CN118879754B

CN118879754B - A method for in situ detection of the quaternary structure of oligomeric enzymes displayed on the yeast surface based on FRET

Info

Publication number: CN118879754B
Application number: CN202410963001.XA
Authority: CN
Inventors: 刘护; 王启彬; 冯旭东; 李春
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2024-07-18
Filing date: 2024-07-18
Publication date: 2025-05-09
Anticipated expiration: 2044-07-18
Also published as: CN118879754A

Abstract

The application discloses a method for in-situ detection of a yeast surface display oligomeric enzyme quaternary structure based on FRET, which comprises the steps of taking a saccharomyces cerevisiae as a chassis strain, knocking out genes PPQ1, ALO1, NAM7 and GAL80, introducing multiple copies of tyrosyl-tRNA synthase and tRNA _CUA ^Tyr, selecting sites at the interface of the oligomeric enzyme to be mutated into cysteine and TAG codons respectively, using an anchoring protein to mediate the surface display of the saccharomyces cerevisiae of the oligomeric enzyme, inducing protein expression by adding galactose and para-azido-phenylalanine fermentation, adding Cy 3-maleimide for coupling with cysteine of the yeast surface display oligomeric enzyme, adding Cy5-DBCO for coupling with azide groups of the yeast surface display oligomeric enzyme, and using a confocal microscope to characterize donor, acceptor and FRET signals. The application can detect the quaternary structure of the oligoenzyme on the surface of the cell in situ without separating the displayed oligoenzyme from the surface of the yeast cell.

Description

Method for displaying four-level structure of oligomeric enzyme on yeast surface based on FRET in-situ detection

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for in-situ detection of a yeast surface display oligomeric enzyme quaternary structure based on FRET (fluorescence resonance energy transfer).

Background

The dissociation of subunits of oligos is a major constraint affecting its application prospect. However, the existing technology for detecting the four-level structure of the yeast surface display oligomeric enzyme mainly adopts the indirect characterization of the structure after the enzyme displayed on the yeast surface is cut off, so that the problems that the enzyme cutting efficiency is low, the oligomeric structure is changed after the enzyme cutting, the structure of the cell surface enzyme cannot be truly reflected and the like are likely to exist. Traditional biophysical techniques (e.g., circular dichroism, fluorescence, FTIR, NMR, crystallography) are sometimes difficult to apply to interface systems (e.g., due to poor signal/background ratios).

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, the invention provides a method for in-situ detection of a yeast surface display oligomeric enzyme quaternary structure based on FRET. According to the invention, a fluorescent probe is introduced into the yeast surface display oligoenzyme, and the FRET technology is utilized to detect the oligomeric structure of the enzyme on the cell surface in situ. The invention avoids the method of cutting enzyme from cell wall, and realizes the real-time and in-situ detection of the oligomeric structure of the yeast surface display oligomeric enzyme. The method using FRET improves the signal-to-noise ratio of the interface system between the protein and the cell.

The invention adopts the technical scheme that:

The invention provides a method for in-situ detection of yeast surface display oligomeric enzyme quaternary structure based on FRET, which comprises the following steps:

(1) Taking saccharomyces cerevisiae as a chassis strain, knocking out genes PPQ1, ALO1, NAM7 and GAL80, and simultaneously introducing multiple copies of tyrosyl-tRNA synthase (OmeRS) and corresponding tRNA _CUA ^Tyr;

(2) Selecting sites at the interface of the oligoenzyme to be mutated into cysteine and TAG codons, respectively, using the anchoring protein to mediate the surface display of the oligoenzyme's Saccharomyces cerevisiae;

(3) Adding a donor fluorescent molecule Cy 3-maleimide for coupling with cysteine of the yeast surface display oligoenzyme, and adding an acceptor fluorescent molecule Cy5-DBCO for coupling with azide of the yeast surface display oligoenzyme;

(4) Characterizing donor, acceptor and FRET signals using confocal microscopy;

Wherein the gene sequence of tyrosyl tRNA synthase is shown as SEQ ID NO.1, and the sequence of tRNA _CUA ^Tyr is shown as SEQ ID NO.2 or SEQ ID NO. 3.

The knockdown of the genes PPQ1, ALO1 and NAM7, which affect the insertion of unnatural amino acids, and the knockdown of GAL80 in step (1) of the method of the invention are used to increase the activity of the GAL1 promoter and to introduce the unnatural amino acid pAzF at the position of the specific amber codon TAG by expressing the tyrosyl-tRNA synthase and tRNA _CUA ^Tyr from e. The side chain of pAzF contains an azide group that is capable of coupling to cyclooctyne functionalized Cy5 fluorescent molecules. Rare cysteine mutations were used for coupling maleimide functionalized Cy3 fluorescent molecules. Further, the four-level structure of the cell surface in-situ detection of the oligoenzyme is realized by characterizing the donor, acceptor and FRET signals through a confocal microscope.

In some embodiments, the saccharomyces cerevisiae is saccharomyces cerevisiae BY4741.

In some embodiments, the promoter that expresses OmeRS is a GPD promoter, the terminator is a GPM1 terminator, the promoter that expresses tRNA _CUA ^Tyr is a SNR52 promoter, and the terminator is a SUP4 terminator, wherein the gene sequence of the GPD promoter is shown in SEQ ID No.4, the gene sequence of the GPM1 terminator is shown in SEQ ID No.5, the sequence of the SNR52 promoter is shown in SEQ ID No.6, and the sequence of the SUP4 terminator is shown in SEQ ID No. 7.

In some embodiments, the two sites of the oligoenzyme interface that are mutated to cysteine and TAG codons, respectively, are located in inactive centers and the distance between the two sites is less than Cy3 and Cy5Distance.

In some embodiments, the inducing protein expression in step (2) is performed in YPG-G418 medium supplemented with pAzF at a final concentration of 0.5-10 mM.

In some embodiments, the donor fluorescent molecule has an excitation wavelength of 500-554nm, a detection wavelength of 546-630nm, the acceptor fluorescent molecule has an excitation wavelength of 582-648nm, a detection wavelength of 650-740nm, and the FRET channel has an excitation wavelength of 500-535nm, and a detection wavelength of 649-740nm.

In a specific embodiment, the excitation wavelength of Cy3 channel is 516nm, the detection wavelength is 550-630 nm, the excitation wavelength of FRET channel is 516nm, the detection wavelength is 650-730nm, the excitation wavelength of Cy5 channel is 640nm, the detection wavelength is 650-730nm, and the laser "smart gain" values of Cy3 channel, cy5 channel and FRET channel are set to 15%, 15% and 20%, respectively.

In some embodiments, when a distinct donor signal, acceptor signal, FRET signal is present, then subunit interface is determined to be present, and when a distinct donor signal and acceptor signal are present, but no FRET signal is shown, then subunit dissociation of the oligoenzyme is determined;

in some embodiments, the method further comprises the step of calculating a FRET ratio, FRET ratio = F _FRET/F_Donor(s), wherein F _FRET is the fluorescence signal of Cy5 at its detection wavelength and F _Donor(s) is the fluorescence signal of Cy3 at its detection wavelength.

In some embodiments, the method further comprises determining the effect of external conditions on the surface display of the yeast oligomer enzyme subunit interface stability based on the change in FRET ratio.

In some embodiments, the method further comprises determining the effect of different expression systems of the yeast surface display oligoenzymes on subunit interface stability based on the change in FRET ratio.

In a specific embodiment, FRET ratios are quantified using ImageJ and Leica Las X software.

In some embodiments, the anchoring protein is selected from one of the Pir1 anchoring protein, sed1 anchoring protein or Ag alpha anchoring protein.

The gene sequence of Pir1 anchoring protein is shown as SEQ ID NO.8, the gene sequence of Sed1 anchoring protein is shown as SEQ ID NO.9, and the gene sequence of Ag alpha anchoring protein is shown as SEQ ID NO. 10.

In a specific embodiment, the oligoenzyme is β -glucuronidase (PGUS), the gene sequence of which has an accession number EU095019 in GeneBank.

In some embodiments, the promoter used for the s.cerevisiae surface display of step (2) using the dockerin mediated oligoenzyme is the GAL1 promoter, the signal peptide is the SED1 signal peptide, and the terminator is the ENO2 terminator. The gene sequence of the GAL1 promoter is shown as SEQ ID NO.11, the gene sequence of the SED1 signal peptide is shown as SEQ ID NO.12, and the sequence of the ENO2 terminator is shown as SEQ ID NO. 13.

The invention has the advantages and beneficial effects.

1. The invention can detect the four-level structure of the oligoenzyme on the surface of the cell in situ without separating the displayed oligoenzyme from the surface of the yeast cell, and has the advantages of less experimental steps, simple operation and the like.

