Light-driven proton concentration regulated engineered yeast

Abstract

The invention provides a light-driven proton concentration regulated engineered yeast strain, and belongs to the technical field of genetic engineering and optogenetics. The invention provides a fusion gene. The fusion gene comprises a coding gene of a visual purple protein and a coding gene of a plasma membrane localization signal peptide. As a light-driven proton pump, the rhodopsin protein can absorb light energy under an illumination condition and pump protons into cells from outside the cells. The fusion gene provided by the invention is constructed to a host bacterium through a genetic engineering means, the rhodopsin is positioned on a plasma membrane of a cell, and the function of a light-driven proton pump can be realized under an illumination condition, so that extracellular protons are pumped into the cell, and accurate regulation and control of intracellular pH are realized. The strategy not only provides a new means for metabolism regulation, but also provides a brand new solution for pH regulation in industrial fermentation and metabolic engineering, and is expected to promote cell growth and production of metabolites.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and optogenetics, and particularly relates to a yeast engineered strain for regulating proton concentration driven by light. Background Art

[0002] With the rapid development of biotechnology, yeast, as an important microorganism, has wide applications in the fields of industrial fermentation, metabolic engineering, environmental protection, drug production, etc. Yeast can carry out efficient metabolism in a complex environment and produce various valuable metabolites (such as ethanol, amino acids, enzymes, and biopharmaceuticals, etc.), thus playing an important role in industries such as food, energy, and pharmaceuticals. However, the metabolic efficiency of yeast is usually significantly affected by the intracellular environment (especially intracellular pH). Changes in intracellular pH often lead to slowed growth, decreased product synthesis efficiency, and may even cause cell death. Therefore, regulating intracellular pH has become a key strategy to improve production efficiency.

[0003] The methods for regulating intracellular pH mainly include external regulation means, such as controlling the pH of the culture medium by adding acids or bases, or genetic engineering modification means, such as enhancing the cell's tolerance to acidic or alkaline environments by overexpressing genes related to proton pumps or knocking out genes for synthesizing acids or bases. However, these methods usually cannot achieve precise regulation of intracellular pH. Summary of the Invention

[0004] The purpose of the present invention is to provide a yeast engineered strain for regulating proton concentration driven by light. The fusion gene, expression cassette, expression vector, and yeast engineered strain of the present invention can be used for precise regulation of the intracellular pH microenvironment of yeast.

[0005] The present invention provides a fusion gene, which includes a coding gene for a rhodopsin protein and a coding gene for a plasma membrane localization signal peptide; the amino acid sequence of the rhodopsin protein is as shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3; the nucleotide sequence of the coding gene for the plasma membrane localization signal peptide is as shown in SEQ ID NO.4.

[0006] Preferably, the coding gene for the plasma membrane localization signal peptide is connected to the 5' end of the coding gene for the rhodopsin protein through linker2; the nucleotide sequence of linker2 is as shown in SEQ ID NO.5.

[0007] Preferably, the fusion gene further includes a coding gene for a fluorescent protein; the coding gene for the fluorescent protein is connected to the 3' end of the coding gene for the rhodopsin protein through linker1; the nucleotide sequence of linker1 is: gaattc.

[0008] The present invention also provides an expression cassette, which is formed by sequentially connecting a constitutive strong promoter endogenous to yeast, the fusion gene described in the above solution, and a terminator.

[0009] The present invention also provides an expression vector containing the expression cassette described in the above solution.

[0010] The present invention also provides a genetically engineered yeast strain, characterized in that the expression cassette described in the above solution is inserted into the genome of the genetically engineered yeast strain.

[0011] Preferably, the coding genes or expression cassettes of CrtE, CrtYB, CrtI, and BCMO are also inserted into the genome of the genetically engineered yeast strain.

[0012] Preferably, the expression cassette includes a first expression cassette and a second expression cassette; the coding genes of CrtE and CrtI are inserted into the first expression cassette; the coding genes of CrtYB and BCMO are inserted into the second expression cassette.

[0013] The present invention also provides the application of the fusion gene, the expression cassette, the expression vector, or the genetically engineered yeast strain described in the above solution in regulating the intracellular pH microenvironment of yeast.

[0014] The present invention also provides a method for regulating the intracellular pH microenvironment of yeast, characterized by including the following steps:

[0015] Culturing the genetically engineered yeast strain described in the above solution in a medium containing retinal; irradiating the genetically engineered yeast strain after or during the culturing.

[0016] The present invention provides a fusion gene, which includes the coding gene of rhodopsin protein and the coding gene of a plasma membrane localization signal peptide. As a light-driven proton pump, rhodopsin protein can absorb light energy under light conditions and pump protons from the extracellular to the intracellular. By constructing the fusion gene of the present invention into a host bacterium through genetic engineering means, rhodopsin protein is localized on the plasma membrane of the cell, and it can achieve the function of a light-driven proton pump under light conditions, thereby pumping extracellular protons into the intracellular to achieve precise regulation of intracellular pH. This strategy not only provides a new means for metabolic regulation, but also provides a brand-new solution for pH regulation in industrial fermentation and metabolic engineering, and is expected to promote cell growth and the production of metabolites. Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0018] Figure 1 Schematic diagram of the principle of the RmXeR / PoXeR / SzR1 engineered bacterium for plasma membrane localization strain;

