WO2023136956A1

WO2023136956A1 - Metal-binding molecule production and use thereof

Info

Publication number: WO2023136956A1
Application number: PCT/US2022/081604
Authority: WO
Inventors: N. Cecilia MARTINEZ-GOMEZ; Nathan Michael Good GOOD; Alexa M. ZYTNICK
Original assignee: The Regents Of The University Of California
Priority date: 2022-01-17
Filing date: 2022-12-15
Publication date: 2023-07-20

Abstract

Genetic elements are provided for the biosynthesis of a novel metal-binding molecule (lanthanophore), that can capture trivalent and tetravalent cations, such as lanthanides and actinides.

Description

Metal-binding molecule production and use thereof

This invention was made with government support under DOE Advanced Research Projects Agency-Energy, grant number DE-AR0001337. The government has certain rights in the invention.

[001] Introduction

[002] Metal ions are essential for life-serving in catalytic and structural roles, metal ions are intrinsically involved in many biological processes including respiration, DNA replication, and biosynthesis of metabolic intermediates. The importance of metal ions for life processes is further underscored by the evolution of elegant systems for acquisition and transport. It is estimated that nearly one third of all enzymes in existence require a metal ion for function, yet metals can be biologically inaccessible in most natural environments.

[003] One such metal is iron, which predominantly exists as insoluble ferric (Fe^') oxides in the environment (1). To increase bioavailability of this essential cofactor, microorganisms have evolved to secrete the Fe-chelating small molecules known as siderophores (Greek: iron carrier). Biosynthesized primarily through nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) systems, siderophores fall into five classes based on their Fe-binding moieties: catecholate, phenolate, hydroxamate, diazeniumdiolate and carboxylate (2). In Gram-negative bacteria, TonB complexes provide the energy for siderophore-bound Fe to be brought into the periplasm via an outer- membrane beta-barrel receptor. An ABC transporter then facilitates the transfer of the complex to the cytoplasm, where the siderophore can be recycled for subsequent iterations of this process or can undergo hydrolysis to release its cargo (3—5).

[004] Like Fe, Ln (light atomic numbers 57-62 and heavy atomic numbers 63-71 ) are established members of the metal ions essential for life. As “new life metals,” little is known of how bacteria acquire these poorly bioavailable elements (6-8). The interaction of siderophores with Ln has been studied extensively in vitro.

[005] Ln must enter the periplasm to be used by Ln:PQQ-dependent enzymes, but the details for acquisition and transport of Ln are not entirely understood. A gene cluster encoding a TonB- dependent receptor and an ABC-transport system important for Ln utilization and transport (lut) was discovered and characterized in M. extorquens AMI (24) and the closely related phyllosphere isolate M. extorquens PAI (25). While PQQ has been shown to directly bind Ln (26) and its production is increased when Ln accumulation is elevated during certain growth conditions in M. extorquens AMI (27), secretion of PQQ for direct Ln binding and uptake during bacterial growth has not been shown. [006] During methylo trophic growth, M. extorquens AMI has been demonstrated to utilize complex, insoluble Ln sources such as computer hard drive magnets to supply these enzymes with their Ln cofactor (23). In natural, non-extreme environments, Ln are most often found in poorly soluble oxide and phosphate forms, usually in minerals and ores such as monazite and bastnasite (42), while extreme environments, such as volcanic mud pot water, with high temperatures and low pH, can contain relatively higher levels of soluble Ln. The ability for methylotrophs to thrive in environments rich in complex and poorly soluble Ln sources suggests that a secreted, highly selective Ln-chelator, a “lanthanophore,” could provide a competitive advantage for acquiring these metals in nature, but the details of Ln solubilization remain unknown.

[007] Summary of the Invention

[008] We assessed and disclose the impact of Ln solubility on the transcriptional response and ability of M. extorquens AMI to grow on methanol and acquire the Ln Nd (atomic number 60) using Ln sources of high and low solubility such as NdCI , and Nd₂O3. We found growth with the oxide source was slower than the chloride salt and used RNAseq transcriptomics to identify the genetic basis for this difference. We identified increased expression of alternative methanol oxidation systems, PQQ biosynthesis, and lut genes, consistent with the reduced solubility of Nd₂O3 negatively impacting growth. We also identified a gene cluster conserved across methylotrophs predicted to encode a siderophore-like molecule that was upregulated during growth with Nd₂C>3. Expression of these genes in trans increased growth with Nd₂C>3 and resulted in increased Ln bioaccumulation not only in pure sources but also in the complex source NedFeB magnet swarf.

[009] The invention provides methods and compositions, including genetic elements, for the biosynthesis of a metal-binding molecule (lanthanophore), that can capture trivalent and tetravalent cations, such as lanthanides and actinides. We disclose methods for production of lanthanophore from genome and genetic constructs, i.e. in trans. We disclose experimental evidence for lanthanophore binding to trivalent/tetravalent cations from soluble and insoluble metal sources. The lanthanophore can be used for capture of trivalent/tetravalent cations (such as lanthanides and actinides), separation of trivalent/tetravalent cations, and sensing trivalent/tetravalent cations.

