included in the fusion protein expressed on the recombinant cells (LBCs) are preferably flexible linkers. Preferably the peptide or polypeptide domains are flexible peptides or polypeptides. A “flexible linker” herein refers to a linker such as a peptide or polypeptide containing two or more amino acid residues joined by peptide bond(s) that provides increased rotational freedom for two polypeptides linked thereby than the two linked polypeptides would have in the absence of the flexible linker. Preferred linkers are flexible linkers such as glycineserine, (GGGGS)n (SEQ ID NO:8), for example, GGGGS (SEQ ID NO: 8), GGGS (SEQ ID NO: 10), GGSS (SEQ ID NO: 11), Thr-Gly-Leu-Thr-Gly-Leu-Asn-Ser-Gly-Leu (SEQ ID NO: 12) and GSE. Other exemplary flexible peptides / polypeptides that can be included in the disclosed fusion proteins include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO:10), Ala-Ser, (Gly4-Ser)3(SEQ ID NO:14), and (Gly4-Ser)4(SEQ ID NO: 15), GSGSGSGS (SEQ ID NO: 13). Additional flexible peptide / polypeptide sequences are well known in the art. In one embodiment, LI is SGSG (SEQ ID NO: 16).

[0133] The surface expressed Set-LBP-PT in Formula I preferably does not include a cleavable linker such as an enzyme cleavable linker.

[0134] 3. Lanthanide-binding protein

[0135] The LPC is rendered lanthanide binding by its expression on its cell surface, of a lanthanide binding protein or a lanthanide binding fragment thereof.

[0136] In some forms, the lanthanide binding protein is lanmodulin (LanM), which is expressed in Methylobacterium extorquens. LanM possesses four metal-binding EF hand motifs, commonly associated with Call-binding proteins. In contrast to other EF hand-containing proteins, however, LanM undergoes a large conformational change from a largely disordered state to a compact, ordered state in response to picomolar concentrations of all Ln111(Ln = La-Lu, Y), whereas it only responds to Call at near-millimolar concentrations.

[0137] Methylorubrum extorquens DM4; EF-hand domain-containing protein

[0138] 45821207.1 14ATTORNEY DOCKET NO. MIT 26099 PCT

[0139] Gene ID: 72989511; 2447861..2448262 of NCB1 Ref.: NC_012988.1

[0140] 1 atggcgttcc gcctctcctc tgccgttctg ctcgctgcgc tcgtcgctgc tccggcctac

[0141] 61 gccgccccga cgacgaccac gaaggtcgat atcgccgcct tcgatccgga caaggacggc 121 acgatcgatc tcaaggaggc cctggccgcc ggctccgctg ccttcgacaa gctcgatccg

[0142] 181 gataaggacg gcacgctcga tgccaaggag ctgaagggcc gcgtcagcga ggccgacctc

[0143] 241 aagaagctcg acccggacaa cgacggcacc ctcgacaaga aggaatacct tgccgccgtt

[0144] 301 gaggcgcagt tcaaggccgc taacccggac aacgatggca ccatcgacgc tagggaactg

[0145] 361 gccagcccgg ccggttcggc cctggtcaat ctgatccgct aa (SEQ ID NO:21).

[0146] Protein Sequence:

[0147] Lanmodulin (Uniprot ID: C5B164 ■ LANM_METEA; 133 amino acids) MAFRLSSAVLLAALVAAPAYAAPTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAF DKLDPDKDGTLDAKELKGRVSEADLKKLDPDNDGTLDKKEYLAAVEAQFKAANPDND GTIDARELASPAGSALVNLIR (SEQ ID NO:22).

[0148] Other lanthanide binding proteins that can be used in the disclosed compositions and methods include lanepsy (LanP), a lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus, (Journal of Biological Chemistry (2023). DOI: 10.1016 / j.jbc.2O23.102940). In vitro characterization of LanP, a PepSY domain-containing protein showing sixfold induction in the presence of La3+, demonstrated the existence of multiple selective binding sites for Ln3+ with affinities of 1 LIM.

[0149] In some forms, the LBCs are provided in a cell culture media. In some embodiments, the compositions further comprise an amount of medium comprising, for example, PYE or LB media as a base media. In other embodiments, the medium can comprise a minimal medium with sugar or yeast extract as additional supplements. The composition can comprise one or more additional substances that can be consumed by the genetically engineered microbe to keep the relevant microbe alive or stimulate its growth. Non-limiting examples of additional substances include mucopolysaccharides, oligosaccharides, polysaccharides, amino acids, vitamins, trace elements, nutrient precursors and proteins. In one embodiment, the additional substance is calcium salt. It is contemplated that the addition of calcium salt in the growth media facilitates, in part, more robust production and / or stability of S -layer protein.

[0150] In some embodiments the growth media composition comprises an agar plate or stab culture.

[0151] In some forms, the LBC composition includes a cryoprotective agent. Media for preservation of cells are known in the art, for example, CRYO-GOLDTM (cryopreservation medium) and CROSSTOR® (cryopreservation freeze media) designed to mitigate temperature- 45821207.1 15ATTORNEY DOCKET NO. MIT 26099 PCT

[0152] induced molecular cell stress responses during freezing and thawing. All CROSSTOR® products are pre-formulated with USP grade DMSO, a permeant solute cryoprotective agent which helps mitigate damage from the formation of intracellular ice. CROSSTOR® is offered in several packages and pre-formulated with DMSO in final concentrations of 2%, 5%, and 10%. A preferred medium for cell preservation includes 5-10 % DMSO, for example, CRYOSTOR® CS10 (a uniquely formulated serum-free, animal component-free, and defined cry opreservation medium containing 10% dimethyl sulfoxide (DMSO)). Additionally, cryoprotectants / cryoprotectant additives which can be include in a cell composition (for cryopreservation) are known in the art and include, ethylene glycol (EG), antioxidants such as taurine, Metformin, gamma amino butyric acid (GABA). Cells may be suspended in a "freeze medium" such as cell culture medium containing 15-20% fetal bovine serum (FBS) and 7-10% DMSO, with or without 5-10% glycerol, at a density, for example, of about 1-10 x 106 cclls / ml. The cells are dispensed into glass or plastic vials, which are then sealed and transferred to a freezing chamber of a programmable or passive freezer. The optimal rate of freezing may be determined empirically. For example, a freezing program that gives a change in temperature of - 1 °C / min through the heat of fusion may be used. Once vials containing the cells have reached - 80 °C, they are transferred to a liquid nitrogen storage area.

[0153] B. Cell-based Lanthanide binding scaffolds

[0154] In some forms, the genetically engineered LBC are non- viable and thus serve as cel-based lanthanide binding scaffolds.

[0155] In some forms, genetically engineered LBC compositions are freeze-dried. Any suitable method known by one of skill in the art can be used. Freeze dry buffers may be included. In other embodiments, the compositions are lyophilized. Lyophilization buffers may be included.

[0156] III. METHODS OF MAKING ANS USING

[0157] A. Methods of Making

[0158] LBC can be obtained by genetically engineering cells to express on their surface, a lanthanide binding protein. In some forms, the lanthanide binding protein is expressed as a fusion protein.

[0159] LBC can be engineered prokaryotic or eukaryotic cells. Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art include, for example, microbes and cultured mammalian cells such as CHO cells. In some embodiments, the microbe is a bacterium or bacteriophage.

[0160] In some embodiments, the bacterium is a Grain-negative bacterium. In other embodiments, the bacterium is a Gram-positive bacterium.

[0161] 45821207.1 16ATTORNEY DOCKET NO. MIT 26099 PCT

[0162] In some embodiments, the bacterium is selected from the group consisting

[0163] of Caulobacter crescentus (C. crescentus), Escherichia coli (E. colt), Bacillus, Caulobacter and Lactobacillus.

