Method for stable inheritance of plasmids in saccharomyces cerevisiae without screening pressure and application

Abstract

The invention discloses a method for stable inheritance of plasmids in saccharomyces cerevisiae under no screening pressure and application, the method comprises the following steps: connecting essential genes and metabolic pathway gene loops in series to the same plasmid through in-vitro one-step assembly and transferring into chassis cells of which the essential genes of a genome are knocked out, and losing rescue plasmids by utilizing different screening markers; therefore, the strains only having the metabolic pathway plasmids are screened. According to the method disclosed by the invention, multiple rounds of gene editing processes of the genome are not needed, the structure of the genome is not damaged, the influence on the growth of a host is reduced, the yield of a target product is favorably improved, the construction period of engineering bacteria is greatly shortened, and the research and development process is accelerated. The engineering strain constructed by using the system does not need to be additionally added with antibiotics or screened in a manner of supplementing auxotrophic genes in production application, so that the safe use of the engineering strain in industrial production is realized, the production cost can be effectively reduced, and the profit is increased.

Description

Technical Field

[0001] The invention relates to the technical field of synthetic biology, and in particular to a method and application of stable inheritance of plasmids in brewer's yeast without screening pressure. Background Art

[0002] Plasmid is a small circular double-stranded DNA molecule that often appears in bacteria and other microorganisms. It can replicate independently in cells and carry certain genetic information. In metabolic engineering, plasmids can be used to carry and deliver target genes to achieve the expression of target genes in host cells. However, since plasmids are circular double-stranded DNA that replicates autonomously outside chromosomes, they need to be stably inherited under screening pressure conditions. The commonly used screening for brewer's yeast plasmids is nutritional deficiency or antibiotics. Natural raw materials cannot provide nutritional deficiency conditions when fermented as substrates, and the use of antibiotics will increase the fermentation cost and may cause safety problems in downstream product applications due to antibiotic residues.

[0003] In addition, in order to obtain a genetically stable strain, metabolic pathways are usually integrated into the yeast genome through multiple rounds of gene editing. This process is not only cumbersome and time-consuming, but also damages the stability of the yeast genome, affecting strain growth and thus the yield of the target product. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes a method and application of stable inheritance of plasmids in Saccharomyces cerevisiae without screening pressure. In order to reduce the fermentation cost and shorten the construction cycle in the "design-build-test-learn" cycle, the present application clones the essential genes of Saccharomyces cerevisiae as rescue plasmids, knocks out the corresponding genes in the genome, constructs the essential genes and metabolic pathway gene circuits on the same plasmid and transfers them into the genome essential gene knockout chassis cells, and obtains strains with only metabolic pathway plasmids by using different screening markers, thereby achieving stable inheritance of metabolic pathways without genome integration and screening pressure conditions.

[0005] The present invention provides a method for stable inheritance of plasmids in Saccharomyces cerevisiae without selection pressure, the method comprising the following steps:

[0006] S1: constructing a first plasmid and a second plasmid carrying essential genes of Saccharomyces cerevisiae, wherein the first plasmid comprises the LEU2 gene and the second plasmid comprises the URA3 gene;

[0007] S2: using the POT2-ccdB plasmid as a template to amplify the URA3 fragment containing the homology arm, the URA3 fragment and the first plasmid of S1 are co-transfected into Saccharomyces cerevisiae, and screened using a plate lacking both uracil and leucine to knock out the essential genes of Saccharomyces cerevisiae in the genome of Saccharomyces cerevisiae, thereby obtaining the recombinant bacteria 1;

[0008] S3: amplifying the upstream and downstream homologous arm sequences of the essential gene of Saccharomyces cerevisiae in the genome of Saccharomyces cerevisiae, fusing the fragment 1 by fusion PCR, transferring the fragment 1 into the recombinant bacteria 1 described in S2, and screening using a plate containing 5FOA to obtain the recombinant bacteria 2 with the URA3 gene removed;

[0009] S4: The second plasmid of S1 is transferred into the recombinant bacteria 2, and screened using a plate lacking uracil to obtain the recombinant bacteria 3;

[0010] S5: construct a third plasmid carrying the target gene, transfer it into the recombinant bacteria 3, and screen it using a plate lacking uracil to obtain the recombinant bacteria 4;

[0011] S6: The recombinant bacteria 4 described in S5 are continuously subcultured without selection pressure.

[0012] In some embodiments, the essential gene of Saccharomyces cerevisiae is TPI1 gene, PGI1 gene, CDC4 gene, CDC9 gene or CDC28 gene, preferably TPI1 gene.

