Yarrowia lipolytica recombinant bacterium capable of enhancing acid production capacity and application of Yarrowia lipolytica recombinant bacterium in bioleaching of rare earth

Abstract

The invention discloses yarrowia lipolytica recombinant bacteria capable of enhancing acid production capacity and application of the yarrowia lipolytica recombinant bacteria in rare earth bioleaching, and belongs to the field of bioengineering and hydrometallurgy. According to the invention, a yarrowia lipolytica Polf strain is modified through genetic engineering: a Polf-pJQ08 strain of an overexpressed homologous YlAMPD gene is constructed, a Polf-pJQ09 strain of an overexpressed heterologous AnYHM2 gene is constructed, and a Po1f-pJQ12 strain co-expressed by the two genes is constructed. The citric acid secretion amount of the modified strain is remarkably increased, and the rare earth leaching rate of fermentation supernate of the strain is remarkably higher than that of a control group. The recombinant strain can efficiently produce acid, improves the rare earth leaching efficiency, and provides a new scheme for green extraction of rare earth elements.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and hydrometallurgical technology, specifically relating to a recombinant Yersinia lipolyticis strain with enhanced acid-producing ability and its application in rare earth bioleaching. Background Technology

[0002] Rare earth elements (REEs), as indispensable strategic resources in modern industry, are widely used in new energy, catalysts, and defense technology. Their efficient recovery is of great significance for sustainable development. Currently, the main extraction process for REEs from rare earth minerals, both domestically and internationally, is chemical leaching. This process requires large amounts of acid, resulting in significant environmental pollution, high energy consumption, and large quantities of chemical reagents. Against this backdrop, biometallurgical technology has become a research hotspot for the green extraction of REEs.

[0003] Acid-producing microorganisms generate acidic metabolites during their growth, which lower the pH of the culture environment. This is beneficial for the acidolysis of rare earth minerals. Furthermore, some acidic metabolites can act as organic ligands to complex rare earth ions. *Yarrowia lipolytica* is a representative acid-producing fungus. Due to its naturally high production of organic acids (such as citric acid and oxalic acid), it can promote the leaching of rare earth ions through acidolysis and complexation. Simultaneously, it utilizes functional groups in its cell wall to achieve specific adsorption of metal ions, giving *Yarrowia lipolytica* a unique advantage in rare earth resource extraction. Citric acid has been proven to be a key organic acid in the bioleaching process; it is more effective than other organic acids in leaching rare earth elements from monazite. Both *Aspergillus niger* and *Yarrowia lipolytica* have been reported as high-potential citric acid-producing strains, but their metabolic characteristics differ. *Aspergillus niger* produces oxalic acid during leaching, which forms insoluble oxalate precipitates with rare earth ions, affecting the leaching efficiency. *Yarrowia lipolytica*, however, does not contain oxalic acid in its metabolites.

[0004] Therefore, developing a lipophilic yeast strain that can efficiently produce acid is of great significance for improving the leaching efficiency of rare earth ions. Summary of the Invention

[0005] Based on the above-mentioned technical problems, the purpose of this invention is to provide a recombinant strain of Yersinia lipolyticis that has been genetically engineered to enhance its acid-producing ability, thereby improving the bioleaching efficiency of rare earth elements.

[0006] On the one hand, the present invention provides a recombinant strain of Yersinia lipolytica, wherein the YlAMPD gene and / or AnYHM2 gene are overexpressed in the Yersinia lipolytica starting strain to obtain the recombinant strain.

[0007] Specifically, the YlAMPD gene is derived from Yersinia lipophila, and its nucleotide sequence is shown in SEQ ID NO.1. The AnYHM2 gene is derived from Aspergillus niger, and its nucleotide sequence is shown in SEQ ID NO.2.

[0008] Specifically, the starting strain of Yersinia lipolytica is Yersinia lipolytica Po1f strain.

[0009] Specifically, the recombinant strain also contains the selection marker gene ura3, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0010] On one hand, the present invention provides a method for preparing a recombinant strain of Yersinia lipolytica with enhanced acid-producing ability, comprising the following steps:

[0011] (1) Construct recombinant plasmids containing the selection marker ura3 gene, the YlAMPD gene expression cassette, and / or the AnYHM2 gene expression cassette;

[0012] (2) The recombinant plasmid described in (1) is treated with restriction endonuclease to obtain a target fragment containing the selection marker ura3 gene, the YlAMPD gene expression cassette and / or the AnYHM2 gene expression cassette. The target fragment is then transformed into the Yersinia lipophila starting strain to obtain the recombinant strain.

