Engineering saccharomycetes for selectively recovering waste valuable metals as well as construction method and application of engineering saccharomycetes

Abstract

The invention discloses engineering saccharomycetes for selectively recovering waste valuable metals as well as a construction method and application of the engineering saccharomycetes. The tested microorganism is saccharomyces cerevisiae and comprises a gene for coding an exogenous metal ion transport protein, an inducible promoter and a terminator, and the promoter is galactose GAL1; and the terminator is CYC1 (Cytochrome CYC1). According to the invention, metal ion transport protein, metabolic pathway regulation, biomineralization and other synthetic biological tools are utilized, so that the recovery selectivity of valuable metals is improved; a biological system is mild in culture, can adapt to a relatively complex wastewater environment, and is beneficial to large-scale industrial operation; and a green and low-cost preparation mode is provided for preparation of high-added-value products. In a coexistence environment, the engineering yeast selectively recovers valuable metal Cd and synthesizes CdSe quantum dots of 92.20 mg / g dry yeast, and the CdSe quantum dots are 1.29 times that of wild saccharomyces cerevisiae. The system has the advantages of high selectivity, low cost, environmental friendliness and the like, and is suitable for metal resource utilization of electronic wastes, industrial wastewater and other scenes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and resource recovery, and particularly relates to an engineered yeast for selectively recovering waste valuable metals, a construction method and application thereof. BACKGROUND

[0002] Valuable metals in traditional industrial wastewater are often recovered by chemical precipitation or flocculation, and are secondarily utilized by building materials, ash landfill, and goaf filling. These methods have problems of high energy consumption, serious secondary pollution, and low efficiency of selective recovery of valuable metals. Although biological methods (such as natural microorganisms of iron-oxidizing Thiobacillus and Bacillus) are environmentally friendly, they have defects of harsh culture conditions and poor selectivity.

[0003] According to a search, an invention patent with a Chinese patent publication number CN101982551A discloses a method for resource recovery of valuable metals on waste PDP glass by using microorganisms, which includes (1) crushing the waste PDP glass, (2) inoculating the bacterial liquid containing iron-oxidizing Thiobacillus and iron-oxidizing Microspira into the Ag-containing culture medium, and obtaining the target bacterial liquid after successive domestication, (3) inoculating the domesticated bacterial liquid into a fermenter for pre-culture, and then adding the culture liquid into the crushed waste PDP glass particles for leaching at 28-35°C for 6-15 days, and (4) filtering the leaching liquid, and obtaining high-purity Ag, Ni, and Zn by extraction-reverse extraction-reduction displacement of the filtrate. The above-mentioned patent needs to culture microorganisms in an acidic environment with a pH of 1.5-2.5, which is not conducive to large-scale operation of industrialized equipment, and the culture time and leaching time are relatively long. In addition, an invention patent with a Chinese patent publication number CN110759454B discloses a method for metal resource recovery and utilization by using Bacillus extracellular polymers, which centrifuges the activated bacterial liquid of Bacillus, freeze-dries the filtrate to obtain the target extracellular polymer powder, and recovers, degrades, or converts metal ions or rare and precious metals by using the powder. The method uses a mild experimental method to recover metal resources by using the obtained bacterial extracellular polymers, but the selectivity for metals is poor.

[0004] In view of this, we propose an engineered yeast for selectively recovering waste valuable metals, a construction method and application thereof, which aims to improve the selective recovery ability of natural microorganisms for waste valuable metals under mild operating conditions by using gene editing technology, and to synthesize biomass quantum dots with high added value in cells on this basis. SUMMARY

[0005] To overcome the shortcomings of existing technologies for recycling valuable metals, such as high energy consumption, serious secondary pollution, and low selective recycling efficiency, this invention provides an engineered yeast strain for selectively recycling waste valuable metals, its construction method, and its application, thus solving the problem of low selective recycling efficiency of waste valuable metals.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0007] An engineered yeast strain for selectively recovering valuable waste metals, the test microorganism being Saccharomyces cerevisiae, comprising a gene encoding an exogenous metal ion transporter, an inducible promoter, and a terminator, wherein the promoter is galactose GAL1 and the terminator is CYC1.