2. The FRET technology used in the invention for detecting the quaternary structure of the cell surface oligoenzyme has the characteristics of high signal-to-noise ratio, high sensitivity and the like.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic diagram of BY 4741-. DELTA.PANG strain construction.

FIG. 2 is a photograph of a successful colony PCR agarose gel electrophoresis verification of BY 4741-delta PANG strain construction.

FIG. 3 is a schematic diagram of pRS41K-EGFP-TAG-mRFP plasmid construction.

FIG. 4 is a photograph of a successful colony PCR agarose gel electrophoresis verification of the construction of the ΔPANG-ERFP strain.

FIG. 5 is a schematic diagram showing construction of pRS41K-PGUS-Pir1 and pRS41K-PGUS-Pir1+ Flag plasmids.

FIG. 6 shows the verification pictures of successful colony PCR agarose gel electrophoresis of LA, LA-W522A, SA-W522A, PGUS-Pir1, S35C-Pir1, D220TAG-Pir1, G153C-Pir1, E488TAG-Pir1, LC and SC strains constructed.

FIG. 7 shows fluorescence of EGFP and mRFP containing reporter strains with or without the addition of 5mM pAzF.

FIG. 8 is a selection of sites at which FRET effects can occur at the interface of the large (a) and small (b) subunits of PGUS. (c) Fluorescent gel images after crosslinking of pAzF-containing mutants with Cy3 and Cy5 were verified.

FIG. 9 shows a subunit interface based on FRET detection surface display PGUS. (a) The FRET signal characterizes the large subunit interface of surface display PGUS in strains LA, LA-W522A and LC. (b) The FRET signal characterizes the small subunit interface of surface display PGUS in strains SA, SA-W522A and SC. (c) The FRET ratios of LA, LA-W522A and LC are shown (F _FRET/F_Donor(s)). (d) shows the FRET ratios of SA, SA-W522A and SC.

FIG. 10 shows the variation of FRET ratio of strains LA and SA with temperature (a) or urea concentration (b).

FIG. 11 shows FRET signals and corresponding FRET ratios of strains BY 4741-. DELTA.PANG, S35C-Pir1, D220pAzF-Pir1, G153C-Pir1 and E488pAzF-Pir1 for excluding the FRET effect of other cell surface proteins with surface display PGUS.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.

The present application has been made based on the fact that FRET (fluorescence resonance energy transfer) is widely used to monitor intermolecular binding between macromolecules and conformational changes within biomolecules. Since the fluorescence of the donor and acceptor fluorophores will vary depending on the distance between the donor and acceptor, a change in spatial position or distance between different domains or subunits is observed. The usual strategy for labeling proteins using FRET fluorescence is to fusion express fluorescent proteins, but they can only be incorporated into the C-or N-terminus of the target protein, but not into the target protein sequence, which may disrupt the structure and function of the protein. The application benefits from the development of genetic code expansion technology, so that a plurality of unnatural amino acids can be introduced into specific sites of yeast cell surface proteins, and various synthetic fluorescent dyes can be coupled through click reaction, so that the influence on the structure and the function of the proteins is reduced.

The following is a term description:

the term "surface display system" refers to the immobilization of a foreign protein or polypeptide on the surface of a cell using cell surface display technology. Surface display systems include, for example, prokaryotic and eukaryotic surface display systems. In one embodiment, the eukaryotic surface display system comprises a yeast surface display system. In one embodiment, eukaryotic surface display systems may be used to display proteins or polypeptides that require post-translational modifications such as phosphorylation, glycosylation, methylation, acetylation, hydroxylation, disulfide isomerization, and the like.

The term "oligomeric enzyme" refers to a multi-subunit enzyme consisting of at least two polypeptide chains that are covalently or non-covalently linked together. The term "oligomeric enzyme" encompasses multi-subunit enzymes in which at least two subunits of the enzyme are linked together covalently or non-covalently. The term "oligoenzyme" includes both homologous oligoenzymes, which are multi-subunit enzymes consisting of only one type of monomer (subunit), and heterologous oligoenzymes consisting of different types of monomers (subunit).

The term "promoter" refers to a nucleic acid sequence, which is typically present upstream (5' to) the coding sequence of a gene of interest, capable of directing transcription of the nucleic acid sequence into mRNA. In general, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.

The term "terminator" refers to a nucleic acid sequence, which is typically present downstream (3' to) of a coding sequence of a gene of interest, capable of terminating transcription of the nucleic acid sequence into mRNA.

Material source

Restriction enzymes were purchased from Takara Bio Inc., DNA polymerase was purchased from Novain, primer synthesis, gene synthesis and gene sequencing were performed by Jin Weizhi, gel recovery kit and plasmid extraction kit were purchased from Tiangen, glycyrrhizic acid was purchased from Xinjiang Tianshan pharmaceutical, his tag ELISA kit was purchased from Kirsrui, para-azido-phenylalanine (pAzF) was purchased from Michael, cy 3-maleimide and Cy5-DBCO were purchased from Simaroubrication.

EXAMPLE 1 construction of Chassis Strain

Expression cassettes P _SNR52-tRNA2_CUA ^Tyr-T_SUP4、P_GPD-OmeRS-T_GPM1 and P _PGK1-3×(P_SUP4-tRNA1_CUA ^Tyr-T_SUP4) are obtained by gene synthesis. tRNA1 _CUA ^Tyr (SEQ ID NO. 2) and tRNA2 _CUA ^Tyr (SEQ ID NO. 3) are the corresponding two tRNA substrates for tyrosyl tRNA synthase, and both tRNA1 _CUA ^Tyr and tRNA2 _CUA ^Tyr bind pAzF under the catalysis of tyrosyl tRNA synthase. The gene sequence of the PGK1 promoter is shown as SEQ ID NO.14, and the sequence of the SUP4 promoter is shown as SEQ ID NO. 15:

The sequence of expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4 was amplified using primers tRNA2-F and tRNA2-R in a system of Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4 gene template 1. Mu.L, primers tRNA2-F and tRNA2-R (10. Mu.M) each 2. Mu. L, phanta Hi-Fi DNA polymerase 1. Mu.L, Double distilled water was supplemented to 50 μl. The amplification conditions were 98℃for 3min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The sequence of expression cassette P _GPD-OmeRS-T_GPM1 was amplified using primers OmeRS-F and OmeRS-R in a system of Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, expression cassette P _GPD-OmeRS-T_GPM1 gene template 1. Mu.L, primers OmeRS-F and OmeRS-R (10. Mu.M) each 2. Mu. L, phanta high-fidelity DNA polymerase 1. Mu.L, Double distilled water was supplemented to 50 μl. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2min (30 cycles) and 72℃for 10 min. His3-F and His3-R primers were used to amplify the His3 gene sequence of the histidine auxotroph selection marker from the Saccharomyces cerevisiae genome in a Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, 1. Mu.L of the template Saccharomyces cerevisiae genome, 1. Mu.L of each of the primers His3-F and His3-R (10. Mu.M) 2. Mu. L, phanta high fidelity DNA polymerase, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The left arm sequence of the PPQ1 gene was amplified from the Saccharomyces cerevisiae genome using primers PPQ1L-F and PPQ1L-R in a Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers PPQ1L-F and PPQ1L-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The right arm sequence of the PPQ1 gene was amplified from the Saccharomyces cerevisiae genome using primers PPQ1R-F and PPQ1R-R in a Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers PPQ1R-F and PPQ1R-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4 gene fragment, the expression cassette P _GPD-OmeRS-T_GPM1 gene fragment, the histidine auxotroph screening marker His3 gene sequence and the PPQ1 left and right arm genes are recovered by a root agarose gel DNA recovery kit, and the primer sequences are as follows:

tRNA2-F:GATGCGTAAGGAGAAAATACCGCATCAGGCTTGATATCGAATTCCTGCAGCCCG(SEQ ID NO.16)

tRNA2-R：CACCCCGCGAATTCGTTCAAGTCTACAAAAAAGATCTCGGCTCTAGAC(SEQ ID NO.17)OmeRS-F：ACTTGAACGAATTCGCGGGGTG(SEQ ID NO.18)

OmeRS-R:CCCGTTGTAACGCTCATTACTGCCGGCTTCTAATCCGTCGAGTTTATCATTATCAATACTGC(SEQ ID NO.19)

His3-F:GAGAAGAAGCCCGTCCAGATCAACTAGTACACTCTATATTTTTTTATGCCTCGG(SEQ ID NO.20)

His3-R：GCAGGAATTCGATATCAAGCCTGATGCGGTATTTTCTCCTTACGCATC(SEQ ID NO.21)PPQ1L-F：CTCTCTGCAGCCGACTACGAGG(SEQ ID NO.22)

PPQ1L-R:CCGAGGCATAAAAAAATATAGAGTGTACTAGTTGATCTGGACGGGCTTCTTCTC(SEQ ID NO.23)