[0164] In eukaryotic host cells, a number of viral-based expression systems can be utilized to express fusion proteins of Formula I. Viral based expression systems are well known in the art and include, but are not limited to, baculoviral, SV40, retroviral, or vaccinia based viral vectors.

[0165] The expressed tagged cells may be recovered from the cell culture by conventional procedures.

[0166] The fusion proteins of Formula I can be obtained by, for example, by chemical synthesis, and more preferably, by recombinant production in a host cell.

[0167] To recombinantly produce a fusion protein such as the fusion protein shown in Formula I, an exogenous nucleic acid containing a nucleotide sequence encoding the polypeptide can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect, yeast, or mammalian cell). As used herein, “exogenous” refers to any nucleic acid sequence that is introduced into the genome of a cell from an external source, where the external source can be the same or a different organism or a nucleic acid generated synthetically. For example, an exogenous nucleic acid can be a nucleic acid from one microorganism that is introduced into a different genus or species of the microorganism. However, an exogenous nucleic acid also can be a nucleic acid from the same organism, that is introduced recombinantly into the same organism as an additional copy despite the presence of a corresponding native nucleic acid sequence.

[0168] In general, nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding the fusion protein of interest. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product, but instead affect the expression of the nucleic acid sequences to which they are operably linked. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter.

[0169] Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site.

[0170] 45821207.1 17ATTORNEY DOCKET NO. MIT 26099 PCT

[0171] An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

[0172] The nucleotide sequences encoding the fusion protein are usually inserted into a recombinant vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.

[0173] Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosomc(s) into which it has been integrated. The vector is preferably an expression vector in which the DNA sequence encoding the fusion protein is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, “operably linked” indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the fusion protein.

[0174] Expression vectors for use in expressing the fusion protein will comprise a promoter capable of directing the transcription of a cloned gene or cDNA. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Expression vectors for use in expressing the fusion protein will include a promoter capable of directing the transcription of a cloned gene or cDNA. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1 81), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), the CMV promoter (Boshart et aL, Cell 41:521-530, 1985) or the adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982), viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalo virus, retroviruses, vaccinia viruses, adenoviruses, and adeno- associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, 45821207.1 18ATTORNEY DOCKET NO. MIT 26099 PCT

[0175] CA), and Invitrogen Life Technologies (Carlsbad, CA). Many common promoters, like CMV, EFl A, and SV40 promoters, are always active and thus referred to as constitutive promoters.

[0176] In some forms, the promoter is an inducible promoter such as a chemically inducible promoter. Chemically regulated promoters are among the most common inducible promoters. The positive inducible tetracycline ON (Tet-On) system, a versatile tool developed for use in prokaryotes and eukaryotes, works via direct activation. In this system, the activator rtTA (reverse tetracycline-controlled transactivator) is normally inactive and cannot bind the tetracycline response elements (TRE) in a promoter. Tetracycline and its derivatives serve as inducing agents to allow promoter activation. One of the most commonly used prokaryotic promoters is the negative inducible pLac promoter. This promoter requires removal of the lac repressor (lacl protein) for transcription to be activated. In the presence of lactose or lactose analog IPTG, the lac repressor undergoes a conformational change that removes it from lacO sites within the promoter and ceases repression of the target gene. A simplified lac inducible system is found in many bacterial expression vectors. Negative inducible promoter pBad is another popular prokaryotic promoter often used for bacterial protein purification. When arabinose is absent, regulatory protein AraC binds O and II sites upstream of pBad, blocking transcription. The addition of arabinose causes AraC to bind II and 12 sites, allowing transcription to begin. In addition to arabinose, cAMP complexed with cAMP activator protein (CAP) can also stimulate AraC binding to II and 12 sites. Supplementing cell growth media with glucose decreases cAMP and represses pBad, decreasing promoter leakiness. Examples include the heat shock-inducible IIsp70 or IIsp90-derived promoters, in which a gene of choice is only expressed following exposure to a brief heat shock. In the case of Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to heat shock elements in the promoter, thereby activating transcription.

[0177] Cloning of lanP for expression in a E. coli

[0178] Cloning of LanP for expression in E. coli can be performed as described in Hemmann, el al, J. Biol. Chem., 299(3): 102940 (2023). Briefly, for N-terminal HislO-tagged expression of lanP (Mfla_0908), the gene without the encoded signal peptide (the first 23 amino acids) was amplified by PCR from genomic DNA of M. flagellatus KT using primers that contained Ndel and BamHI restriction sites (See Table S4, in Hemmann, et al , J. Biol. Chem., 299(3): 102940 (2023) for suitable primers). The PCR product can be cloned into the expression vector pET-16b (Novagen) using restriction enzyme cloning. Ligation products were transformed into E. coli DH5a, and correct plasmids were identified by colony PCR and sequencing.

[0179] 45821207.1 19ATTORNEY DOCKET NO. MIT 26099 PCT

[0180] LanM and EanP can be recombinantly expressed for surface expression as disclosed herein and the resultant LBCs isolated.

[0181] Isolation of LBC

[0182] Suitable means for isolation of the resulting lanthanide-binding cells include, but are not limited to centrifugation, filtration, dialysis, or a combination thereof.

[0183] Histidine readily forms coordination bonds with immobilized transition metal ions. Immobilized Co2+, cu2+, Ni2+, Zn2+, Ca2+, and Fe3+can all be used to purify polyhistidine fusion proteins, but Ni2+is the most commonly used. If purification by Ni2+is unsatisfactory, empirical determination of the most effective transition metal ion for purification of a specific polyhistidine fusion protein can be performed. There are several companies that offer IMAC resin. The matrix most widely used for IMAC is Ni(II)-nitrilotriacetic acid (Ni-NTA), available from Qiagen. Other resins used for the immobilization of transition metal ions include iminodiacetic acid agarose (chelating Sepharose, GE Healthcare) and carboxymethylaspartate agarose (Talon resin, Clontech). A His tagged fusion protein can then be purified from its medium (e.g. cell lysate) using suitable purification systems, such as an affinity column (e.g. a HisTrap™ affinity column). HisTrap™ HP is a ready to use column, prepacked with precharged Ni Sepharose™ High Performance which has high binding capacity and low nickel ion leakage that ensures reliable capture of target protein. Ni Sepharose High Performance (HP) affinity resists consists of highly crosslinked agarose beads to which a chelating group has been coupled. This chelating group is precharged with nickel, which selectively retains proteins with exposed histidine groups.

[0184] GST fusion proteins can be purified by affinity chromatography (UNIT 6.6) on commercially available glutathione (y-glutamylcysteinylglycine) Sepharose (Ka=0.6 nM), which is affected by y-glutamyl transpeptidase activity in crude cell lysates. Glutathione affinity chromatography is amenable to low concentrations of denaturing agents (2 to 3 M urea or guanidine hydrochloride), reducing agents (<10 mM 2-mercaptoethanol or dithiothreitol), and nonionic detergents (2% v / v Tween 20), depending on the nature of the fusion protein. GST fusion proteins are incubated with glutathione Sepharose in order to facilitate crosslinking between the two. Elution with 10 mM glutathione is relatively mild, often preserving protein function and antigenicity. A 70 kDa E. coll heat-shock-induced chaperonin often copurifies with eluted GST fusion proteins. This contaminant can be removed by treatment of cell lysates with 5 mM MgCh and 5 mM ATP prior to purification. Furthermore, GST can be cleaved from its fusion protein while still bound to glutathione agarose, providing a convenient method for separating the 26 kDa GST from the protein of interest.

