Saccharomyces cerevisiae with a deleted gene and use thereof in modifying proteins with unnatural amino acids

Abstract

The application discloses a gene-knocking-out Saccharomyces cerevisiae and application of the Saccharomyces cerevisiae in modification of proteins by using non-natural amino acids; the gene-knocking-out Saccharomyces cerevisiae is constructed by the following steps: knocking out genes of Saccharomyces cerevisiae; the genes are knocked out as genes YEL014C are knocked out; or genes YEL013W are knocked out; or genes YEL014C and genes YEL013W are knocked out; introducing recombinant plasmids pWJR077 and pWJR066 into the above-mentioned strains; the application realizes the improvement of the coding efficiency of the non-natural amino acids in the modification of the proteins in the Saccharomyces cerevisiae by using the gene-knocking-out; and a new model is provided for the research of the production of therapeutic proteins.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of synthetic biology, in particular to the application of gene-deleted Saccharomyces cerevisiae in efficiently utilizing non-natural amino acid modified proteins. BACKGROUND

[0002] The functional diversity of proteins is mainly determined by their amino acid sequences. Although chemical modification can endow proteins with new functions, there are still limitations in precisely realizing macromolecular interactions, regulating immune responses, and deeply understanding biological mechanisms. Through gene coding expansion technology, existing technologies integrate non-natural amino acids into specific sites of proteins by modifying engineered aminoacyl-tRNA synthetases (aaRS) and tRNA gene sequences, thereby significantly increasing protein diversity and achieving precise modification of target proteins. This method has attracted widespread attention in the fields of antibody drug conjugation, protein localization, and vaccine production, providing new possibilities for improving regulatory capacity and rationally designing biological systems.

[0003] Although non-natural amino acid modification has been achieved in prokaryotic host cells such as Escherichia coli, there may be problems such as incorrect folding of functional proteins. Although eukaryotic cells such as mammalian cells avoid incorrect folding of functional proteins, there are still major problems in protein production. Saccharomyces cerevisiae is superior in protein expression, folding, and post-translational modification, and is an excellent host cell for protein production. However, Saccharomyces cerevisiae still faces challenges such as low coding efficiency in efficiently expressing proteins with precise non-natural amino acid modification.

[0004] Therefore, there is an urgent need for a strain that efficiently utilizes non-natural amino acid modified proteins to meet future needs for producing non-natural amino acid modified therapeutic proteins, vaccine production, and fluorescently labeled proteins in Saccharomyces cerevisiae, and to have applications in basic research and drug development, biological engineering, and other fields. Moreover, exploring new targets can serve as a new approach to improving the coding efficiency of non-natural amino acids. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art and provide the application of gene deletion in Saccharomyces cerevisiae for efficiently utilizing non-natural amino acid modified proteins.

[0006] The second purpose of the present application is to provide a gene-deleted Saccharomyces cerevisiae.

[0007] The third purpose of the present application is to provide the application of a gene-deleted Saccharomyces cerevisiae in efficiently utilizing non-natural amino acid modified proteins.

[0008] The technical solutions of the present application are summarized as follows:

[0009] Application of deletion of genes in Saccharomyces cerevisiae for efficient utilization of unnatural amino acids to modify proteins

[0010] The deletion of genes is:

[0011] 1) deletion of gene YEL014C; or

[0012] 2) deletion of gene YEL013W; or

[0013] 3) deletion of genes YEL014C and YEL013W.

[0014] The Saccharomyces cerevisiae with deletion of genes is constructed by the following steps:

[0015] 1) the Saccharomyces cerevisiae gene is knocked out; the gene is knocked out is that the gene YEL014C is knocked out; or the gene YEL013W is knocked out; or the gene YEL014C and the gene YEL013W are knocked out;

[0016] 2) introducing recombinant plasmid pWJR077 and recombinant plasmid pWJR066 into the strain obtained in step 1); the recombinant plasmid pWJR077 is LeuOmeRS and 2x gene is obtained by connecting pRS413 as a carrier; the recombinant plasmid pWJR066 is EGFP 40UAG gene is obtained by connecting pRS425 as a carrier;

[0017] The nucleotide sequence of the LeuOmeRS gene is shown in SEQ ID No. 1;

[0018] The nucleotide sequence of the gene is shown in SEQ ID No. 2;

[0019] The nucleotide sequence of the EGFP 40UAG gene is shown in SEQ ID No. 3;

[0020] The nucleotide sequence of the recombinant plasmid pWJR077 is shown in SEQ ID No. 4;

[0021] The nucleotide sequence of the recombinant plasmid pWJR066 is shown in SEQ ID No. 5.