[0013] In some embodiments, S1 the first plasmid is obtained by connecting a plasmid containing an open reading frame of an essential gene of Saccharomyces cerevisiae, a promoter plasmid and a terminator plasmid to a POT5-LEU2-ccdB vector.

[0014] In some embodiments, S1 the second plasmid is obtained by connecting a plasmid containing an open reading frame of an essential gene of Saccharomyces cerevisiae, a promoter plasmid and a terminator plasmid to a POT3-URA3-RFP vector.

[0015] In some embodiments, the third plasmid described in S5 is obtained by connecting a plasmid containing an open reading frame of an essential gene of Saccharomyces cerevisiae, a target gene plasmid, a promoter plasmid and a terminator plasmid to a POT3-URA3-RFP vector.

[0016] In some embodiments, the connection is made using Golden Gate technology.

[0017] In some embodiments, the nucleotide sequence of the POT3-URA3-RFP vector is shown as SEQ ID NO.5.

[0018] In some embodiments, the nucleotide sequence of the POT5-LEU2-ccdB vector is shown as SEQ ID NO.6.

[0019] In some embodiments, the nucleotide sequence of fragment 1 of S3 is as shown in SEQ ID NO.17.

[0020] The invention also provides application of the method in metabolic engineering production or biosynthesis.

[0021] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0022] 1. The present invention uses the stable genetic system of Saccharomyces cerevisiae without screening plasmids to construct metabolic pathways. It does not require multiple rounds of genome editing and will not damage the structure of the genome. It will reduce the impact on the growth of the host and help increase the yield of the target product. The plasmid can be stably inherited for more than 125 generations without screening pressure. When constructing metabolic pathways, there is no need to go through the genome integration step. The use of the plasmid without screening pressure system to construct chassis strains will greatly shorten the construction cycle and accelerate the research and development process. The engineered strains constructed using this system do not need to use screening pressure reagents in production applications, which will effectively reduce production costs and increase production profits.

[0023] 2. The present invention can reduce the risk of drug-resistant microorganisms and the cost of using antibiotics by reducing the reliance on antibiotic screening markers and metabolites. It realizes a low-cost and high-efficiency strain construction method and provides a stable and reliable tool system for the practical application of synthetic biology and industrial biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a flowchart of the stable genetic system of Saccharomyces cerevisiae plasmid of the present invention;

[0026] Figure 2 This is the electrophoresis diagram for identifying the TPI1 gene amplified by PCR in Example 1 of the present invention;

[0027] Figure 3 This is the electrophoresis diagram of the restriction enzyme digestion identification of the HCKan-O-TPI1 plasmid BglII+XhoI in Example 1 of the present invention;

[0028] Figure 4 This is the electrophoresis diagram of BsaI restriction enzyme digestion identification of the POT3-URA3-pACF2-TPI1-tADH1 plasmid assembly in Example 1 of the present invention;

[0029] Figure 5 This is the electrophoresis diagram of BsaI restriction enzyme digestion identification of the POT5-LEU2-pACF2-TPI1-tADH1 plasmid assembly in Example 1 of the present invention;

[0030] Figure 6This is the electrophoresis diagram of the fragment of TPI1 knocked out by URA3 in Example 2 of the present invention;

[0031] Figure 7 This is the electrophoresis diagram for the verification of knocking out TPI1 in Example 2 of the present invention; from left to right are the left arm, right arm, full length, and control;

[0032] Figure 8 This is the verification electrophoresis diagram of the upstream and downstream homology arms of the URA3 removal fragment in Example 2 of the present invention;

[0033] Fig. 9 This is the verification electrophoresis diagram of the PCR results of the upstream and downstream homology arm fusion in Example 2 of the present invention;

[0034] Fig.10 This is a verification electrophoresis diagram of the amplification of the verification primer TPI1-A / D for URA3 removal in Example 2 of the present invention; from left to right are full length and control;

[0035] Fig.11 This is the electrophoresis diagram for verifying the amplification of the green fluorescent protein GFP in Example 4 of the present invention;

[0036] Fig.12 This is the electrophoresis diagram for verification of EarI and NdeI restriction enzyme digestion of the HCKan-GFP plasmid in Example 4 of the present invention;

[0037] Fig.13 This is the electrophoresis diagram of BsaI restriction enzyme digestion verification of the plasmid of POT2-pGPD-GFP-tADH1 in Example 4 of the present invention;

[0038] Fig.14 The electrophoresis diagrams of two groups of enzyme digestion identification KpnI and NheI of POT-CK-HIS-GFP-TPI1 plasmid in Example 4 of the present invention;