[010] In aspects and embodiments the invention provides:

[Oil] 1. A plasmid or expression vector encoding gene: METAlp4132. [012] 2. A plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4132 through METAlp4138.

[013] 3. A plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4129 through METAlp4138.

[014] 4. A method of producing an iron uptake chelate synthetase (an lucA homolog), comprising expressing in trans a plasmid or expression vector encoding gene: METAlp4132. [015] 5. A method of producing a lanthanophore comprising expressing in trans a plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4132 through METAlp4138. [016] 6. A method of producing a lanthanophore comprising expressing in trans a plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4129 through METAlp4138. [017] 7. A method of binding, separating and/or recovering a trivalent or tetravalent cation, such as lanthanides and actinides, in a medium, comprising contacting the medium with an engineered microbe expressing in trans a plasmid or expression vector encoding gene: METAlp4132.

[018] 8. A method of binding, separating and/or recovering a trivalent or tetravalent cation, such as lanthanides and actinides, in a medium, comprising contacting the medium with an engineered microbe expressing in trans a plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4132 through METAlp4138.

[019] 9. A method of binding, separating and/or recovering a trivalent or tetravalent cation, such as lanthanides and actinides, in a medium, comprising contacting the medium with an engineered microbe expressing in trans a plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4129 through METAlp4138.

[020] 10. A genetically engineered microbe comprising a plasmid or expression vector herein. [021] 11. A composition comprising a lanthanophore produced by a method or microbe herein. [022] 12. A medium comprising a trivalent or tetravalent cation and genetically engineered microbe herein.

[023] 13. A method herein further comprising detecting a resultant lanthanophore.

[024] 14. A method herein wherein the lanthanide is selected from: La, Nd, Sm, Gd, and Dy. [025] 15. A method herein wherein medium is from soluble or insoluble metal sources, including but not limited to, electronic waste, magnet swarf, wastewater, medical waste, and mining tailings.

[026] 16. A method herein further comprising isolating the cation (e.g. lanthanide), for example, from the microbe. [027] 17. Use of a microbe herein for the acquisition, storage and use of the cations, particularly heavy lanthanides.

[028] 18. A method herein wherein the microbe is a yeast (e.g. .S', cerevisiae) or bacterium (e.g. E. coli and Streptomyces or a Methylobacterium (Methylorubrum) species such as M. extorquens or a methylotroph.

[029] 19. A method herein wherein the microbe is a Methylobacteriaceae species, including such as Methylobacterium adhaesivum, Methylobacteriiun aminovorans, Methylobacteriiun aquaticum, Methylobacterium chloromethanicum, M. dichloromethanicum, Methylobacterium extorquens, Methylobacterium fujisawaense, Methylobacterium hispanicum, Methylobacterium isbiliense, Methylobacterium lusitanum, Methylobacterium mesophilicum, Methylobacterium nodulans, Methylobacterium organophilum, Methylobacterium podarium, Methylobacterium populi, Methylobacterium radiotolerans, Methylobacterium rhodesianum, Methylobacterium rhodinum, Methylobacterium suomiense, Methylobacterium thiocyanatum, Methylobacterium variabile, Methylobacterium zatmanii; and Methylorubrum species, such as Methylorubrum aminovorans, Methylorubrum extorquens, Methylorubrum podarium, Methylorubrum populi, Methylorubrum pseudosasae, Methylorubrum rhodesianum, Methylorubrum rhodinum, Methylorubrum salsuginis, Methylorubrum suomiense, Methylorubrum thiocyanatum and Methylorubrum zatmanii.

[030] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[031] Brief Description of the Drawings

[032] Fig. 1A-C. Differences in Ln source solubility affect growth and response of M. extorquens. A) Growth rate of tsmxaF is significantly increased during growth rate and yield on soluble NdCh (purple) compared to insoluble Nd Oi (green) (p values = 0.000095 and 0.024892, respectively). Individual data points represent the mean of three replicates. B) Volcano plot of DEGs identified between NdCh and Nd2Og conditions. Vertical lines signify log2FC cutoffs of < -1 and > 1, while the horizontal line signifies a cutoff of p value < 0.0000005. Select lut cluster, PQQ biosynthesis, Ln-dependent ADH, and uncharacterized biosynthetic cluster genes are highlighted in green, red, purple, and blue, respectively. C) Table depicting transcriptomic differences between ADH, PQQ biosynthesis, and lut cluster genes between NdCU and Nd₂O3, and no Ln conditions.

[033] Fig. 2A-B. LCC bioinformatic characterization. A) Table depicting upregulation of METAlp4129 through METAlp4138 during growth with Nd₂O3 compared to growth with NdCIg, local alignment of each gene, and HMM prediction of the gene products of the cluster. B) The METAlp4129 through METAlp4138 cluster shares high percent identity with BGCs in other methylotrophs and proteobacteria, but shares no similarity with the aerobactin BGC of G. hollisae.