[0019] Figure 2 Confocal laser scanning microscopy image of the RmXeR / PoXeR / SzR1 engineered bacterium; where the scale bar is 10 μm;

[0020] Figure 3 Verification result diagram of the proton pump function of the RmXeR / PoXeR / SzR1 engineered bacterium in cells;

[0021] Figure 4 Map of plasmid pUC19-int9L-TDH3p-PM-linker2-RmXeR-linker1-EGFP-PGK1t-BleoCase-int9R;

[0022] Figure 5 Map of plasmid pUC19-YPRC3L-TEF1p-CrtE-TEF1t-FBA1p-CrtI-FBA1t-TEF2p;

[0023] Figure 6 Map of plasmid pUC19-TEF2p-CrtYB-TEF2t-TDH3p-BCMO-PGK1t-KanMX-YPRC3R; Figure 7 Detection result diagram of the positive bacteria in liquid phase in Example 6. Detailed implementation manners

[0024] The present invention provides a fusion gene, including the coding gene of rhodopsin protein and the coding gene of plasma membrane localization signal peptide; the amino acid sequence of the rhodopsin protein is as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; the nucleotide sequence of the coding gene of the plasma membrane localization signal peptide is as shown in SEQ ID NO.4.

[0025] Rhodopsin protein, as a light-driven proton pump, can absorb light energy under light illumination and pump protons from the extracellular side into the intracellular side. In the specific implementation process of the present invention, the plasma membrane localization signal peptide can enable the rhodopsin protein to be expressed on the plasma membrane, and the rhodopsin protein expressed on the plasma membrane can activate the proton transfer function through light illumination. By constructing the fusion gene of the present invention into a host bacterium by genetic engineering means, the rhodopsin protein is introduced into the cell and localized on the cytoplasmic membrane. The rhodopsin protein can achieve the function of a light-driven proton pump under light illumination, thereby achieving precise regulation of intracellular pH. This strategy not only provides a new means for metabolic regulation but also provides a brand-new solution for pH regulation in industrial fermentation and metabolic engineering.

[0026] In the specific implementation process of the present invention, the rhodopsin protein is the RmXeR protein from Rubricoccus marinus, the PoXeR protein from Parvularcula oceani, or the SzR1 protein from Candidatus Lokiarchaeota archaeon, which are exogenously expressed in yeast by genetic engineering means. In the specific implementation process of the present invention, the full length of the RmXeR protein is 233 amino acids (aa). In the specific implementation process of the present invention, the full length of the PoXeR protein is 223 aa. In the specific implementation process of the present invention, the full length of the SzR1 protein is 201 aa.

[0027] In the specific implementation process of the present invention, the amino acid sequence of the RmXeR protein is shown in SEQ ID NO.1 respectively, specifically: MSLETLLYTVYIASMAAGALLFLSWMRDPKGVPVWEYIVAALIPVWSGLAYLAMALGLGTAEIAGQTTYWARYADWVVTTPLLLTALWMTAVTRSDKSKHVPLLLGLVSADIIMILCGLVGDLSSGPARYVYFGIGVAALVVVFALTWGPLRRVAEQDPEIGGIYRKVAAYLALFWIGYPLTWILGPSGLGIVGQTVDTTLFILLPIFSKVGFSIVDLGMLRAAGGTGCAAHE.

[0028] In the specific implementation process of the present invention, the amino acid sequences of the PoXeR protein are shown in SEQ ID NO.2 respectively, specifically: MTEAFWLWLYVAVMAAGACLFLYWSRKPRGVPGYEYTIAAVIPIWSGLAYLAMALGQGTVMIDGREVYYARYLDWLVTTPLLLWLLGSTATFYRANDTRLIGSLMFADVVMILSGLFADLTAEQSVRWLWYTIGCVSFLLILWQVWGPLRRIAGEQGEALSKTYTRVAAYLTVFWVSYPLVWLISPSGIGIVGPTVSIALFVILPAFSKVGFSILDLYELRRL.

[0029] In the specific implementation process of the present invention, the amino acid sequences of the SzR1 protein are shown in SEQ ID NO.3 respectively, specifically: MEEIIFYIGAGVFTLTSITFFLLKKKNLEVASLNMIVNFVTIASYLLMVSGLFVASAVSGDSIYWTRWAFYAVSCSFLMVEISMLLSIDKSIKLEIIVFNCLVMITGLLASVSEGIIKWLFFTLSSVAYLYVLFQIIKHRSNEKFIVAFVAIFWSGFPIIWILSPAGLMLIDAFWTALFYLVLDLITKVYFGYHTTLKFSK.

[0030] In the specific implementation process of the present invention, the nucleotide sequence of the coding gene of the plasma membrane localization signal peptide (PM) is shown in SEQ ID NO.4 respectively, specifically: atgctttcactacgtcaatctataagatttttcaagccagccacaagaactttgtgtagctctagatatctgcttcag.

