1. Overview

Throughout the engineering design cycles of our project, team HAOLVE-Nanjing designed and characterized 4 new basic parts and 6 new composite parts. Our goal was to enhance the rare earth element (REE) adsorption capacity of Pseudomonas kunmingensis (P. kunmingensis) HL22-2^T by genetically reprogramming its inorganic polyphosphate (polyP) metabolic network. This synthetic biology approach aims to advance more efficient and sustainable REE recovery processes.

1.1. New Basic Parts

We registered and uploaded basic parts BBa_25WRSMIP, BBa_25CVSUA1, BBa_25TC4IDA.

We also uploaded basic parts BBa_259STERK as a backbone and proved it worked as we expected.

1.2. New Composite Parts

We have designed eight composite parts (BBa_2502RTST, BBa_253PFMUM, BBa_25J1CVVU, BBa_25WN4TIV, BBa_259D2DLA, BBa_2566HVYF) in order to overexpress the corresponding genes in cells.

1.3. Updated parts

2. Pre-experiments-Screening of Microbial Chassis with Moderate Phosphate-Solubilizing Capacity

2.1 The native phosphate-solubilizing assay

Rare earth elements (REEs) are traditionally extracted using chemical methods like sulfate roasting and alkaline leaching, which are environmentally damaging. In contrast, bacterial biosorption offers a sustainable and eco-friendly alternative due to its low cost, ease of cultivation, and biodegradability. Functional groups on bacterial cells—particularly phosphate, carboxyl, and hydroxyl groups—serve as key binding sites for REE ions, forming stable complexes through coordination bonds. Among these, phosphate groups exposed by cell wall components such as teichoic acids and lipopolysaccharides are especially effective, under high local ion concentrations even leading to the formation of REE phosphate nanoparticles (e.g., REEPO₄) on or within the cells.

In REE tailings and mining regions, Pseudomonas species frequently emerge as dominant microbiota due to their remarkable environmental adaptability and metabolic diversity, making them particularly suitable for REE biosorption and recovery applications. Pseudomonas kunmingensis (P. kunmingensis) HL22-2^T, isolated from a phosphate mining area in Kunming, Yunnan, represents a promising candidate within this genus. This Gram-negative, exopolysaccharide-producing, strictly aerobic, monotrichous bacterium has demonstrated significant potential for REE adsorption.

Phosphate-solubilizing microorganisms (PSMs) secrete organic acids to dissolve inorganic phosphate from minerals, and their cell surfaces, abundant in phosphate and carboxyl groups, simultaneously enable efficient REE adsorption. To evaluate these properties, P. kunmingensis HL22-2^T was cultured in PVK medium with tricalcium phosphate as the sole phosphorus source, along with reference PSMs: Shewanella oneidensis (S. oneidensis) MR-1, Escherichia coli (E. coli) DH5α, and Corynebacterium glutamicum (C. glutamicum) ATCC 14067. After two days of incubation, phosphate solubilization capacity toward Ca₃(PO₄)₂ was quantified using the molybdenum-antimony anti-spectrophotometric method. The results revealed the following efficiency ranking: C. glutamicum ATCC 14067 > E. coli DH5α > P. kunmingensis HL22-2^T > S. oneidensis MR-1, indicating P. kunmingensiss possesses moderate phosphate-solubilizing capability among the tested strains.

Figure 1. Assessment of phosphate-solubilizing capabilities among selected microbial strains

2.2 REE Adsorption Capacity of wild-type P. kunmingensis

Strains with moderate phosphate-solubilizing activity secrete optimal amounts of organic acids, enabling sufficient phosphate release for growth while avoiding excessive acidification or abrupt PO₄³⁻ release. This controlled phosphate availability allows more rare earth ions to bind directly to surface functional groups—primarily phosphate and carboxyl groups—promoting effective biosorption and enrichment of REEs on the biomass. Then, we employed inductively coupled plasma mass spectrometry (ICP-MS) to evaluate the biosorption capacity of P. kunmingensis HL22-2^T for the REEs— Lanthanum (La³⁺), Praseodymium (Pr³⁺), and Neodymium (Nd³⁺). The results confirmed the strain’s inherent ability to adsorb REEs, establishing P. kunmingensis HL22-2^T as a promising host for subsequent genetic engineering studies.

Figure 2. ICP-MS analysis of REE adsorption by wild-type P. kunmingensis
(RE ion CONC. (ng/g): The mass of adsorbed rare earth ions per gram of wet biomass)

2.3 Pre-experimental conclusion

Through comparative analysis with other PSMs including C. glutamicum, we determined that P. kunmingensis HL22-2^T exhibits moderate phosphate-solubilizing activity—sufficient to supply bioavailable phosphorus for growth while avoiding excessive acidification or abrupt phosphate release. This balanced metabolic characteristic enables the strain to maintain environmental pH stability while providing adequate surface functional groups (e.g., phosphate and carboxyl groups) for efficient complexation with REEs. These characteristics distinguish P. kunmingensis HL22-2^T from other common strains and make it an ideal host for subsequent genetic engineering aimed at enhancing REE biosorption.