[034] Fig. 3A-B. Characterization of aerobactin as a Ln-chelator in vitro and in vivo. A) CAS/HDTMA assay with aerobactin at three different pH values (6.4, 7.2, 8.1) in MOPS/HEPES buffer (1.5 mM/1.5 mM) with NdCIg. The concentrations of buffer, metal (18.75 pM), and CAS/HDTMA (18.75 pM/50 pM) were kept constant while the concentration of aerobactin varied from 0 pM to 56.25 pM. B) Effect of 2 pM aerobactin on growth of \mxaF with NdCIg or Nd₂O3. Individual data points represent the mean of three replicates. Aerobactin does not significantly affect growth rate (NdCIg p value = 0.4144, Nd₂Og p value = 0.1886) or growth yield (NdCIg p value = 0.8931, Nd₂Og p value = 0.4487) of fsmxaF with either NdCIg or Nd₂Og.

[035] Fig. 4A-D. Effect of expression of LCC in trans during Ln-dependent growth. A) Plasmid map of pAZOOl with METAlp4129 through METAlp4138 (blue) and segments for expression in 5. cerevisiae (grey), M. extorquens (green), and E coli (yellow). B) Growth rate of tsmxciF is significantly increased (p value = 0.045141) during growth with Nd₂Og when its supernatant is replaced with supernatant from AmxaF/p AZ001 (light blue triangles) compared to a control that was resuspended in its own supernatant (dark blue circles). Asterisk and arrow denote the time at which supernatants were manipulated. C) Growth rate of AmxaF/p AZ001 (dark purple) is significantly decreased (p value = 0.000564) during growth on NdCIg compared to csmxaF (light purple). D) Growth rate of AmxaF/pAZOOl (light green) is significantly increased (p value = 0.00054) during growth on Nd₂O3 compared to kmxaF (dark green).

Individual data points represent the mean of three replicates.

[036] Fig. 5A-B. LCC product increases Ln bioaccumulation in M. extorquens.

Bioaccumulation of Lns and Fe³⁺ in AmxaF (solid bars) and AmvaF/p AZ00I (checkered bars) cell pellets as quantified by ICP-MS. Values represent the mean of three replicates. A) A/nw/F/p AZ001 shows significantly increased bioaccumulation of Nd³⁺ (blue) with Nd₂Og and magnet swarf as Ln sources (p values = 0.0073 and 0.0005, respectively), but not with NdCIg (p value = 0.1631). A/mv/F/p AZ001 also shows significantly increased bioaccumulation of the heavy Ln Dy³⁺ (green) and the light Ln Pr³⁺ (yellow) with magnet swarf as a Ln source (p values = 0.0002 and 0.0004, respectively). B) AmxaF/p AZ001 does not significantly increase the bioaccumulation of Fe³⁺ (red) when grown with Nd₂Og, NdCIg, or magnet swarf (p values = 0.0809, 0.0910, and 0.0214 respectively). [037] Fig. 6A-B. Growth of \mxaF is affected by Nd concentration. Growth of \mxaF with 2000 nM, 200 nM, 100 nM, or 50 nM Nd using NdCl₃ and Nd₂O3. Values represent the mean of three replicates. A) When using NdCl₃, growth rate of kmxaF is significantly improved when increasing [Nd] from 50 nM to 100 nM (p value = 0.000011) and from 100 nM to 200 nM (p value = 0.000139), while growth rate is significantly decreased when increasing [Nd] from 200 nM to 2000 nM (p value = 0.001782). Growth yield is also affected, with a significant increase in yield between 2000 nM and 200 nM Nd (p value = 0.043417) and between 50 nM and 100 nM (p value = 0.000236). No significant difference in growth yield is seen between 100 nM and 200 nM (p value = 0.076903). B) When using Nd₂O₃, growth rate of fxmxaF is significantly improved when increasing [Nd] from 50 nM to 100 nM (p value = 0.007329), 100 nM to 200 nM (p value = 0.013001), and 200 nM to 2000 nM (p value = 0.000225). Growth yield is also positively affected, increasing between 50 nM to 100 nM Nd (p value = 0.002288), 100 nM and 200 nM Nd (p value = 0.000564), and 200 nM to 2000 nM Nd (p value = 0.000221).

[038] Fig. 7. HCl-treated Nd₂C>3 improves \mxaF growth compared to Nd₂O₃. Growth of txmxaF with either Nd₂O₃ (green), HC1 treated Nd₂O₃ (blue), or NdCl₃ (purple). Values represent the mean of three replicates. Growth rate is significantly increased when Nd₂O₃ is acidified (p value < .00001) while growth yield remains unaffected (p value = 0.277216). Growth rate with acid treated Nd₂O₃ is significantly slower than growth with NdCl₃ (p value = 0.014747) while growth yield is unaffected (p value = 0.083836).

[039] Fig. 8A-C. Ln solubility affects gene expression in M. extorquens AMI. (A) Principal component analysis (PCA) shows small intragroup and large intergroup variance between reads from kmxaF Nd₂O₃, i^mxaF NdCl₃, and WT no Ln conditions. (B) Genes upregulated in WT no Ln (log2(FC) > 0) or AmxaF Nd₂O₃ (log2(FC) < 0). (C) Genes upregulated in WT no Ln (log2(FC) > 0) or \mxaF NdCl₃ (log2(FC) < 0). Vertical lines signify log₂FC cutoffs of < -1 and > 1, while the horizontal line signifies a cutoff of p value < 0.0000005.