[0031] In the specific implementation process of the present invention, the rhodopsin protein is located at the N-terminus of the fusion protein encoded by the fusion gene; the coding gene of the plasma membrane localization signal peptide is connected to the 5'-end of the coding gene of the rhodopsin protein through linker2; the fusion gene further includes the coding gene of a fluorescent protein; the coding gene of the fluorescent protein is connected to the 3'-end of the coding gene of the rhodopsin protein through linker1; both linker1 and linker2 are flexible linkers; the nucleotide sequence of linker1 is: gaattc; the nucleotide sequence of linker2 is as shown in SEQ ID NO.5, specifically: ggtggtggttctggtggtggtggttctggtggtggtggtggttc; the fluorescent protein is EGFP; the nucleotide sequence of the coding gene of EGFP is as shown in SEQ ID NO.As shown in Figure 6, specifically: atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag。.

[0032] The present invention also provides an expression cassette, which is composed of a constitutive strong promoter endogenous to yeast, the fusion gene described in the above solution, and a terminator connected in sequence.

[0033] There are no requirements for the promoters and terminators used in the present invention, and the sequences are all known sequences, which can be retrieved from the database. The database is Saccharomyces Genome Database | SGD (yeastgenome.org).

[0034] In one embodiment of the present invention, the constitutive strong promoter endogenous to the yeast includes the constitutive promoter TDH3p; the nucleotide sequence of the constitutive promoter TDH3p is as shown in SEQ ID NO.7, specifically: aacagtttattcctggcatccactaaatataatggagcccgctttttaagctggcatccagaaaaaaaaagaatcccagcaccaaaatattgttttcttcaccaaccatcagttcataggtccattctcttagcgcaactacagagaacaggggcacaaacaggcaaaaaacgggcacaacctcaatggagtgatgcaacctgcctggagtaaatgatgacacaaggcaattgacccacgcatgtatctatctcattttcttacaccttctattaccttctgctctctctgatttggaaaaagctgaaaaaaaaggttgaaaccagttccctgaaattattcccctacttgactaataagtatataaagacggtaggtattgattgtaattctgtaaatctatttcttaaacttcttaaattctacttttatagttagtcttttttttagttttaaaacaccaagaacttagtttcgaataaacacacataaacaaacaaaa。

[0035] In the specific implementation process of the present invention, the terminator is PGK1t; the nucleotide sequence of the terminator is shown in SEQ ID NO.8, specifically: aagctttcccatgtctctactggtggtggtgcttctttggaattattggaaggtaaggaattgccaggtgttgctttcttatccgaaaagaaataaattgaattgaattgaaatcgatagatcaa tttttttcttttctctttccccatcctttacgctaaaataatagtttattttattttttgaatattttttatttatatacgtatatatagactattatttatcttttaatgattattaagatttttattaaaaaaaaattc gctcctcttttaatgcctttatgcagtttttttttcccattcgatatttctatgttcgggttcagcgtattttaagtttaataactcgaaaattctgcgttcgtt.

[0036] In the specific implementation process of the present invention, the construction method steps of the expression cassette are as follows: constructing the expression cassette by Gibson assembly of the promoter, targeting peptide PM, linker2, RmXeR / PoXeR / SzR1, linker1, and EGFP; or constructing the expression cassette by Gibson assembly of the homologous sequence, promoter, targeting peptide PM, linker2, RmXeR / PoXeR / SzR1, linker1, and EGFP; the homologous sequence is the homologous arm int9L.

[0037] The present invention also provides an expression vector containing the expression cassette described in the above solution.

[0038] In one embodiment of the present invention, the rhodopsin protein is RmXeR. Using the yeast S288C genome as a template, gene fragments of the homologous arm int9L, TDH3p, PGK1t, and int9R are obtained by PCR amplification. The RmXeR and EGFP coding gene fragments are synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. Using pUC19 as the backbone, the backbone fragments of int9L, TDH3p, RmXeR / PoXeR / SzR1, EGFP, PGK1t, and int9R are assembled by Gibson assembly. On this basis, the BleoR selection marker is inserted to obtain the pUC19-int9L-TDH3p-PM-linker2-RmXeR-linker1-EGFP-PGK1t-BleoCase-int9R plasmid, and the plasmid map is asFigure 4 as shown

[0039] In another embodiment of the present invention, the rhodopsin protein is PoXeR. Using the yeast S288C genome as a template, gene fragments of homologous arms int9L, TDH3p, PGK1t, and int9R were obtained by PCR amplification. The gene fragments encoding PoXeR and EGFP were synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. Using pUC19 as the backbone, the backbone fragments of int9L, TDH3p, RmXeR / PoXeR / SzR1, EGFP, PGK1t, and int9R were assembled by Gibson assembly. On this basis, a BleoR selection marker was inserted to obtain pUC19-int9L-TDH3p-PM-linker2-PoXeR-linker1-EGFP-PGK1t-BleoCase-int9R.

[0040] In another embodiment of the present invention, the rhodopsin protein is SzR1. Using the yeast S288C genome as a template, gene fragments of homologous arms int9L, TDH3p, PGK1t, and int9R were obtained by PCR amplification. The gene fragments encoding SzR1 and EGFP were synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. Using pUC19 as the backbone, the backbone fragments of int9L, TDH3p, RmXeR / PoXeR / SzR1, EGFP, PGK1t, and int9R were assembled by Gibson assembly. On this basis, a BleoR selection marker was inserted to obtain the plasmid pUC19-int9L-TDH3p-PM-linker2-SzR1-linker1-EGFP-PGK1t-BleoCase-int9R.