3. Cycle one: Engineering P. kunmingensis for Enhanced REE Biosorption

3.1 Design: Construction of Plasmids for Phosphate Metabolic Engineering

Polyphosphate (PolyP) serves as a dynamic intracellular reservoir and source of soluble phosphate, playing a central role in cellular phosphate metabolism. Key enzymes such as polyphosphate kinases (PPK1 and PPK2) and exopolyphosphatase (PPX) tightly regulate PolyP turnover, maintaining phosphate homeostasis and influencing the levels of critical metabolites including ATP, GTP, and free phosphate.

To systematically reprogram the phosphate metabolism regulatory network, we constructed three recombinant expression plasmids based on the pCM28 plasmid backbone to heterologously express codon-optimized ppk2-I from Pseudomonas aeruginosa (P. aeruginosa)-P.KPPK2-I, ppx from Escherichia coli (E. coli)-P.KPPX, and ppk1 from Citrobacter freundii (C. freundii)- P.KPPK1 in P. kunmingensis HL22-2^T. These enzymes complementarily regulate phosphate metabolism: PPK2 drives GTP and ATP regeneration, PPX hydrolyses polyphosphate to release inorganic phosphate, and PPK1 promotes polyphosphate synthesis. This design enables systematic perturbation of the phosphate metabolic network, thereby screening for engineered strains with enhanced rare earth element biosorption capacity.

Figure 3. Plasmid construction schemes

3.2 Build-Construction of Engineered P. kunmingensis Strains

To enhance translation efficiency in P. kunmingensis HL22-2^T, a ribosome binding site (RBS) sequence (BBa_K4604004 from the iGEM Parts Registry) was inserted upstream of each target gene. The three composite parts were amplified by polymerase chain reaction (PCR) and cloned into the pCM28 backbone, resulting in plasmids: pCM28-Modified T7 RBS-P.KPPK2-I, pCM28-Modified T7 RBS-P.KPPX, and pCM28-Modified T7 RBS-P.KPPK1. Successful assembly of each recombinant plasmid was confirmed through agarose gel electrophoresis.

Figure 4. A-C. pCM28-Modified T7 RBS-P.KPPK2-I；D-F. pCM28-Modified T7 RBS-P.KPPK1; G-I. pCM28-Modified T7 RBS-P.KPPX (Targeted gene amplification; Plasmid double-enzyme digestion; Colony PCR verification)

3.3 Test- Characterization of Engineered P. kunmingensis Strains

3.3.1 Protein Expression Analysis

The verified plasmids were then individually transformed into competent P. kunmingensis cells, generating the engineered strains KPPK2-I, KPPX, and KPPK1. Protein expression analysis using sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) detected specific bands at approximately 45.1 kDa and 80 kDa, confirming the successful expression of P.KPPX and P.KPPK1, respectively. In contrast, no band was observed at the expected molecular weight of 40.8 kDa for PPK2-I, indicating unsuccessful expression of this protein in P. kunmingensis HL22-2^T. Furthermore, alterations in intracellular polyP content also revealed successful expression of both P.KPPX and P.KPPK1 proteins within P. kunmingensis.

Figure 5. SDS-PAGE analysis of recombinant proteins in engineered strains

Figure 6. Detection of intracellular polyP content in bacteria

3.3.2 REE Adsorption Performance

To evaluate the enhancement of REE adsorption capacity, engineered strains KPPX and KPPK1 were co-cultured with REE solutions, respectively. After incubation, bacterial cells were harvested by centrifugation and the adsorbed REEs were quantified using inductively coupled plasma mass spectrometry (ICP-MS). The results demonstrated that, compared to the WT strain, the KPPX strain exhibited significantly reduced adsorption capacity for La3+, Pr3+ and Nd3+. In contrast, the KPPK1 strain showed markedly enhanced REE adsorption, with increases of 207%, 48%, and 26% for La3+, Pr3+ and Nd3+, respectively. These findings confirm that expression of polyphosphate kinase PPK1 effectively improves the adsorption capacity of P. kunmingensis HL22-2^T for REEs.

Figure 7. ICP-MS analysis of REE adsorption by engineered P. kunmingensis
(RE ion CONC. (ng/g): The mass of adsorbed rare earth ions per gram of wet biomass)

3.4 Learn

Based on the structural properties of polyP as a phosphate polymer and the high affinity of inorganic phosphate for REEs, we postulated that strain KPPK1 might mediate changes in intracellular and extracellular phosphate levels. Consequently, we systematically monitored the dynamic changes in intracellular polyP content and extracellular phosphate concentration during the bacterial REE adsorption process, aiming to elucidate the mechanism underlying the enhanced REE adsorption capacity observed in the engineered strain KPPK1.