[0013] Preferably, the YlAMPD gene expression cassette further includes a promoter and a terminator, preferably, the promoter is TEF1 and the terminator is PGK; preferably, the AnYHM2 gene expression cassette further includes a promoter and a terminator, preferably, the promoter is GPD and the terminator is PEX20.

[0014] Specifically, the target fragment is integrated into the genome of the Yersinia lipolyticis starting strain via homologous recombination. Preferably, the Yersinia lipolyticis starting strain is the Yersinia lipolyticis Po1f strain.

[0015] Specifically, the target fragment is transferred using the lithium acetate conversion method.

[0016] The present invention also provides the application of the recombinant strain of Yersinia lipolyticis in increasing citric acid secretion or in the extraction of rare earth elements.

[0017] Specifically, the application includes fermenting the recombinant strain, mixing the fermentation supernatant with rare earth ore, and leaching rare earth elements by shaking. Preferably, the rare earth ore is an ion-adsorption rare earth ore.

[0018] Specifically, the fermentation conditions are: 27-35℃, 4-7 days, and a rotation speed of 150-300 rpm; the leaching conditions are: 40-48℃, 5-9 days, and a rotation speed of 150-300 rpm. Preferably, the fermentation conditions are: 28-31℃, 4-6 days, and a rotation speed of 180-250 rpm; the leaching conditions are: 43-46℃, 6-8 days, and a rotation speed of 180-250 rpm.

[0019] Beneficial Effects: This invention utilizes genetic engineering to obtain recombinant *Yersinia lipophila* strains, constructing the Polf-pJQ08 strain overexpressing the homologous YlAMPD gene, the Polf-pJQ09 strain overexpressing the heterologous AnYHM2 gene, and the Po1f-pJQ12 strain co-expressing both genes. The modified strains exhibit significantly increased citric acid secretion compared to the control strain. In particular, the co-expressed Po1f-pJQ12 strain showed a 62.84% increase in citric acid secretion and a 70.54% increase in rare earth element leaching rate, making it more environmentally friendly and efficient, providing a new approach for rare earth element extraction. Attached Figure Description

[0020] Figure 1 This is a diagram of the pJQ08 plasmid and the results of its electrophoresis verification. Figure 1 Image A shows the pJQ08 plasmid pattern, and image B shows the pJQ08 electrophoresis verification results.

[0021] Figure 2 This is a diagram of the pJQ09 plasmid and the results of its electrophoresis verification. Figure 2 Image A shows the pJQ09 plasmid pattern, and image B shows the pJQ09 electrophoresis verification results.

[0022] Figure 3 These are PCR verification diagrams for different strains, where lanes 1-3 represent strain Po1f-pJQ03, lanes 4-6 represent strain Po1f-pJQ08, and lanes 7-9 represent strain Po1f-pJQ09.

[0023] Figure 4 These are the pJQ12 plasmid map and electrophoresis verification results. Figure 4 Image A shows the pJQ12 plasmid pattern, and image B shows the pJQ12 electrophoresis verification results.

[0024] Figure 5 This is a PCR verification image of strain Po1f-pJQ12.

[0025] Figure 6 It refers to the concentration of citric acid in the fermentation broth of strains Po1f-pJQ08, Po1f-pJQ09, Po1f-pJQ12 and the blank control strain Po1f-pJQ03.

[0026] Figure 7 This is a graph showing the leaching rate of rare earth elements. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials and reagents used are commercially available.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Example 1: Construction of YlAMPD and AnYHM2 single-gene overexpression strains Po1f-pJQ08 and Po1f-pJQ09