[0008] The gene encoding the exogenous metal ion transporter is the codon-optimized TpNramp5 gene, and the nucleotide sequence of the codon-optimized TpNramp5 gene is shown in SEQ ID No. 2.

[0009] The codon-optimized TpNramp5 gene is located on the Saccharomyces cerevisiae expression vector pYES2.0 plasmid, named pYES2.0-TpNramp5 plasmid, and the nucleotide sequence of the pYES2.0-TpNramp5 plasmid is shown in SEQ ID No. 3.

[0010] A method for constructing an engineered yeast strain capable of selectively recovering valuable waste metals involves using homologous recombination technology to edit the genes of wild-type Saccharomyces cerevisiae, cloning the gene encoding an exogenous metal ion transporter into the Saccharomyces cerevisiae expression vector pYES2.0 plasmid, inducing expression with an inducible promoter, and terminating transcription with a terminator to obtain the engineered yeast strain.

[0011] The gene encoding the exogenous metal ion transporter is the codon-optimized TpNramp5 gene, the nucleotide sequence of which is shown in SEQ ID No. 2. The codon-optimized TpNramp5 gene is cloned into the Saccharomyces cerevisiae expression vector pYES2.0 plasmid to form the pYES2.0-TpNramp5 plasmid, the nucleotide sequence of which is shown in SEQ ID No. 3. The promoter is galactose GAL1, and the terminator is CYC1.

[0012] The method for selectively recovering valuable waste metals using the engineered yeast includes the following steps:

[0013] S1: Construct engineered yeast according to the method described in claim 5;

[0014] S2: Engineered yeast culture: The engineered yeast was activated using defective solid culture plates, and colonies were picked and transferred to a defective liquid culture medium at pH 5.8 and cultured at 30°C until the stationary phase. The above microbial culture was centrifuged at 4°C, and then resuspended in fresh defective liquid culture medium and concentrated to OD. 600 = 1.5 ± 0.2;

[0015] S3: Selenization process: Add fresh Na2SeO3 to the concentrated engineered yeast culture obtained in step S2, incubate for 24 hours and then centrifuge to obtain selenized engineered yeast cells;

[0016] S4: Recovery reaction: The selenized engineered yeast cells obtained in step S3 are added to the target valuable metal ion working solution for incubation to generate engineered yeast containing high value-added products.

[0017] S5: Extraction process: The engineered yeast containing high value-added products obtained in step S4 is subjected to cell wall lysis by lysing enzymes to dissolve the cell wall and break the cells to obtain high value-added products.

[0018] The defective culture medium mentioned in step S2 is galactose-induced uracil-deficient culture medium SG / -Ura.

[0019] The final concentration of Na2SeO3 in step S3 is 5 mmol / L, and the incubation time is 24 h.

[0020] The target valuable metal ion working solution described in step S4 was prepared using MES buffer with a final concentration of 1 mmol / L and an incubation time of 24 h.

[0021] In step S5, the concentration of the cell lysin is 0.1 mg / mL, the reaction temperature is 30°C, and the reaction time is 10 h; the cell disruptor operates at a temperature of 4°C, a power of 300 W, and a duration of 40 min.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention utilizes gene editing technology to construct engineered bacteria that efficiently enrich valuable metals. By employing synthetic biology tools such as metal ion transporters, metabolic pathway regulation, and biomineralization, the selective recovery of valuable metals (e.g., Cd) is enhanced. The biological system exhibits mild cultivation characteristics, adapting to complex wastewater environments and facilitating large-scale industrial operations. It provides a green and low-cost preparation method for high-value-added products, reducing the consumption of high-purity chemical reagents. In a coexisting environment, the engineered yeast selectively recovers the valuable metal Cd and synthesizes 92.20 mg / g CdSe quantum dots in dry yeast, 1.29 times that of wild-type Saccharomyces cerevisiae. This system possesses advantages such as high selectivity, low cost, and environmental friendliness, making it suitable for the resource utilization of metals in scenarios such as electronic waste and industrial wastewater. Attached Figure Description

[0024] Figure 1 A flowchart illustrating the construction process of genetically engineered bacteria;

[0025] Figure 2 This is a schematic diagram of a bioreactor structure. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. These embodiments are merely exemplary and do not limit the scope of protection of the present invention.

