Methods and compositions related to engineered bacterial spores to capture rare earth elements

Abstract

Engineered bacterial spores have been engineered to express a lanthanide binding protein. This binding protein is capable of capturing rare earth elements (REEs). There are various methods which can be used to capture REEs by using these spores.

Description

METHODS AND COMPOSITIONS RELATED TO ENGINEERED BACTERIALSPORES TO CAPTURE RARE EARTH ELEMENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 652,401, filed May 28, 2024, incorporated herein by reference in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant numbers W912HZ- 23-P-0154 and W912HZ-24-F-0147 awarded by the U. S. Army Engineer Research And Development Center. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] This sequence listing submitted on May 23, 2025, as an .XML file entitled “10046- 610WO1 Sequence Listing” created on May 23, 2025 and having a file size of 5,031 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUND

[0004] Rare earth elements (REEs) comprise seventeen elements in the periodic table, specifically the 15 lanthanide elements plus scandium and yttrium. REEs are a group of metallic elements with unique chemical, catalytic, magnetic, metallurgical and phosphorescent properties, and as such find use in a wide variety of modern devices including high-strength magnets, batteries, displays, lighting, and high performance metal alloys.

[0005] REEs are relatively plentiful in the earth's crust. However, REEs are typically highly dispersed and are not often found as concentrated rare earth minerals in economically exploitable ore deposits.

[0006] Lanthanides (Lns) are essential cofactors in certain enzymes. Lanmodulin (LanM) is a metal binding protein found in several lanthanide-utilizing, methylotrophic bacteria like Methylorubrum sp. The protein exhibits unique metal-binding properties and binds REEs with very high affinity, often better than many synthetic chelators. LanM-REE complexes are stable at extreme conditions like high temperature, repeated acid treatments down to pH 2.5 and up to molar amounts of competing non-REE metal ions.

[0007] Previously, others have studied and purified LanM from E. coli and physically attached LanM to inert styrene beads to capture and release REEs from solution. In addition, others have shown that E. coli and other bacterial cells can display LanM on the surface to capture REEs at neutral pH and low toxicity environments. However, bacterial cells are far less capable of withstanding the types of environments that REEs are often found in.

[0008] Thus, there exists a need in the art for inexpensive and accurate REE capture methods that provides the flexibility to capture REEs in extreme environmental conditions. This is satisfied by the present disclosure.DESCRIPTION OF DRAWINGS

[0009] Figure 1 shows a B. subtilis spore engineered with LanM. Coat and crust proteins, such as CotVWXYZ, are shown on the surface. N-terminal and C-terminal LanM fusion occurs with FLAG. LanM is displayed onto the surface of the modified spores, with up to ~ 20,000 copies of LanM per spore. LanM can be a luminescent variant for detection purposes. Flow cytometry can be used to evaluate LanM display. LanM spores are exposed to REE and detection can be done with DPA.

[0010] Figure 2 shows flow cytometry showing LanM spore display. B. subtilis were engineered to express different spore coat proteins (CotE, CotV-Z) with LanM and FLAG tag on the N- and C-terminal of the protein. Each cell line expressing one of these proteins were sporulated and the spores treated with anti-FLAG-FITC conjugated antibody to determine spore surface display of the coat protein fused LanM. All coat proteins showed display with some variability. Wild type spores were used as a negative control. All the following experiments were done using CotY_FLAG_LanM engineered spores.

[0011] Figure 3 shows an assay where both wild type and LanM engineered Bacillus subtilis spores are exposed to REE at different pHs. Supernatant is captured to measure unbound REE. Pellets are treated with pH 1.5 to release REE from spores. REE is then measured with DPA assay. Sample in pH or buffer conditions neutralized in pH 7.42 HEPES buffer before assay.

[0012] Figures 4A and 4 B show wild type (A) and LanM engineered (B) Bacillus subtilis spores exposed to Dy3 at different pHs. Almost no capture occurs at pH 1. At pH 2-3 capture of the element using LanM spores increases significantly. And pH 3-7 show robust capture and 60-70% release with one single acid wash.

[0013] Figures 5 A and 5B show acid wash to release Dy3 at various time intervals at pH 5 for wild type (A) and LanM mutants (B). For the WT spores, after 10 min of the wash, all the bound REE was released. For LanM spores, the first 5 min of wash showed the release ofalmost 70% of the bound REE with more time (30 min) being necessary to release all the bound REE. 100 |iM DPA and 2.5 pM Dy3+ used. Total Released: WT 2.926 pM (117.04%) / LanM 2.195 pM (87.79%).

