Broad-spectrum and effective rare earth enriching via Lanmodulin-displayed Yarrowia lipolytica.

The traditional mining processes of rare earth elements (REEs) are accompanied by the production of a large number of acid mine drainage rich in REEs. A wide-adaptive, low-cost and environmentally friendly biosorbent is an attractive technology to enrich and recycle REEs from the liquid wastes. To construct a broad-spectrum and efficient biosorbent, a novel REEs-binding protein Lanmodulin (LanM) is successfully displayed on the cell surface of a fungus, Yarrowia lipolytica, for the first time, and the adsorption capacities for various REEs are studied. The LanM-displayed Y. lipolytica shows significantly enhanced adsorption capacities for multiple REEs, achieving the highest reported values of 49.83 ± 2.87 mg Yb /g DCW, 50.38 ± 1.46 mg Tm /g DCW, 49.94 ± 3.61 mg Er /g DCW and 48.72 ± 3.09 mg Tb/g DCW, respectively. Moreover, the LanM-displayed Y. lipolytica possesses a high selectivity for REEs over other common metal cations and excellent suitability under acidic conditions. The kinetics and equilibrium analysis of biosorption processes agree well with the pseudo-first kinetic and Langmuir isotherm model. Based on the FTIR and SEM-EDS analysis, the chelation with phosphate/carboxylate groups dominates the Yb binding in LanM-displayed cells, and LanM enhances the adsorption performances by introducing more binding sites with high selectivity towards a wide range of REEs. Thus, the LanM-displayed Y. lipolytica investigated in this study exhibits prosperous potential for the enriching/removal of REEs from acid mine drainage.

Effectively auto-regulated adsorption and recovery of rare earth elements via an engineered E. coli.

Conventional mining processes of rare earth elements (REEs) usually produce REEs-rich industrial waterwastes, which leads to a significant waste of REEs resources and causes serious environmental pollution. Biosorption using engineered microorganisms is an attractive technology for the recovery of REEs from aqueous solution. To regulate the REEs' adsorption and recovery by sensing extraneous REEs, an engineered cascaded induction system, pmrCAB operon containing a lanthanide-binding tag (LBT) for sensing REEs, was incorporated into E. coli in conjunction with a silica-binding protein (Si-tag) and dLBT anchored onto the cell membrane. The sensing and adsorption capacities for Terbium (Tb), a typical study subject of REEs, were enhanced by screening an effective LBT and increasing the dLBT copy number. The adsorption capacity for Tb reached the highest reported value of 41.9 mgg-1 dry cell weight (DCW). After adhering the engineered cells onto the silica column surface through overexpressed Si-tag, a high recovering efficiency (> 90%) of Tb desorption could be obtained with 3 bed volumes of citrate solution. In addition, the engineered cells also possessed fairly good adsorption capacity of other tested REEs. Our findings showed that the recovery of REEs with high efficiency, selectivity and controllability from aqueous solution can be well achieved via specifically bio-engineered strains.

Employing Engineered Enolase Promoter for Efficient Expression of Thermomyces lanuginosus Lipase in Yarrowia lipolytica via a Self-Excisable Vector.

Yarrowia lipolytica is progressively being employed as a workhouse for recombinant protein expression. Here, we expanded the molecular toolbox by engineering the enolase promoter (pENO) and developed a new self-excisable vector, and based on this, a combined strategy was employed to enhance the expression of Thermomyces lanuginosus lipase (TLL) in Y. lipolytica. The strength of 11 truncated enolase promoters of different length was first identified using eGFP as a reporter. Seven of the truncated promoters were selected to examine their ability for driving TLL expression. Then, a series of enolase promoters with higher activities were developed by upstream fusing of different copies of UAS1B, and the recombinant strain Po1f/hp16e100-tll harboring the optimal promoter hp16e100 obtained a TLL activity of 447 U/mL. Additionally, a new self-excisable vector was developed based on a Cre/loxP recombination system, which achieved efficient markerless integration in Y. lipolytica. Subsequently, strains harboring one to four copies of the tll gene were constructed using this tool, with the three-copy strain Po1f/3tll showing the highest activity of 579 U/mL. The activity of Po1f/3tll was then increased to 720 U/mL by optimizing the shaking flask fermentation parameters. Moreover, the folding-related proteins Hac1, Pdi, and Kar2 were employed to further enhance TLL expression, and the TLL activity of the optimal recombinant strain Po1f/3tll-hac1-pdi-kar2 reached 1197 U/mL. By using this combined strategy, TLL activity was enhanced by approximately 39.9-fold compared to the initial strain. Thus, the new vector and the combined strategy could be a useful tool to engineer Y. lipolytica for high-level expression of heterologous protein.

A Novel Cre/lox-Based Genetic Tool for Repeated, Targeted and Markerless Gene Integration in Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

Cohesively Enhancing the Conductance, Mechanical Robustness, and Environmental Stability of Random Metallic Mesh Electrodes via Hot-Pressing-Induced In-Plane Configuration.

Flexible transparent conductive electrode (FTCE) is highly desirable due to the fast-growing flexible optoelectronic devices. Several promising FTCEs based on metal material have been developed to replace conventional indium tin oxide (ITO). The random metal mesh is considered to be one of the competitive candidates. However, obtaining feasible random metal mesh with low sheet resistance, high transparency, good mechanical durability, and strong environmental stability is still a great challenge. Here, a random metal mesh-based FTCE with an in-plane structure, achieved by a facile hot-pressing process, is demonstrated. The hot-pressing process enables the fabrication of highly conductive FTCE with improved mechanical robustness and environmental stability. The in-plane FTCE shows a low sheet resistance of 1.63 Ω·sq-1 with an 80.6% transmittance, low relative resistance increase (RRI) of 7.9% after 240 h 85 °C/85% RH test, and low RRI of 8.0% after 105 cycles of bending test. Besides, various applications of the in-plane FTCE were demonstrated, including the flexible heater, flexible touch screen, and flexible electroluminescence. We anticipate that these results will spark interest in in-plane random metal mesh electrodes and enable the application of random metal mesh in flexible optoelectronic devices.