3.4.1 Detection of intracellular polyP levels

A two-day biosorption experiment was conducted in an aqueous solution containing 100 μM REEs, during which dynamic changes in intracellular polyP levels and extracellular phosphate concentrations were systematically monitored. The results revealed that, due to the absence of external phosphate sources, the bacteria were compelled to degrade their polyP reserves to obtain inorganic phosphate for maintaining essential cellular functions, leading to a continuous decline in intracellular polyP over the 48-hour period. Notably, the presence of REEs significantly accelerated polyP hydrolysis, with La3+ showing the most pronounced effect.

Figure 8. Detection of intracellular polyP content in bacteria

3.4.2 Detection of extracellular free inorganic phosphate levels

This enhanced degradation promoted the release of inorganic phosphate, which subsequently demonstrated strong binding affinity with REEs in the solution. This interaction was confirmed by the significantly lower accumulation of free phosphate in the rare earth solution compared to the control group. Particularly noteworthy was the observation that in the 100 μM La³⁺ solution experiment, the increase in supernatant phosphate was only 10% of that in the control group. These findings are consistent with previous ICP-OES analytical results, collectively demonstrating the exceptional La³⁺ adsorption capacity of the engineered KPPK1 strain.

Figure 9. Detection of phosphate content in culture

3.4.3 Morphological Characterization of KPPK1 after Rare Earth Element Adsorption

To further validate the mechanism of REE adsorption, we characterized the morphological and elemental composition of P. kunmingensis following incubation in La³⁺ solution. Transmission electron microscopy (TEM) revealed a distinct electron-dense granular coating surrounding bacterial cells in the La³⁺-exposed group, which was absent in the untreated control. Energy-dispersive X-ray spectroscopy (EDS) confirmed the presence of lanthanum in these surface deposits, indicating that La³⁺ ions underwent coordination and precipitation with free phosphate to form rare earth phosphate complexes predominantly deposited on bacterial surfaces.

Figure 10. A) TEM image of the naked KPPK1 strain. B) TEM image of the KPPK1 strain after co-cultivation with La³⁺. C) EDS elemental analysis of the KPPK1 strain after La³⁺ exposure

3.4.4 Conclusion

While heterologous expression of P.KPPK2-I was not achieved in P. kunmingensis, our study yielded key insights into polyP-mediated REE biosorption. The P.KPPK1-expressing strain exhibited markedly enhanced REE adsorption, attributed to its ability to accumulate intracellular polyP and supply abundant surface phosphate groups for REE binding. In contrast, P.KPPX expression led to premature polyP hydrolysis and reduced adsorption, underscoring the importance of temporal control in phosphate metabolism.

Mechanistic studies revealed that P.KPPK1 expression elevated intracellular polyP levels in P. kunmingensis HL22-2^T. During REE exposure, stored polyP was hydrolyzed and released as inorganic phosphate, which complexed with REE cations (e.g., La³⁺) to form surface-associated precipitates. This localized precipitation mechanism drove the enhanced REE adsorption observed in the KPPK1 strain.

These findings highlight the synergy between phosphate metabolism and REE biosorption, establishing engineered polyP management as an effective strategy for improving microbial REE recovery in bioremediation and resource recycling applications.

4.Next Plan

4.1 Optimization of PPK2-I Expression

The heterologous expression of P.KPPK2-I was not detected in P. kunmingensis, which may be due to improper protein folding, rapid proteolytic degradation, or translational incompatibility within the host. Future work will explore alternative codon optimization algorithms tailored to Pseudomonas, test PPK2 orthologs from different microbial sources, and evaluate the use of weaker promoters or fusion tags to enhance soluble expression. Additionally, co-expression of chaperone proteins may be applied to assist proper folding and stability.

4.2 Exploration of Endogenous Polyphosphate Kinases

Given the presence of native polyphosphate metabolism machinery in P. kunmingensis, we plan to construct strains overexpressing endogenous polyphosphate kinases (PPKs) under inducible promoters. This approach will help elucidate the specific role of native enzymes in REE biosorption and avoid potential incompatibility issues associated with heterologous expression. Systematic comparison of REE uptake between strains expressing endogenous and exogenous PPKs will provide insights into the evolutionary adaptation of host-specific phosphate regulation.

4.3 Directed Evolution and Host Engineering of PPK1

Building on the success of polyphosphate kinase PPK1 in enhancing REE adsorption, we will implement a directed evolution campaign to generate PPK1 variants with improved polymerization activity and stability. A high-throughput screening platform will be established based on polyP accumulation or phosphate release assays. In parallel, we will extend our engineering strategy to other phosphate-solubilizing hosts to evaluate the transferability of the polyP-enhanced REE biosorption phenotype. These efforts will help identify universal and host-specific determinants for efficient REE recovery.