[0014] Figures 6A and 6B show Bacillus subtilis wild type (A) and LanM mutant (B) spores exposed to Eu3 at different pHs.

[0015] Figures 7A and 7B shows Dy3+ recovery. 7A shows wild type Bacillus subtilis spores Dy3+recovery under different conditions. 7B shows LanM Bacillus subtilis spores Dy3+recovery under different conditions. The LanM spores performed better at capturing the Dy3+in these conditions than WT, consistently releasing -70% Dy3+on one single wash. NaCl and KC1 had no impact on WT and LanM spores capturing Dy3+, but CaC12 rather than decreasing performance of REE binding to LanM, more than likely inhibited the ability of the DPA assay to quantify Dy3+. 100 pM DPA and 10 pM Dy3+was used.

[0016] Figure 8 shows wild type (WT) and LanM spores at OD10 concentration were exposed to 200 pM REE (Dy) at pH 5 for 1 hour. The unbound REE (UB) was measured and the bound REE (W) measured after released from the spores with treatment with pH 1.5 for 5 min. The spores were then treated again with the same amount of metals to determine reusability and the same process was repeated 3 times. WT completely stops binding after a single pH 1.5 wash, while LanM capture and release capabilities aren’t hindered with multiple washes and stay fairly consistent.SUMMARY

[0017] Disclosed herein is an engineered bacterial spore, wherein the spore has been engineered to express a lanthanide binding protein.

[0018] Also disclosed is a method of capturing a rare earth element (REE), the method comprising: a) providing an engineered bacterial spore, wherein the spore has been engineered to express a lanthanide binding protein; b) exposing the engineered bacterial spore to an REE under conditions which allow the lanthanide binding protein to bind the REE; c) providing conditions suitable for releasing the REE from the spore; and d) capturing the REE.

[0019] Further disclosed is a system for purifying, enriching, or extracting rare earth elements (REEs), the system comprising an engineered bacterial spore, wherein the spore has been engineered to express a lanthanide binding protein.

[0020] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detaileddescription. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.DETAILED DESCRIPTION

[0021] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0022] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0023] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0024] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0025] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself.For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0026] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y ’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0027] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0028] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at orabout” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0029] The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non- nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0030] As used herein, “expression” 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.

[0031] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0032] As used herein, “introduced” refers to the introduction by means of modern biotechnology, and not a naturally occurring introduction.

[0033] In some embodiments, the bacteria of the present disclosure have been modified such that they are not naturally occurring bacteria.

[0034] As used herein, the term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the disclosure can be operably linked, either directly or indirectly, 5’ to the target mRNA, or 3’ to the target mRNA, or within the target mRNA, or a first complementary region is 5’ and its complement is 3’ to the target mRNA.

[0035] As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms, used interchangeably, include but are not limited to, the two prokaryotic domains, Bacteria and Archaea. The term may also encompass eukaryotic fungi and protists.

[0036] The term “microbial consortia” or “microbial consortium” refers to a subset of a microbial community of individual microbial species, or strains of a species, which can be described as carrying out a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest.

[0037] The term “microbial community” means a group of microbes comprising two or more species or strains. Unlike microbial consortia, a microbial community does not have to be carrying out a common function, or does not have to be participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest.

[0038] As used herein, “isolate,” “isolated,” “isolated microbe,” and like terms, are intended to mean that the one or more microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (for example soil, water, plant tissue, etc.). Thus, an “isolated microbe” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain). In aspects, the isolated microbe may be in association with an acceptable carrier.

[0039] In certain aspects of the disclosure, the isolated microbes exist as “isolated and biologically pure cultures.” It will be appreciated by one of skill in the art, that an isolated and biologically pure culture of a particular microbe, denotes that said culture is substantially free of other living organisms and contains only the individual microbe in question. The culture can contain varying concentrations of said microbe. The present disclosure notes that isolated andbiologically pure microbes often “necessarily differ from less pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microbes), see also, Parke- Davis & Co. v. H. K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned Hand discussing purified adrenaline), affd in part, rev'd in part, 196 F. 496 (2d Cir. 1912), each of which are incorporated herein by reference. Furthermore, in some aspects, the disclosure provides for certain quantitative measures of the concentration, or purity limitations, that must be found within an isolated and biologically pure microbial culture. The presence of these purity values, in certain embodiments, is a further attribute that distinguishes the presently disclosed microbes from those microbes existing in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limitations for vitamin B 12 produced by microbes), incorporated herein by reference.

[0040] As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms.