PPQ1R-F：GATAAACTCGACGGATTAGAAGCCGGCAGTAATGAGCGTTACAACGGG(SEQ ID NO.24)PPQ1R-R：CGTATTACGCCGATTAGGCTGACAC(SEQ ID NO.25)

The amplified fragments were mixed in equimolar ratio and 10. Mu.L of electric shock was converted into fresh Saccharomyces cerevisiae BY4741 competent. Spread on SD solid medium lacking histidine (SD-his) and cultured until a single apparent colony of yeast appears. Single colonies were picked and streaked onto SD-his plates for storage. Recombinant bacteria were randomly picked from the stored SD-his streak plates, 3 parallel control groups were each picked, and inoculated into test tubes containing 5mL of SD-his liquid medium for cultivation for about 24 hours. 2mL of bacterial liquid is taken to extract a yeast genome, then primers PPQ1L-F and tRNA2-R are used for carrying out PCR verification and amplification on target strips, the recombinant bacteria amplified from the target strips are preliminarily judged to be positive bacteria, the construction schematic diagram of the bacterial strain is shown in figure 1, and the colony PCR verification result is shown in figure 2. A positive strain was obtained in which PPQ1 was knocked out and one copy of tRNA2 _CUA ^Tyr and OmeRS was introduced simultaneously, and was designated BY 4741-. DELTA.P.

A tandem expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4-P_GPD-OmeRS-T_GPM1 gene fragment was amplified from BY 4741-. DELTA.P strain using primers tRNA2-OmeRS-F and tRNA2-OmeRS-R in a system of Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, BY 4741-. DELTA.P genomic template 1. Mu.L, primers tRNA2-OmeRS-F and tRNA2-OmeRS-R (10. Mu.M) each 2. Mu.L, phanta high-fidelity DNA polymerase 1. Mu.L, double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2 min (30 cycles) and 72℃for 10 min. Leucine auxotroph selection marker Leu2 gene sequence was amplified from Saccharomyces cerevisiae genome using primers Leu2-F and Leu2-R in PhantaBuffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers Leu2-F and Leu2-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The left arm sequence of the ALO1 gene was amplified from the Saccharomyces cerevisiae genome using primers ALO1L-F and ALO1L-R in a Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers ALO1L-F and ALO1L-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The right arm sequence of the ALO1 gene was amplified from the Saccharomyces cerevisiae genome using primers ALO1R-F and ALO1R-R in a Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers ALO1R-F and ALO1R-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The gene fragment of the expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4-P_GPD-OmeRS-T_GPM1, the leucine auxotroph screening mark Leu2 gene sequence and the left and right arm sequences of the ALO1 locus are recovered by a root agarose gel DNA recovery kit, and the primer sequences are as follows:

tRNA2-OmeRS-F:CCTAATTTGATATTGGAGGGCTTGATATCGAATTCCTGCAGCCCG(SEQ ID NO.26)

tRNA2-OmeRS-R：CCTTCAATGCCATCCCCCTCAGGCTTCTAATCCGTCGAGTTTATCATTATCAATACTG(SEQ ID NO.27)Leu2-F：CGATATTATCAGGTTTTTCACCCCATGTCAACTGTGGGAATACTCAGGTATCGTAAG(SEQ ID NO.28)

Leu2-R:CCCGGGCTGCAGGAATTCGATATCAAGCCCTCCAATATCAAATTAGGAATCGTAGTTTCATG(SEQ ID NO.29)

ALO1L-F:GTGGGTGCCGACTCATACGC(SEQ ID NO.30)

ALO1L-R:CTTACGATACCTGAGTATTCCCACAGTTGACATGGGGTGAAAAACCTGATAATATCGG(SEQ ID NO.31)

ALO1R-F：ATGATAAACTCGACGGATTAGAAGCCTGAGGGGGATGGCATTGAAGG(SEQ ID NO.32)ALO1R-R：GCATGTCTCTACTATCGACATTGTTGCG(SEQ ID NO.33)

The amplified fragments were mixed in equimolar ratio and 10. Mu.L of electric shock was converted into fresh Saccharomyces cerevisiae BY 4741-DeltaP competent. Spread on SD solid medium lacking leucine (SD-leu) and cultured until a single apparent colony of yeast appears. Single colonies were picked and streaked onto SD-his plates for storage. Recombinant bacteria were randomly picked from the stored SD-leu streak plates, 3 parallel control groups were each picked, and inoculated into test tubes containing 5mL of SD-leu liquid medium for cultivation for about 24 hours. 2mL of bacterial liquid is taken to extract a yeast genome, then primers ALO1L-F and tRNA2-R are used for carrying out PCR verification and amplification on target strips, the recombinant bacteria amplified from the target strips are preliminarily judged to be positive bacteria, the construction schematic diagram of the bacterial strain is shown in figure 1, and the colony PCR verification result is shown in figure 2. A positive strain was obtained, in which PPQ1 and ALO1 were knocked out, and two copies of tRNA2 _CUA ^Tyr and OmeRS were introduced simultaneously, designated BY 4741-. DELTA.PA.

A tandem expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4-P_GPD-OmeRS-T_GPM1 gene fragment was amplified from BY 4741-. DELTA.PA strain using primers tRNA2-OmeRS-2F and tRNA2-OmeRS-2R in a system of Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, BY 4741-. DELTA.PA genome template 1. Mu.L, primers tRNA2-OmeRS-2F and tRNA2-OmeRS-2R (10. Mu.M) 2. Mu.L each, phanta high-fidelity DNA polymerase 1. Mu.L, double distilled water was supplemented to 50. Mu.L. the amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2 min (30 cycles) and 72℃for 10 min. The hygromycin resistance gene HygR sequence was amplified from the Saccharomyces cerevisiae genome using primers hyg-F and hyg-R in a PhantaBuffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers hyg-F and hyg-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. Using primers tRNA1-F and tRNA1-R, the expression cassette P _PGK1-3×(P_SUP4-tRNA1_CUA ^Tyr-T_SUP4 sequence was amplified in Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, 1. Mu.L of plasmid template containing P _PGK1-3×(P_SUP4-tRNA1_CUA ^Tyr-T_SUP4, 2. Mu.L each of primers tRNA1-F and tRNA1-R (10. Mu.M), phanta high-fidelity DNA polymerase 1. Mu.L, double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. NAM7 site gene left arm sequence was amplified from Saccharomyces cerevisiae genome using primers NAM7L-F and NAM7L-R in Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers NAM7L-F and NAM7L-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. the amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. NAM7 locus gene right arm sequence was amplified from Saccharomyces cerevisiae genome using primers NAM7R-F and NAM7R-R in Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers NAM7R-F and NAM7R-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The gene fragment of the expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4-P_GPD-OmeRS-T_GPM1, the HygR sequence of the hygromycin resistance gene, the sequence of the expression cassette P _PGK1-3×(P_SUP4-tRNA1_CUA ^Tyr-T_SUP4) and the left and right arm sequences of NAM7 sites which are connected in series are recovered by a Tiangen agarose gel DNA recovery kit, and the primer sequences are as follows:

tRNA2-OmeRS-2F:CGTCCGAGGGCAAAGGAATAATGACACCGATTATTTAAAGCTGCAGCATACG

(SEQ ID NO.34)

tRNA2-OmeRS-2R:GGGAGGGACACCTTTATACGCTTTCGGGCTTCTAATCCGTCGAGTTTATCATTATCAATACTG(SEQ ID NO.35)

hyg-F：GTAGAAATGGATCCCCCACACACCGACATGGAGGCCCAGAATACCCTC(SEQ ID NO.36)hyg-R：GCTGCAGCTTTAAATAATCGGTGTCATTATTCCTTTGCCCTCGGACGAG(SEQ ID NO.37)tRNA1-F:ATGACTGGGCCCAGTTGCTGGTGGGTCATGCATCGATTTGGGCGC(SEQ ID NO.38)tRNA1-R:GAGGGTATTCTGGGCCTCCATGTCGGTGTGTGGGGGATCCATTTCTAC(SEQ ID NO.39)NAM7L-F：CCAATGGCCTGTTGTTCATTGGC(SEQ ID NO.40)

NAM7L-R：GCGCCCAAATCGATGCATGACCCACCAGCAACTGGGCCCAGTC(SEQ ID NO.41)NAM7R-F：TAAACTCGACGGATTAGAAGCCCGAAAGCGTATAAAGGTGTCCCTCCC(SEQ ID NO.42)NAM7R-R：CCACTATCACAGAGAACGAACGCC(SEQ ID NO.43)

The amplified fragments were mixed in equimolar ratio and 10. Mu.L of electric shock was converted into fresh Saccharomyces cerevisiae BY4741- ΔPA competent. Spread on YPD plates (YPD-hyg) containing 7/1000 hygromycin and incubated until a single apparent colony of yeast was present. And (5) picking single colonies, and scribing and preserving the single colonies on a YPD-hyg plate. Recombinant bacteria were randomly picked from the stored YPD-hyg streaking plates, 3 parallel control groups were each picked, and inoculated into test tubes containing 5mL of YPD-hyg liquid medium and cultured for about 24 hours. 2mL of bacterial liquid is taken to extract a yeast genome, then primers NAM7L-F and tRNA2-R are used for carrying out PCR verification and amplification on target strips, the recombinant bacteria amplified from the target strips are preliminarily judged to be positive bacteria, the construction schematic diagram of the bacterial strain is shown in figure 1, and the colony PCR verification result is shown in figure 2. A positive strain was obtained that knocked out PPQ1, ALO1 and NAM7, with the simultaneous introduction of three copies of tRNA1 _CUA ^Tyr、tRNA2_CUA ^Tyr and OmeRS, designated BY 4741-. DELTA.PAN.