[0027] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; the experimental methods described are conventional methods unless otherwise specified. Wild-type Saccharomyces cerevisiae BY4742, the expression vector pYES2.0 plasmid, and the SG / -Ura-deficient culture medium premix powder are all commercially available.

[0028] Example 1: Construction and culture of engineered yeast:

[0029] Homologous recombination technology was used to construct an engineered yeast strain for gene overexpression. The amino acid sequence (SEQ ID No. 1) of the Nramp5 protein (GenBank: ANT73693.1, hereinafter referred to as TpNramp5) from Polish dwarf wheat (Triticum polonicum) was searched in the NCBI database (https: / / www.ncbi.nlm.njh.gov / ). Reverse translation and codon optimization were performed by Suzhou Genewise Biotechnology Co., Ltd., and the codon-optimized TpNramp5 gene sequence is SEQ ID No. 2, which is a gene encoding a metal ion transporter derived from Polish dwarf wheat. The TpNramp5 (SEQ ID No. 2) gene was cloned into the Saccharomyces cerevisiae expression vector pYES2.0 (ampicillin-tagged) plasmid using 5' HindIII and 3' EcoRI. The TpNramp5 gene was induced by the Saccharomyces cerevisiae inducible promoter galactose GAL1, and transcription was terminated by the CYC1 terminator. After successful sequencing verification, the pYES2.0-TpNramp5 plasmid (SEQ ID No. 3) was obtained.

[0030] Saccharomyces cerevisiae BY4742 was cultured in a yeast extract-tryptone-glucose (YPD) liquid medium containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose, under shaking conditions of 30°C, 180 rpm, and 24 h.

[0031] Take 10 μL of the above yeast culture and streak it on a YPD solid culture plate (i.e., YPD medium with 2% agar added) to obtain a single yeast colony.

[0032] Pick a single yeast colony from a YPD solid culture plate and transfer it to 5 mL of YPD liquid culture medium. Incubate overnight at 30°C and 180 rpm with shaking.

[0033] The yeast culture was centrifuged at 6000 rpm for 5 min to obtain cells, and then washed three times with a certain amount of sterile water (i.e., resuspended in sterile water and centrifuged three times at 6000 rpm for 5 min) to achieve a cell concentration of 1×10⁻⁶. 8 The sample was then aliquoted into 1.5 mL centrifuge tubes, 100 μL per tube.

[0034] Take one tube of the above-dispensed bacterial culture, centrifuge at 6000 rpm for 5 min, discard the supernatant, and place the bacterial cells on ice for inversion.

[0035] Preconversion solution was prepared on ice: 240 μL of 50% polyethylene glycol (PEG) 3350, 36 μL of 1 mol lithium acetate, 10 μL of 10 mg / mL salmon sperm DNA, and 1 μg of pYES2.0-TpNramp5 plasmid DNA.

[0036] Add the above pre-conversion solution to the yeast to be converted, repeatedly blow and aspirate, and then heat shock at 42°C for 40 min.

[0037] The heat-shocked yeast cells were centrifuged at 12,000 rpm for 30 s, the supernatant was discarded, and 1 mL of sterile water was added to resuspend the cells. Then, 10 μL of the resuspended cells were diluted with 1 mL of sterile water, and 100 μL of the diluted solution was evenly spread onto galactose-induced uracil (Ura)-deficient SG / -Ura solid culture plates. The plates were incubated at 30°C inverted for 3–5 days to obtain engineered yeast transformants.

[0038] A single transformant was selected for PCR verification using primers TpNramp5-F (SEQ ID No. 4) and TpNramp5-R (SEQ ID No. 5). The PCR product (expected to be 1273 bp) was subjected to agarose gel electrophoresis (1.5% agarose). Obtaining the correct band indicated that the engineered yeast had been successfully constructed.

[0039] The culture medium SG / -Ura used for engineered yeast is a glucose-free yeast induction medium containing galactose. It is a fully synthetic yeast culture medium with a defined composition. SG / -Ura is missing only uracil and contains 15 essential amino acids other than uracil and adenine. It can be used for the induction expression of pYES2.0 plasmid.

[0040] The specific components of SG / -Ura are: amino acid-free yeast basal nitrogen source medium: 6.7 g / L; amino acid mixture (uracil-free, DO Supplement-Ura): 1.15 g / L; galactose: 20 g / L.