[040] Fig. 9. NdCl₃ increases internal PQQ concentrations. Absorbance at 360 nM of PQQ spiked water (yellow) was used to calculate the internal concentration of PQQ in \mxaF NdCl₃ (purple) and kmxaF Nd₂O₃ (green) samples.

[041] Fig. 10A-B. Kanamycin affects the influence of pAZOOl on \mxaF growth with both Nd₂O₃ and NdCl₃. Values represent the mean of three replicates. (A) When grown with NdCl₃ without kanamycin (squares), A/nvuF/p AZ001 exhibits a small but significant decrease in growth rate (p value = 0.002575) with no effect on growth yield (p value = 0.410508) compared to L\mxaF (circles). When grown with NdCl₃ with kanamycin (triangles), AmwF/pAZOO I exhibits significant decreases in both growth rate (p value = 0.00054) and growth yield (p value = 0.002274) compared to AmxaF (circles). (B) When grown with Nd₂O3 without kanamycin (squares), AmxaF/pAZOOl exhibits a small but significant increase in growth rate (p value = 0.000088) with no effect on growth yield (p value = 0.260195) compared to L\mxaF (circles). When grown with Nd₂O3 with kanamycin (triangles), A/n.w/F/p AZ001 exhibits significant increases in both growth rate (p value = 0.000564) and growth yield (p value = 0.002679) compared to AmxaF (circles).

[042] Fig. 11. Expression of LCC in trans affects growth yield during non-methylotrophic growth. Values represent the mean of four replicates. Compared to MnxaF, AmxaFbpNZMM does not affect growth yield on succinate with no added Ln (p value = 0.076677), significantly increases growth on succinate with NdCl₂ (p value = 0.001075), and significantly increases growth on succinate with Nd₂C>3 (p value ~ 0.00025).

[043] Fig. 12A-B. LCC product affects Ln concentration in AmruF supernatants.

Concentrations of Lns and Fe^J’in hmxaF (solid bars) and AnuaF/pAZOOl (checkered bars) supernatants as quantified by ICP-MS. Values represent the mean of three replicates. A) AmxaF/p AZ001 does not affect supernatant levels of Nd^J+ (blue) with Nd₂C>3 or NdCL (p values “ 0.077328 and 0.135465, respectively), but it significantly increases supernatant levels of Nd³¹' with magnet swarf (p value ~ 0.043313). AmxuF/pAZOOl supernatants also show significantly increased levels of the heavy Ln Dy³’ (green) but not the light Ln Pr^J+ (yellow) with magnet swarf as a Ln source (p values = 0.023595 and 0.05118, respectively). B) AmxaF/p AZ001 does not significantly increase the bioaccumulation of Fe³⁺ (red) when grown with Nd₂O₃, NdClj, or magnet swarf (p values = 0.24253, 0.378189, and 0.464906, respectively).

[044] Description of Particular Embodiments of the Invention

[045] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes. [046] Example: Identification of a biosynthetic gene cluster encoding a novel lanthanide chelator in Methylorubrum extorquens AMI

[047] In this example we describe the changes in the metabolic machinery of Methylorubrum extorquens AMI in response to poorly soluble Nd₂C>3, including 4-fold increases in orphan pqqA genes and the Ln-dependent ADHs xoxFl and exaF compared to growth with soluble NdCI₃. We report the first description of a Ln-chelator biosynthetic gene cluster, encoded by METAlp4129 through METAlp4138, which we named the Lanthanide Chelation Cluster (LCC). The LCC encodes a TonB dependent receptor and NRPS biosynthetic enzymes and is predicted to produce a metal-chelating molecule. As some LCC enzymes share similarity to biosynthetic enzymes producing the Fe-chelating siderophore aerobactin, the capacity of aerobactin for binding lanthanides was tested. It was found that while aerobactin can bind La and Nd at physiological pH, providing exogenous aerobactin did not affect growth rate or yield. The LCC is highly upregulated when AMI is grown using Nd₂O₃ and its expression in trans enabled an increase of neodymium bioaccumulation by over 50%. Expression of the LCC in trans did not affect iron bioaccumulation, providing further evidence that its product is a novel Ln-chelator. Finally, expression of the LCC in trans increased neodymium, dysprosium, and praseodymium bioaccumulation by over 60% using the complex Ln source NdFeB magnet swarf, enabling new strategies for sustainable recovery of these critical Rare Earth Elements. [048] Ln solubility differentially regulates the Ln metabolic network. The presence of light Ln in methanol growth medium effects the regulatory switch from the Ca-dependent MxaFI MDH to the Ln-dependent XoxF MDH (28), but otherwise generates only modest alterations to growth rate, growth yield, and down-regulation of PQQ biosynthesis genes (28, 29). In these studies, however, only soluble chloride compounds of light Ln were tested. To better understand the impacts of Ln solubility on methylotrophy in M. extorquens AMI, we measured methanol growth with soluble NdCl₃, poorly soluble Nd₂O₃, or no Ln source using a strain that lacks catalytically active MxaFI. Ln solubility affected methanol growth of M. extorquens AMI, resulting in significantly faster growth and better yield (p values = 0.000095 and 0.024892, respectively) when grown with a soluble Ln source when comparing growth on NdCl₃ and Nd₂O₃ (Fig. 1A). To gain further insight into how methanol oxidation and Ln utilization and transport machinery respond to Ln solubility, RNA-seq transcriptomics was employed to uncover mechanistic differences between these conditions. Using stringent cut-offs of llog2(FC)l > 1 and p < 0.0000005, we identified 1,468 differentially expressed genes (DEGs) between the NdCl₃ and Nd₂O₃ conditions (Fig. IB).