[0041] Microbes of the present disclosure include spores, which are different than vegetative cells. In some embodiments, microbes of the present disclosure include microbes in a viable but non-culturable (VBNC) state. As used herein, “spore” or “spores” refer to structures produced by bacteria and fungi that are adapted for survival and dispersal. Spores are generally characterized as dormant structures; however, spores are capable of differentiation through the process of germination. Germination is the differentiation of spores into vegetative cells that are capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single fungal or bacterial vegetative cell. Fungal spores are units of asexual reproduction, and in some cases are necessary structures in fungal life cycles. Bacterial spores are structures for surviving conditions that may ordinarily be non- conducive to the survival or growth of vegetative cells.

[0042] As used herein, “microbial composition” refers to a composition comprising one or more microbes of the present disclosure.GENERAL DESCRIPTION

[0043] Rare Earth Elements (REEs) (Lanthanide series of metals) from dilute or concentrated environmental (rivers) and industrial mixed chemical environments (e-waste). This invention utilizes a natural Lanthanide Binding Protein, Lanmodulin (LanM) fused to a bacterial spore coat protein (e.g. CotE, CotV-Z), which is ultimately displayed onto the surfaceof a bacterial spore, such as Bacillus subtilis bacterial spores. During sporulation of living cells, the LanM::CotY (e.g.) is produced and is self-assembled in the spore surface, exposing the LanM to the external environment. Depending on which spore coat protein, the number of LanMs displayed on the surface can be controlled. For example, 1,000-20,000 copies or more per spore can be displayed.

[0044] In the presence of REEs, the LanM selectively binds three ions in the metal binding hands and holds onto them. They can be subsequently released in the presence of low pH (for example, pH less than 2.0) or other strong metal chelators (e.g. EDTA). This allows the harvesting of REEs from a liquid solution, and subsequent titrated release on command.

[0045] The major advantages of using this system are: a) spores are inactive (and can be made permanently dead via engineering); b) spores are highly resistant to temperature, pH, drying, radiation, toxins etc., allowing them to operate in environments naturally rich in REEs (e.g. Acid Mine Drainage), without impacting function; c) spores can bind REEs at pH 2-3 where living bacterial cells would not function; d) due to extreme stability, after releasing the REEs using low pH, the LanM functionalized spores can be reused to capture and release more REEs; and e) they are an inexpensive method to produce and display LanM, compared to other state of the art on purifying LanM from E. coli and attaching to styrene beads.Spores

[0046] Specifically, disclosed herein is an engineered bacterial spore, wherein the spore has been engineered to express a lanthanide binding protein. An example of the lanthanide binding protein is lanmodulin (LanM). Lanthanide binding proteins, such as LanM, can bind rare earth elements (REEs). For example, LanM undergoes a conformational change upon binding to lanthanides and has a 100 million- fold higher affinity for Ln3+than for Ca2+(Daumann, L. CS Cent. Sci. 2021, 7, 11, 1780-1782; October 21, 2021).

[0047] LanM is a small protein of around 12 kDa produced by some methylotrophic organisms and is a natural lanthanide-modulated protein. Wild type M. extorquens LanM protein has a sequence of SEQ ID NO: 1, and can optionally be tagged or otherwise mutated or engineered when used with the spores described herein. In addition to the wild type M. extorquens LanM protein, also contemplated herein are homologs from other organisms. For example, contemplated herein are LanMs having at least four EF hand motifs, with at least one EF hand having at least 3 carboxylate residues, and at least 2 of the EF hand motifs being separated by a space of 10-15 residues. The LanM protein can be further engineered oroptimized to better capture REEs or for ease-of-use with the spores with which they are associated.

[0048] There is a wide range of lanthanide binding proteins which are also contemplated for use with the invention disclosed herein, such as those found in Martin LJ et al., Doublelanthanide-binding tags: design, photophysical properties, and NMR applications, J Am Chem Soc. 2007 Jun 6; 129(22) :7106-13, and CS Synth. Biol. 2024, 13, 3, 958-962.

[0049] Publication Date:February 20, 2024, both of which are herein incorporated by reference in their entirety for their teachings concerning lanthanide binding proteins.

[0050] Reference herein will be made generally to “lanmodulin,” “LanM” or “LanM protein” and should be understood to include the wild type and homologs described herein. “LanM” can include full proteins having one or more LanM units or portions thereof comprising the one or more LanM units. LanM units include at least two EF hand motifs, with at least one EF hand motifs having at least 3 carboxylate residues, and at least 2 of the EF hand motifs being separated by a space of 10-15 residues. For ease of reference, discussion will be made with reference to lanmodulin, LanM or LanM protein and should be understood to include both the full proteins and portions of full proteins having the suitable LanM unit.