[0031] (1) Construction of integrative plasmid pJQ08

[0032] Using the genome of *Y. lipolytica* as a template, PCR amplification was performed using primers JQoli_19 / JQoli_20 to obtain the YlAMPD gene fragment to be overexpressed (its nucleotide sequence is shown in SEQ ID No. 1). Then, using the pJQ03 plasmid stored in our laboratory as a template, PCR amplification was performed using primers JQoli_21 / JQoli_22 to obtain the target fragment homologous upstream and downstream to the aforementioned YlAMPD gene fragment. The linearized pJQ03 and YlAMPD gene fragments were seamlessly ligated to construct the recombinant vector pJQ04. Further PCR amplification was performed using primers JQoli_33 / JQoli_35 and plasmid pJQ04 as a template to obtain the YlAMPD expression cassette TEF1p-YlAMPD-PGKt containing a promoter and terminator. Using the pQHF03 plasmid stored in our laboratory as a template, the target fragment homologous to the upstream and downstream of the YlAMPD expression cassette gene fragment was amplified using primer pair JQoli_34 / JQoli_36. The target fragment contains the selection marker ura3 gene (its nucleotide sequence is shown in SEQ ID NO.3) and upstream and downstream homologous arms of the integration site. The YlAMPD expression cassette and the above target fragment were fused using seamless cloning to finally construct the integrative plasmid pJQ08. The plasmid map is shown below. Figure 1 As shown in Figure A. Plasmid pJQ08 was validated by PCR using the validation primer pair JQoli_C7 / JQoli_C8. The gel electrophoresis results showed a band size of approximately 2500 bp (e.g., ...). Figure 1 As shown in Figure B, the length of the validation fragment is consistent with the expected 2768bp, proving that the pJQ08 plasmid was successfully constructed.

[0033] (2) Construction of integrative plasmid pJQ09

[0034] Using plasmid pJQ02 containing the codon-optimized Aspergillus niger AnYHM2 gene (nucleotide sequence as shown in SEQ ID No. 2) as a template, PCR amplification was performed using primers JQoli_26 / JQoli_27 to obtain the AnYHM2 expression cassette GPDp-AnYHM2-PEX20t containing a promoter and terminator. Then, using plasmid pQHF03 as a template, primers JQoli_38 / JQoli_39 amplified a target fragment homologous upstream and downstream to the ANYHM2 expression cassette gene fragment. This target fragment also contained the selection marker ura3 gene and homologous arms upstream and downstream of the integration site. Seamless cloning was used to fuse the target fragment with the two fragments of the AnYHM2 expression cassette, ultimately constructing the integrative plasmid pJQ09. The plasmid map is shown below. Figure 2As shown in Figure A. Plasmid pJQ09 was validated by plasmid PCR using the validation primer pair JQoli_C15 / JQoli_C16, and the validation was confirmed by gel electrophoresis as shown below. Figure 2 As shown in Figure B, the results show that the band size is approximately 2000 bp, which is consistent with the expected validation fragment length of 2281 bp, proving that the pJQ09 plasmid was successfully constructed.

[0035] (3) Construction of YlAMPD and AnYHM2 single gene overexpression strains

[0036] The pJQ08 and pJQ09 plasmids constructed in (1) and (2), as well as the pJQ03 plasmid preserved in the laboratory, were digested with the restriction endonuclease PmeI to obtain three target fragments: one containing the selection marker ura3 gene and the expression cassette TEF1p-YIAMPD-PGKt, one containing the ura3 gene and the expression cassette GPDp-AnYHM2-PEX20t, and one containing only the ura3 gene. After purifying the three gene fragments by gel extraction, the three target fragments were transformed into three initial Y. lipolytica Po1f strains using the lithium acetate transformation method. Each expression cassette was integrated into the genome of the Po1f strain through homologous recombination. Possible positive clones from the three transformed plates were picked and inoculated into YPD medium (20 g / L glucose, 20 g / L Typtone, 10 g / L Yeast extract) for culture. Genomic DNA was extracted and PCR verification was performed. After successful verification, Y overexpression was obtained. The strains Po1f-pJQ08, which expressed the lipolytica homologous gene YIAMPD, Po1f-pJQ09, which expressed the gene AnYHM2 from A. niger, and the blank control strain Po1f-pJQ03 were used. The PCR verification electrophoresis results of the DNA from each strain are shown in the figure. Figure 3 As shown, lanes 1-3 represent strain Po1f-pJQ03, lanes 4-6 represent strain Po1f-pJQ08, and lanes 7-9 represent strain Po1f-pJQ09. M represents molecular weight marker.