[049] The upregulation of Ln-dependent MDH xox genes generated by the presence of exogenous LaCl₃ has been studied (29-32). Here, when Nd₂O₃ was provided the genes xoxFl, xoxG, and xoxJ, which respectively encode the primary Ln-dependent MDH, its cognate cytochrome, and a MxaJ-like accessory protein, were among the most upregulated DEGs, exhibiting 5-fold increases when compared to the soluble Ln and no Ln conditions (Fig. 1C). exaF encoding the Ln-dependent ethanol dehydrogenase was shown to have a 3-fold increase in expression during growth with Nd₂C>3 compared to growth with NdCh suggesting that alternative Ln-ADHs are important when Ln are less bioavailable. The use of alternative Ln- ADHs during growth with heavier Ln, e.g. gadolinium (Gd), has been shown previously (27). Additionally, mxaG, mxaJ, and mxal, encoding the Ca-dependent MDH MxaFl’s cognate cytochrome, a periplasmic solute binding protein, and the small MDH subunit, were downregulated 2-fold on average during growth with NdCL compared to the no Ln control.

[050] Previous studies have shown that although upregulation of xoxFl occurs as a response to soluble Ln, concomitant upregulation of the PQQ biosynthetic genes does not occur (29, 31). Similarly, most of the genes encoding the PQQ biosynthetic machinery were not significantly upregulated in the NdCla condition in this study. Interestingly, pqqA2 andpqqA3 were upregulated 4-fold in the NdCf? condition compared to the Nd₂O₃ condition (Fig. 1 C). Internal concentrations of PQQ were measured in cell extracts from cultures grown in both conditions to determine the correlation between the transcript profile and production of PQQ. Cell lysates from cultures grown with NdCl₃ contained significantly more PQQ (120 mM ± 29 mM) than lysates of cultures grown with Nd₂O₃ (24 mM + 28 mM) (Fig. 8A-C). This result suggests that pqqA levels and activity are likely limiting factors for PQQ biosynthesis.

[051] The hit cluster, which encodes a TonB dependent transporter, an ABC transport system, and various periplasmic proteins, is essential for Ln transport. Previous work has shown expression of lutH does not change in response to soluble LaCh (29). In this study, lutH was downregulated 9-fold in the NdCfi condition compared to the no Ln condition and upregulated 9-fold to the Nd₂O3 condition denoting tight regulation of the TonB dependent transporter based on solubility and Ln species (Fig. 1C). The remaining hit genes exhibited 2-fold upregulation on average during growth with Nd₂O3.

[052] Ln solubility differentially regulates a putative siderophore biosynthetic gene cluster. Of the DEGs identified in RNAseq analysis, the genes METAlp4129 through METAlp4138 were the most highly upregulated in the Nd₂O3 condition, with an average increase in expression of 32-fold compared to growth with NdCh (Fig. 2A). Based on local alignment and HMM modeling, the cluster is composed of a TonB dependent receptor, regulatory elements, biosynthetic machinery, and hypothetical proteins (Fig. 2A). antiSMASH predicts the product of the biosynthetic gene cluster (BGC) to be a siderophore, yet the cluster lacks high homology to other siderophore BGCs in the antiSMASH repository. Phyre2 homology modeling of these gene products corroborates antiSMASH analysis, identifying genes in the cluster as siderophore biosynthesis components (Fig. 2A ). This cluster encodes a NRPS system, which is the most abundant biosynthesis pathway type for siderophores. Furthermore, METAlp4132 has 98% sequence identity to the aerobactin synthesis gene iucA and Phyre2 homology modeling exhibits 100% confidence in this classification (Fig 2A).