[0185] 45821207.1 20ATTORNEY DOCKET NO. MIT 26099 PCT

[0186] Maltose binding protein (MBP) is often used to increase the expression level and / or solubility of its fusion partner, with typical yields of 10 to 40 mg fusion protein per liter culture. pMAL vectors are available for cytoplasmic or periplasmic expression in all three reading frames, with factor Xa, enterokinase, or genenase I protease cleavage sequences (New England Biolabs). Other MBP fusion vectors include pIVEX (Roche), which can be used for coupled in vitro transcription / translation. MBP fusion proteins can be purified by affinity chromatography on cross-linked amylose resin. Amylose resins are commercially available but are affected by amylase activity in crude cell lysates.

[0187] The calmodulin binding peptide (CBP) purification system utilizes a C-terminal fragment from muscle myosin light-chain kinase in order to purify proteins of interest from bacteria. With low levels of calcium present at physiological pH, this 26 amino acid fragment displays a fairly strong affinity (Kd=10“9M) for the protein calmodulin. Removal of calcium causes calmodulin to undergo a conformational change resulting in the release of its ligand. T7 -based pET expression vectors have been engineered to allow attachment of the CBP affinity tag to either the C- or N-terminal of the fusion protein.

[0188] Optionally, the isolated cells can be subsequently washed with a solvent and dried by a suitable means.

[0189] One or more washings of the bacteria-based catalysts can be performed to remove impurities, i.e. media components, present in the resulting lanthanide-binding cells. Suitable solvent for washing includes, but is not limited to water, ethanol or a combination thereof.

[0190] Drying and lyophilization of LB Cs

[0191] Freeze-drying, also called lyophilization or cryodesiccation, is the process of removing water from a product after it's frozen and placing it in a vacuum. This allows ice to change from a solid to a vapor, without going through a liquid phase. Ice (or other frozen solvents) are removed from a product through the process of sublimation and bound water molecules are removed through the process of desorption.

[0192] Drying the lanthanide- binding cells can be accomplished by any suitable means, which includes, but is not limited to, heating to a suitable temperature, lyophilizing the bacteria cells, and air-dry. Suitable temperature for drying can be a temperature between about 20 °C and about 45 °C, between about 25 D and about 45 °C , between about 35 °C and about 45 °C. The drying step can be performed in a period between about 2 and about 24 hours, between about 5 and about 20 hours, between about 5 and about 15 hours, and between about 10 and about 15 hours.

[0193] Preserving bacteria by lyophilization requires that the bacteria are suspended in a medium that helps to maintain their viability through freezing, water removal, and subsequent 45821207.1 21ATTORNEY DOCKET NO. MIT 26099 PCT

[0194] storage. The ideal solution will have a component that helps to form a solid "cake" which gives body to the bacterial suspension once freeze dried. Common ingredients for this include mannitol, skim milk and bovine serum albumin (BSA). A second component of a good medium is a lyoprotectant which will help to preserve the structure of biomolecules throughout the lyophilization process. The classic lyoprotectant used with bacteria is sucrose, which is a glucose linked to fructose via its Cl (anomeric) carbon. A similar molecule, trehalose (glucose linked Cl to Cl with another glucose) is very popular for freeze drying proteins, but can be less effective for preserving bacteria. Salts are one component which should not be used in freeze drying media as the salts will concentrate during water sublimation which can destroy cells via severe localized dehydration.

[0195] Several basic freeze drying solutions are commonly used to produce good results. Skim milk at a concentration of 20% is a very traditional medium, but viability after processing may decrease up to 90% or more (depending upon the strain). However, if the sample originally contained upwards of 109cells then plenty of viable cells remain. Alternatively, a solution of 5-10% sucrose is a traditional freeze drying medium. Cake formation with sucrose is not as good as solutions containing BSA or mannitol, but the lyoprotecting property of the sugar yields good viability. Several solutions are suggested by the American Type Culture Collection (ATCC). They have published several recipes for freeze drying solutions, such as Reagent 18, which combines sucrose and BSA that together generate samples that freeze dry well and are effectively preserved. The Microbial Freeze Drying Buffer formulated by OPS Diagnostics is built off the Reagent 18 formulation, but substitutes plant protein for BSA.

[0196] B. Methods of Using

[0197] Methods for preferentially separating lanthanide from a lanthanide containing material are disclosed. The methods include the steps of: (a) providing a cell population genetically engineered to include an exogenous nucleic acid sequence encoding at least one lanthanide-binding protein and expressing the lanthanide-binding protein on the cell surface; (b) contacting the genetically engineered cells with a lanthanide containing material, whereupon the lanthanide-binding protein specifically binds at least a portion of the lanthanide form a cell- lanthanide complex; and (c) separating the cell- lanthanide complex from at least a portion of the material.

[0198] In some forms the methods include the steps of: (a) contacting one or more LBCs of Formula I with the lanthanide containing material at an effective pH (provided by a binding buffer) to form a LBC-Lanthanide complex; (b) contacting the LBC-Lanthanide complex with a composition containing a binding partner to the purification tag (PT) (biosorption / adsorption media) on the LBC; and (c) separating the LBC-Lanthanide complex based on affinity of the PT 45821207.1 22ATTORNEY DOCKET NO. MIT 26099 PCT

[0199] for its binding partner. The method further comprises separating the lanthanide from the LBC-Lanthanide complex by contacting the LB C -Lanthanide complex with a washing buffer with an acidic pH for example, pH less than 3, for example, PH 1, 2, 3 or 4, exemplified herein using 0.25 mM HC1 in acetate buffer (pH = 1.7) alone or in combination with four additions of HC1 solution (0.25 mM, 20 mM NaOAc, 100 mM KC1, pH 1.7). In some forms, the LBC includes an inducible promoter and the method further includes inducing the inducible promoter. For example, if the inducible promoter isopropyl-0-D-thiogalactopyranosid (IPTG)-inducible, the method further includes contacting the LBC with an effective amount of IPTG.

[0200] In some forms, the effective pH for binding of the LBC to lanthanide is between a pH of about 2.5- about 7, preferably from about 3 to about 6, and more preferably from a pH or 4-5. Lanthanide binding is exemplified in the Examples using 20 mM NaOAc, 100 mM KC1, pH 5.0.

[0201] Lanthanide can be released from the LBC-lanthanidc complex by contacting the LBC-lanthanide complex with a washing buffer at an effective pH for example, an acidic pH.

[0202] For isolation of the LBC-Lanthanide complex, the LBC-Lanthanide complex can be attached to a solid support, for example, a column, a membrane, a bead, or the like. The solid support can be any suitable composition known to one of skill in the art including, for example, a polymer, alginate, acrylamide, regenerated cellulose, cellulose ester, plastic, or glass.

[0203] The biosorption media, which include, for example, biofilm, microbe beads, and carbon nanotube embedded membranes can be used for adsorption under continuous flow. It is contemplated that LBC immobilization in biosorption media for use in flow through setups allows for complete (or substantially complete) separation of lanthanide from lanthanide-containing mixed metal solutions in a single step and, for example, without the need of centrifugation, filtration, or both. Methods of making biosorption media that immobilize LBC are known in the art and are disclosed for example, in U.S. Patent No. 11713493.

[0204] Materials from which lanthanide can be extracted include any material containing lanthanides, for example, materials including rare earth elements (REE). As REEs are used in the manufacture of many technological equipments, industrial and domestic waste from these equipments and, in particular, from electrical and electronic equipment, also known as WEEE, represents a source of access to REE. This electrical and electronic waste comes from the urban mine, i.e., from the collection of post-consumer waste, or is industrial waste. Permanent magnets represent the application that consumes the largest tonnage of REEs and generates the highest market value. They are found in wind turbine generators, computer hard drives, air conditioner compressors, etc. The NdFeB type magnet is the most widely used type of REEs magnet due to its high magnetic performance. These magnets contain the following REEs: neodymium (Nd), 45821207.1 23ATTORNEY DOCKET NO. MIT 26099 PCT

[0205] praseodymium (Pr) and dysprosium (Dy), to which scandium, cerium, lanthanum, gadolinium are sometimes added.