A tandem expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4-P_GPD-OmeRS-T_GPM1 gene fragment was amplified from BY 4741-. DELTA.PAN genome using primers tRNA2-OmeRS-3F and tRNA2-OmeRS-3R in a system of Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, BY 4741-. DELTA.PAN genome template 1. Mu.L, primers tRNA2-OmeRS-3F and tRNA2-OmeRS-3R (10. Mu.M) 2. Mu.L each, phanta high-fidelity DNA polymerase 1. Mu.L, double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2 min (30 cycles) and 72℃for 10 min. Uracil auxotroph marker gene Ura3 sequence was amplified from Saccharomyces cerevisiae genome using primers Ura3-F and Ura3-R in Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers Ura3-F and Ura3-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. the amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. GAL80 locus gene left arm sequence was amplified from Saccharomyces cerevisiae genome using primers GAL80L-F and GAL80L-R in Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers GAL80L-F and GAL80L-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The GaL locus gene right arm sequence was amplified from the Saccharomyces cerevisiae genome using primers GAL80R-F and GAL80R-R in Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, template Saccharomyces cerevisiae genome 1. Mu.L, primers GAL80R-F and GAL80R-R (10. Mu.M) each 2. Mu. L, phanta Hi-Fi DNA polymerase 1. Mu.L, supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. The gene fragment of the expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4-P_GPD-OmeRS-T_GPM1, uracil auxotroph screening marker Ura3 gene sequence and NAM7 site left and right arm sequences are recovered by a root agarose gel DNA recovery kit, and the primer sequences are as follows:

tRNA2-OmeRS-3F:CGTCCGAGGGCAAAGGAATAATGACACCGATTATTTAAAGCTGCAGCATACG

(SEQ ID NO.44)

tRNA2-OmeRS-3R:GGGAGGGACACCTTTATACGCTTTCGGGCTTCTAATCCGTCGAGTTTATCATTATCAATACTG(SEQ ID NO.45)

ura3-F：GCTAATCCGGTCACTGCCGTACTGAGAGTGCAGCTTCAATTCATC(SEQ ID NO.46)ura3-R：GGCTGCAGGAATTCGATATCAAGCGGTATTTCACACCGCATAGGGTAATAACTG(SEQ ID NO.47)

GAL80L-F:CTCACTCTGCACTGCCGTGC(SEQ ID NO.48)

GAL80L-R:GATGAATTGAAGCTGCACTCTCAGTACGGCAGTGACCGGATTAGCATTGG(SEQ ID NO.49)

GAL80R-F：GGAGTACAGTCACTTTGAGAACCCCTTTTTCGTGCATGCGGGTG(SEQ ID NO.50)GAL80R-R：CATCTTGAAATGCTGCAATTATGTCGGG(SEQ ID NO.51)

Mixing the amplified fragments according to an equimolar ratio, and taking 10 mu L of electric shock to convert into fresh Saccharomyces cerevisiae BY 4741-delta PAN competent. Spread on uracil-deficient SD solid medium (SD-ura) until a single apparent colony of yeast appears. Single colonies were picked and streaked on SD-ura plates for storage. Recombinant bacteria were randomly picked from the stored SD-ura streaking plates, 3 parallel control groups were each picked, and inoculated into test tubes containing 5mL of SD-ura broth, and incubated for about 24 hours. 2mL of bacterial liquid is taken to extract a yeast genome, then primers GAL80L-F and tRNA2-R are used for carrying out PCR verification and amplification on target strips, the recombinant bacteria amplified from the target strips are preliminarily judged to be positive bacteria, the construction schematic diagram of the bacterial strain is shown in figure 1, and the colony PCR verification result is shown in figure 2. Positive strains were obtained that knocked out PPQ1, ALO1, NAM7 and GAL80, with the simultaneous introduction of three copies of tRNA1 _CUA ^Tyr, four trnas 2 _CUA ^Tyr and OmeRS, designated BY4741- Δpang.

Example 2 construction of a Strain containing a fluorescent protein reporter Gene and verification of pAzF introduction

2.1 Construction of reporter Gene plasmids and Strain containing fluorescent proteins

The plasmid selected was pRS41K and contained the G418 selection marker kMX gene, the prokaryotic replicons ColE1 and f1, the self-replicating sequences CEN/ARS in Saccharomyces cerevisiae and the ampicillin selection marker AmpR gene. The linearized plasmid was amplified using primers pRS41K-F and pRS41K-R in Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, pRS41K plasmid template 1. Mu.L, primers pRS41K-F and pRS41K-R (10. Mu.M) 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min pre-denaturation, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 3 min (35 cycles) and 72℃for 10 min.

A tandem expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4-P_GPD-OmeRS-T_GPM1 gene fragment was amplified from BY 4741-. DELTA.PANG genome using primers tRNA2-OmeRS-4F and tRNA2-OmeRS-4R in a system of Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, BY 4741-. DELTA.PANG genome template 1. Mu.L, primers tRNA2-OmeRS-4F and tRNA2-OmeRS-4R (10. Mu.M) 2. Mu.L each, phanta high-fidelity DNA polymerase 1. Mu.L, double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2 min (35 cycles) and 72℃for 10 min. pGal1 promoter and tENO2 terminator were amplified from BY 4741-. DELTA.PANG genome using primers pGal1-F and pGal1-R, tENO2-F and tENO-R, respectively. The amplification system was Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, BY 4741-. DELTA.PANG genomic template 1. Mu.L, primers pGal1-F and pGal1-R or tENO2-F and tENO2-R (10. Mu.M) each 2. Mu. L, phanta high-fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2 min (35 cycles) and 72℃for 10 min. EGFP was amplified using primers EGFP-F and EGFP-R. The amplification system was Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, EGFP gene fragment template 1. Mu.L, primers EGFP-F and EGFP-R (10. Mu.M) each 2. Mu. L, phanta high fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1min (35 cycles) and 72℃for 10 min. mRFP was amplified using the primers mRFP-F and mRFP-R. The amplification system was PhantaBuffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, mRFP gene fragment template 1. Mu.L, primers mRFP-F and mRFP-R (10. Mu.M) each 2. Mu. L, phanta high-fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1min (35 cycles) and 72℃for 10 min. The linearized pRS41K plasmid fragment, the tandem expression cassette P _SNR52-tRNA2_CUA ^Tyr-T_SUP4-P_GPD-OmeRS-T_GPM1 gene fragment, the pGal1 promoter gene fragment, the EGFP gene fragment, the mRFP gene fragment and the tENO terminator gene fragment are recovered by a Tiangen agarose gel DNA recovery kit, and the primer sequences are pRS41K-F GAGAACGAGCTCCAGCTTTTGTTCCCTTTAGTG (SEQ ID NO. 52)

pRS41K-R:CCCGGGCTGCAGGAATTCG(SEQ ID NO.53)

tRNA2-OmeRS-4F:CGAATTCCTGCAGCCCGGGTCTTTGAAAAGATAATGTATGATTATGCTTTCACTC(SEQ ID NO.54)

tRNA2-OmeRS-4R:GCTCGGCGGCTTCTAATCCGTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACG(SEQ ID NO.55)

pGal1-F:CGGATTAGAAGCCGCCGAGC(SEQ ID NO.56)

pGal1-R：CCTCGCCCTTGCTCACCATGTTTTTTCTCCTTGACGTTAAAGTATAGAGG(SEQ ID NO.57)tENO2-F：CACTCCACCGGTGCTTGAAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGC(SEQ ID NO.58)

tENO2-R：GGAACAAAAGCTGGAGCTCGTTCTCAAAGTGACTGTACTCCATG(SEQ ID NO.59)EGFP-F：ATGGTGAGCAAGGGCGAGG(SEQ ID NO.60)

EGFP-R:CCGCTGCCGCTCTTGTACAGCTCGTCCATGCCGAG(SEQ ID NO.61)

mRFP-F:GCTGTACAAGAGCGGCAGCGGTAGCGGCAGCGGTAGCTAAATGGCTTCCTCCGAAGACG(SEQ ID NO.62)

mRFP-R:CGTCACTCCACCGGTGCTTGAAGTGCTTTTAACTAAG(SEQ ID NO.63)

Gibbsen assembly System the vector and the volume of the vector to be added were calculated so that the concentration ratio of the vector to the fragment was 2:1, gibson Mix was removed from-20℃with 6.5. Mu.L per tube, 2.5. Mu.L of LDNA fragment was added, 1. Mu. LTAQ DNALIGASE. EP tube was placed in a PCR apparatus at 50℃for 1 hour, gibbsen assembly products were transformed into E.coli Trans1-T1 competent cells, respectively, plated on solid LB medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agarose 20 g/L) containing 100mg/L ampicillin, and cultured at 37 ℃.