[0022] Preferably, the type of Saccharomyces cerevisiae is BY4741, BY4742, BY4743 or yXZX846.

[0023] The application of the above-mentioned Saccharomyces cerevisiae with deletion of genes for efficient utilization of unnatural amino acids to modify proteins.

[0024] The above-mentioned Saccharomyces cerevisiae with deletion of genes is a new model for protein production research.

[0025] The present application has the following beneficial effects compared with the prior art:

[0026] 1. The use of gene knockout realizes the improvement of the non-natural amino acid modification protein coding efficiency in Saccharomyces cerevisiae.

[0027] 2. A new model is provided for the production of therapeutic proteins. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Gene knockout verification and fluorescence intensity determination of YEL014C, wherein A is a gene-specific primer for PCR verification of YEL014C gene knockout in the strain, and if YEL014C gene knockout is confirmed, no PCR product can be formed; and B is the fluorescence intensity after YEL014C gene knockout in the strain.

[0029] Figure 2 Gene knockout verification and fluorescence intensity determination of YEL013W, wherein A is a gene-specific primer for PCR verification of YEL013W gene knockout in the strain, and if YEL013W gene knockout is confirmed, no PCR product can be formed; and B is the fluorescence intensity after YEL013W gene knockout in the strain. DETAILED DESCRIPTION

[0030] The raw materials and reagents used in the present application can be purchased from the market.

[0031] The SC-His-Leu medium involved in the present application: glucose 20 g / L, YNB 6.7 g / L; four amino acid powder 2 g / L (-Ura, -His, -Trp, -Leu), Ura 20 mg / L, Trp 20 mg / L, pH adjusted to 6.0.

[0032] The SC-Leu-Ura medium involved in the present application: glucose 20 g / L, YNB 6.7 g / L; four amino acid powder 2 g / L (-Ura, -His, -Trp, -Leu), His 20 mg / L, Trp 20 mg / L, pH adjusted to 6.0.

[0033] The DNA polymerase used in each embodiment of the present application is Max Super-Fidelity DNA Polymerase.

[0034] Each embodiment of the present application utilizes agarose gel electrophoresis to test the size of the fragment, and the correct band is purified using the agarose gel recovery kit of Tiangeng Biochemical Technology Co., Ltd.

[0035] The initial strain yXZX846 is provided by the laboratory of Yuan Yingjin of Tianjin University.

[0036] The strain construction is described in Xie, Z.X. ; Li, B.Z. ; Mitchell, L.A. ; Wu, Y. ; Qi, X. ; Jin, Z. ; Jia, B. ; Wang, X. ; Zeng, B.X. ; Liu, H.M. et al. "Perfect" designer chromosome V and behavior of a ring derivative. Science 2017, 355 (6329). http: / / doi.org / 10.1126 / science.aaf4704 .

[0037] For further understanding of the present application, the plasmids, vectors and the like described in the present application can be purchased through commercial channels, unless otherwise specified.

[0038] The present application utilizes the characteristics of homologous recombination of Saccharomyces cerevisiae, and obtains the corresponding strain by knocking out YEL014C or YEL013W gene, and can also knock out YEL014C and YEL013W genes at the same time to obtain the corresponding strain.

[0039] The nucleotide sequence of pWJR077 (SEQ ID No. 4).

[0040] The present application is further described below through specific examples.