[0041] The present invention also provides a yeast engineering bacterium, in which the expression cassette described in the above solution is inserted into the genome of the yeast engineering bacterium.

[0042] Compared with the starting strain, the extracellular H + concentration change of the yeast engineering bacterium of the present invention is significantly greater than that of the starting strain. The rhodopsin protein plays an inward proton pump function on the plasma membrane, pumping H + in the solution into the cell. Therefore, the yeast engineering bacterium of the present invention has the function of using light to drive the rhodopsin protein on the plasma membrane to absorb protons. The yeast engineering bacterium of the present invention can pump extracellular protons into the cell under light conditions, thereby regulating the cytoplasmic pH microenvironment, and is expected to promote cell growth and the production of metabolites.

[0043] In the specific implementation process of the present invention, the starting bacteria of the yeast include but are not limited to Saccharomyces cerevisiae S288C or Yarrowia lipolytica W29.

[0044] In the specific implementation process of the present invention, the present invention realizes the insertion of the fusion gene into the genome of the starting bacterium by culturing chemically competent cells, and the insertion site is the int9 site of the starting bacterium's competent cells. In one embodiment of the present invention, a DNA fragment int9L-TDH3p-PM-linker2-RmXeR / PoXeR / SzR1-linker1-EGFP-PGK1t-BleoCase-int9R with homologous arms and a resistance screening marker gene BleoR is obtained from a plasmid by PCR amplification. The fragment is integrated into the int9 site of the starting bacterium's competent cells by homologous recombination through the lithium acetate chemical transformation method. The localization of the fusion protein is observed using a laser confocal microscope to obtain a yeast engineering bacterium in which the rhodopsin protein is localized to the plasma membrane.

[0045] In the present invention, the nucleotide sequence of the int9L is as shown in SEQ ID NO.9, specifically: gttgtaacaccacttgatgacaaagcaaatttgctcaattttctatatccctttaatgcatgaacggttctttattttgttttctgtcgacattctagtttttcggaatctcaaaataattaattacatatacgtacataagtagcccagataaacaaaaataccttaaaaaagagttactagccgtatatggatgtttgaagatacatggaaaccgtctctggtgtcgtgtatataagaaacttctagttttattcagacgcactcattatctttgctacataacatttctctctgatttgactgcgcatcttacccctcccccatgcatgtggagtcataggagtaattttaaaggtagaatttcatattaaatatcgctgcttgattattttgtagcaaatcaaaagagtgtttcaagtaagtaaaaacatttgagcctccccatttgttgaaaggagagaaattaaacttggttggggttaattatttgatggg.

[0046]

[0047] In the present invention, the nucleotide sequence of int9R is as shown in SEQ ID NO.11, specifically: ctacaaaacctttatcaatagtggtgaagtctttagtgcgatctacctggggttaatgaacgagaagttcttgagatatctttcctgtttacctccgtgcatcctgtaaggaattgggtttatcattttatcatttattttagtacaaacttttttttttggcccgggcgcactttttcaagcggtgggaactcatcaaaatgaaaaactagatacttttagacttattaaatggtttaaatattttgagatgttcgttatatcagaaacttccttacttctatcttttattccaatacaaagaagtcacaagattacttggtaagaaagaagcagttaatttttaattttgccgacaagccaag。

[0048] In the specific implementation process of the present invention, the construction method steps of the engineered yeast strain are as follows: The obtained expression cassette is transformed into the starting strain, integrated into the int9 locus of the genome, and an engineered yeast strain with rhodopsin protein membrane localization is constructed. After the construction, it also includes the localization characterization of the engineered yeast strain, and the steps are as follows: The localization of the RmXeR / PoXeR / SzR1-EGFP fusion protein in the strain is observed through a laser confocal microscope, and it is obtained that the PM-RmXeR / PoXeR / SzR1-EGFP fusion protein with the PM connected to the N-terminus is successfully localized on the plasma membrane.

[0049] In the specific implementation process of the present invention, the coding genes or expression cassettes of CrtE, CrtYB, CrtI, and BCMO are also inserted into the genome of the engineered yeast strain, enabling the engineered yeast strain to endogenously synthesize retinal; the UniProt database number of the coding gene of CrtE is: Q1L6K3; the UniProt database number of the coding gene of CrtYB is: Q7Z859; the UniProt database number of the coding gene of CrtI is: A0A0P0KMF9; the UniProt database number of the coding gene of BCMO is: Q4PNI0; the coding gene fragments of CrtE, CrtYB, CrtI, and BCMO are synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. In the specific implementation process of the present invention, the insertion of CrtE, CrtYB, CrtI, and BCMO into the genome is achieved by culturing chemically competent cells; the expression cassette includes a first expression cassette and a second expression cassette; the coding genes of CrtE and CrtI are inserted into the first expression cassette; the coding genes of CrtYB and BCMO are inserted into the second expression cassette. In the specific implementation process of the present invention, the insertion sites of the obtained first expression cassette and second expression cassette on the genome of the starting strain are the YPRC3 sites. In an embodiment of the present invention, the structure of the first expression cassette is YPRC3L-TEF1p-CrtE-TEF1t-FBA1p-CrtI-FBA1t-TEF2p; the structure of the second expression cassette is TEF2p-CrtYB-TEF2t-TDH3p-BCMO-PGK1t-KanMX-YPRC3R.