[0206] The disclosed compositions and methods can be further understood by the following non limiting paragraphs and examples.

[0207] 1. A genetically engineered cell comprising an exogenous nucleic acid encoding a binding protein (EBP) or a lanthanide binding fragment thereof, or comprising EBP, wherein the EBP is expressed on the cell surface, wherein the LBP optionally expressed as a fusion protein comprising a surface expression tag and a purification tag.

[0208] 2. The cell of paragraph 1, generally represented by the formula:

[0209] B Li (Set L2LBP - El PT) / (LBP- L2Set L3.PT) / (PT- L2LBP- L3. Set)

[0210] Formula I, where,

[0211] B represents a cell,

[0212] Set represents a surface expression tag,

[0213] LBP represent a lanthanide-binding protein, and

[0214] PT represents a purification tag, preferably, where the portion including Set, LBP and PT is expressed on the surface of the cell B, as a fusion protein.

[0215] Li, L2, L3are optional linkers.

[0216] The slashes (-(Set- L2. LBP - L3.PT) / (LBP- L2.Set- L3PT) / (PT- L2. LBP- L3. Set)) in Formula I represent alternative arrangements of Set, LBP and PT.

[0217] 3. The cell of paragraph 1 or 2, wherein the LBP is lanmodulin or lanepsy.

[0218] 4. The cell of any one of paragraphs 1-3, wherein the cell is a prokaryotic or a eukaryotic cell.

[0219] 5. The cell of paragraph 4, wherein the cell is a microbe selected from the group consisting of Caulobacter crescentus (C. crescentus), Escherichia coli (E. coli), Bacillus, and Lactobacillus.

[0220] 6. The cell of any one of paragraphs 1-5, comprising an inducible promoter.

[0221] 7. The cell of any one of paragraphs 1-6, wherein the purification tag is selected from the group consisting of His-tag wherein the His-tag is [His]n, where n is an integer from 2-10, preferably, where n is 6; FLAG tags, which typically include the sequence DYKDDDDK (SEQ ID NO2): haemagglutinin (HA) for example, YPYDVP (SEQ ID NO:3); MYC tag for example ILKKATAYIL (SEQ ID NO:4 or EQKLISEEDL (SEQ ID NO:5); Strep-tag WSHPQFEK (SEQ ID NO: 6), glutathione S-transferase (GST); Maltose binding protein (MBP), calmodulin binding peptide (GBP); the intein-chitin binding domain (intein-CBD), the streptavidin tag and AWSHPQFEK (SEQ ID NO:23).

[0222] 45821207.1 24ATTORNEY DOCKET NO. MIT 26099 PCT

[0223] 8. The cell of claim 7, wherein PT is H6 (SEQ ID NO:1).

[0224] 9. The cells of any one of paragraphs 1-8, wherein the fusion protein comprises linker, wherein the linker is selected from the group consisting of (GGGGS)n (SEQ ID NO:8), for example, GGGGS (SEQ ID NO: 8), GGGS (SEQ ID NO:6), GGSS (SEQ ID NO: 11), Thr-Gly-Leu-Thr-Gly-Leu-Asn-Ser-Gly-Leu (SEQ ID NO: 12), GSE, Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO: 10), Ala-Ser, (Gly4-Ser)3(SEQ ID NO: 14), (Gly4-Ser)4(SEQ ID NO: 15), GSGSGSGS (SEQ ID NO: 13 and SGSG (SEQ ID NO: 16).

[0225] 10. The cell of any one of paragraphs 1-9, wherein the cell is E. coli.

[0226] 11. A composition comprising the cell of any one of paragraphs 1-10, in (a) cell culture medium or (b) a cryoprotectant agent.

[0227] 12. The composition of claim any one of paragraphs 1-11, where the cells have been freeze-dried or lyophilized.

[0228] 13. A method for preferentially separating lanthanide from an REE containing material, the method comprising the steps of:

[0229] a) contacting the cell of any one of paragraphs 1-10 or the composition of any one of paragraphs 11-12 with a lanthanide containing material to form a cell-lanthanide-complex; and

[0230] b) separating the cell-lanthanide complex from at least a portion of the material by contacting the LBC-Lanthanide complex with a composition containing a binding partner to the purification tag (PT) expressed on the LBC.

[0231] 14. The method of paragraph 13, comprising adding a binding buffer in step (a) at an effective pH for binding of the LBC to lanthanide.

[0232] 15. The method of paragraph 14, wherein the pH is between about 2.5- 6.

[0233] 16. The method of paragraph 15, wherein the pH is between about 4 and 5.

[0234] 17. The method of any one of paragraphs 13-16, wherein the LBC comprises an inducible promoter, the method further comprising contacting the LBC with an inducing agent effective to induce the inducible promoter.

[0235] 18. The method of paragraph 17, wherein the inducible promoter is promoter isopropyl-[3-D-thiogalactopyranosid (IPTG)-inducible, the method comprising contacting the LBC with IPTG in an effective amount to induce the IPTG-inducible promoter.

[0236] 19. The method of any one of paragraphs 13-18, further comprising separating the lanthanide from the LBC-Lanthanide complex by contacting the LBC-Lanthanide complex with washing buffer at an acidic pH.

[0237] 20. The method of paragraph 19, wherein the washing buffer is an acetate buffer at a pH of about 2.

[0238] 45821207.1 25ATTORNEY DOCKET NO. MIT 26099 PCT

[0239] EXAMPLES

[0240] Materials and Methods

[0241] Chemicals and Instruments

[0242] All chemicals and reagents used in these studies were purchased from Sigma Aldrich, TCI, or Thermo Fisher Scientific as reagent grade and were used without further purification unless otherwise stated. 18 MQ ultrapure water was obtained from a Sartorius Arium Mini unit.

[0243] Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

[0244] Inductively Coupled Plasma Mass Spectrometry (ICP-MS) measurements were conducted at the core facilities of the Center for Environmental Health Sciences (CEHS) at MIT. An Agilent 7900 ICP-MS system in gas-mode was used for all measurements. Samples were diluted 1:1000 into 2% nitric acid to a final volume of 2 mL. Germanium was added as an internal standard, and all samples were filtered using

[0245] 0.22 pm cellulose acetate filters prior to ICP-MS analysis (Thermo Scientific).

[0246] UV-Visible (UV-Vis) Absorbance Measurements

[0247] ODeoo was measured in 1 mL plastic cuvettes using a NanoDrop One (Thermo). A cell density of ODeoo

[0248]

[0249] 1 was set to being equivalent to 8 x 108cells mlf for further data analysis. A microplate reader (BioTek, Synergy Hl) was used for acquisition of full spectral UV-Vis absorbance measurements (300 - 700 nm). For relative absorbance measurements (A490 / 405 nm), single-wavelength measurements at 405 nm and 490 nm were acquired on a BioTek, 800 TS microplate reader. All microplate measurements were taken using 96- well, polystyrene, flat-bottom plates.

[0250] Antibody Labeling of LanM-Expressing Cells

[0251] The LanM surface-expressing cells (ODeoo = 6) were incubated with an aqueous solution of 6-His Tag Polyclonal Antibody-FITC conjugate (A190-114F, 0.1 mg mL’1, PBS, 1% BSA, 0.1% Tween) for 36 h to selectively bind the Hise-tag of the INPNC-LanM-Hise construct. Prior to analysis, the cell suspension was washed with PBS (supplemented with 1% BSA, 0.1% Tween) three times.