Transformants were identified by colony PCR and sequencing methods. Colony PCR System template LB plates were single-colony, 1. Mu.L each of the upstream and downstream primers tRNA2-OmeRS-4F and EGFP-R, 2-xrTaq mix (Takara Bio Inc.) 10. Mu.L, and 20. Mu.L was made up with double distilled water. PCR conditions were pre-denatured at 94℃for 5 minutes, denatured at 94℃for 30 seconds, annealed at 60℃for 30 seconds, extended at 72℃for 3 minutes, cycled 30 times, 72℃for 10 minutes, and stored at 4 ℃. Transformants containing the target band were confirmed by colony PCR for DNA sequencing and plasmid construction was confirmed to be successful, and the plasmid was designated pRS41K-EGFP-TAG-mRFP.

10. Mu.L of shock from pRS41K-EGFP-TAG-mRFP above was transferred into fresh Saccharomyces cerevisiae BY 4741-. DELTA.PANG competent. Spread on YPD-G418 plates containing 2/1000G418 and incubated until a single apparent colony of yeast is present. Single colonies were picked and streaked onto YPD-G418 plates for storage. Recombinant bacteria were randomly picked from the stored YPD-G418 streak plates, 3 parallel control groups were each picked, and inoculated into test tubes containing 5mL of YPD-G418 liquid medium and cultured for about 24 hours. 2mL of bacterial liquid is taken to extract yeast genome, then corresponding upstream and downstream primers are used for PCR verification and amplification of target strips, the recombinant bacteria amplified from the target strips are preliminarily judged to be positive bacteria, the strain construction schematic diagram is shown in figure 3, and the colony PCR verification result is shown in figure 4. The positive strain successfully constructed above was designated as ΔPANG-ERFP.

2.2 Verification of pAzF introduction Using fluorescent proteins

Three single colonies were inoculated from the plates into 5mLYPD-G418 medium. The liquid culture was grown to saturation and then diluted to OD ₆₀₀ = 1 in 5mL of the same medium. The diluted medium was grown overnight at 30℃and then induced in 5mL galactose medium (YPG-G418) with OD ₆₀₀ = 1 containing 2/1000G418, pAzF (1M stock solution, dissolved in 2M NaOH, ready to use) was added at a final concentration of 5mM, and the wild type control was induced without pAzF addition and incubated with shaking at 200rpm at 30℃for 2 days.

As can be seen from fig. 7, most of the yeasts added with pAzF exhibited green fluorescence and red fluorescence at the same time, while most of the yeasts not added with pAzF exhibited green fluorescence only, demonstrating that the yeast strain Δpang-ERFP was able to specifically introduce pAzF into the position of TAG codon between green fluorescent protein EGFP and red fluorescent protein mRFP.

EXAMPLE 3 construction of Yeast surface display Strain

(1) Plasmid pRS41K-EGFP-TAG-mRFP was linearized using primers pRS-F and pRS-R with Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, pRS41K-EGFP-TAG-mRFP plasmid template 1. Mu.L, primers pRS-F and pRS-R (10. Mu.M) 2. Mu. L, phanta each high fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min pre-denaturation, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2.5 min (35 cycles) and 72℃for 10 min. The β -glucuronidase PGUS gene and the ankyrin Pir1 gene were amplified from laboratory preservation plasmid pGAPZ-Pir-PGUS (the preparation of the plasmid is described in patent 201910023085.8) using primers PGUS-F and Pir 1-R. The amplification system was Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, pGAPZ-Pir-PGUS plasmid template 1. Mu.L, primers PGUS-F and Pir-R (10. Mu.M) each 2. Mu. L, phanta Hi-Fi DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2 min (35 cycles) and 72℃for 10 min. The linearized plasmid fragment and PGUS-Pir1 gene fragment are recovered by a Tiangen agarose gel DNA recovery kit, and the primer sequences are as follows:

pRS-F:GCGCCCATCATCATCATCATCACTGAAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGC

(SEQ ID NO.64)

pRS-R:CCGGCAGATAATAGGACAGTTGATAATTTCATGTTTTTTCTCCTTGACGTTAAAGTATAGAGG(SEQ ID NO.65)

PGUS-F:CCTATTATCTGCCGGTTTAGCCTCGACTACTTTGGCCCAAATGCTCAAACCGCAGCAAACTAC(SEQ ID NO.66)

Pir1-R:CAGTGATGATGATGATGATGGGCGCTATTCAGATCCTCTTCTGAG(SEQ ID NO.67)

Transformants were identified by colony PCR and sequencing methods. Colony PCR System template LB plate single colony, upstream and downstream primers PGUS-F and Pir1-R each 1. Mu.L, 2xrTaq mix (Takara Bio Inc.) 10. Mu.L, and 20. Mu.L was made up with double distilled water. PCR conditions were pre-denatured at 94℃for 5 minutes, denatured at 94℃for 30 seconds, annealed at 60℃for 30 seconds, extended at 72℃for 3 minutes, cycled 30 times, 72℃for 10 minutes, and stored at 4 ℃. Transformants containing the target band were confirmed by colony PCR for DNA sequencing and plasmid construction was confirmed to be successful, and the plasmid was designated pRS41K-PGUS-Pir1.

The PGUS sequence was amplified using primers S35C-F and D220TAG-R in a Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, pRS41K-PGUS-Pir1 plasmid template 1. Mu. L, S35C-F and D220TAG-R primers (10. Mu.M) each 2. Mu. L, phanta Hi-Fi DNA polymerase 1. Mu.L supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. pRS41K-PGUS-Pir1 was linearized using primers D220TAG-F and S35C-R with Phanta Buffer (2×) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, pRS41K-PGUS-Pir1 plasmid template 1. Mu. L, D220TAG-F and S35C-R primers (10. Mu.M) 2. Mu. L, phanta each of high fidelity DNA polymerase 1. Mu.L supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min pre-denaturation, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 3 min (30 cycles) and 72℃for 10 min. The linearized plasmid fragment and PGUS gene fragment are recovered by a root agarose gel DNA recovery kit, and the primer sequences are as follows:

S35C-F:CAACAATACGCAACCATGGACATGCCAACTAAAAACGTC(SEQ ID NO.68)

D220TAG-R:GCTACGGTTGTGCCCTACTCATCTATCACGGC(SEQ ID NO.69)

D220TAG-F:GTGATAGATGAGTAGGGCACAACCGTAGCGACAAGC(SEQ ID NO.70)

S35C-R:GGCATGTCCATGGTTGCGTATTGTTG(SEQ ID NO.71)

Transformants were identified by colony PCR and sequencing methods. Colony PCR System template LB plate single colony, upstream and downstream primers PGUS-F and Pir1-R each 1. Mu.L, 2xrTaq mix (Takara Bio Inc.) 10. Mu.L, and 20. Mu.L was made up with double distilled water. PCR conditions were pre-denatured at 94℃for 5 minutes, denatured at 94℃for 30 seconds, annealed at 60℃for 30 seconds, extended at 72℃for 3 minutes, cycled 30 times, 72℃for 10 minutes, and stored at 4 ℃. Transformants containing the target band were confirmed by colony PCR for DNA sequencing and plasmid construction was confirmed to be successful, and the plasmid was designated pRS41K-S35C-D220TAG-Pir1.