[0041] The SG / -Ura defective screening medium requires sterilization at 121°C for 15 min.

[0042] A single engineered yeast cell was transferred to 100 mL of SG / -Ura liquid medium and cultured at 30°C and 180 r / min until the stationary phase. It was then centrifuged at 6000 rpm for 5 min, resuspended in fresh SG / -Ura medium, and concentrated to OD. 600 = 1.50 ± 0.02.

[0043] Example 2: Application of engineered yeast in the selective recovery of valuable waste metals:

[0044] 1. Construct and culture engineered yeast according to the method in Example 1.

[0045] 2. Selenization process

[0046] Take 50 mL of the above-mentioned engineered yeast concentrate, add 1 mL of fresh Na2SeO3 stock solution (250 mmol / L, prepared from deionized water with resistivity >18.2 MΩ·cm) to make a final concentration of 5 mmol / L, incubate for 24 h, and then centrifuge at 6000 rpm for 5 min to obtain selenized engineered yeast cells.

[0047] The specific preparation steps for the Na2SeO3 mother liquor are as follows:

[0048] 2.16 g of Na2SeO3 was dissolved in 50 mL of deionized water with a resistivity >18.2 MΩ·cm to obtain a 250 mmol / L Na2SeO3 stock solution.

[0049] The main reactions that occur during the selenization of engineered yeast include:

[0050] SeO3 2- + 4GSH + 2H + → GSSeSG + GSSG + 3H2O

[0051] GSSeSG + NADPH + H + GSSeH + GSH +NADP +

[0052] GSSeH → Se 0 + GSH

[0053] GSSeH + GSH → H2Se + GSSG

[0054] 3. Recovery reaction

[0055] The 50 mL selenized engineered yeast obtained by centrifugation was resuspended in 50 mL of 10 mmol / L MES buffer (pH 7.0), and 0.5 mL of Cd was added at once. 2+ and 0.05 mL Zn 2+ Metal ion mother liquor, with final concentrations of Cd 2+ = 1mmol / L, Zn 2+= 0.1 mmol / L, reacted for 24 h, and then centrifuged at 6000 rpm for 5 min to obtain engineered yeast containing CdSe quantum dots.

[0056] The metal ion mother liquor is prepared from deionized water, and the specific operation is as follows:

[0057] Dissolve 0.92 g CdCl2 or 0.68 g ZnCl2 in 50 mL of deionized water with a resistivity >18.2 MΩ·cm (the ZnCl2 stock solution needs to be supplemented with 1% hydrochloric acid to promote Zn...). 2+ Dissolve) to obtain a mother liquor of 100 mmol / L corresponding metal ions.

[0058] The main reactions that occur during the biomineralization process of engineered yeast include:

[0059] Cd 2+ + 2GSH → Cd(GS)2 + 2H +

[0060] HSe - + Cd(SG)2 + H + → CdSe quantum dots (high value-added product) + 2GSH

[0061] 4. Extraction process

[0062] The recovered engineered yeast cells containing CdSe quantum dots were resuspended in a 0.1 mg / mL lysozyme solution and reacted at 30°C and 180 r / min for 10 h. The cell wall-digested engineered yeast cells were then obtained by centrifugation at 6000 rpm for 5 min.

[0063] The engineered yeast, after cell wall digestion, was added to a cell disruptor and disrupted at 4°C and 300 W for 40 min. Then, it was centrifuged at 6000 rpm for 5 min to obtain a supernatant containing CdSe quantum dots.

[0064] The supernatant containing CdSe quantum dots was concentrated by adding it to an ultrafiltration tube with a molecular weight cutoff of 100 KD. The concentrate was then purified by adding it to a dialysis bag with a molecular weight cutoff of 100 KD to obtain a high value-added product.

[0065] In this embodiment, the CdSe quantum dot yield synthesized by engineered yeast in the coexisting environment was 92.20 mg / g dry yeast, which is 1.29 times that of wild-type Saccharomyces cerevisiae (wild-type Saccharomyces cerevisiae CdSe quantum dot yield 71.47 mg / g dry yeast).

[0066] The foregoing has shown and described the basic design, main features, and advantages of this invention. Through the above steps, this invention has established an engineered yeast strain (BY4742::TpNramp5) and applied it to the selective recycling and resource utilization of valuable waste metals.