[0252] Confocal Laser-Scanning Microscope (CLSM)

[0253] Confocal fluorescence images were recorded on a Zeiss CLSM 710 using FITC-antibody labeled cells. Excitation was performed with an Ar laser at a wavelength of 488 nm, and emission was monitored at 493- 556 nm.

[0254] Fluorescently Assisted Cell Sorting (FACS).

[0255] Following antibody labeling, cell suspensions were diluted to an ODeoo of -0.01 prior to FACS measurement. FACS data was acquired on a BD FACS Melody. Excitation was 45821207.1 26ATTORNEY DOCKET NO. MIT 26099 PCT

[0256] performed at a wavelength of 488 nm, and the emission was monitored using a filter at 527 / 32 nm and a mirror at 560 LP. 100,000 events were acquired and analyzed using the Floreada.io online analysis tool (https: / / floreada.io / analysis).

[0257] Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray (EDX) (SEM-EDX) SEM images and EDX spectra were acquired with a Zeiss Merlin High-Resolution Scanning Electron Microscope (SEM, Jena, German). Lyophilized, reconstituted cells were dropcasted on double-sided carbon tape that was mounted on a standard, aluminum pin stub mount.

[0258] Binding Data Analysis

[0259] For quantitation of the colorimetric data, the ratio of absorbance at 490 nm and 405 nm (A490 / 405 nm) was used. To determine the concentration, measured values were compared to linear fits derived from standard titration curves. To calculate relative binding, measured concentrations were compared with control samples (no cells, Equation SI and S2).

[0260] For ICP-MS data, the “relative bound” values were calculated by dividing experimental data with values obtained from control samples (no cells, Equation SI).

[0261] Statistical Analysis

[0262] All error bars included in Figures are lx standard error. Comparison of means were performed with a paired two sample two-tailed Student’s t-test using Excel. Statistically significant changes are displayed with * P

[0263] < 0.05, ** P < 0.01, *** P < 0.001, and n.s. (not significant) > 0.05. All experiments were performed in biological or technical triplicates, as indicated. All data was fit using GraphPas Prism 10 software.

[0264] Protein Expression

[0265] For gel analysis, overnight cultures were diluted in LB media (50 pg mL1kanamycin), and expression was induced with isopropyl-P-D-thiogalactopyranosid (IPTG, 100 pM) at an ODeoo = ~0.8. Cells were then incubated at 18 °C, 200 rpm for 20 h. Proteins were extracted by sonificarion of washed cells (3 x PBS, 10,000 g for 3 min) according to established protocols. The lysed cells were centrifuged (13’000 g for 3 min at 4 °C) and the crude protein extract was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS page) analysis using standard protocols.

[0266] Lanthanide binding with surface-expressed LanM

[0267] Relative binding values were determined using Equation S 1.

[0268] % bound =[Ln3+]m

[0269] [Ln ] (Equation SI)

[0270] 45821207.1 27ATTORNEY DOCKET NO. MIT 26099 PCT

[0271] With [Ln3+]mindicating the measured and in Equation S 1 calculated concentration and [ Ln3+]1being defined as the initial, loaded concentration.

[0272] Experimental Section

[0273] Plasmid Design and Purification

[0274] Lanmodulin in a pET24a backbone was obtained from the Cotruvo Lab.30Gibson assembly was used to integrate the LanM-His6 gene block into an INPNC-containing pSKB3 vector with kanamycin resistance. Gibson assembly reactions were performed with the Gibson Assembly Cloning Kit from New England Biolabs (E5510S, Ipswitch, MA). Once assembled, plasmids were transformed into chemically-competent E. coli using standard protocols. The LanM- INPNC-His6 plasmid was transformed into Novagen NovaBlue competent cells. The transformed cells were grown on 50 pg mL1kanamycin agar plates. Single colonies were picked and inoculated in Luria Broth Miller (LB) media (50 pg mL1kanamycin). Cells were grown overnight at 37 °C with shaking (250 rpm), followed by lysis and plasmid purification according to the Novagen plasmid prep procedure. The purified plasmid was frozen and stored at -20 °C for future use.

[0275] All sequences used were previously published;29’30,34’3’ a plasmid map of the final assemblies is displayed in FIG. 6.

[0276] LanM expression and purification

[0277] Expression of the LanM was performed according to published procedures.50The amplified LanM-INPNC-His6 plasmid was transformed into Novagen BL21(DE3) chemically competent cells. Single colonies were picked and inoculated in LB media (10 mL, 50 pg mb'1kanamycin), followed by growth overnight at 37 °C with shaking (250 rpm). The cells were again diluted into LB media (50 mL, 50 pg mL1kanamycin) and incubated at 37 °C until an OD600 of 0.4 -0.6 was reached (~1 hour). Protein expression was then induced by addition of IPTG (100 pM). Cells were then maintained at 37 °C with shaking for an additional 5 hours. The cells were pelleted by centrifugation at 4,000 x g for 5 min. The cells were then washed with PBS (2 x 15 mL) and resuspended in PBS supplemented with trehalose (100 mM, 2.5 mL, OD600 ~30 - 50). Cells were snap frozen in liquid nitrogen and lyophilized overnight. The resulting solid was stored at -20 °C until further use.

[0278] Lanthanide binding to surface expressed LanM

[0279] Lyophilized INPNC-LanM cells (induced and uninduced) stored at -20 °C were thawed and resuspended in water. Cell density of the suspension was determined by OD600 measurements. For titrations, a constant number of cells (9.6 x 109cells) was aliquoted, and cells were pelleted by centrifugation (4,000 x g, 5 min). The cells were washed with acetate buffer (2 x 0.5 mL, 20 45821207.1 28ATTORNEY DOCKET NO. MIT 26099 PCT

[0280] mM NaOAc, 100 mM KC1, pH 5.0) and pelleted by centrifugation (4,000 x g, 5 min). Cells were subsequently incubated with increasing amounts of metal mixture (equimolar GdC13, LaC13, TbC13, and YC13, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.6, and 2 mM total in 500 pL, 20 mM NaOAc, 100 mM KC1, pH 5.0) for 5 min, with tube inversion every 1 - 2 min. Between each binding step cells were pelleted by centrifugation (6,000 x g, 5 min), and a sample was taken for ICP-MS analysis (data not shown FIG. 8A-8D and Equation SI). Fluorescence images of LanM- expressing E. coli. Cells treated with 6-His Tag Polyclonal Antibody-FITC conjugate (A190- 114F) for 36 h prior to imaging, were taken. Fluorescent images of induced and La3+ treated cells, induced, and uninduced and La3+ treated E. coli, were taken (data not shown)

[0281] For the calcium / magnesium challenge, cells (1.2 x 109cells) were aliquoted and pelleted by centrifugation (4,000 x g, 5 min). The cells were washed with acetate buffer (2 x 0.5 mL, 20 mM NaOAc, 100 mM KC1, pH 5.0) and pelleted by centrifugation (4,000 x g, 5 min). Cells were incubated with metal mixtures (equimolar GdC13, LaC13, TbC13, and YC13, 0.5 mM, 125 pL, 62.5 nmol total, 20 mM NaOAc, 100 mM KC1, pH 5.0) supplemented with varying amounts of Mg2+and Ca2+(0, 20, or 200 eq., 0, 1.25 pmol, 12,4 pmol) for 5 min, with tube inversion every 1 - 2 min. Cells were pelleted by centrifugation (6,000 x g, 5 min), and the M3+concentration of the resulting supernatant or effluent was determined by ICP-MS measurements (FIG.3A, FIG. 13A-13D, and FIG. 12).