[0051] “Binding tags” are also contemplated herein, and can include 12-20 amino acids, for example. Examples of binding tags which can be used with the invention include, but are not limited to, those found in Martin LJ et al. Double-lanthanide-binding tags: design, photophysical properties, and NMR applications. J Am Chem Soc. 2007 Jun 6;129(22):7106- 13 which is herein incorporated by reference in its entirety for its teaching concerning binding tags.

[0052] The lanthanide binding protein can be expressed on the surface of the spore, such as on the spore coat. Spore coats are comprised of protein, have ordered arrays of protomeric subunits, exhibit self-assembly and have protective properties (Driks A. Bacillus subtilis spore coat. Microbiol Mol Biol Rev. 1999;63:1-20; Ricca E, Cutting SM. Emerging Applications of Bacterial Spores in Nanobiotechnology. J Nanobiotechnology. 2003 Dec 15;1(1):6). As dormant metabolically inactive life forms, spores can survive indefinitely in a desiccated state.

[0053] Mature spores are about 1.0 pm in length and can have a spherical or ellipsoidal shape. A single bacterial chromosome is condensed within the center of the spore, and this is known as the “core.” Layers of lipid membrane and modified peptidoglycan can surround the spore core, as well as the spore coat.

[0054] The spore coat proteins can be used to express the lanthanide binding protein In Bacillus subtilis, for example, as many as 25 different coat proteins are present in two distinctcoat layers. For example, Bacillus spores can be comprised of the spore coat proteins cotE, cotV, cotW, cotX, cotY, and cotZ. The lanthanide binding protein can be expressed on one of these, or more than one, or all of these proteins. It can be expressed on the C-terminal or the N-terminal, and the spore coat proteins can also be engineered to accommodate the lanthanide binding protein.

[0055] The engineered spore contemplated herein can be from any bacteria which produces spores. Examples include, but are not limited to, spores from Bacillus, Alicyclobacillus, Sporosarcina or Clostridium. Specific examples include Bacillus subtilis, Bacillus cereus, Bacillus Bacillus coagulans, Alicyclobacillus acidoterrestris, Sporosarcina pasteurii, Sporosarcina ureae, Clostridium perfringens or Clostridium acetobutylicum.

[0056] A variety of methods can be used to express the lanthanide binding protein to the spore coat. For example, a surface display system can be used (Isticato et al. Surface display of recombinant proteins on Bacillus subtilis spores. J Bacteriol. 2001 Nov;183(21):6294-301.) The surface display technique can locate exogenous proteins on the surfaces of spores through fusion vectors. After transformation, heterologous protein together with anchor proteins can be expressed on the surfaces of spores by inducing sporulation (Guoyan et al. Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications. J Microbiol Biotechnol. 2019 Feb 28;29(2): 179-190). Other ways to express the lanthanide binding proteins on the spore coat include, but are not limited to, covalently linking portions to the surface via click chemistry, e.g. use maleimide functionalized LanM and target thiol groups on cysteine to attach the proteins to the spore surface.

[0057] In addition to being engineered to express lanthanide binding protein, the spores disclosed herein can also be engineered so that the expression of other genes is also modulated. By “modulated” is meant that expression of a gene can be upregulated, downregulated, or “knocked out” (removed) entirely. This can be done to alter one or more properties of the spore, such as when or if it returns to a vegetative state, or what conditions are required for such a vegetative state, or to express or repress expression of genes which may assist in the binding or release of REEs.

[0058] Specifically, disclosed herein are spores which have been further engineered so that they are inactive or dead. More specifically, disclosed herein are spores which have been engineered so that they are not capable of returning to a vegetative cell. This can be done in a multitude of ways, such as knocking out, or reducing expression of, a gene or genes encoding receptors for triggering germination. These genes can include, for example, one or more of the following: gerD, gerF, gerA, gerB, or gerK. Further contemplated is an engineered spore wherea germination-specific lytic enzyme is knocked out or removed. These genes can include, for example, one or more of the following: cwlJ and sleB. Multiple other genes can be similarly manipulated to alter their expression.

[0059] The REEs which can be captured by the spores disclosed herein constitute the “f- block” of the periodic table. They’re alternatively referred to as rare-earth metals. Lutetium is often included in the group, despite being a d-block transition metal, as it has common properties with the lanthanides. Thus there are either 14 or 15 members of the group, depending on definitions, with atomic numbers ranging from 57 to 71. The REEs disclosed herein comprise Cerium (Ce), Europium (Eu), Neodymium (Nd), Ytterbium (Yb), Terbium (Tb), Praseodymium (Pr), Dysprosium (Dy), Gadolinium (Gd), Erbium (Er), Holmium (Ho), Lutetium (Lu), Promethium (Pm), Samarium (Sm), Thulium (Tm), and Lanthanum (La). Also disclosed are isotopes of these REEs. The REE binding ligands can bind any of the elements in any oxidation state (e.g., Ln2+, Ln3+, Ln4+, etc.)