[0039] Fig.15 This is an electrophoresis diagram for verifying the existence of the POT-CK-HIS-GFP-TPI1 plasmid in Example 5 of the present invention;

[0040] Fig.16 This is the test result of the fluorescent reporter gene without screening pressure passage in Example 5 of the present invention. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0042] The invention discloses a method for stable inheritance of plasmids in saccharomyces cerevisiae without screening pressure. The specific steps are as follows: by cloning the essential gene TPI1 of saccharomyces cerevisiae into POT3-URA3-RFP and POT5-LEU2-ccdB vectors respectively, two rescue plasmids POT3-URA3-pACF2-TPI1-tADH1 and POT5-LEU2-pACF2-TPI1-tADH1 are obtained, POT5-LEU2-pACF2-TPI1-tADH1 is transferred into saccharomyces cerevisiae BY4741 strain, the strategy of URA3 / 5FOA is used to realize the traceless knockout of genomic TPI1, and then POT3-URA3-pACF2-TPI1-tADH1 is transferred into a strain with the traceless knockout of genomic TPI1, and the strain containing only the POT3-URA3-pACF2-TPI1-tADH1 plasmid is obtained by using the processes of different screening markers and plasmid loss. The TPI1 gene, GFP and the receiving vector POT-CK-HIS were assembled in vitro in one step through Golden Gate to obtain the POT-CK-HIS-GFP-TPI1 fluorescent reporter circuit. The metabolic pathway plasmid was transferred into the TPI1 knockout strain, and the knockout strain was screened at 5FOA.

[0043] The POT3-URA3-pACF2-TPI1-tADH1 plasmid was lost, thus obtaining a strain with only the POT-CK-HIS-GFP-TPI1 plasmid. Continuous subculture was performed under conditions without screening pressure to test the stability of the plasmid in this system.

[0044] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0045] Example 1 Construction of rescue plasmid

[0046] (1) Construction of HCKan-TPI1 plasmid

[0047] The genome of Saccharomyces cerevisiae BY4741 was used as a template to amplify the open reading frame sequence of the TPI1 gene by PCR. The primer sequences are as follows:

[0048] Hc-TPI1-F: SEQ ID NO.1;

[0049] Hc-TPI1-R: SEQ ID NO.2.

[0050] Amplification program: 95°C, 5 min; 95°C, 15 s, 55°C, 15 s, 72°C, 30 s, 30 cycles; 72°C, 5 min; 16°C, forever.

[0051] The PCR product sequence is shown in SEQ ID NO.3, and the electrophoresis diagram is shown in Figure 2 The product size is 775 bp. Then, it was connected with HCKan-O-RFP vector (sequence as shown in SEQ ID NO.4) through Golden Gate assembly, and the ligation product was transformed into DH5α Escherichia coli competent cells, screened with LB plates containing 50 μg / mL kanamycin, and the recombinant plasmid was identified by BglII and XhoI restriction enzyme digestion and sequencing. Figure 3 As shown, sequencing was subsequently performed and the correctly sequenced plasmid was named HCKan-O-TPI1, and the sequence is shown in SEQ ID NO.26.

[0052] (2) Construction of POT3-URA3-pACF2-TPI1-tADH1 and POT5-LEU2-pACF2-TPI1-tADH1 plasmids

[0053] The open reading frame plasmid containing the TPI1 gene (HCKan-O-TPI1), the promoter plasmid (HCKan-P-ACF2) and the terminator plasmid (HCKan-T-ADH1) were assembled with two different vectors POT3-URA3-RFP (sequence as shown in SEQ ID NO.5) and POT5-LEU2-ccdB (sequence as shown in SEQ ID NO.6) to construct plasmids for URA3 and LEU2 screening, respectively. Subsequently, the recombinant plasmids were screened on LB plates containing 100 μg / mL ampicillin, and the recombinant plasmids were identified by enzyme digestion, as shown in FIG. Figures 4-5 As shown in Figure 2, the correct plasmids were named POT3-URA3-pACF2-TPI1-tADH1 and

[0054] POT5-LEU2-pACF2-TPI1-tADH1, the sequences are shown in SEQ ID NOs. 27 and 28 respectively.

[0055] Example 2 TPI1 gene seamless knockout

[0056] (1) URA3 knockout of TPI1

[0057] Using POT2-ccdB plasmid (sequence as shown in SEQ ID NO.7) as a template, TPI1-del-F and TPI1-del-R primers were used to PCR amplify the URA3 fragment containing 50 bp homology arms (sequence as shown in SEQ ID NO.8). The primer sequences are as follows:

[0058] TPI1-del-F: SEQ ID NO.9;

[0059] TPI1-del-R: SEQ ID NO.10.