[0282] For pH dependent binding studies, cells (2.4 x 109cells) were aliquoted and pelleted by centrifugation (4,000 x g, 5 min). The cells were washed with acetate buffer (2 x 0.5 mL, 20 mM NaOAc, 100 mM KC1, pH 5.0) and pelleted by centrifugation (4,000 x g, 5 min). Cells were incubated with metal mixtures (equimolar GdC13, LaC13, TbC13, and YC13, 1 mM, 125 pL, 125 nmol total, 20 mM NaOAc, 100 mM KC1) at varying pH (pH 5, 4, 3.5, and 2.5) for 5 min, with tube inversion every 1 - 2 min. Cells were pelleted by centrifugation (6,000 x g, 5 min), and the M3+concentration of the resulting supernatant or effluent was determined by ICP-MS measurements(FIG. 3B, and FIG. 15A-15D).

[0283] Lanthanide concentration by colorimetric alizarin assay

[0284] Standards were prepared using two-fold serial dilutions of GdC13, La 03, Tb 03, Y 03, CaC12, and MgC12 (50 pL, 0.0156 - 1.0 mM, 20 mM NaOAc, 100 mM KO, pH 5.0) or an equimolar mixture of GdC13, LaC13, TbC13, and YC13 (50 pL, 0.0039 - 0.25 mM, 20 mM NaOAc, 100 mM KO, pH 5.0) were mixed with alizarin-3-methyliminodiacetic acid (100 pL, 0.5 mM, 150 mM NaOAc, pH 5) in a 96-well plate. UV-Vis absorbance spectra were measured using a microplate reader (BioTek Synergy Hl, or BioTek 800 TS) at either the full spectra (300 45821207.1 29ATTORNEY DOCKET NO. MIT 26099 PCT

[0285] - 700 nm) or at two specific wavelengths (405 nm and 490 nm). The relative absorbance ratio of A490 / 405 nm was plotted against concentration, with linear regression analysis used to establish a linear relationship between concentration and absorbance (FIG. 4A-4D, FIG. 12A-12D, Table 2, and Equation S2).

[0286] Table 2. Equations for linear fits of colorimetric quantification of lanthanides.

[0287]

[0288] Linear fits plotted against the standard curves of relative absorption at A490 / 405 nm for the dilution series of mixtures of constant alizarin-3-methyliminodiacetic acid concentration (0.5 mM, 150 mM NaOAc, pH 5.0) and varying concentrations of GdCL, LaC.li, TbCL, YCI3, and an equimolar mixture of the four metals. All reported values were calculated using following equation and were fit using GraphPad Prism 10 software.

[0289] [Ln3+] = A490 / 405 nm - B

[0290] A (Equation S2)

[0291] With A490 / 405 nm being the measured values and A and B being the factors derived from the linear fits (Table 1).

[0292] Lanthanide concentrations of effluent samples were measured using an analogous protocol.

[0293] Samples (50 pL) were mixed with alizarin-3-methyliminodiacetic acid (100 pL, 0.5 mM, 150 mM NaOAc, pH 5.0), and absorbances were measured using a microplate reader. Concentrations of the lanthanide metal were calculated using the linear relationship between

[0294] concentration and absorbance found in the standard curve analysis (Equation S2).

[0295] Lanthanide binding and release from packed-bed filter

[0296] Lyophilized INPNC-LanM cells (induced and uninduced) stored at -20 °C were thawed and resuspended in water. OD600 values of 24 were loaded onto a pre- wetted Steriflip® Vacuum Tube Top Filter (Millipore Sigma, PES, 0.22 pm). The filter was washed with 5 mL acetate buffer before adding an equimolar mixture of GdC13, LaC13, TbC13, and YC13 (0.6 mL, 0.25 mM of each Ln3+; 1 mM total metal concentration, 600 nmol, 20 mM NaOAc, 100 mM KC1, pH 5.0). Effluent was collected, and the filter was washed 3 times using acetate buffer (0.6 mL) with collection of each fraction. To release, four additions of HC1 solution (0.6 mL, 0.25 mM, 20 mM NaOAc, 100 mM KC1, pH 1.7) were performed, with subsequent effluent 45821207.1 30ATTORNEY DOCKET NO. MIT 26099 PCT

[0297] collection. To regenerate the LanM, the filter was flushed with acetate buffer (5 x 0.6 mE, pH 5.0). Ln3+concentrations, as well as binding capacities of all collected samples were determined by colorimetric alizarin assay using Equations S3 - 5 (FIG. 5A-5D, and FIG. 14).

[0298] Results and Discussion

[0299] Lanmodulin (LanM) surface-expression

[0300] Because of the challenges associated with lanthanide recovery from systems with internally- expressed binding proteins,1417the disclose platform was designed with LanM (from Methylobacterium extorquens) on the surface of the cell. The ice nucleation protein from Pseudomonas syringae (INPNC),34,35was incorporated this sequence at the A'-lerminus of the LanM gene. The final plasmid contains both the INPNC and LanM sequences that are optimized for expression in BL21 E. coli (FIG. 5A-5D). The system is isopropyl-0-D-thiogalactopyranosid (IPTG)-induciblc, and INPNC-LanM expression is estimated to yield -50,000 proteins per cell using these conditions.34,35Following induction optimization for LanM, overexpression was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie staining. Despite being membrane -bound, a clear overexpression band at the expected molecular weight for LanM is observed (FIG.6).

[0301] Subsequent studies sought to verify that the LanM is on the surface of the cells by characterizing the protein in situ using confocal microscopy, flow cytometry, and scanning electron microscopy (SEM). Cells that surface-express LanM were evaluated against uninduced controls. As the LanM sequence contained a His6 tag at the C-terminus, an anti-His6 antibody- FITC was employed conjugate to label the surface displayed LanM.

[0302] Following incubation of cells with the fluorescently-labeled antibody, fluorescence was observed only on the induced population of cells. Further, the intensity of the fluorescence increased upon addition of lanthanum ions (La3+), which is consistent with protein stabilization upon lanthanide binding as well as improved access to the LanM C-terminus (FIG. IA-IC and data not shown, , and Table I).30Fluorescent evaluation of LanM expression was conducted. Cells were treated with an anti-His6-antibody-FITC conjugate for 36 h prior to imaging. Confocal microscopy images induced and La3+bound, induced, and uninduced cellswere obtained (data not shown). FACS was performed on LanM expressing E. coli. Cells were treated with 6-His Tag Polyclonal Antibody-FITC conjugate (A190-114F) for 36 h prior to analysis. Forward v.y. side scatter of all recorded events of induced and La3+-treated cells; uninduced and La3+treated induced cells, and induced and La3+treated cells, without addition of 6-His Tag Polyclonal Antibody-FITC conjugate were obtained. Histograms of FITC signal of analyzed population Pl of induced and La3+treated uninduced and La3+treated induced cells, and 45821207.1 31ATTORNEY DOCKET NO. MIT 26099 PCT

[0303] induced and Ea3+treated cells, without addition of 6-His lag Polyclonal Antibody-FITC conjugate were obtained. Histograms of FITC signal of analyzed population Pl .

[0304] Table 1. Quantification of FACS analysis of Pl.

[0305]

[0306] Cells were treated with 6-His Tag Polyclonal Antibody-FETC conjugate (Al 90-114F) for 36 h prior to analysis. A threshold for FITC positive cells was set, the relative values are the integration of defined populations over all recorded events in Pl.

[0307] To further confirm that the metals were bound to the surface of microbes with displayed LanM, cells were imaged by Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray Spectroscopy (EDX). As EDX only measures the surface layer of the sample, any observed ions are surface-associated. By EDX, induced cells incubated with either La3+or Gd3+showed the respective cation, indicating surface association (FIG. 7A-7C). Taken together, the results confirm that LanM is expressed on the cell surface.