The PGUS sequence was amplified using primers G153C-F and E488TAG-R in Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, pRS41K-PGUS-Pir1 plasmid template 1. Mu. L, G153C-F and E488TAG-R primers (10. Mu.M) 2. Mu. L, phanta each of high fidelity DNA polymerase 1. Mu.L, supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 1 min (30 cycles) and 72℃for 10 min. pRS41K-PGUS-Pir1 was linearized using primers E488TAG-F and G153C-R with Phanta Buffer (2×) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, pRS41K-PGUS-Pir1 plasmid template 1. Mu. L, D220TAG-F and S35C-R primers (10. Mu.M) 2. Mu. L, phanta each of high fidelity DNA polymerase 1. Mu.L supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min pre-denaturation, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 3 min (30 cycles) and 72℃for 10 min. The linearized plasmid fragment and PGUS gene fragment are recovered by a root agarose gel DNA recovery kit, and the primer sequences are as follows:

G153C-F:CTCGAGGCGACGTGCAAGAAGGTGCAGACTTATCAGCATG(SEQ ID NO.72)

E488TAG-R:CGAAGTTCCTATTCCAGCGCAGCTTCTGCCTCG(SEQ ID NO.73)

E488TAG-F:GCTGCGCTGGAATAGGAACTTCGCGGATGGACCGAG(SEQ ID NO.74)

G153C-R:CCTTCTTGCACGTCGCCTCGAGAATCTCC(SEQ ID NO.75)

Transformants were identified by colony PCR and sequencing methods. Colony PCR System template LB plate single colony, upstream and downstream primers PGUS-F and Pir1-R each 1. Mu.L, 2xrTaq mix (Takara Bio Inc.) 10. Mu.L, and 20. Mu.L was made up with double distilled water. PCR conditions were pre-denatured at 94℃for 5 minutes, denatured at 94℃for 30 seconds, annealed at 60℃for 30 seconds, extended at 72℃for 3 minutes, cycled 30 times, 72℃for 10 minutes, and stored at 4 ℃. Transformants containing the target band were confirmed by colony PCR for DNA sequencing and plasmid construction was confirmed to be successful, and the plasmid was designated pRS41K-G153C-E488TAG-Pir1.

The PGUS sequence of the above pRS41K-S35C-D220TAG-Pir1 or pRS41K-G153C-E488TAG-Pir1 plasmid was subjected to point mutation using primers W522A-F and W522A-R. The amplification system was Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, pRS41K-S35C-D220TAG-Pir1 or pRS41K-G153C-E488TAG-Pir1 plasmid template 1. Mu. L, W522A-F and W522A-R primers (10. Mu.M) each 2. Mu. L, phanta Hi-Fi DNA polymerase 1. Mu.L supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2 min (30 cycles) and 72℃for 10 min. The amplified product was digested with DpnI enzyme in a 10 XDpnI buffer 1. Mu.L, and PCR amplified product 8. Mu.L, dpnI enzyme 1. Mu.L. Digestion conditions were incubated for 1h at 37 ℃. The digested product was transformed into E.coli Trans1-T1 competent cells, plated on solid LB medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agarose 20 g/L) containing 100mg/L ampicillin, and cultured at 37 ℃. The primer sequences were as follows:

W522A-F:GGTTACTCCCGCGAGCGAGGAGTTCCAGGTCGAG(SEQ ID NO.76)

W522A-R:GGGAGTAACCATGACCGAGTGC(SEQ ID NO.77)

Transformants were identified by colony PCR and sequencing methods. Colony PCR System template LB plate single colony, upstream and downstream primers PGUS-F and Pir1-R each 1. Mu.L, 2xrTaq mix (Takara Bio Inc.) 10. Mu.L, and 20. Mu.L was made up with double distilled water. PCR conditions were pre-denatured at 94℃for 5 minutes, denatured at 94℃for 30 seconds, annealed at 60℃for 30 seconds, extended at 72℃for 2 minutes, cycled 30 times, 72℃for 10 minutes, and stored at 4 ℃. Transformants containing the target band were confirmed by colony PCR to be subjected to DNA sequencing, and the success of plasmid construction was confirmed, and the plasmids were designated pRS41K-S35C-D220TAG-W522A-Pir1 or pRS41K-G153C-E488TAG-W522A-Pir1, respectively.

The PGUS sequence in plasmid pRS41K-PGUS-Pir1 was subjected to single point mutation using primers S35C-F and S35C-R, D TAG-F and D220TAG-R, G C-F and G153C-R, E488TAG-F and E488TAG-R, respectively. The amplification system was Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu. L, pRS41K-PGUS-Pir1 plasmid templates 1. Mu. L, S35C-F and S35C-R, D220TAG-F and D220TAG-R, G C-F and G153C-R or E488TAG-F and E488TAG-R primers (10. Mu.M) 2. Mu. L, phanta each of high-fidelity DNA polymerase 1. Mu.L supplemented with double distilled water to 50. Mu.L. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2 min (30 cycles) and 72℃for 10 min. The amplified product was digested with DpnI enzyme in a 10 XDpnI buffer 1. Mu.L, and PCR amplified product 8. Mu.L, dpnI enzyme 1. Mu.L. Digestion conditions were incubated for 1h at 37 ℃. The digested product was transformed into E.coli Trans1-T1 competent cells, plated on solid LB medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agarose 20 g/L) containing 100mg/L ampicillin, and cultured at 37 ℃.

Transformants were identified by colony PCR and sequencing methods. Colony PCR System template LB plate single colony, upstream and downstream primers PGUS-F and Pir1-R each 1. Mu.L, 2xrTaq mix (Takara Bio Inc.) 10. Mu.L, and 20. Mu.L was made up with double distilled water. PCR conditions were pre-denatured at 94℃for 5 minutes, denatured at 94℃for 30 seconds, annealed at 60℃for 30 seconds, extended at 72℃for 2 minutes, cycled 30 times, 72℃for 10 minutes, and stored at 4 ℃. Transformants containing the target band were confirmed by colony PCR for DNA sequencing, confirming successful plasmid construction, and the plasmids were designated pRS41K-S35C-Pir1, pRS41K-D220TAG-Pir1, pRS41K-G153C-Pir1 and pRS41K-E488TAG-Pir1, respectively.

The Pir1 anchor protein of plasmid pRS41K-S35C-D220TAG-Pir1 or pRS41K-G153C-E488TAG-Pir1 was removed using primers Flag-F and Flag-R and replaced the Flag immune TAG (underlined as Flag TAG sequence). The amplification system was Phanta Buffer (2X) 25. Mu. L, dNTP (2.5 mM) 1. Mu.L, plasmid pRS41K-S35C-D220TAG-Pir1 or pRS41K-G153C-E488TAG-Pir1 template 1. Mu. L, flag-F and Flag-R primer (10. Mu.M) 2. Mu. L, phanta each of high-fidelity DNA polymerase 1. Mu.L, and double distilled water was supplemented to 50. Mu.L. The amplification conditions were 98℃for 3 min pre-denaturation, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 3 min (35 cycles) and 72℃for 10 min. The amplified product was digested with DpnI enzyme in a 10 XDpnI buffer 1. Mu.L, and PCR amplified product 8. Mu.L, dpnI enzyme 1. Mu.L. Digestion conditions were incubated for 1h at 37 ℃. The digested product was transformed into E.coli Trans1-T1 competent cells, plated on solid LB medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agarose 20 g/L) containing 50mg/L bleomycin, and cultured at 37 ℃. The primer sequences were as follows:

Flag-F:CGGTAGCGATTACAAGGATGACGACGATAAGTGAAGTGCTTTTAACTAAGAATTATTAGTCTTTTCT GC(SEQ ID NO.78)

Flag-R:CGTCGTCATCCTTGTAATCGCTACCGCTGCCGCTCTGGAAGGTCTTCCCTCCCTCAGC(SEQ ID NO.79)

Transformants were identified by colony PCR and sequencing methods. Colony PCR System template LB plates were single-colony, and 1. Mu.L each of the upstream and downstream primers PGUS-F and tENO-R, 2xrTaq mix (Takara Bio Inc.) was 10. Mu.L, and 20. Mu.L was made up with double distilled water. PCR conditions were pre-denatured at 94℃for 5 minutes, denatured at 94℃for 30 seconds, annealed at 60℃for 30 seconds, extended at 72℃for 2 minutes, cycled 30 times, 72℃for 10 minutes, and stored at 4 ℃. Transformants containing the target band were confirmed by colony PCR to be subjected to DNA sequencing, and the success of plasmid construction was confirmed, and the plasmids were designated pRS41K-S35C-D220TAG-Flag or pRS41K-G153C-E488TAG-Flag, respectively.