[0067] The embodiments of the present invention are also applicable to Ca 2+ Mg 2+ Ni 2+ Coexistence scenarios.

[0068] The above embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope thereof, but all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. An engineered yeast fungus for selective recovery of spent valuable metals, characterized in that, The test microorganism is Saccharomyces cerevisiae, which comprises a gene encoding an exogenous metal ion transporter, an inducible promoter and a terminator, wherein the promoter is galactose GAL1, and the terminator is CYC1.

2. The engineered yeast of claim 1, wherein the engineered yeast is capable of selectively recovering the waste valuable metal, and The gene encoding the exogenous metal ion transporter is a codon-optimized TpNramp5 gene, and the nucleotide sequence of the codon-optimized TpNramp5 gene is shown in SEQ ID No.

2.

3. The engineered yeast of claim 2, wherein the engineered yeast is capable of selectively recovering the waste valuable metal, and The codon-optimized TpNramp5 gene is located on a Saccharomyces cerevisiae expression vector pYES2.0 plasmid, and is named as pYES2.0-TpNramp5 plasmid, and the nucleotide sequence of the pYES2.0-TpNramp5 plasmid is shown in SEQ ID No.

3.

4. A method for constructing an engineered yeast for selective recovery of waste valuable metals, characterized by, The gene encoding the exogenous metal ion transporter is cloned into the Saccharomyces cerevisiae expression vector pYES2.0 plasmid by using the homologous recombination technology, and the gene is expressed by the inducible promoter and terminated by the terminator to obtain the engineered yeast.

5. The method for constructing an engineered yeast for selective recovery of waste valuable metals according to claim 4, wherein, The gene encoding the exogenous metal ion transporter is a codon-optimized TpNramp5 gene, and the nucleotide sequence of the codon-optimized TpNramp5 gene is shown in SEQ ID No. 2; the codon-optimized TpNramp5 gene is cloned into the Saccharomyces cerevisiae expression vector pYES2.0 plasmid to form the pYES2.0-TpNramp5 plasmid, and the nucleotide sequence of the pYES2.0-TpNramp5 plasmid is shown in SEQ ID No. 3; the promoter is galactose GAL1, and the terminator is CYC1.

6. The method for selectively recovering waste valuable metals using the engineered yeast of claim 1, characterized in that, The method comprises the following steps: S1: constructing the engineered yeast according to the method of claim 5; S2: engineering yeast culture: the engineering yeast was activated by using a deficient solid culture plate, and the colony was picked to a deficient liquid culture medium with pH 5.8 and cultured at 30°C to the stable phase; the microbial culture liquid was centrifuged at 4°C, and then resuspended and concentrated to OD 600 = 1.5± 0.2 with fresh deficient liquid culture medium. S3: seleniumization process: adding fresh Na2SeO3 into the concentrated solution of the engineered yeast obtained in step S2, and centrifuging after incubation for 24 h to obtain the seleniumized engineered yeast; S4: recovery reaction: adding the seleniumized engineered yeast obtained in step S3 into a target valuable metal ion working solution to generate the engineered yeast containing high-value-added products; S5: extraction process: dissolving the cell wall of the engineered yeast containing high-value-added products obtained in step S4 by lysozyme, and crushing the cells to obtain the high-value-added products.

7. The method for selective recovery of waste valuable metals according to claim 6, characterized in that, The deficient solid culture plate and the deficient liquid culture medium in step S2 are both galactose-induced uracil-deficient culture medium SG / -Ura.

8. The method for selective recovery of waste valuable metals according to claim 6, characterized in that, The final concentration of Na2SeO3 in step S3 is 5 mmol / L, and the incubation time is 24 h.

9. The method for selective recovery of waste valuable metals according to claim 6, characterized in that, The target valuable metal ion working solution in step S4 is prepared by MES buffer, and the final concentration is 1 mmol / L, and the incubation time is 24 h.

10. The method for selective recovery of waste valuable metals according to claim 6, characterized in that, In step S5, the concentration of lysozyme is 0.1 mg / mL, the reaction temperature is 30°C, and the reaction time is 10 h; the working temperature of the cell disrupter is 4°C, the working power is 300 W, and the working time is 40 min.

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