[0037] Example 2: Construction of the strain Po1f-pJQ12 co-expressing YlAMPD and AnYHM2 genes

[0038] (1) Using pJQ04 as a template, PCR amplification was performed using primer pair JQoli_46 / JQoli_47 to obtain the target fragment containing the expression cassette TEF1p-YlAMPD-PGKt. Then, using the pJQ02 plasmid stored in our laboratory as a template, primer pair JQoli_48 / JQoli_49 was used to amplify the target fragment homologous to the upstream and downstream components of the above-mentioned expression cassette TEF1p-YlAMPD-PGKt. The two fragments were fused through homologous recombination to construct the recombinant vector pJQ11. Further PCR amplification was performed using primer pair JQoli_50 / JQoli_51 with plasmid pJQ11 as a template to obtain the target fragment containing the co-expression modules of TEF1p-YlAMPD-PGKt and GPDp-AnYHM2-PEX20t. Using the pQHF03 plasmid stored in our laboratory as a template, PCR amplification was performed using primer pairs JQoli_52 / JQoli_53 to amplify the target fragment containing homologous upstream and downstream segments of the co-expression module. Through seamless cloning, the co-expression template fragment was fused with a linearized pQHF03 fragment containing homologous arms, ultimately constructing the integrative plasmid pJQ12. The plasmid map is shown below. Figure 4 As shown in Figure A. Plasmid pJQ12 was validated by PCR using the validation primer pair JQoli_C20 / JQoli_C21. ​​Gel electrophoresis results showed a band size of approximately 3000-4000 bp. Figure 4 As shown in Figure B, the length of the validation fragment is consistent with the expected 3653bp, proving that the pJQ12 plasmid was successfully constructed.

[0039] (2) The pJQ12 constructed in (1) was digested with the restriction endonuclease PmeI to obtain the target fragment containing the selection marker ura3 gene, the expression cassette TEF1p-YlAMPD-PGKt, and the expression cassette GPDp-AnYHM2-PEX20t. After gel purification of the gene fragment, the target fragment was transformed into the initial Po1f strain using the lithium acetate transformation method. Possible positive clones from the transformation plate were picked and inoculated into YPD medium for culture. Genomic DNA was extracted for PCR verification. After successful verification, the strain Po1f-pJQ12 overexpressing YlAMPD and AnYHM2 was obtained. The PCR verification electrophoresis results of strain Po1f-pJQ12 are shown in the figure. Figure 5 As shown, the verification band is 6514bp.

[0040] Example 3: Identification of the citric acid secretion capacity of the modified strain

[0041] The modified strains Po1f-pJQ08 and Po1f-pJQ09 from Example 1, the modified strain Po1f-pJQ12 from Example 2, and the blank control strain Po1f-pJQ03 were all inoculated into 250 mL Erlenmeyer flasks containing 50 mL of citric acid production medium YNBN0.2B6 (1.5 g / L Least Nitrogen Base (Without (NH4)2SO4), 2.0 g / L (NH4)2SO4, 90 g / L glucose). The cell concentration in the medium after inoculation was 1 × 10⁻⁶. 5 The cells / mL were approximately [number] cells / mL. After fermentation for 5 days at 200 rpm and 30℃, the fermentation broth of each strain was collected, and the citric acid concentration and cell dry weight were measured. The results are as follows: Figure 6 As shown in the figure. The results indicated that the citric acid concentrations in the fermentation broths of strains Po1f-pJQ08, Po1f-pJQ09, Po1f-pJQ12, and the blank control strain Po1f-pJQ03 were 14.63 g / L, 14.24 g / L, 18.32 g / L, and 11.25 g / L, respectively. Compared with the control strain, the citric acid secretion of strains Po1f-pJQ08, Po1f-pJQ09, and Po1f-pJQ12 increased by 30.04%, 26.57%, and 62.84%, respectively. Furthermore, the cell dry weight of the four strains did not change significantly. Therefore, it can be concluded that the strain Po1f-pJQ12, which co-expresses the YlAMPD and AnYHM2 genes, can significantly increase citric acid secretion.

[0042] Example 4: Application of YlAMPD and AnYHM2 gene co-expression strains in rare earth element bioleaching

[0043] (1) Strains Po1f-pJQ12 and blank control strain Po1f-pJQ03 were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of citric acid production medium YNBN0.2B6 (1.5 g / L Yeast Nitrogen Base (Without (NH4)2SO4), 2.0 g / L (NH4)2SO4, 90 g / L glucose). The cell concentration in the medium after inoculation was 1 × 10⁻⁶. 5 Fermentation was carried out at approximately 200 rpm and 30°C for 5 days, with cells / mL.

[0044] (2) On day 5, transfer the fermentation broth from (1) to a centrifuge tube and centrifuge at 15000 rpm for 10 min. Collect the supernatant and use 0.22... Membrane filtration for sterilization.