[0060] Amplification program: 95°C, 5 min; 95°C, 15 s, 55°C, 15 s, 72°C, 45 s, 12 cycles; 95°C, 15 s, 62°C, 15 s, 72°C, 45 s, 18 cycles; 72°C, 5 min; 16°C, forever.

[0061] The fragment size is 1296 bp, and the electrophoresis diagram is as follows Figure 6 shown.

[0062] The above-amplified fragment and the POT5-LEU2-pACF2-TPI1-tADH1 plasmid constructed in Example 1 were co-transformed into the BY4741 strain, and the transformants containing the URA3 and LEU2 screening markers were screened using SC-URA-LEU plates. The transformants were screened and identified by colony PCR and genomic PCR. TPI1-A and URA3-B were used to verify the left arm, with a size of 1094bp; URA3-C and TPI1-D were used to verify the right arm, with a size of 921bp. TPI1-A and TPI1-D were used to verify the full length, with a size of 2618bp after URA3 insertion and 2169bp without insertion, to verify whether TPI1 was knocked out and whether the URA3 gene was successfully inserted. The primer sequences are as follows:

[0063] TPI1-A: SEQ ID NO.11;

[0064] URA3-B: SEQ ID NO.12;

[0065] URA3-C: SEQ ID NO. 13;

[0066] TPI1-D: SEQ ID NO.14.

[0067] Electrophoresis diagram Figure 7 As shown, the successfully constructed strain was named 4741-tpi1-del-URA3.

[0068] (2) Removal of URA3

[0069] In order to achieve a seamless knockout of the TPI1 gene, the URA3 gene needs to be removed. Using the yeast BY4741 genome as a template, tpi1-del-up-F and tpi1-del-up-R primers were used to PCR amplify the upstream homology arm fragment tpi1-del-up-500 (sequence as shown in SEQ ID NO.15), del-tpi1-DN-F and tpi1-del-DN-R primers were used to PCR amplify the downstream homology arm fragment tpi1-del-ND-643 (sequence as shown in SEQ ID NO.16), and then the tpi1-del-up-500 and tpi1-del-ND-643 fragments were fused by fusion PCR to obtain the tpi1-Markerless-1118 fragment (sequence as shown in SEQ ID NO.17), which was used to replace the previously inserted URA3 marker gene. It was transferred into the 4741-tpi1-del-URA3 yeast strain.

[0070] The primer sequences are as follows:

[0071] tpi1-del-up-F: SEQ ID NO.18;

[0072] tpi1-del-up-R: SEQ ID NO.19;

[0073] del-tpi1-DN-F: SEQ ID NO.20;

[0074] tpi1-del-DN-R: SEQ ID NO. 21.

[0075] Negative screening was performed using SC+5FOA plates (strains with the URA3 gene could not grow on 5FOA), transformants were screened and identified by colony PCR and genomic PCR, and TPI1-A and TPI1-D were used to verify whether URA3 was successfully removed (the size after URA3 removal was 1422, and the size before removal was 2618). The electrophoresis diagram is shown in Figures 8 to 10 As shown, the knockout strain was named 4741-tpi1-del.

[0076] Example 3 Replacement of rescue plasmid

[0077] The constructed POT3-URA3-pACF2-TPI1-tADH1 plasmid was transferred into the strain 4741-tpi1-del, and the strain containing URA3 was screened by SC-URA plates. After 48 hours, a single clone was picked from the plate and streaked on the SC-URA plate for POT5-LEU2-pACF2-TPI1-tADH1 plasmid loss screening. After 48 hours of culture, the strain was transferred to new YPD, SC-LEU, and SC-URA plates. The single clones that grew on the SC-URA plate but not on the SC-LEU plate were selected and dissolved in sterile water, and then dropped on new YPD, SC-LEU, and SC-URA plates for secondary verification of plasmid loss. Finally, a TPI1 knockout strain containing only the POT3-URA3-pACF2-TPI1-tADH1 plasmid was obtained to ensure that only the POT3-URA3 plasmid existed in the strain. The strain was named 4741-tpi1-del+URA3-TPI1.