[0308] Lanthanide recovery with surface-expressed LanM

[0309] As it was confirmed that LanM was scaffolded on the surface of lyophilized cells and demonstrated a straightforward colorimetric assay for lanthanide quantification, subsequent studies then sought to determine the binding capacity of these materials. A constant number of lyophilized cells were reconstituted (9.6 x 109cells), followed by treatment with increasing amounts of REE (equimolar GdC13, LaC13, TbC13, and YC13, 0.2 to 2 mM total metal concentration, 500 yL; representative REEs, 20 mM NaOAc, 100 mM KC1, pH 5.0) for five minutes. With all REEs, increasing metal concentration showed a linear increase in binding for cells with LanM expressed (FIGs. 2B, and 9A-9D). Conversely, metal adsorption with uninduced cells decays exponentially, confirming non-specific binding.

[0310] At a ratio of 52 nmol metal to 109cells, all tested REEs bound equivalently (76.5 ± 7.8 % Y3+, 77.0 ± 5.8 % La3+, 82.2 ± 4.6 % Gd3+, and 83.2 ± 4.4 % Tb3+, FIG. 2B grey dotted line, and FIG.

[0311] 2B, dark). Additionally, across all conditions, lower overall capture for La3+and Y3+are observed, consistent with previous reports of LanM affinity for different metals.7’30’31’37In comparison, both uninduced (41.0 ± 2.1 % Y3+, 37.5 ± 3.2 % La3+, 46.5 ± 4.0 % Gd3+, and 48.9 45821207.1 32ATTORNEY DOCKET NO. MIT 26099 PCT

[0312] ± 3.0 % Tb3+) and BE21 control samples (49.8 ± 5.5 % Y3+, 45.5 ± 3.0 % La3+, 56.6 ± 3.1 % Gd3+, and 57.4 ± 3.2 % Tb3+FIG. 2B, light and striped) adsorbed significantly less. Some nonspecific adsorption of REEs to both uninduced cells and unmodified BL21 cells is expected based on known non-specific interactions between microbial surfaces and REEs.9,12 19The observed differences in binding capacity can be exploited to minimize non-specific binding when selecting microbial concentrations for REE recovery.

[0313] Salt concentration and pH dependent REE recovery

[0314] LanM contains four metal binding sites, with three that have high affinity for lanthanides and one that has low affinity for these metals. Many sources of REEs, including acid mine drainage, contain high concentrations of Mg2+and Ca2+, and Ca2+has been reported to decrease non- specific cell surface REE binding.19Thus, the specificity of the system for REE capture in the presence of other cations was investigated. REE mixtures were doped with Mg2+and Ca2+(in 20 mM NaOAc, 100 mM KC1, pH 5.0) followed by the addition of this mixture to a fixed number of cells (1.2 x 109cells) and incubation for five minutes. No significant differences in REE binding are observed in the presence of competing ions at up to 100-fold excess (FIG. 3 a, FIG. 9A-9D, and FIG. 10). In contrast to prior work, in which constant REE binding was observed in the presence of Mg2+and Ca2+ion concentrations as high as 1 M,7,31decreased REE binding capacity was observed at high Mg2+and Ca2+concentrations (200-fold excess, 0.1 M of each Mg2+and Ca2+). Similarly, no decrease in non-specific adsorption was observed upon addition of these cations, in contrast to previous reports.19Each approach to deploy LanM has employed a different system to do so, making direct comparisons between materials challenging. Overall, LanM shows the capacity to bind to REEs in highly complex matrices and high salt concentrations.

[0315] LanM is an extremely stable protein, even at low pl I.729 31Thus, the impact of pH on binding was evaluated to determine if lower pH could decrease non-specific adsorption. A constant concentration of cells (2.4 x 109cells) was exposed to REE mixtures (equimolar GdC13, LaC13, TbC13, and YC13, 1 mM, 125 pL, 125 nmol total) at pH’s ranging from 5.0 to 2.5 for 5 min. Metal binding remained consistent down to pH 4.0 (-70-75 %, FIG. 3A-3B, and FIG. 11A-11D) for LanM- expressing cells, with non-specific adsorption to uninduced cells significantly reduced (37.5 ± 3.2 % La3+to 26.1 ± 1.5 % La3+, FIG. 3A-3B, light purple). At pH 3.5, both LanM-expressing and uninduced cells showed a significant decrease in overall binding while maintaining differential binding (51.3 ± 3.4 % La3+induced vs. 14.6 ± 1.5 % La3+uninduced). At pH 2.5, selective binding is compromised for LanM-expressing cells, though other LanM systems show efficient binding at pH’s as low as 2.3,7'31These studies suggest that non-specific 45821207.1 33ATTORNEY DOCKET NO. MIT 26099 PCT

[0316] REE interactions with E. coli can be reduced by lowering the pH of the binding buffer to a point where REE binding to LanM remains intact but background binding is decreased. Taken together, this work shows that surface-displayed LanM can be applied in complex matrices at relevant pH’s and salt concentrations similar to brine.

[0317] Rapid colorimetric quantification for field deployment

[0318] The most prevalent quantification method for REEs is TCP-MS-based elemental analysis, often necessitating sending samples to costly and slow external laboratories for testing. Thus, to ensure that the workflow for capture with the disclosed materials is compatible with field deployment, rapid colorimetric quantification was incorporated with the filtration.38-41Colorimetric methods are simple and can be performed with portable equipment. Though colorimetric REE quantification can be performed with compounds such as Arsenazo III39and xylenol orange,38challenges such as toxicity and pH limitations limit their use.38-41Studies therefore sought to expand the library of compounds for colorimetric REE quantification using alizarin-3- methyliminodiacetic acid (alizarin Complexone).42-44Upon REE chelation, the peak absorbance of alizarin Complexone shifts sufficiently to observe the color change with the naked eye and enables REE quantification (FIG. 4A-4D).45’46Using the ratio of A490 / 405 nm, the linear range of detection for REEs was 0 mM and 0.5 mM for Tb3+and Y3+(R2> 0.97, FIGs. 4C, 14A-14D) and 0 mM to 1 mM for Gd3+and La3+(R2> 0.97, FIG. 4C, 14A-14D). In contrast, no color change is observed for either Ca2+or Mg2+, indicating selectivity for the tested REEs over the alkaline earth’s calcium and magnesium, though there is the potential for this molecule to complex with other (post-)transition metals (FIG. 4C black and grey). The detection window for this assay is within the relevant concentration ranges for REEs, expanding the toolbox of colorimetric REE assays.

[0319] To employ the colorimetric assay with actual samples, different concentrations of lyophilized cells were reconstituted (2.4, 4.8, and 9.6 x 109cells), followed by treatment with a constant amount of REE (either GdC13, LaC13, TbC13, or YC13, 250 nmol) for five minutes. Cation capture was evaluated by both colorimetric and ICP-MS analysis (FIGs. 5D, 5E and FIG.

[0320] 13A-13B). For all conditions, results obtained by colorimetric assay were consistent with ICP-MS, demonstrating the practical applicability of the assay.

[0321] Rapid recovery of REEs through packed-bed filtration

[0322] For scalable deployment of decorated cells, flow-through systems are advantageous to enable processing of large volumes in short amounts of time. Thus, the lyophilized microbes were incorporated in a flow-through system for REE capture and recovery. Cells were loaded into a 0.22 pm filter unit, and a mixture of REEs was then applied to the cells (FIG. 5A). Metal 45821207.1 34ATTORNEY DOCKET NO. MIT 26099 PCT

[0323] concentrations in the effluent were analyzed following binding and again after a low-pH rinse (pH 1.7). Initial effluent analysis by colorimetric analysis showed significantly depleted REEs following passage through the packed bed, showing a filter capture capacity of 83.5% ± 4.3% for the LanM-expressing cells (n = 10, FIG. 5B), with only 48.4% ± 8.4% binding for uninduced cells (n = 10, FIG. 14). Further, almost complete release of adsorbed metal ions (81.1% ± 5.1%, n = 10) was achieved by washing the filter with acidified buffer (pH 1.7), as monitored by colorimetric effluent analysis. Binding capacity was restored after a rebuffering wash (pH 5), and the filters could be reused over six days across ten cycles without a noticeable decrease in recovery efficiency. The ability to reuse the recovery materials without losing specificity further demonstrates the compatibility of this material with inexpensive, scalable processes.