[0041] Example 1

[0042] The construction of Saccharomyces cerevisiae strain yWJR109 includes the following steps:

[0043] 1. Constructing a guide-RNA plasmid with a target site of YEL014C, and the construction steps are as follows:

[0044] a) Selecting protospacer (SEQ ID No. 6) as a template;

[0045] b) Synthesizing primers (SEQ ID No. 7) and (SEQ ID No. 8);

[0046] c) Annealing and bonding the two primers to obtain double-stranded DNA;

[0047] d) Digesting the plasmid pRS426-SNR52p- gRNA-SUP4t with restriction endonuclease NotI and calf alkaline phosphatase (CIP) to linearize it;

[0048] gRNA-SUP4t, linearizing it;

[0049] e) Gibson assembly to assemble linearized plasmid and double-stranded DNA;

[0050] f) Transform the reaction system into competent cells such as DH5a E. coli, spread on LB+Carb plates, and incubate at 37°C

[0051] for 12h;

[0052] g) Pick 3 single colonies and inoculate in 5mL LB+Carb liquid medium, incubate overnight at 37°C, then extract the plasmid and perform Sanger sequencing;

[0053]

[0054] h) The correct sequencing plasmid is named pRS426-SNR52P-gRNA.YEL014C.

[0055] 2. Construction of YEL014C knockout donor DNA, the construction steps are as follows:

[0056] a) Synthesis of primers:

[0057] oWJR0563 (SEQ ID No. 9); oWJR0564 (SEQ ID No. 10); oWJR0566 (SEQ ID

[0058] No. 12); oWJR0565 (SEQ ID No. 11);

[0059] b) Using the upstream primer oWJR0566 (SEQ ID No. 12), the downstream primer oWJR0565 (SEQ ID No. 11), and amplifying the promoter region of YEL014C, a fragment of 571bp is obtained using the genome of strain yXZX846 as a template;

[0060] c) Using the upstream primer oWJR0563 (SEQ ID No. 9) and the downstream primer oWJR0564 (SEQ ID No. 10), a fragment of 556bp is obtained by amplifying the terminator region of YEL014C using the genome of strain yXZX846 as a template;

[0061] d) The PCR products of steps b) and c) are amplified by overlap extension PCR, using the upstream primer oWJR0566 and the downstream primer oWJR0564, to obtain a YEL014C knockout donor DNA of 1087bp in length.

[0062]

[0063] 3. Saccharomyces cerevisiae transformation, the steps are as follows:

[0064] ​​a) Pick a single colony of yXZX846 into 5 mL YPD liquid medium, incubate at 30°C overnight;

[0065] b) Measure the OD of the overnight culture of S. cerevisiae 600 , inoculate the overnight culture into 5 mL YPD liquid medium (0.125 OD 600 / mL), incubate at 30°C, 220 rpm until the OD 600 reaches 0.5 (about 3.5-4.5 h);

[0066] c) Collect the cells by centrifuging the S. cerevisiae culture from step b) at 5000 rpm for 1 min, resuspend the cells with 1 mL sterile water, centrifuge again, collect the cells, resuspend the cells with 1 mL 0.1 M LiOAc, centrifuge again, collect the cells, remove 900 μL supernatant with a pipette, resuspend the cells with the remaining 100

[0067] μL LiOAc, and place on ice to obtain competent cells.

[0068] d) Prepare the transformation system, in which

[0069] the plasmid DNA is pRS426-SNR52p-gRNA.YEL014C, 200 ng;

[0070] the plasmid DNA is pRS415-Cas9, 200 ng;

[0071] the donor DNA for YEL014C knockout, 200 ng, add water to 50 μL;

[0072] Volume (μL)

[0073] Component Volume (μL) 50% PEG 3350 620 10 mg / mL ssDNA 40 1 M LiOAc 90 DNA 50

[0074] e) Add 100 μL competent cells to the transformation system, mix by pipetting, vortex at the highest speed for 10 s, incubate in a 30°C incubator for 30 min, add 90 μL DMSO, vortex for 10 s, heat shock at 42°C for 18 min, centrifuge at 3600 rpm for 30 s to collect the cells, remove the supernatant, resuspend the cells with 400 μL 5 mM CaCl2, stand for 10 min, centrifuge at 3600 rpm for 30 s, remove the supernatant, resuspend in sterile water, and then spread on SC-Leu-Ura plates for screening.

[0075]

[0076] f) After the yeast grows on the screening plate for 2 days, pick a single colony and streak on an SC-Leu-Ura plate for purification.