The expression cassettes pGal1-S35C-D220TAG-Flag and pGal1-G153C-E488TAG-Flag-tENO2 were amplified from the plasmids pRS41K-S35C-D220TAG-Flag or pRS41K-G153C-E488TAG-Flag using primers PGUS-Flag-F and PGUS-Flag-R in a system of PhantaBuffer (2X) 25 μ L, dNTP (2.5 mM) 1 μ L, pRS K-S35C-D220TAG-Flag or pRS41K-G153C-E488TAG-Flag plasmid templates 1 μL, primers PGUS-Flag-F and PGUS-Flag-R (10 μM) each 2 μ L, phanta high fidelity DNA polymerase 1 μL supplemented with double distilled water to 50 μL. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 2 min (35 cycles) and 72℃for 10 min. Plasmids pRS41K-S35C-D220TAG-Pir1 or pRS41K-G153C-E488TAG-Pir1 were linearized with primers pRS-Pir-Flag-F and pRS-Pir-R, respectively, using Phanta Buffer (2X) 25 μ L, dNTP (2.5 mM) 1 μ L, pRS K-S35C-D220TAG-Pir1 or pRS41K-G153C-E488TAG-Pir1 plasmid template 1 μl, primers PGUS-Flag-F and PGUS-Flag-R (10 μM) 2 μ L, phanta each of high fidelity DNA polymerase 1 μl supplemented with double distilled water to 50 μl. The amplification conditions were 98℃for 3 min, 98℃for 10 seconds, 60℃for 30 seconds, 72℃for 5 min (35 cycles) and 72℃for 10 min. The expression cassettes pGal1-S35C-D220TAG-Flag-tENO2 and pGal1-G153C-E488TAG-Flag-tENO2 gene fragments and linearized pRS41K-S35C-D220TAG-Pir1 and pRS41K-G153C-E488TAG-Pir1 plasmid fragments were recovered by a root agarose gel DNA recovery kit, the primer sequences were as follows:

PGUS-Flag-F:AGGAGCCGGAAGCGTTCTCAAAGTGACTGTACTCCATGTTTTCTTATCATCC(SEQ ID NO.80)

PGUS-Flag-R:CACCGGATTAGAAGCCGCCGAGC(SEQ ID NO.81)

pRS-Pir-Flag-F：CGGCGGCTTCTAATCCGGTGTAAAGCCTGGGGTGCC(SEQ ID NO.82)pRS-Pir-Flag-R：GAGAACGCTTCCGGCTCCTATGTTGTGTG(SEQ ID NO.83)

Transformants were identified by colony PCR and sequencing methods. Colony PCR System template LB plate single colony, upstream and downstream primers PGUS-Flag-F and PGUS-Flag-R were each 1. Mu.L, 2xrTaq mix (Takara Bio Inc.) 10. Mu.L, and 20. Mu.L was made up with double distilled water. PCR conditions were pre-denatured at 94℃for 5 minutes, denatured at 94℃for 30 seconds, annealed at 60℃for 30 seconds, extended at 72℃for 3 minutes, cycled 30 times, 72℃for 10 minutes, and stored at 4 ℃. Transformants containing the target band were confirmed by colony PCR to be subjected to DNA sequencing, and the success of plasmid construction was confirmed, and the plasmids were designated pRS41K-S35C-D220TAG-Pir1+flag or pRS41K-G153C-E488TAG-Pir1+flag, respectively.

Mu.L of each of the above pRS41K-S35C-D220TAG-Pir1、pRS41K-S35C-D220TAG-W522A-Pir1、pRS41K-S35C-D220TAG-Pir1+Flag、pRS41K-G153C-E488TAG-Pir1、pRS41K-G153C-E488TAG-W522A-Pir1 and pRS41K-G153C-E488TAG-Pir1+ Flag was shock-transferred into fresh Saccharomyces cerevisiae BY4741- ΔPANG competent. Spread on YPD-G418 plates containing 2/1000G418 and incubated until a single apparent colony of yeast is present. Single colonies were picked and streaked onto YPD-G418 plates for storage. Recombinant bacteria were randomly picked from the stored YPD-G418 streak plates, 3 parallel control groups were each picked, and inoculated into test tubes containing 5mL of YPD-G418 liquid medium and cultured for about 24 hours. 2mL of bacterial liquid is taken to extract yeast genome, then corresponding upstream and downstream primers are used for PCR verification and amplification of target strips, the recombinant bacteria amplified from the target strips are preliminarily judged to be positive bacteria, a bacterial strain construction schematic diagram is shown in figure 5, and a colony PCR verification result is shown in figure 6. The positive strains constructed successfully above were designated as LA, LA-W522A, LC, SA, SA-W522A and SC, respectively.

Plasmids pRS41K-PGUS-Pir1, pRS41K-S35C-Pir1, pRS41K-D220TAG-Pir1, pRS41K-G153C-Pir1 and pRS41K-E488TAG-Pir1 were also shock-transformed into fresh Saccharomyces cerevisiae BY 4741-. DELTA.PANG competent cells in 10. Mu.L, respectively. Spread on YPD-G418 plates containing 2/1000G418 and incubated until a single apparent colony of yeast is present. Single colonies were picked and streaked onto YPD-G418 plates for storage. Recombinant bacteria were randomly picked from the stored YPD-G418 streak plates, 3 parallel control groups were each picked, and inoculated into test tubes containing 5mL of YPD-G418 liquid medium and cultured for about 24 hours. 2mL of bacterial liquid is taken to extract yeast genome, then corresponding upstream and downstream primers are used for PCR verification and amplification of target strips, the recombinant bacteria amplified from the target strips are preliminarily judged to be positive bacteria, a bacterial strain construction schematic diagram is shown in figure 5, and a colony PCR verification result is shown in figure 6. The positive strains constructed successfully above were designated PGUS-Pir1, S35C-Pir1, D220pAzF-Pir1, G153C-Pir1 and E488pAzF-Pir1, respectively.

As can be seen from fig. 8, a crystal structure according to PGUS is selected to be large (interface area is) And small (interface area is) Is defined by the subunit interface of (a). The S35 site in the A chain, which is close to the large subunit interface and does not participate in the interface interaction, and the D220 site in the B chain are selected to be mutated to cysteine and the stop codon TAG, respectively. The distance between the two sites is aboutCompared with Cy3 and Cy5Distance ofShort. Distance between adjacent FRET pairs (respectively And) Compared with Cy3 and Cy5Distance ofLong. Likewise, the surface residues G153 in chain a and E488 in chain C were located near the small interface, away from the active center, and were selectively mutated to cysteine and amber codons, respectively, to incorporate pAzF (b in fig. 8). The distance between the two sites is aboutAlso shorter than Cy3 and Cy5Distance ofThus, a FRET signal will be generated. Distance of different FRET pairs (respectivelyAnd) Greater than or near Cy3 and Cy5Distance ofFRET signals from different FRET pairs are negligible. (2) Surface display contains pAzF PGUS activity and display quantity characterization

Three single colonies were inoculated from the plates into 5mLYPD-G418 medium. The liquid culture was grown to saturation and then diluted to OD ₆₀₀ = 1 in 5mL of the same medium. The diluted medium was grown overnight at 30℃and then induced in 5mL galactose medium (YPG-G418) with OD ₆₀₀ = 1 containing 2/1000G418, and pAzF (1M stock solution, dissolved in 2M NaOH, ready to use) was added at a final concentration of 5mM, and the wild type control was induced without pAzF addition and incubated with shaking at 200rpm at 30℃for 2 days. PGUS Glycyrrhizinic acid hydrolysis Activity 100. Mu.L of the washed yeast cell suspension (OD ₆₀₀ =20) was added to 300. Mu.L of ammonium glycyrrhizinate solution. After incubation at 40 ℃ for 10 minutes, 900 μl of methanol was added to terminate the reaction, and then all impurities were removed using a filter and the substrate and product were analyzed by HPLC. Yeast surface display PGUS amounts were determined according to the instructions using a commercial His-tag ELISA kit (gold-srey).

As can be seen from Table 1, recombinant s.cerevisiae cells containing the stop codon mutation had glycyrrhizic acid hydrolysis activity, and His-TAG on the cell surface could be detected by ELISA, indicating that pAzF could be introduced into the position of TAG codon of PGUS sequence, so that PGUS-Pir1 sequence was expressed intact and secreted outside the cell for surface display. In addition, the introduction of pAzF had no effect on the specific enzymatic activities of LA and SA, indicating that the introduction of pAzF did not affect the oligomeric structure of surface display PGUS.

3. Yeast surface chemical markers

Cell pellet was induced, washed 3 times with PBS (pH 7.2), and resuspended in 199. Mu.L ice-cold PBS (pH 7.2). Then 1. Mu.L of 20mM Cy3-maleimide (dissolved in DMSO) was added to the sample and vortexed briefly. Thiol-maleimide reaction was reacted at 4 ℃ for 4 hours, then the samples were diluted in PBS (pH 7.2), precipitated by centrifugation, and then washed three times with ice-cold PBS (pH 7.2). Cells were then resuspended in 199 μ LPBS (pH 7.2), 1 μl of 20mM DBCO-Cy5 (dissolved in DMSO) was added, and the cells were incubated at 4 ℃ for 2.5 hours, after which the samples were diluted in PBS (pH 7.2), pelleted by centrifugation, and then washed three times with ice-cold PBS (pH 7.2). As can be seen from FIG. 8C, the purified proteins were labeled with Cy5-DBCO and Cy 3-maleimide, respectively, and the SDS-PAGE gel had a single fluorescent band corresponding to PGUS, further demonstrating successful incorporation of pAzF and correct modification of the fluorophore.