[0324] Based on the filter recovery data, the binding capacity of the scaffolded protein materials is 11.8 ± 0.9 mg g-1dry cell weight (dew, n = 10) with an overall recovery of -70% of the applied metal. In contrast, uninduced cells have a total capacity of 6.2 ± 2.3 mg g1dew (n = 10) and low overall recovery (39%), consistent with reported non-specific binding (e.g., cation exchange resin,47ligand grafted silica,48activated carbon49) and alternative surface expression platforms.24The binding capacity of surface-expressed LanM is nearly twice that of control material and comparable to that of other REE capture materials.7This binding capacity further confirms that the disclosed materials provide unique advantages over established materials for facile implementation. The ease with which these surface-expressed proteins are integrated into a filter for both specific capture and facile release of REEs demonstrates their potential for scalable recovery of these metals from aqueous waste streams and mining ores without the need for the harsh conditions or expensive equipment currently used.31Moreover, recent advances in increased selectivity between REE with next generations of LanM37could be easily deployed to enable enhanced metal separation.

[0325] Conclusions

[0326] REEs, including lanthanides, are important components of many clean energy technologies, but less than one percent are recycled, and the remainder are obtained through environmentally harmful mining and extraction. Protein-decorated cell materials have been developed based on the surface expression of LanM with an INPNC tag. The cells are freeze-dried. creating a material with a binding capacity of almost 12 mg g1dew. A simple and fast colorimetric assay for lanthanide quantification was implemented, which complements ICP-MS analyses for rare earth metal monitoring. This assay was used to quantify lanthanide binding to bio- scaffolded LanM. Four REE, Gd3+, La3+, Tb3+, and Y3+, were evaluated, and surface-expressed LanM captured over 80% of these metals from solution. Subsequently, nearly 45821207.1 35ATTORNEY DOCKET NO. MIT 26099 PCT

[0327] complete release of the lanthanides was achieved through the simple addition of low-pH solution. This capture and release were shown over six days and ten cycles, demonstrating the potential of these materials to enable the sustainable recovery and recycling of lanthanides. This material can be prepared cost-effectively without requirements for protein purification or immobilization chemistries, significantly decreasing the potential cost of deployment. Applying this technology to mining and e-waste recycling could drastically reduce the environmental impact of lanthanide recovery and ensure sufficient supply of these metals as demand for them grows exponentially.

[0328] References

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[0383] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0384] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

[0385] 45821207.1 37

Claims

ATTORNEY DOCKET NO. MIT 26099 PCTCLAIMSWe claim:

1. A population of genetically engineered cells, comprising an expression vector comprising an exogenous nucleic acid encoding a lanthanide binding protein (LBP), a surface expression tag and a purification tag.

2. The cells of claim 1 , (a)wherein the lanthanide binding protein (LBP), the surface expression tag and a purification tag are expressed as a fusion protein on the surface of the cell or (b) comprising SEQ ID NO:19, 20 and 21.

3. A population of genetically engineered cells comprising cells expressing a fusion protein comprising LBP, a surface expression tag and a purification tag on the cell surface, optionally, wherein the LBP is comprises SEQ ID NO:22.

4. The cells of any one of claims 1-3, each represented by the formula:B L1 (Set L2LBP L3PT)Formula II, wherein,B represents a cell,Set represents a surface expression tag,LBP represents a lanthanide-binding protein, andPT represents a purification tag, wherein Set, LBP and PT are on the surface of the cell B, and wherein Li, L2, Lt are optional linkers.

5. The cells of any one of claims 1-4, wherein the LBP is lanmodulin or lanepsy.

6. The cells of any one of claims 1-5, wherein the cell is a prokaryotic or a eukaryotic cell.

7. The cell of claim 6, wherein the cell is a microbe selected from the group consisting of Caulobacter crescentus (C. crescentus), Escherichia coli (E. coli), Bacillus, and Lactobacillus.

8. The cell of any one of claims 1-7, comprising an inducible promoter.

9. The cell of any one of claims 1-8, wherein the purification tag is selected from the group consisting of His-tag wherein the His-tag is [His]n, where n is an integer from 2-10, preferably, where n is 6; DYKDDDDK (SEQ ID NO:2); YPYDVP (SEQ ID NO:3);ILKKATAYIL (SEQ ID NO:4); EQKLISEEDL (SEQ ID NO:5); WSHPQFEK (SEQ ID NO: 6), glutathione S-transferase (GST); Maltose binding protein (MBP), calmodulin binding peptide (CBP); the intein-chitin binding domain (intein-CBD), the streptavidin tag and AWSHPQFEK (SEQ ID NO:23).

10. The cell of claim 9, wherein PT is H6 (SEQ ID NO:1).45821207.1 38ATTORNEY DOCKET NO. MIT 26099 PCT11. The cell of any one of claims 1-10, wherein the fusion protein comprises linker, wherein the linker is selected from the group consisting of (GGGGS)n (SEQ ID NO:8), for example, GGGGS (SEQ ID NO: 8), GGGS (SEQ ID NO:6), GGSS (SEQ ID NO:11), Thr-Gly-Leu-Thr-Gly-Leu-Asn-Ser-Gly-Leu (SEQ ID NO: 12), GSE, Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO:10), Ala-Ser, (Gly4-Ser)3(SEQ ID NO:14), (Gly4-Ser)4(SEQ ID NO:L5), GSGSGSGS (SEQ ID NO: 13 and SGSG (SEQ ID NO: 16).

12. The cell of any one of claims 1-11, wherein the cell is E. coli.

13. A composition comprising the cell of any one of claims 1-12, in (a) cell culture medium or (b) a cryoprotectant agent.

14. The composition of claim any one of claims 1-13, where the cells have been freeze-dried or lyophilized.

15. A method for preferentially separating lanthanide from an REE containing material, the method comprising the steps of:a) contacting the cell of any one of claims 1-14 or the composition of any one of claims 13-14 with a lanthanide containing material to form a cell-lanthanide-complex; andb) separating the cell-lanthanide complex from at least a portion of the material by contacting the LBC-Lanthanide complex with a composition containing a binding partner to the purification tag (PT) expressed on the LBC.

16. The method of claim 15, comprising adding a binding buffer in step (a) at an effective pH for binding of the LBC to lanthanide.

17. The method of claim 16, wherein the pll is between about 2.5- 6.

18. The method of claim 17, wherein the pH is between about 4 and 5.

19. The method of any one of claims 15-18, wherein the LBC comprises an inducible promoter, the method further comprising contacting the LBC with an inducing agent effective to induce the inducible promoter.

20. The method of claim 19, wherein the inducible promoter is promoter isopropyl- -D-thiogalactopyranosid (IPTG)-inducible, the method comprising contacting the LBC with IPTG in an effective amount to induce the IPTG-inducible promoter.

21. The method of any one of claims 15-20, further comprising separating the lanthanide from the LBC-Lanthanide complex by contacting the LBC-Lanthanide complex with washing buffer at an acidic pH.

22. The method of claim 21, wherein the washing buffer is an acetate buffer at a pH of about 2.45821207.1 39

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