[0050] In the specific implementation process of the present invention, the homologous sequences, promoters, CrtE, CrtYB, CrtI, and BCMO are assembled into an expression cassette through Gibson assembly. In an embodiment of the present invention, using the yeast S288C genome as a template, gene fragments of homologous arms YPRC3L, TEF1p, TEF1t, FBA1p, FBA1t, TEF2p, TEF2t, TDH3p, PGK1t, and YPRC3R are amplified by PCR. Using pUC19 as a backbone, the backbone fragments of YPRC3L, TEF1p, CrtE, TEF1t, FBA1p, CrtI, FBA1t, TEF2p, CrtYB, TEF2t, TDH3p, BCMO, PGK1t, and YPRC3R are assembled by Gibson assembly. On this basis, a KanMX selection marker is inserted to obtain the pUC19-YPRC3L-TEF1p-CrtE-TEF1t-FBA1p-CrtI-FBA1t-TEF2p plasmid (the plasmid map is as Figure 5as shown) and the plasmid pUC19-TEF2p-CrtYB-TEF2t-TDH3p-BCMO-PGK1t-KanMX-YPRC3R (plasmid map as Figure 6 shown). DNA fragments YPRC3L-TEF1p-CrtE-TEF1t-FBA1p-CrtI-FBA1t-TEF2p and TEF2p-CrtYB-TEF2t-TDH3p-BCMO-PGK1t-KanMX-YPRC3R, which carry homologous arms and the resistance screening marker gene KanMX, were obtained from the plasmid by PCR amplification, with lengths of 6600bp and 7165bp respectively. The fragments were integrated into the YPRC3 locus of the competent cells of the starting strain by homologous recombination through the lithium acetate chemical transformation method.

[0051] In the specific implementation process of the present invention, the obtained first expression cassette and second expression cassette were co-transformed into a positive bacterium expressing the coding gene of rhodopsin protein and the coding gene of the plasma membrane localization signal peptide, and integrated into the genomic YPRC3 locus to construct a yeast engineering bacterium for endogenous synthesis of retinol; the insertion order of the first expression cassette and the second expression cassette was simultaneous insertion.

[0052] In the present invention, the transformation operation can be carried out according to the conventional technical means in the art.

[0053] The present invention also provides the application of the fusion gene, the expression cassette, the expression vector or the yeast engineering bacterium described in the above scheme in regulating the intracellular pH microenvironment of yeast.

[0054] In the present invention, the regulation of the intracellular pH microenvironment of yeast includes light-driven regulation of the intracellular proton concentration; the regulation of the intracellular proton concentration includes pumping protons from outside the cell into the cell to achieve precise regulation of the intracellular pH.

[0055] The present invention also provides a method for regulating the intracellular pH microenvironment of yeast, including the following steps:

[0056] Culturing the yeast engineering bacterium described in the above scheme in a medium containing retinol; irradiating the yeast engineering bacterium after or during the culturing.

[0057] Under light conditions, the method of the present invention regulates the cytoplasmic pH through the protons pumped into the cell by the yeast engineering bacterium.

[0058] In the specific implementation process of the present invention, the wavelength of the irradiation includes but is not limited to 380 - 780nm; the intensity of the irradiation includes but is not limited to 2 - 10mW / cm 2 , and further 2 - 5mW / cm 2, at this light intensity, it is possible to avoid the side effects on cell growth and metabolism caused by the over-strong function of the proton pump (i.e., over-alkalinity inside the cell); the light type of the light is white light.

[0059] In the specific implementation process of the present invention, the addition amount of retinal in the culture medium includes but is not limited to 10 μM. When retinal is added exogenously, the heterologously expressed rhodopsin protein will bind to retinal, and the bound retinal will undergo a conformational change upon illumination, transferring protons outside the cell into the cell.

[0060] In the specific implementation process of the present invention, the method also includes the characterization of the proton pump function of rhodopsin; the characterization of the proton pump function is achieved by monitoring the pH change of the solution under illumination conditions when the engineered yeast strain is resuspended in the buffer-free solution SS; the buffer-free solution SS is composed of 10 mM NaCl, 10 mM MgSO4·7H2O, 0.1 mM CaCl2·2H2O, and 0.2 M D-sorbitol. Through characterization, it is found that under illumination, the H + concentration in the buffer-free solution where the engineered yeast strain is located decreases significantly, showing an obvious change compared with the starting strain. It indicates that under the combined action of light and retinal, the rhodopsin RmXeR / PoXeR / SzR1 in the engineered yeast strain exerts the proton pump function, effectively pumping protons in the extracellular solution into the cell, verifying that rhodopsin RmXeR / PoXeR / SzR1 can be correctly expressed and exert the expected proton pump function in the engineered yeast strain ( Figure 3 ).

[0061] Through synthetic biology means, the present invention has successfully constructed an engineered yeast strain with membrane localization of rhodopsin protein, and under the conditions of illumination and exogenous addition of retinal or by expressing CrtE, CrtYB, CrtI, and BCMO proteins to enable the yeast to endogenously synthesize retinal by itself, the intracellular pH of the strain is regulated, which is expected to promote cell growth and the production of metabolites. The method of the present invention provides a brand-new strategy for regulating the intracellular pH microenvironment of microorganisms and has important industrial application prospects.