[053] Presence and similarity of the cluster across the RefSeq non-redundant protein record was accomplished using the cblaster and clinker pipelines (33, 34). The gene cluster is highly conserved across Methylorubrum and Methylobacterium species. Some elements of the cluster are conserved in distantly related proteobacteria, such as Rhodopseudomonas palustris and Hyphomicrobiales bacterium. Interestingly, the cluster is also present in Phreatobacter stygius, a non-methylotroph member of Rhizobiales which lacks the remaining necessary genes for aerobactin synthesis (Fig. 2B). The insights that the gene cluster is a) conserved across methylotrophs, b) predicted to encode a metal chelator, and c) upregulated during growth with an insoluble Ln source all suggest that the cluster can play a critical role in Ln solubilization. For these reasons we have named this gene cluster LCC for “Lanthanide Chelation Cluster” [054] The LCC produces an uncharacterized siderophore-like compound, a novel lanthanophore. Phyre2 modeling suggests some gene products of the LCC resemble those for aerobactin biosynthesis, thus we investigated binding of aerobactin to Ln. Using an indirect dyebased assay with the triphenylmethane dye chrome azurol S (CAS) and the cationic surfactant hexadecyl trimethylammonium bromide (HDTMA), we obtained quantitative measurements of the ability of aerobactin to bind Nd and La under different conditions (pH 6.4, 7.2, 8.1). The assay is based on the findings of Gladilovich and Kuban who used CAS/HDTMA for the determination of Ln³⁺ and Y³⁺ concentrations (35, 36). These assays show that at physiological pH aerobactin is able to bind Nd, La, and Lu, but aerobactin’s Ln binding efficiency is affected by pH (Fig. 3A and Fig. 11). The higher the pH of the solution, the stronger the initial interaction with CAS/HDTMA and the more effective aerobactin is in binding the Ln. Interestingly, differences in the binding capacities with different Ln was observed, as three equivalents of aerobactin was enough to completely displace Lu from CAS/HDTMA at pH 8, but not La or Nd. [055] Because aerobactin can bind Nd at physiological pH, we determined the effect of including aerobactin in our growth conditions. When methanol growth is dependent on the Ln- dependent machinery including XoxF and ExaF, addition of aerobactin to the growth media has no significant effect on growth rate or growth yield when using Nd₂O3 or NdCL (Fig 3B).

[056] Expression of the LCC in trans enables secretion of a compound that improves Ln- dependent growth. A plasmid containing the putative biosynthetic genes of the cluster, METAlp4132 through METAlp4138, was constructed via the DNA assembler method developed by Shao et al. (37) (Fig. 4A). This plasmid was transformed into a AmxaF background, generating A/nvuF/p AZ001 > To determine if the product of the LCC is secreted, AmxaF/pAZOOl was grown with Nd₂O3 to an OD of 0.3, after which the supernatant was collected, filter sterilized, and used to resuspend \mxaF cells grown with Nd₂O3. Growth was compared to a kmxaF culture grown in methanol with Nd₂C>3 that had been resuspended in its own supernatant. Exchange with the AmxaF/pAZOOl supernatant increased \mxaF growth by over 40% (Fig. 4B). Furthermore, expression of this cluster in trans improved the growth on Nd₂O3, increasing growth rate by 47% and growth yield by 20%. Conversely, growth on NdCL was diminished, with growth rate and yield decreasing by 42% and 44%, respectively (Fig. 4CD).

[057] LCC expression in trans improves Ln recovery using the low grade Ln-source NdFeB magnet swarf. Ln bioaccumulation was measured via ICP-MS of AmxaF and AmxaF/p AZ001 cultures grown in methanol minimal medium with either Nd₂O3, NdCp, or 1% pulp density NdFeB magnet swarf. NdFeB magnet swarf is a complex, insoluble Ln source that has commercial value for Ln recovery. Expression of the LCC in trans significantly increased Nd³⁺ bioaccumulation during growth with the insoluble sources Nd₂C>3 and magnet swarf by 56% and 68%, respectively. NdFeB magnet swarf also contains significant amounts of the light Ln Pr³⁺ and the heavy Ln Dy³⁺; bioaccumulation of these Lns, both of which have high technological value, was increased on average by 71% with expression of the LCC in trans (Fig. 5A). Despite these effects with insoluble sources, expression of the LCC did not significantly increase Nd³⁺ with the soluble source NdCL (Fig. 5A). Finally, we probed the specificity of the LCC- generated lanthanophore by measuring Fe³⁺ content in the same cells. Expression of the LCC in trans resulted in no significant differences in Fe³⁺ bioaccumulation between NnxaF and AmroF/p AZ001 with any of the Ln sources (Fig. 5B).

[058] DISCUSSION

[059] Ln, along with scandium and yttrium, are a group of chemically and physically similar elements known as “Rare Earth Elements” (REEs). REEs were named as such due to their insolubility and the lack of their presence in concentrated mineral deposits and rich ores. Despite their name, total concentrations of REE in the Earth’s crust are similar to those of copper, zinc, and lead (38-40) and together these metals comprise about 0.015% of the total material in the Earth’s crust (41). REE are most frequently found in highly insoluble phosphate and oxide mineral forms, restricting bioavailability (42). Though the number of studies investigating various aspects of REE biochemistry, including Ln metabolism in bacteria, have increased considerably over the past decade, nearly all of these studies have used soluble Ln chloride salts as a REE source. Therefore, it is important to understand how solubility and bioavailability of REE impact metabolism and physiology. Here, we leveraged RNAseq transcriptomics to measure changes in gene expression that indicate REE uptake mechanisms related to REE bioavailability and identified the LCC: the first reported BGC encoding a secreted Ln-chelating molecule, a lanthanophore.