[0060] The REEs which have been captured by the engineered spore can be released upon a change of conditions. These conditions can include, for example, pH, temperature, pressure, vibration, or exposure to a chelating agent. For example, LanM can release REEs at low pH. For example, the lanthanide binding protein can capture REEs at a pH of about 2.0 to about 7. Specifically, capture can occur at a pH of about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about9.5, or about 10.

[0061] The REE can then be separated from the spore by contacting the spore with a solution having a pH of less than about 2.4. For example, the solution can have a pH of less than about 2.4, less than about 2.3, less than about 2.2, less than about 2.1 , less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about1 .5, or less. In accordance with the embodiments described herein, a solution having a specific or specified pH is generated by using any solution or composition known in the art to produce that pH, e.g., an HCL solution, an H2S04 solution, an HN03 solution, a solution having a mixture of HCI / NaCI, H2S04 / KHS04 / Na2S04, or other mixture, a solution of glycine, or any other compatible solvent capable of producing the desired solution. The engineered spores disclosed herein can further comprise a cleavable moiety which can allow for the release of the captured REE. Examples of cleavable moieties include, but are not limited to, a 3C tag, enterokinase, Factor X; SUMO protease, TEV protease, or thrombin.

[0062] The engineered spores can also comprise a linker which joins the lanthanide binding protein to the spore coat. This linker can be any length which is appropriate for tethering the lanthanide binding protein to the spore and allowing it to access REEs in the environment. For example, the linker can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long. Further disclosed is that the engineered spore can comprise a FLAG tag between the spore coat and the lanthanide binding protein.Methods and Systems

[0063] Disclosed herein are methods of using the engineered spores described above. For example, disclosed is a method of capturing an REE, the method comprising: a) providing an engineered bacterial spore, wherein the spore has been engineered to express a lanthanide binding protein; b) exposing the engineered bacterial spore to an REE under conditions which allow the lanthanide binding protein to bind the REE; c) providing conditions suitable for releasing the REE from the spore; and d) capturing the REE. These methods can be used to capture REEs under a variety of environmental conditions or in the lab.

[0064] In some embodiments, the steps described are executed once. In other embodiments, the steps or a portion of the steps are executed more than once, for example, 2, 3, 4, 5, or more times. The engineered spores described herein can be recycled, so that they can be reused multiple times. Therefore, contemplated herein is re-using the same spores to repeat the method described above. The spores can be reused 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more times while also maintaining their high REE binding capacity. Also contemplated is using different spores each time.

[0065] In some embodiments, the steps or portions of the steps are repeated until at least about 100%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, or at least about 10% of the REEs are separated from the spore.

[0066] The engineered spores can be added to a column prior to contacting the spores with the REE-containing material. This enables a continuous flow system in which REE-containing material is introduced to the column, and flows through the column. In some embodiments, the flow is from the top to the bottom of the column. In some embodiments, the flow is reversed and the flow is from the bottom to the top of the column.

[0067] Disclosed herein is a method for harvesting REEs from REE-containing material comprising one or more REE binding cycles. In some embodiments, the methods disclosedherein may include two or more REE binding cycles. In some embodiments, each cycle includes steps where the REEs are adsorbed to and desorbed from the spore separately. For example, in some embodiments, upon contacting the engineered spores with the REE- containing material, the REEs are bound to the spore, and then under changing conditions, the REEs are released from the spore.

[0068] The REE can be separated from the spore by contacting the REE-spore complex with a solution comprising a chelator. A chelating agent can include any compound comprising a functional group capable of binding non-REE metal or a REE. For example, in certain embodiments, the chelator or chelating agent can be a mono-carboxylic acid, a di-carboxylic acid, or a tri-carboxylic acid. Non-limiting examples of chelators or chelating agents include EDTA, citrate, dimercaprol, malonate, iminodiacetate, diglycolic acid, hydroxyisobutyric acid, polyaminocarboxylates, hydroxypyridinones, catechols, hydroxamates, acetate, nitrilotriacetate, dipicolinic acid, or a-hydroxyisobutyric acid.