[0077] ​g) Using the yeast colony PCR method, the primers oWJR0543 (SEQ ID No. 13) and oWJR0545 (SEQ ID No. 14) inside the open reading frame of the gene were selected to verify whether the YEL014C gene was knocked out in the purified strain (Huxley, C., et al. Trends Genet (1990), 6, 236). The verification of whether the YEL014C gene was knocked out is shown in Figure 1 A. Subsequently, the YEL014C gene knockout strain was selected and preserved.

[0078] 4. Construction of the pWJR077 plasmid (the pWJR077 plasmid is a pRS413-LeuOmeRS-2x ), the construction steps of which are as follows:

[0079] a) Using the plasmid pWJR012 (pRS413-LeuOmeRS-1x ) as a template, the SNR52p-tRNA-SUP4 expression cassette was amplified using oWJR0313

[0080] (SEQ ID No. 15) and oWJR0322 (SEQ ID No. 16), and XhoI and EcoRI enzyme digestion sites were introduced, digested, and gel purified.

[0081] b) The pWJR012 was treated with XhoI and EcoRI restriction enzymes at 37°C for 3 h, and after enzyme digestion and purification, the linearized vector was obtained.

[0082] c) The SNR52p-tRNA-SUP4 and the linearized vector pWJR012 were connected by Gibson assembly to achieve the connection of the target fragment, and then Escherichia coli assembly was performed. The obtained single colony was verified by PCR using oWJR323 (SEQ ID No. 17) and oWJR324

[0083] (SEQ ID No. 18), the correct strain was verified for plasmid extraction and sequencing verification, and the pWJR077 plasmid (shown in SEQ ID No. 4) was obtained.

[0084] 5. The constructed pWJR077 and pWJR066 (pRS425-EGFP 40UAG ) (shown in SEQ ID No. 5) were used

[0085] to transform the YEL014C gene knockout strain obtained in the above step 3 by lithium acetate transformation.

[0086] 6. Single colonies on SC-Leu-His plates were picked and after streaking to purity, S. cerevisiae strain yWJR109 was obtained.

[0087] wherein: the nucleotide sequence of the LeuOmeRS gene is shown in SEQ ID No. 1 ;

[0088] the nucleotide sequence of the EGFP gene is shown in SEQ ID No. 3;

[0089] EGFP 40UAG the nucleotide sequence of the EGFP gene is shown in SEQ ID No. 3;

[0090] 7. EGFP fluorescence intensity measurement, steps as follows:

[0091] a) yWJR068 was obtained by transforming the strain yXZX846 with two plasmids pWJR012 (pRS413-LeuOmeRS-1x

[0092] and pWJR066 (pRS425-EGFP 40UAG ). yWJR068 and yWJR109 strains were grown in SC-His-Leu liquid medium at 30°C for 24h.

[0093] b) The cultures were passaged into 6mL SC-His-Leu medium with 0mM or 1mM O-methyl-L-

[0094] tyrosine (OMeY) to 0.05 (OD 600 / mL) respectively and incubated at 30°C for 24h.

[0095] c) The cultures were harvested by centrifugation at 5000rpm for 5min and resuspended in 2mL ddH2O.

[0096] d) 200μL of the resuspended cell culture was transferred to each well of a 96-well plate and subsequently the optical density (OD 600 ) and green fluorescence (λex= 488nm, λem= 520nm) were measured using a microplate reader.

[0097] e) To normalize the measured data, the relative fluorescence intensity was normalized to OD 600 and compared to the fluorescence intensity of a positive control (wild-type

[0098] EGFP fluorescence intensity) and a negative control (no plasmid, 0mM OMeY).

[0099] The results showed that, using the EGFP fluorescence intensity detection method, the fluorescence intensity of yWJR109 (YEL014C knockout) was significantly improved, reaching 114.52% of wild-type EGFP, and compared to 8.41% of yWJR068, the fluorescence intensity increased by 106.11%. Figure 1 As shown in B.

[0100] Example 2: Construction of Saccharomyces cerevisiae strain yWJR110, including the following steps:

[0101] 1. Construct a guide-RNA plasmid with the target site YEL013W. The construction steps are as follows:

[0102] a) Select the protospacer (pre-spacer sequence neighbor motif) as the template (SEQ ID No. 19);

[0103] b) Synthetic primers: (SEQ ID No. 20) and (SEQ ID No. 21);

[0104] c) Annealing binds the two primers together to obtain double-stranded DNA;

[0105] d) Digest plasmid pRS426-SNR52p- using restriction endonuclease NotI and calf alkaline phosphatase (CIP).