[060] We surveyed the RefSeq non-redundant protein record for organisms that encode clusters similar in identity to the LCC, METAlp4129 through METAlp4138, and found that the LCC is conserved across methylotrophs from diverse environments such as the phyllosphere, drinking water, and the human oral cavity (Fig. 2B). The LCC is also present in Beijerinckiaceae indica A TCC9039, which is closely related to methanotrophs and methylotrophs but is itself a generalist (43). B. indica ATCC9039 was isolated from rice paddies, an environment with poorly bioa vailable Lns. The genome of the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV does not contain the LCC genes; as SolV resides in an extreme environment where Ln are more bioavailable, it would not require the LCC product to solubilize Ln.

Mirroring its presence in organisms that need to solubilize Ln in their environments and its absence in organisms that already have soluble Ln available in their environments, we found the LCC to be highly upregulated in response to insoluble Ln compared to soluble Ln (Fig. 2A).

[061] METHODS

[062] Strains and cultivation

[063] .S. cere visiae HZ848 was grown on Yeast YAPD medium at 30C, shaking at 250rpm. Yeast transformants were maintained on synthetic complete drop-out medium lacking uracil. TransforMAX EPI300 E. coli was grown on SOB medium at 37C, shaking at 250rpm. E. coli transformants were maintained on LB medium. E. coli were induced to express pAZOOl using CopyControl Induction Solution (Lucigen). M. extorquens AMI strains were grown in a variant of Hypho medium with half the concentration of PO4. 3 mL cultures with 15 mM succinate were grown overnight in round-bottom glass culture tubes at 30C, shaking at 200rpm. These cultures were then subcultured into fresh media containing 50 mM methanol (volume and vessel defined by the experiment; see below). NdjO? and NdCF were added to final neodymium concentrations of 2 uM unless otherwise indicated. NdFeB magnet swarf (source) was added to a final pulp density of 1%. When necessary, kanamycin, chloramphenicol, ampicillin, and aerobactin were added to final concentrations of 50 ug/mL, 12.5 ug/mL, 50 ug/mL, and 2 uM, respectively.

Growth curves were obtained with 650 uL cultures grown in transparent 48 well plates (Corning) incubated at 30C, shaking at XXX using a Synergy HTX plate reader (BioTek).

[064] RNA-seq transcriptomics [065] 50 mL cultures were grown in 250 mL erlenmeyer flasks to an OD600 of 0.8, which corresponded to mid-exponential growth. Total RNA samples were generated as described previously (Good 2015 ). rRNA depletion, library preparation, and Illumina Hi-Seq sequencing were performed by the Microbial Genome Sequencing Center (MiGS, Pittsburgh, PA). Using KBase (Arkin a/ 2018), reads were aligned with H1STAT2, transcripts were assembled with StringTie, and DEGs were identified using DESeq2.

[066] DNA manipulation

[067] To prepare plasmid pAZOOl, we designed a yeast in vivo DNA assembly strategy based on DNA assembler (Shao et al). Primers used to construct fragments can be found in Table 2. First, the structural genes METAlp4132 through METAlp4133 and METAlp4134 through METAlp4137 were PCR-amplified as two 4.5 kb fragments with 800 bp of overlap from M. extorquens AMI gDNA isolated using the DNeasy PowerMax Soil Kit (Qiagen). 800 bp chimeric joint fragments consisting of 400 bp flanks of the 5. cerevisiae backbone and the structural genes, the E. coli backbone and the structural genes, the M. extorquens backbone and the E. coli backbone, and the M. extorquens backbone and the S', cerevisiae backbone were constructed through overlap extension PCR (OE-PCR) of 400 bp fragments from the provided backbone, M. extorquens AMI gDNA, and pCM66T. To prevent competition with the E coli backbone, the colE locus of pCM66T was removed with OE-PCR through amplification of the components up and downstream of colE locus. A list of primers used to construct these fragments can be found in Table 2. Following electrophoresis, PCR products were purified from a 1% agarose gel using the Thermo Fisher Scientific GeneJET Gel Extraction Kit. 150 ng of each PCR product was combined, dried under N2, and the final mixture was resuspended in 4 uL of Milli-Q double deionized water. Electrocompetent, uracil auxotrophic S. cerevisiae HZ848 were freshly prepared and transformed with this mixture and spread on synthetic complete medium minus uracil (SC-ura) plates to select for homologous recombination of the DNA mixture. Eight prototrophic colonies were grown in liquid SC-ura, lysed using the Zymoprep Yeast Plasmid MiniPrep II Kit (Zymo Research); the plasmid was purified from lysate using the Zymo Research BAC DNA Miniprep Kit (Zymo Research) according to die manufacturer’s protocol. The plasmid was then transformed into electrocompetent Lucigen TransforMax EPI300TM E. coli, which were plated on chloramphenicol to select for successful transformation. Resultant colonies were grown in liquid media overnight and were induced to express the construct the following morning by passaging into fresh media with CopyControl Induction Solution (Lucigen). After five hours of growth, the plasmid was purified using Zymo Research BAC DNA Miniprep Kit and verified via Sanger sequencing (Barker Sequencing). The plasmid was then used in the electroporation of kmxaF to generate strain AntvaF/p AZ001 .