[0069] The REE-containing material can be any material known to contain or suspected to contain REEs. This can be, for example, a solid material, a semi-solid material, or an aqueous medium. Non-limiting examples of suitable materials for use in extraction of REE include leachates derived from rare earth ores (e.g., bastnasite, monazite, loparite, xenotime, allanite, and the lateritic ion-adsorption clays), geothermal brines, coal, coal byproducts, mine tailings, phosphogypsum, acid leachate of solid source materials, REE solution extracted from solid materials through ion-exchange methods, or other ore materials, such as REE-containing clays, volcanic ash, organic materials, and any solids / liquids that react with igneous and sedimentary rocks.

[0070] The spores described herein can also be used for recovering REE from recycled REE- containing products such as, com pact fluorescent light bulbs, electroceramics, fuel cell electrodes, NiMH batteries, permanent magnets, catalytic converters, camera and telescope lenses, carbon lighting applications, computer hard drives, wind turbines, hybrid cars, x-ray and magnetic image systems, television screens, computer screens, fluid cracking catalysts, phosphor-powder from recycled lamps, and the like. In some embodiments, the REEs are recovered from a liquid waste stream from a given industry (e.g., an effluent from a factory that needs to be decontaminated from its REEs or hospital effluents for potential recovery of gadolinium) or a leachate solution coming from these REE containing materials.

[0071] Disclosed herein is a system for purifying, enriching, or extracting REEs, the system comprising using an engineered bacterial spore, wherein the spore has been engineered to express a lanthanide binding protein. As described above, the engineered spore can be affixedto a solid surface, such as a column. In another example, the spores can be affixed to other spores so as to form a complex. The spores can also be free-floating, such as in a liquid media. When affixed, the spores can be affixed by electrostatic or covalent bonds to surfaces, crosslinking to hydrogels, or cross-linking spores to themselves. The spores can be contained via dialysis membrane or crossflow filtration system. Contemplated herein is any system which comprises the engineered spores described herein. This system can be used to capture REEs from a variety of environments or conditions, some of which are disclosed above.Sequences

[0072] The LanM protein can be any of the following:Wild-type M. extorquens LanMPTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEA KKLDPDNDGTLDKKEYLAAVEAQFKAANPDNDGTIDARELASPAGS ALVNLIR (SEQ ID NO: 1)

[0073] Some examples of homologs of the wild type include:

[0074] Example A: RH AL1AKMDMKAIDPDSDGTVSLAEAQDAAAKKFAAMDPDNDGTIDLKEAKGKMAKAKF KKTDADNDGTVDKAEYSALVESAFKAADPDGDGTLDAKELKTPAGQKLLSLIQ (SEQ ID NO: 2)In this protein, one of the EF hands lacks a proline, and EFl lacks a carboxylate at positions 9 and 11 , but it still undergoes a conformational response at free concentrations of rare earth elements in the picomolar range.

[0075] Example B: Hansschlegelia sp.ASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWA RANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK (SEQ ID NO: 3)In this protein (31 % identity), only one of the EF hands has a proline at the second position, and only one has an Asp residue at the first position. However, it still undergoes a conformational response to free concentrations of rare earth elements in the picomolar range and much more weakly to other metals (e.g. calcium).

[0076] Example C: Xanthomonas axonopodisAQAQVQVQDSQQYLQRMDTDGDGRVSLDEYLAWMSYAFDQRDTDHDGVLQGDELPGRRGKPITRAAHRATLIARFARQDANGDGYLSARELLAPPR (SEQ ID NO: 4)EXAMPLESExample 1: Using Engineered Spores to Capture Rare Earth Elements

[0077] Rare earth elements (REE) have become essential and critical elements for renewable energy technologies. Bacillus subtilis wild-type spores were found to be capable of biosorption of REE because of phosphate and carboxylic acid groups on the outermost layers of the spore coat layer (Dong, W et al. 2019. Accumulation and Release of Rare Earth Ions by Spores of Bacillus Species and the Location of These Ions in Spores. Applied and environmental microbiology, 85(17), e00956-19). However, it has also been speculated that the inside of the spore can also influence binding due to the natural dipicolinic acid (DPA) released by germinating spores (Shuster, B., et al. 2019. Contributions of crust proteins to spore surface properties in Bacillus subtilis. Molecular microbiology, 111(3), 825-843). Lanmodulin (LanM) protein is also known to undergo a conformational change once bound to REE with 108-fold selectivity for Ln+3 over Ca+2 (Mattocks, J. A. et al. 2019. A Selective, Protein-Based Fluorescent Sensor with Picomolar Affinity for Rare Earth Elements. lournal of the American Chemical Society 141 (7), 2857-2861). Additionally, while LanM is capable of functioning at low pH’s such as pH 2.5 - 3, LanM binding capabilities decrease by 50% once the pH is 2.2 and desorbing bound REE at pH 1.5, which allows for release and capture (Dong, Z. et al. 2021. Bridging Hydrometallurgy and Biochemistry: A Protein-Based Process for Recovery and Separation of Rare Earth Elements. ACS Central Science 7(11), 1798-1808). Optimal binding conditions were selected based on acid mine drainage effluent, and REE containing groundwater as feedstock (Goodman, A. J. et al. 2023. Rare earth element recovery in hard- rock acid mine drainage and mine waste: A case study in Idaho Springs, Colorado. Applied Geochemistry, 150). B. subtilis LanM engineered spores give a promise for efficient recovery, detection, and purification of REE.