[0106] gRNA-SUP4t was used to linearize it;

[0107] e) Use Gibson assembly to assemble linearized plasmids and double-stranded DNA;

[0108] f) Transform the reaction system into DH5α Escherichia coli competent cells, spread it on LB+Carb plates, and incubate at 37°C.

[0109] Incubate for 12 hours;

[0110] g) Pick 3 single colonies and inoculate them into 5 mL of LB+Carb liquid medium. Incubate overnight at 37°C, then extract the spores.

[0111] The particles were subjected to Sanger sequencing.

[0112] h) The plasmid that was correctly sequenced was named pRS426-SNR52P-gRNA.YEL013W.

[0113] 2. Construct the YEL013W knockout donor DNA. The construction steps are as follows:

[0114] a) Synthetic primers: oWJR0545: (SEQ ID No. 14); oWJR0543: (SEQ ID No. 13);

[0115] oWJR0567: (SEQ ID No. 22); oWJR0568: (SEQ ID No. 23).

[0116] b) using the upstream primer oWJR0545 (SEQ ID No. 14) and the downstream primer oWJR0543 (SEQ ID No. 13) to amplify the promoter region of YEL013W from the genome of the strain yXZX846, to obtain a fragment of 443 bp in length;

[0117] c) using the upstream primer oWJR0567 (SEQ ID No. 22) and the downstream primer oWJR0568 (SEQ ID No. 23) to amplify the terminator region of YEL013W from the genome of the strain yXZX846, to obtain a fragment of 431 bp in length;

[0118] d) using the PCR products of step b) and step c) to amplify by overlap extension PCR, using the upstream primer oWJR0545 and the downstream primer oWJR0568, to obtain the donor DNA for YEL013W knockout of 854 bp in length.

[0119] 3. The Saccharomyces cerevisiae transformation, the steps of which are as follows:

[0120] a) picking a single colony of yXZX846 in 5 mL YPD liquid medium, and culturing overnight at 30°C;

[0121] b) measuring the OD 600 of the overnight culture of Saccharomyces cerevisiae, and inoculating the overnight culture into 5 mL YPD liquid medium (0.125 OD 600 / mL), and culturing at 30°C, 220 rpm, until the OD 600 reaches 0.5 (about 3.5

[0122] h-4.5 h);

[0123] c) collecting the cells in a 1.5 mL EP tube by centrifuging the Saccharomyces cerevisiae culture of step b) at 5000 rpm for 1 min, resuspending the cells with 1 mL sterile water, centrifuging as above, collecting the cells, resuspending the cells with 1 mL 0.1M LiOAc, centrifuging as above, collecting the cells, and resuspending the cells with 100

[0124] μL LiOAc, and placing on ice to obtain competent cells.

[0125] d) preparing a transformation system, wherein

[0126] Plasmid DNA is pRS426-SNR52p-gRNA.YEL013W, 200 ng;

[0127] Plasmid DNA is pRS415-Cas9, 200 ng;

[0128] Donor DNA for YEL013W knockout, 200 ng, add water to 50 μL.

[0129] Volume (μL)

[0130] Component Volume (μL) 50% PEG 3350 620 10 mg / mL ssDNA 40 1 M LiOAc 90 DNA 50

[0131] e) Add 100 μL competent cells to transformation system, mix by pipetting, vortex at highest speed for 10 s; incubate in 30 °C incubator for 30 min; add 90 μL DMSO, vortex for 10 s; heat shock at 42 °C for 18 min;

[0132] Centrifuge at 3600 rpm for 30 s, collect cells; pipette out supernatant, add 400 μL 5 mM CaCl2, resuspend cells, stand for 5-15 min; centrifuge at 3600 rpm for 30 s, pipette out supernatant, resuspend in sterile water, then spread on SC-Leu-

[0133] Ura plate for screening.

[0134] f) After the yeast grows on the screening plate for 2 days, pick single colonies and streak on SC-Leu-Ura plate for purification.