[068] Determining secretion of LCC product

[069] 3 mL cultures were grown in round-bottom glass culture tubes at 30C, shaking at 200rpm. After 40 hours, AmxaF/p AZ001 and AmxaF cultures were spun down at 2000xg and supernatants were collected. The AmxaF control condition was resuspended in its own supernatant that had been 0.22 um filtered, while the kmxaF test condition was resuspended in AmxaF/p AZ001 supernatant that had been 0.22 um filtered.

[070] Chrome Azurol S (CAS) Assay for Spectrophotometric Determination of Ln-binding Ligands

[071] All solutions used for the assay described herein were prepared in ultrapure water (type 1, pH 5.5; Synergy® UV system from Merck Millipore®). Chrome azurol S (Sigma Aldrich), Hexadecyltrimethylammonium bromide (HDTMA, >99%, ACROS Organics), LaCL x 7 H2O (99.999%, Sigma Aldrich) and NdCh x 6 H2O (99.9%, abcr) were commercially obtained and used without further purification. pH measurements were carried out with a FiveEasy pH-meter (Mettler Toledo) which was calibrated prior to use. The assay was performed in transparent 96- well-plates (FALCON®) using an epoch2 plate reader (BioTek). Data was pathlength and baseline corrected using the software Gen5 3.03 and plotted with Origin 2018. Stock solutions were freshly prepared in water on the day of the measurement. 0.25 mM metal solutions (LaCR x 7 H₂O, NdCR x 6 H₂O, LuCR x 6 H₂O) were prepared from 10 mM stock solutions in water which had been stored in aliquots at -25 °C. Aerobactin was prepared as 0.25 mM solution in water. Final concentrations of MOPSO/HEPES buffer (1.5 mM/1.5 mM), CAS (18.75 pM), HDTMA (50 pM), and metal (18.75 pM) were kept constant while the concentration of aerobactin (0 uM - 56.25 uM) was varied. pH values of buffer were adjusted to 6.4, 7.2, and 8.1 with NaOH (6 M) before the solutions were made up to the final volume. The final volume in each well was 200 pL. The reagents were added in the following order: water, buffer, CAS/HDTMA Assay-Mix, metal solution, aerobactin. The plate was then incubated for 5 minutes at room temperature (180 cpm, orbital shake). The UV-Vis spectra were recorded in the range from 350 nm to 750 nm in 1 nm intervals with a scan rate of 8 measurements per data point.

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[074] Table 1. RefSeq accession numbers of organisms with LCC homologs

[075] Table S2. Primers and resulting fragments used to generate pAZOOl

Claims

CLAIMS:

1. A plasmid or expression vector encoding gene: METAlp4132.

2. A plasmid or expression vector encoding a biosynthetic cluster of genes: METAlp4132 through METAlp4138.

3. A plasmid or expression vector encoding a biosynthetic cluster of genes: METAlp4129 through METAlp4138.

4. A method of producing an iron uptake chelate synthetase, comprising expressing in trans a plasmid or expression vector encoding gene: METAlp4132.

5. A method of producing a lanthanophore comprising expressing in trans a plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4132 through METAlp4138.

6. A method of producing a lanthanophore comprising expressing in trans a plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4129 through METAlp4138.

7. A method of binding, separating and/or recovering a trivalent or tetravalent cation, such as lanthanides and actinides, in a medium, comprising contacting the medium with an engineered microbe expressing in trans a plasmid or expression vector encoding gene: METAlp4132.

8. A method of binding, separating and/or recovering a trivalent or tetravalent cation, such as lathanides and actinides, in a medium, comprising contacting the medium with an engineered microbe expressing in trans a plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4132 through METAlp4138.

9. A method of binding, separating and/or recovering a trivalent or tetravalent cation, such as lathanides and actinides, in a medium, comprising contacting the medium with an engineered microbe expressing in trans a plasmid or expression vector encoding biosynthetic cluster of genes: METAlp4129 through METAlp4138.

10. A genetically engineered microbe comprising a plasmid or expression vector of any of claims 1-4.

11. A composition comprising a microbe of claim 10 and a lanthanophore produced by the microbe.

12. A medium comprising a trivalent or tetravalent cation and genetically engineered microbe of claim 10.

13. A method of any of claims 6-9, further comprising detecting a resultant lanthanophore.

14. A method of any of claims 6-9, wherein the lanthanide is selected from: La, Nd, Sm, Gd, and Dy.

15. A method of any of claims 6-9, wherein the medium is from soluble or insoluble metal sources, such as electronic waste, magnet swarf, wastewater, medical waste, and mining tailings.

16. A method of any of claims 6-9, further comprising isolating the cation or lanthanide.

17. Use of a microbe of claim 10 for the acquisition, storage and use of the cations, particularly heavy lanthanides.

18. A method of any of claims 6-9, wherein the microbe is a yeast (e.g. S. cerevisiae).

19. A method of any of claims 6-9, wherein the microbe is a bacterium (e.g. E. coli, Streptomyces or a Methylobacterium (Methylorubrum) species such as M. extorquens).

20. A method of any of claims 6-9, wherein the microbe is a methylotroph.