4. Confocal microscope

Cell fluorescence was detected by 63 x objective using LEICA STELLARIS system. Considering direct excitation of the acceptor and leakage of donor fluorescence into the acceptor channel, the Cy3 and FRET channels use 516nm excitation at detection wavelengths of 550-590nm and 650-730nm, respectively. The excitation wavelength and the detection wavelength of the Cy5 channel are 640nm and 650-730nm respectively. To obtain a clear signal and avoid overexposure, the laser "smart gain" values for the Cy3 channel, cy5 channel and FRET channel were set to 15%, 15% and 20%, respectively. For all fluorescence channels, a laser of 15%, pinhole of 1AU was used. FRET ratio (F _FRET/F_Donor(s)) was quantified using ImageJ and Leica Las X software.

As can be seen in FIG. 9, strains LA and LA-W522A showed clear and bright FRET signals, indicating that when PGUS or dimer W522A (W522A was PGUS small subunit interface at the W522 site mutated to alanine to change PGUS tetramer to dimer ,Tetramerization of GH2β-Glucuronidases is Essential for Catalyzing the Hydrolysis ofthe Large Substrate Glycyrrhizin) for display on the yeast surface, the large subunit interface of PGUS was stably present strain SA showed FRET signals from small subunit interface, while negative control SA-W522A did not show any FRET signals, as no small subunit interface was present in dimer mutant W522A these results demonstrated the feasibility of FRET strategy detection surface to show PGUS subunit interface.

To compare the structural differences between the yeast surface display PGUS anchor expression system and the co-expression system, yeast surface display strains (designated LC and SC, respectively) were constructed that co-expressed the S35C-D220pAzF and G153C-E488pAzF anchor and free subunits, respectively. These two strains show FRET signals at the large and small subunit interfaces, respectively. Since the difference in the number of donor fluorophores between different strains is excluded, FRET ratio may represent the proportion of protein subunits forming a large or small interface. As can be seen from FIG. 9, the co-expression strain LC had a FRET ratio of 0.764, which was unchanged from LA (FRET ratio of 0.763) and LA-W522A (FRET ratio of 0.788). The large subunit interface was demonstrated to exist stably in all yeast surface display PGUS systems. Whereas the FRET ratio of SA is much lower (0.337). Compared with it, the FRET ratio of the coexpression strain SC was increased by 94.4% (the FRET ratio was 0.655). An increase in FRET ratio for SC indicates an increase in small subunit interface and indicates that co-expression of the anchor and free subunits is beneficial for maintaining the small subunit interface.

As can be seen from fig. 10, at high temperatures (above 40 ℃) and urea concentrations (above 4M), the FRET ratio of the small interface in SA is significantly reduced, indicating instability of the PGUS small subunit interface.

As can be seen from FIG. 11, all 4 single point mutant strains S35C-Pir1, D220pAzF-Pir1, G153C-Pir1, E488pAzF-Pir1 and control strain BY 4741-. DELTA.PANG exhibited distinct donor and acceptor signals, but little FRET signal was observed, demonstrating that the detected FRET signal was derived from the subunit interface of yeast surface display PGUS, rather than being produced BY surface display PGUS between other surface proteins.

TABLE 1 Saccharomyces cerevisiae surface display of pAzF-containing PGUS Activity and display characterization

The terms "one embodiment," "some embodiments," "examples," and the like, herein, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A method for detecting yeast surface display oligomeric enzyme quaternary structure based on FRET in situ, which is characterized by comprising the following steps:

(1) Taking saccharomyces cerevisiae as a chassis strain, knocking out genes PPQ1, ALO1, NAM7 and GAL80, and simultaneously introducing multicopy tyrosyl-tRNA synthase (OmeRS) and two corresponding tRNA _CUA ^Tyr;

(2) Two sites at the interface of the selected oligos are mutated into cysteine and TAG codons, respectively, and the surface display of the oligos is mediated by using an anchoring protein;

(4) Characterizing donor, acceptor and FRET signals using confocal microscopy;

Wherein the gene sequence of tyrosyl-tRNA synthase is shown as SEQ ID NO.1, the sequence of tRNA _CUA ^Tyr is shown as SEQ ID NO.2 and SEQ ID NO. 3;

two sites that are mutated to cysteine and TAG codons, respectively, at the interface of the oligoenzyme are located in the inactive center and the distance between the two sites is less than the foster distance of Cy3 and Cy 5.

2. The method for in situ detection of yeast surface display of quaternary structure of oligomeric enzyme based on FRET according to claim 1, wherein said Saccharomyces cerevisiae is Saccharomyces cerevisiae BY4741.

3. The method for displaying the quaternary structure of the oligomerase on the surface of the yeast based on FRET in situ detection according to claim 1, wherein the promoter for expressing OmeRS is a GPD promoter, the terminator is a GPM1 terminator, the promoter for expressing tRNA _CUA ^Tyr with the gene sequence shown as SEQ ID NO.2 is a SUP4 promoter, the terminator is a SUP4 terminator, the promoter for expressing tRNA _CUA ^Tyr with the gene sequence shown as SEQ ID NO.3 is a SNR52 promoter, the terminator is a SUP4 terminator, the gene sequence of the GPD promoter is shown as SEQ ID NO.4, the gene sequence of the GPM1 terminator is shown as SEQ ID NO.5, the sequence of the SNR52 promoter is shown as SEQ ID NO.6, the sequence of the SUP4 terminator is shown as SEQ ID NO.15, and the sequence of the SUP4 terminator is shown as SEQ ID NO. 7.

4. The method for in situ detection of yeast surface display of quaternary structure of oligomerase based on FRET according to claim 1, wherein the induction of protein expression in step (2) is carried out in YPG-G418 medium supplemented with pAzF at a final concentration of 0.5-10 mM.

5. The method for in situ detection of yeast surface display of oligomeric enzyme based on FRET according to claim 1, wherein the excitation wavelength of the donor fluorescent molecule is 500-554nm, the detection wavelength is 546-630nm, the excitation wavelength of the acceptor fluorescent molecule is 582-648nm, the detection wavelength is 650-740nm, the excitation wavelength of the FRET channel is 500-535nm, and the detection wavelength is 649-740nm.

6. The method for in situ detection of yeast surface display of oligomeric enzyme quaternary structure based on FRET according to claim 5, wherein excitation wavelength of Cy3 channel is 516 nm, detection wavelength is 550-780 nm, excitation wavelength of FRET channel is 516 nm, detection wavelength is 650-730nm, excitation wavelength of Cy5 channel is 640nm, detection wavelength is 650-730nm, and laser "smart gain" values of Cy3 channel, cy5 channel and FRET channel are respectively set to 15%, 15% and 20%.

7. The method according to claim 1, wherein the presence of a significant donor signal, acceptor signal, FRET signal and the dissociation of the subunits of the oligoenzyme are determined when a significant donor signal and acceptor signal are present but no FRET signal is present.

8. The method of claim 1 or 7, further comprising the step of calculating a FRET ratio, FRET ratio = F _FRET/F_Donor(s), wherein F _FRET is a fluorescence signal of Cy5 at its detection wavelength and F _Donor(s) is a fluorescence signal of Cy3 at its detection wavelength.

9. The method for in situ detection of yeast surface display of quaternary structure of oligomerase according to claim 8, wherein,

The method further comprises determining the effect of external conditions on the stability of the subunit interface of the yeast surface display oligoenzyme based on the change in FRET ratio;

alternatively, the method further comprises determining the effect of the different expression systems of the yeast surface display oligoenzymes on the stability of the subunit interface based on the change in FRET ratio.

10. The method for FRET-based in situ detection of yeast surface display of quaternary structure of oligomerase according to claim 1, wherein the anchoring protein is selected from one of Pir1 anchoring protein, sed1 anchoring protein or Ag alpha anchoring protein;

the gene sequence of Pir1 anchoring protein is shown as SEQ ID NO.8, the gene sequence of Sed1 anchoring protein is shown as SEQ ID NO.9, and the gene sequence of Ag alpha anchoring protein is shown as SEQ ID NO. 10;

the oligoenzyme is beta-glucuronidase (PGUS), and the accession number of the gene sequence of the beta-glucuronidase in GeneBank is EU095019;

The promoter used for displaying the surface of the saccharomyces cerevisiae using the ankyrin mediated oligoenzyme in the step (2) is a GAL1 promoter, the signal peptide is an SED1 signal peptide, and the terminator is an ENO2 terminator, wherein the gene sequence of the GAL1 promoter is shown as SEQ ID NO.11, the gene sequence of the SED1 signal peptide is shown as SEQ ID NO.12, and the sequence of the ENO2 terminator is shown as SEQ ID NO. 13.