[0135] g) Use yeast colony PCR method to verify whether the purified strain knocks out YEL013W gene by selecting internal primer oWJR0546 (SEQ ID No. 24) and external primer oWJR0541 (SEQ ID No. 25) in the open reading frame of the gene (Huxley, C., et al. Trends Genet (1990), 6, 236), see Figure 2 A. Then select the YEL013W gene knockout strain for preservation.

[0136] 4. Transform the built pWJR077 plasmid (pRS413-LeuOmeRS-2x ) and pWJR066 (pRS425-

[0137] EGFP 40UAG ) into the above YEL013W gene knockout strain using lithium acetate transformation method.

[0138] 5. Pick single colony on SC-Leu-His plate, and get yWJR110 after streaking and purification.

[0139] 6. EGFP fluorescence intensity measurement, steps as follows:

[0140] a) yWJR068 is obtained by introducing two plasmids pWJR012 (pRS413-LeuOmeRS-1x ) and pWJR066 (pRS425-EGFP 40UAG ) into the strain of yXZX846. yWJR068 and yWJR110 cells are cultured in SC-His-Leu at 30℃ for 24h, respectively.

[0141] b) The culture is subcultured into 6mL SC-His-Leu medium, and 0mM or 1mM O-methyl-L-tyrosine (OMeY) is added to 0.05 (OD 600 / mL), respectively, and incubated at 30℃ for 24h.

[0142] c) Centrifuge at 5000rpm for 5min to harvest the bacterial solution, and resuspend with 2mL ddH2O.

[0143] d) Transfer 200μL of resuspended cell culture solution into each well of a 96-well plate, and then use an enzyme marker to measure optical density (OD 600 ) and green fluorescence (λex=488nm, λem=520nm).

[0144] e) For standardized measurement data, the relative fluorescence intensity is normalized to OD 600 , and compared with the fluorescence intensity of positive control (wild-type EGFP fluorescence intensity) and negative control (no plasmid, 0mM OMeY).

[0145] The results show that the fluorescence intensity of yWJR110 (knockout YEL013W) is significantly improved by the method of EGFP fluorescence intensity detection, reaching 137.60% of the wild-type EGFP, and the fluorescence intensity is improved by 129.19% compared with 8.41% of yWJR068, as shown in Figure 2 B.

[0146] The microorganism used in the present application is Saccharomyces cerevisiae, preferably BY4741, BY4742, BY4743 or yXZX846; other Saccharomyces cerevisiae strains can also be used in the present application.

[0147] The experiment proves that the fluorescence intensity of the strain constructed by simultaneously knocking out the genes YEL014C and YEL013W and then introducing the recombinant plasmid pWJR077 and the recombinant plasmid pWJR066 is stronger than that of yWJR110.

[0148] Substituting the gene encoding other protein (for example, superoxide dismutase) for EGFP in Example 1 40UAG The gene, other than Example 1, the coding efficiency of the obtained recombinant bacteria is similar to that of Example 1.

[0149] The above only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A yeast strain lacking a gene, characterized in that... Build it using the following steps: (1) The Saccharomyces cerevisiae gene was knocked out; the gene knocked out is a gene YEL014C Knocked out; or gene YEL013W Knocked out; (2) Introduce recombinant plasmids pWJR077 and pWJR066 into the strain obtained in step (1); the recombinant plasmid pWJR077 is ligated using pRS413 as a vector. LeuOmeRS and The recombinant plasmid pWJR066 was obtained by using pRS425 as a vector. Gene acquisition; The LeuOmeRS The nucleotide sequence of the gene is shown in SEQ ID No. 1; The The nucleotide sequence is shown in SEQ ID No. 2; The The nucleotide sequence of the gene is shown in SEQ ID No. 3; The nucleotide sequence of the recombinant plasmid pWJR077 is shown in SEQ ID No. 4; The nucleotide sequence of the recombinant plasmid pWJR066 is shown in SEQ ID No. 5; The type of brewing yeast is BY4741, BY4742, BY4743 or yXZX846.

2. The application of the gene-deficient Saccharomyces cerevisiae of claim 1 in the modification of proteins using non-natural amino acids, wherein the non-natural amino acid is O-methyl-L-tyrosine.

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