[0062] To further illustrate the present invention, the following describes in detail an engineered yeast strain for regulating proton concentration driven by light provided by the present invention in combination with the accompanying drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0063] The experimental methods not specifically described in the following examples are all conventional methods, and the materials and reagents not specifically described can be obtained from commercial channels. The materials, reagents, etc. used in the following examples can be obtained from commercial channels without special instructions.

[0064] Term

[0065] As used herein, the "engineered bacteria" refers to a bacterial cell line in which foreign genes are highly expressed by genetic engineering methods.

[0066] As used herein, the "starting strain" refers to the original strain used for modification.

[0067] Example 1 Construction of a strain with RmXeR localized to the yeast plasma membrane

[0068] Gene fragments of int9L (SEQ ID NO.9), TDH3p (SEQ ID NO.7), PGK1t (SEQ ID NO.8), and int9R (SEQ ID NO.11) were amplified by PCR using the yeast S288C genome as a template. The coding gene fragments of RmXeR (SEQ ID NO.1) and EGFP (SEQ ID NO.6) were synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. Using pUC19 as the backbone, the backbone fragments of int9L, PM, TDH3p, RmXeR, EGFP, PGK1t, and int9R were assembled by Gibson assembly, and a BleoR selection marker was inserted on this basis. The assembled plasmid was transferred into Escherichia coli, and after correct sequencing, the plasmid was extracted to obtain the pUC19-int9L-TDH3p-PM-linker2-RmXeR-linker1-EGFP-PGK1t-BleoCase-int9R plasmid. The primers used are shown in Table 1. The target fragment with a size of 4303 bp was amplified using the primers int9L-F1 / int9R-R1 and reserved.

[0069] The fragment int9L-TDH3p-PM-linker2-RmXeR-linker1-EGFP-PGK1t-BleoCase-int9R was integrated into the int9 locus of the genome of the yeast starting strain competent cells (prepared freshly) by homologous recombination using the lithium acetate transformation method. The transformed bacteria were spread on a YPD solid plate supplemented with 0.8‰ Bleo and cultured in a 30 °C incubator for 2 - 3 days until obvious single colonies appeared. Single colonies were picked and streaked on a YPD plate containing 0.8‰ Bleo for preservation, and cultured in a 30 °C incubator for one day. A small amount of the bacterial cells was picked and added to 30 μL of lysis buffer (5 mL / L Triton, 0.292 g / L EDTA, and 2 g / L NaOH), heated at 100 °C for 20 min, and then 60 μL of ddH2O was added. Then, PCR amplification was performed using the corresponding upstream and downstream primers int9L-wF1 / TDH3p-pR1, BleoCase-pF1 / int9R-wR1 (Table 1). The recombinant bacteria that amplified the target band were initially determined to be positive bacteria.

[0070] Table 1 Primers used in the examples of the present invention

[0071]

[0072]

[0073]

[0074] Construction of Yeast Strains with PoXeR Localized to the Plasma Membrane in Example 2

[0075] Gene fragments of int9L, TDH3p, and PGK1t int9R were amplified by PCR using the yeast S288C genome as a template. The gene fragments of PoXeR and EGFP were synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. Using pUC19 as the backbone, the backbone fragments of int9L, PM, TDH3p, PoXeR, EGFP, PGK1t, and int9R were assembled by Gibson assembly, and a BleoR selection marker was inserted on this basis. The assembled plasmid was transformed into Escherichia coli, and after correct sequencing, the plasmid was extracted to obtain

[0076] the pUC19-int9L-TDH3p-PM-linker2-PoXeR-linker1-EGFP-PGK1t-BleoCase-int9R plasmid. The primers used are shown in Table 1. The target fragments were amplified using primers int9L-F1 / int9R-R1, with sizes of 4273 bp respectively, for later use.

[0077] The fragment int9L-TDH3p-PM-linker2-PoXeR-linker1-EGFP-PGK1t-BleoCase-int9R was integrated into the int9 locus of the genome of the yeast starting strain competent cells (prepared freshly before use) by homologous recombination using the lithium acetate transformation method. The transformed bacteria were spread on a YPD solid plate supplemented with 0.8‰ Bleo and cultured in a 30°C incubator for 2 - 3 days until obvious single colonies appeared. Single colonies were picked and streaked on a YPD plate containing 0.8‰ Bleo for preservation, and cultured in a 30°C incubator for one day. A small amount of bacterial cells were picked and added to 30 μL of lysis buffer (5 mL / L Triton, 0.292 g / L EDTA, and 2 g / L NaOH), heated at 100°C for 20 min, and then 60 μL of ddH2O was added. Then, PCR amplification was performed using the corresponding upstream and downstream primers int9L-wF1 / TDH3p-pR1, BleoCase-pF1 / int9R-wR1 (Table 1). The recombinant bacteria that amplified the target band were initially determined to be positive bacteria.