[0045] (3) Take 20 mL of the filtered supernatant and add it to a 50 mL conical flask. Add 0.1 g of rare earth mineral and leach at 45 °C and 200 rpm for 7 days.

[0046] (4) Take a sample on the 7th day of leaching, centrifuge at 15000 rpm for 10 min, and use 0.22 ml of the supernatant. Membrane filtration.

[0047] (5) The rare earth element content of the filtrate was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). (The rare earth elements were then leached.)

[0048] (6) The ion-adsorption rare earth ore was sent to the company for digestion, and the rare earth element content was determined using inductively coupled plasma atomic emission spectrometry.

[0049] (7) The leaching rate of rare earth elements was calculated. The leaching rates of rare earth elements in the fermentation supernatant of strain Po1f-pJQ12 and the blank control strain Po1f-pJQ03 were shown in Table 1 and 2. Figure 7 As shown, the elements are: erbium (Er), europium (Eu), cerium (Ce), lanthanum (La), praseodymium (Pr), ytterbium (Yb), neodymium (Nd), terbium (Tb), holmium (Ho), samarium (Sm), dysprosium (Dy), yttrium (Y), and REEs represent the above 12 rare earth elements. Compared with the leaching of rare earth elements by the fermentation supernatant of the blank control strain Po1f-pJQ03, the leaching rate of REEs by strain Po1f-pJQ12 increased to 70.54%.

[0050] Table 1. Leaching rates of rare earth elements in strains Po1f-pJQ12 and Po1f-pJQ03

[0051]

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant strain of Yarrowia lipolytica, characterized in that, In the Yarrowia lipolytica starting strain, YlAMPD gene and / or AnYHM2 gene are overexpressed to obtain a recombinant strain.

2. The recombinant bacterial strain of claim 1, wherein, The YlAMPD gene is derived from Yarrowia lipolytica, and its nucleotide sequence is shown as SEQ ID NO. 1; the AnYHM2 gene is derived from Aspergillus niger, and its nucleotide sequence is shown as SEQ ID NO.

2.

3. The recombinant bacterial strain of claim 1, wherein, The Yarrowia lipolytica starting strain is Yarrowia lipolytica Po1f strain.

4. The recombinant bacterial strain of claim 1, wherein, The recombinant strain further contains a selection marker gene ura3, and its nucleotide sequence is shown as SEQ ID NO.

3.

5. A process for the preparation of a recombinant strain of Yarrowia lipolytica according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: (1) constructing a recombinant plasmid containing a selection marker ura3 gene, a YlAMPD gene expression cassette, and / or an AnYHM2 gene expression cassette; (2) treating the recombinant plasmid in (1) with a restriction enzyme to obtain a target fragment containing a selection marker ura3 gene, a YlAMPD gene expression cassette, and / or an AnYHM2 gene expression cassette, and transforming the target fragment into a Yarrowia lipolytica starting strain to obtain a recombinant strain; Preferably, the YlAMPD gene expression cassette further comprises a promoter and a terminator, preferably the promoter is TEF1 and the terminator is PGK; preferably, the AnYHM2 gene expression cassette further comprises a promoter and a terminator, preferably the promoter is GPD and the terminator is PEX20.

6. The production method according to claim 5, wherein The target fragment is integrated into the genome of the Yarrowia lipolytica starting strain by homologous recombination, and preferably the Yarrowia lipolytica starting strain is Yarrowia lipolytica Po1f strain.

7. The preparation method according to claim 5, characterized in that, The target fragment is transformed by lithium acetate transformation method.

8. The Yarrowia lipolytica recombinant strain of any one of claims 1-4 in the application of improving citric acid secretion or in the extraction of rare earth elements.

9. Use according to claim 8, characterized in that, The recombinant strain is subjected to fermentation culture, the fermentation supernatant is mixed with rare earth ore, and the rare earth elements are leached by oscillation, and preferably the rare earth ore is ionic rare earth ore.

10. Use according to claim 9, characterised in that, The fermentation culture conditions are 27-35℃, 4-7 days, and the rotation speed is 150-300 rpm; the leaching conditions are 40-48℃, 5-9 days, and the rotation speed is 150-300 rpm; preferably, the fermentation culture conditions are 28-31℃, 4-6 days, and the rotation speed is 180-250 rpm; the leaching conditions are 43-46℃, 6-8 days, and the rotation speed is 180-250 rpm.

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