[0078] The effects of pH and salt concentrations on both Bacillus subtilis wild-type and LanM engineered spores were evaluated. A novel luminescent assay was established to detect lanthanides and established ideal pH conditions for adsorbing and releasing Dy+3from wild type and LanM-expressing spores. Concentrations of NaCl and KC1 did not have a significant effect on the adsorption of Dy+3, whereas CaCh seems to interfere with the capturing of the REE. The results are presented within the figures. Overall, the findings show that LanM-expressing spores have a high affinity for REE and can be used for the efficient recovery and purification of these critical metals.METHODS and RESULTS

[0079] Engineering B. subtilis spores for LanM display, genetically expressing lanM fused with coat proteins of B. subtilis (cotE, cotV-Z) using in house designed plasmids. The sequence of the coat proteins of B. subtilis as well as LanM of Methylobacterium extorquens were attained from the GeneBank database and the plasmid design reflected expression of LanM at the N- or C-terminus of the coat protein, spaced by a FLAG tag for easy identification. The plasmids were constructed with a B. subtilis promoter, pCotYZ, with a T7 terminator. The parts were gathered with a GFP dropout shuttle vector containing kanamycin and tetracycline resistance genes using Golden Gate assembly. After amplified in E. coli, the plasmids were then transformed into B. subtilis and selection of the cells with the plasmid was made by antibiotic resistance. After growth and once the sporulation of B. subtilis is induced via nutrient starvation, the coat protein with LanM is displayed on the spores’ surface, with -20,000 copies of LanM per spore.

[0080] LanM display was evaluated by treating the spores with anti-FLAG-FITC conjugated antibody for 1 h shaking at 4°C. Excess and unbound antibody was washed with 1% BSA in PBS and the samples analyzed by flow cytometry. All coat proteins showed display with some variability, with wild type spores were used as a negative control. All the following experiments were done using CotY_FLAG_LanM engineered spores.

[0081] WT and LanM spores REE capture at different pHs. For REE capturing and release assay WT and LanM spores were concentrated to 1 ml of OD600 10 and exposed to the REE at different pHs (1 - 7) for 1 h at room temperature. The spores were then centrifuged, and the supernatant and pellet separated. The supernatant was neutralized to pH 7 for DPA assay to detect how much metal was not bound to the spores. The pellet was incubated in pH 1.5 for 5 min for REE release, and after centrifugation the supernatant which contains the released REEs neutralized to pH 7 for DPA assay. The DPA assay was performed mixing 100 ul of the neutralized supernatants and 100 ul of 100 uM DPA in a 96-well plate. After 5 min incubation, the samples were excited at 262 nm and the emission was set for each REE.

[0082] The WT spores show less efficiency at binding REEs, with the majority of REE being accounted as unbound and binding capacity starting at pH 4. The LanM spores, on the other hand, were able to bind REEs at pH as low as 2, with better capturing capacity starting at pH 3, with 60-70% release of the REEs after one single acid wash.Example 2: LanM spores recyclability using pH and EDTA for REE release.

[0083] LanM spores at a concentration of 10 OD600 units were concentrated by centrifugation at 9,000 x g for 10 minutes. The supernatant was removed and the pellet treated with 200 mg Dy+3 at pH 5. After 1 hour the spores were centrifuged, and the supernatant saved to determine the amount of REE unbound. The pellet containing spores and bound REE was treated with 0.15 M HC1 (pH 1.5) for 5 min, and after centrifugation the supernatant was saved to determine amount of REE released from the spores. The spores were then treated again with the same amount of metals to determine reusability and the same proc was repeated 3 times. The DPA assay for the quantification of unbound and released REE was performed mixing 100 ul of the neutralized supernatants and 100 ul of 100 uM DPA in a 96- well plate. After 5 min incubation, the samples were excited at 262 nm and the emission was set for each REE.