[0078] Construction of Yeast Strains with SzR1 Localized to the Plasma Membrane in Example 3

[0079] The gene fragments of int9L, TDH3p, PGK1t int9R were amplified by PCR using the yeast S288C genome as a template. The gene fragments of SzR1 and EGFP were synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. Using pUC19 as the backbone, the backbone fragments of int9L, PM, TDH3p, SzR1, EGFP, PGK1t, and int9R were assembled by Gibson assembly, and on this basis, the BleoR selection marker was inserted. The assembled plasmid was transformed into Escherichia coli, and after correct sequencing, the plasmid was extracted to obtain the pUC19-int9L-TDH3p-PM-linker2-SzR1-linker1-EGFP-PGK1t-BleoCase-int9R plasmid. The primers used are shown in Table 1. The target fragments were amplified using the primers int9L-F1 / int9R-R1, with sizes of 4207 bp respectively, and reserved for later use.

[0080] The fragment int9L-TDH3p-PM-linker2-SzR1-linker1-EGFP-PGK1t-BleoCase-int9R was integrated into the int9 locus of the genome of the yeast starting strain competent cells (prepared freshly before use) by homologous recombination using the lithium acetate transformation method. The transformed bacteria were spread on a YPD solid plate supplemented with 0.8‰ Bleo and cultured in a 30 °C incubator for 2 - 3 days until obvious single colonies appeared. Single colonies were picked and streaked on a YPD plate containing 0.8‰ Bleo for preservation, and cultured in a 30 °C incubator for one day. A small amount of bacterial cells were picked and added to 30 μL of lysis buffer (5 mL / L Triton, 0.292 g / L EDTA, and 2 g / L NaOH), heated at 100 °C for 20 min, and then 60 μL of ddH2O was added. Then, PCR amplification was performed using the corresponding upstream and downstream primers int9L-wF1 / TDH3p-pR1, BleoCase-pF1 / int9R-wR1 (Table 1). The recombinant bacteria that amplified the target band were initially determined as positive bacteria.

[0081] Example 4 Localization Characterization of the RmXeR / PoXeR / SzR1 Engineered Bacteria

[0082] To verify that rhodopsin RmXeR / PoXeR / SzR1 was successfully localized to the plasma membrane of yeast, the positive bacteria of Examples 1 to 3 were inoculated into a test tube containing 3 mL of YPD liquid medium and cultured overnight at 30 °C with 220 rpm. Then, the seed solution was inoculated into 2 mL of YPD liquid medium at an inoculation ratio of seed solution:YPD = 1:10 and cultured at 30 °C with 220 rpm for 4.5 h. Centrifuge at 5000 rpm for 2 min, discard the supernatant, wash 3 times with 1 mL of 0.01 M PBS buffer (pH = 7.4), and finally resuspend the cells in 200 μL of 0.01 M PBS buffer (pH = 7.4). Spot 3 μL of the suspension on a glass slide, cover with a coverslip, and invert it on the lens of a laser confocal microscope. Select a 100× objective lens. In the observation wavelength setting of the laser confocal microscope, the excitation wavelength of EGFP was selected as 488 nm, and the emission wavelength was selected as 507 nm. The laser confocal microscope observed obvious green fluorescence distributed on the plasma membrane ( Figure 2 ), indicating that RmXeR / PoXeR / SzR1 was successfully localized to the plasma membrane of the engineered bacteria.

[0083] Example 5 Characterization of the function of rhodopsin in RmXeR / PoXeR / SzR1 engineered bacteria

[0084] The RmXeR / PoXeR / SzR1 engineered bacteria of Examples 1 to 3 were respectively inoculated into a test tube containing 3 mL of YPD liquid medium and cultured overnight at 30 °C with 220 rpm. Then, the seed solution was inoculated into 50 mL of YPD liquid medium containing 10 μM retinal at an inoculation ratio of seed solution:YPD = 1:10 and cultured at 30 °C with 220 rpm for 18 h. Transfer the bacterial solution into a centrifuge tube, centrifuge at 4000 rpm for 3 min to collect the cells, resuspend the cells with 20 mL of buffer-free solution SS (composed of 10 mM NaCl, 10 mM MgSO4·7H2O, 0.1 mM CaCl2·2H2O, 0.2 M D-sorbitol), centrifuge at 4000 rpm for 3 min and discard the supernatant, then resuspend the cells with 20 mL of buffer-free solution SS again, centrifuge at 4000 rpm for 3 min and discard the supernatant. Resuspend the cells in 60 mL of buffer-free solution SS to make its OD 600 value 3, and detect the change in the pH value of the solution during 4.5 h of illumination ( Figure 3 ). It was found that the concentration of H + in the solution of RmXeR / PoXeR / SzR1 engineered bacteria decreased significantly, indicating that the rhodopsin RmXeR / PoXeR / SzR1 bound to retinal in the engineered bacteria during illumination exerted the proton pump function to pump protons from the buffer-free solution into the cells.

[0085] Example 6 Construction of an endogenous retinal-synthesizing strain

[0086] On the basis of the positive bacteria constructed in Example 3, which express the coding gene of rhodopsin protein and the coding gene of plasma membrane localization signal peptide, an expression cassette for synthesizing retinal is further inserted.

[0087] Using the yeast S288C genome as a template, gene fragments of homologous arms YPRC3L, TEF1p, TEF1t, FBA1p, FBA1t, TEF2p, TEF2t, TDH3p, PGK1t, YPRC3R were obtained by PCR amplification. The gene fragments of CrtE, CrtYB, CrtI, and BCMO were synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. Using pUC19 as the backbone, the backbone fragments of YPRC3L, TEF1p, CrtE, TEF1t, FBA1p, CrtI, FBA1t, TEF2p, CrtYB, TEF2t, TDH3p, BCMO, PGK1t, YPRC3R were assembled by Gibson assembly. On this basis, a KanMX selection marker was inserted. The assembled plasmid was transferred into Escherichia coli. After correct sequencing, the plasmid was extracted to obtain the plasmids pUC19-YPRC3L-TEF1p-CrtE-TEF1t-FBA1p-CrtI-FBA1t-TEF2p and pUC19-TEF2p-CrtYB-TEF2t-TDH3p-BCMO-PGK1t-KanMX-YPRC3R. The primers used are shown in Table 1. The target fragments with sizes of 6600bp and 7165bp were amplified using the primers YPRC3L-F1 / TEF2p-R1 and TEF2p-F1 / YPRC3R-R1 and reserved for use.

[0088] The fragments YPRC3L-TEF1p-CrtE-TEF1t-FBA1p-CrtI-FBA1t-TEF2p and TEF2p-CrtYB-TEF2t-TDH3p-BCMO-PGK1t-KanMX-YPRC3R were integrated into the YPRC3 locus of the genome of the positive bacterium constructed in Example 3 by homologous recombination using the lithium acetate transformation method. The transformed bacteria were spread on a YPD solid plate supplemented with 3‰ KanMX and cultured in an incubator at 30 °C for 2 - 3 days until obvious single colonies appeared. Single colonies were picked and streaked on a YPD plate containing 3‰ KanMX for preservation, and then cultured in an incubator at 30 °C for one day. A small amount of the bacterial cells was picked and added to 30 μL of lysis solution (5 mL / L Triton, 0.292 g / L EDTA, and 2 g / L NaOH), heated at 100 °C for 20 min, and then 60 μL of ddH2O was added. Then, PCR amplification was performed using the corresponding upstream and downstream primers YPRC3L-wF1 / TEF1p-pR1, FBA1t-pF1 / CrtYB-pR1, KanMX-pF1 / YPRC3R-wR1 (Table 1). The recombinant bacteria that amplified the target band were initially determined to be positive bacteria.

[0089] Sample treatment and detection method: The cultured cells were centrifuged to harvest the bacterial cells, resuspended in 1 ml of acetone in a 2 ml screw-cap tube, and the cells were lysed using a grinder. After centrifugation, the supernatant was filtered into a new brown glass bottle to measure the retinol level. HPLC detection was used, and a C18 column (250 mm × 4.6 mm, 5 μm) was used to analyze the acetone extract. The flow rate of HPLC analysis was 1.5 ml / min, the mobile phase was 95:5 methanol and acetonitrile, the column temperature was 40 °C, and the detection wavelength was set at: 370 nm. The liquid phase result diagram is as Figure 7 shown. It can be Figure 7 seen that a substance with the same retention time as the retinol standard can be detected in the culture product of the positive bacteria, and the retention time is 11.119. This indicates that the positive bacteria can synthesize retinol by themselves.

[0090] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fusion gene, characterized in that, Comprising a coding gene of rhodopsin protein and a coding gene of a plasma membrane localization signal peptide; the amino acid sequence of the rhodopsin protein is as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; The nucleotide sequence of the coding gene of the plasma membrane localization signal peptide is as shown in SEQ ID NO.

4.

2. The fusion gene according to claim 1, characterized in that, The coding gene of the plasma membrane localization signal peptide is connected to the 5' end of the coding gene of the rhodopsin protein through linker2; the nucleotide sequence of linker2 is as shown in SEQ ID NO.

5.

3. The fusion gene according to claim 1, wherein The fusion gene further comprises a coding gene of a fluorescent protein; the coding gene of the fluorescent protein is connected to the 3' end of the coding gene of the rhodopsin protein through linker1; the nucleotide sequence of linker1 is: gaattc.

4. An expression cassette, characterized in that, Composed of a constitutive strong promoter endogenous to yeast, the fusion gene according to any one of claims 1 to 3 and a terminator connected in sequence.

5. An expression vector, characterized in that, Containing the expression cassette according to claim 4.

6. A genetically engineered yeast strain, characterized in that, The expression cassette according to claim 4 is inserted into the genome of the engineered yeast strain.

7. The engineered yeast strain according to claim 6, wherein, The coding genes or expression cassettes of CrtE, CrtYB, CrtI and BCMO are also inserted into the genome of the engineered yeast strain.

8. The engineered yeast strain according to claim 6, characterized in that, The expression cassette comprises a first expression cassette and a second expression cassette; the coding genes of CrtE and CrtI are inserted into the first expression cassette; the coding genes of CrtYB and BCMO are inserted into the second expression cassette.

9. Use of the fusion gene according to any one of claims 1 to 3, the expression cassette according to claim 4, the expression vector according to claim 5 or the engineered yeast strain according to any one of claims 6 to 8 in regulating the intracellular pH microenvironment of yeast.

10. A method for regulating the intracellular pH microenvironment of yeast, characterized in that, Comprising the following steps: Culturing the engineered yeast strain according to claim 6 in a medium containing retinal; irradiating the engineered yeast strain after or during the culturing.

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