[0084] Firstly, the LanM spores outperformed the WT by capturing virtually all the REEs present in the solution. WT spores completely stops binding after a single pH 1.5 wash, while LanM capture and release capabilities aren’t hindered with multiple washes and stay fairly consistent.REFERENCES FROM EXAMPLE 1:1. Dong, W., Li, S., Camilleri, E., Korza, G., Yankova, M., King, S. M., & Setlow, P. (2019). Accumulation and Release of Rare Earth Ions by Spores of Bacillus Species and the Location of These Ions in Spores. Applied and environmental microbiology, 85(17), e00956- 19.2. Shuster, B., Khemmani, M., Abe, K., Huang, X., Nakaya, Y., Maryn, N., Buttar, S., Gonzalez, A. N., Driks, A., Sato, T., & Eichenberger, P. (2019). Contributions of crust proteins to spore surface properties in Bacillus subtilis. Molecular microbiology, 111(3), 825-843.3. Mattocks, J.A., Ho, J.V., & Cotruvo, J.A. (2019). A Selective, Protein-Based Fluorescent Sensor with Picomolar Affinity for Rare Earth Elements. Journal of the American Chemical Society 141 (7), 2857-2861.4. Dong, Z., Mattocks, J.A., Deblonde, G.J.P., Hu, D., Jiao, Y., Cotruvo J.A., & Park, D.M. (2021) Bridging Hydrometallurgy and Biochemistry: A Protein-Based Process for Recovery and Separation of Rare Earth Elements. ACS Central Science 7(11), 1798-1808.5. Goodman, A. J., Bednar, A. J., Ranville, J. F. (2023). Rare earth element recovery in hard- rock acid mine drainage and mine waste: A case study in Idaho Springs, Colorado. Applied Geochemistry, 150.6. Barela, T.D., Dean Sherry, A. (1976). A simple, one-step fluorometric method for determination of nanomolar concentrations of terbium. Analytical Biochemistry, 71(2), 351- 352.

Claims

CLAIMSWhat is claimed is:

1. An engineered bacterial spore, wherein the spore has been engineered to express a lanthanide binding protein.

2. The spore of claim 1, wherein the lanthanide binding protein is lanmodulin (LanM).

3. The spore of claim 1 or 2, wherein the spore is Bacillus, Alicyclobacillus, Sporosarcina or Clostridium.

4. The spore of claim 3, wherein the spore is Bacillus subtilis, Bacillus cereus, Bacillus Bacillus coagulans, Alicyclobacillus acidoterrestris, Sporosarcina pasteurii, Sporosarcina ureae, Clostridium perfringens or Clostridium acetobutylicum.

5. The spore of any one of claims 1-4, wherein the spore has been engineered so that it is inactive or dead.

6. The spore of claim 5, wherein a gene or genes encoding receptors for triggering germination are knocked out or removed so that the spore cannot be germinated.

7. The spore of claim 6, wherein at least one of these genes are knocked out or removed: gerD, gerF, gerA, gerB, or gerK.

8. The spore of claim 6, wherein a gene encoding a germination- specific lytic enzyme is knocked out or removed.

9. The spore of claim 7, wherein at least one of these genes are knocked out or removed: cwlJ and SleB.

10. The spore of any one of claims 1-9, wherein the lanthanide binding protein is expressed on a spore coat protein.

11. The spore of claim 10, wherein lanthanide binding protein is expressed on at least one of cotE, cotV, cotW, cotX, cotY, and / or cotZ.

12. The spore of claim 10 or 11, wherein the lanthanide binding protein is expressed on either an N or C terminal of the spore coat protein.

13. The spore of any one of claims 1-12, wherein the lanthanide binding protein can bind a rare earth element (REE).

14. The spore of claim 13, wherein the REE comprises lanthanides.

15. The spore of claim 12 or 13, wherein the REE can be released upon a change in environmental conditions.

16. The spore of any one of claims 1-15, wherein the spore comprises a cleavable moiety.

17. The spore of claim 16, wherein the cleavable moiety can be cleaved in order to release the REE.

18. The spore of any one of claims 1-17, wherein a linker joins the lanthanide binding protein to the spore coat.

19. The spore of any one of claims 1-18, wherein a FLAG tag is between the spore coat and the lanthanide binding protein.

20. A method of capturing a rare earth element (REE), the method comprising: a. providing an engineered bacterial spore, wherein the spore has been engineered to express a lanthanide binding protein; b. exposing the engineered bacterial spore to an REE under conditions which allow the lanthanide binding protein to bind the REE; c. providing conditions suitable for releasing the REE from the spore; and d. capturing the REE.

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