[0078] Example 4 Construction and further tandem assembly of GFP-TPI1 plasmid

[0079] Construction of POT2-pGDP-GFP-tADH1 plasmid:

[0080] Using the pYG032-reporter2.7.2 plasmid (sequence shown in SEQ ID NO.22) as a template, a green fluorescent protein (GFP) fragment (GFP-748) was amplified by PCR, and the primers were as follows:

[0081] GFP-F: SEQ ID NO. 23;

[0082] GFP-R: SEQ ID NO.24.

[0083] The fragment of GFP-748 is shown in SEQ ID NO.25, and the electrophoresis diagram is shown in Fig.11 As shown, the product is correct, the PCR product is connected to the HCKan-O-RFP vector (sequence as shown in SEQ ID NO.29) through Golden Gate assembly, the connection product is transformed into DH5α Escherichia coli competent cells, and screened on LB plates containing 50 μg / mL kanamycin. The recombinant plasmid is identified by restriction digestion and sequencing, as shown in FIG. Fig.12 As shown, the plasmid with correct sequencing was named HCKan-O-GFP.

[0084] The three plasmids containing the open reading frame plasmid HCKan-O-GFP, the promoter plasmid HCKan-P-GPD, and the terminator plasmid HCKan-T-ADH1 were connected to the POT2-URA3-ccdB vector through Golden Gate assembly, and the ligation products were transformed into DH5α Escherichia coli competent cells and screened on LB plates containing 100 μg / mL ampicillin. The recombinant plasmids were identified by enzyme digestion, such as Fig.13 The correct plasmid was named POT2-URA3-pGPD-GFP-tADH1, and the sequence was shown in SEQ ID NO.30.

[0085] One-step tandem assembly of GFP-TPI1:

[0086] The POT2-URA3-pGPD-GFP-tADH1 and POT3-URA3-pACF2-TPI1-tADH1 plasmids were assembled with the receiving vector POT-CK-HIS (sequence as shown in SEQ ID NO.31) by GoldenGate assembly in one step. The ligation products were transformed into DH5α Escherichia coli competent cells and screened on LB plates containing 50 μg / mL kanamycin. The recombinant plasmids were identified by enzyme digestion. The electrophoresis results were as follows: Fig.14 The correct plasmid was named POT-CK-HIS-GFP-TPI1, and the sequence was shown in SEQ ID NO.32.

[0087] Example 5 Genetic stability test

[0088] POT-CK-HIS-GFP-TPI1 was transferred into 4741-tpi1-del+URA3-TPI1 yeast strain and screened using SC-HIS plates. After 48 hours, single colonies were picked and streaked onto SC+5FOA plates to lose the POT3-URA3-pACF2-TPI1-tADH1 plasmid.

[0089] Select the monoclonal clones on the SC+5FOA plate, dissolve them in sterile water, and drop them on the SC-URA and SC-HIS plates for secondary verification of the loss of the POT3-URA3-pACF2-TPI1-tADH1 plasmid. Select the monoclonal clones that grow on the 3 SC-HIS plates but not on the SC-URA plates and inoculate them with 5 mL YPD liquid medium for culture. After 24 hours, take 5 μL of the culture and transfer it to fresh 5 mL YPD culture. After 24 hours of culture, transfer the culture in the same way until 10 transfers are completed. Take the 10th transfer culture to measure the OD600 value, dilute it and spread it on the YPD plate so that the number of cells is 200 / plate. After 24 hours of culture, the cells were photocopied to YPD and SC-HIS plates, and the number of fluorescent clones on the photocopied plates was counted. Ten single clones were randomly selected from YPD to extract the genome and then PCR was performed to detect the presence of the POT-CK-HIS-GFP-TPI1 plasmid. Two sets of primers were used for amplification. The first set was POT-JF and TPI1-B, with a size of 2235, and the second set was POT-JR and TPI1-C, with a size of 519 bp. The primer sequences are as follows:

[0090] POT-JF: SEQ ID NO.33;

[0091] TPI1-B: SEQ ID NO. 34;

[0092] POT-JR: SEQ ID NO.35;

[0093] TPI1-C: SEQ ID NO.36.

[0094] The number of fluorescent clones in the replica plate is shown in Table 1:

[0095] Table 1 Statistics of colony numbers on photocopied SC-HIS and YPD plates after 10 transfers (about 125 generations)

[0096] strain YPD SC-HIS 4741-tpi1-del+GFP-TPI1 110 110

[0097] As shown in Table 1, after the parent strain was transferred 10 times in YPD liquid medium without screening pressure for about 125 generations, the number of colonies on the YPD plate was the same as the number of colonies on the YPD plate after the colonies on the YPD plate were copied to the SC-HIS plate, which preliminarily indicated that POT-CK-HIS-GFP-TPI1 was not lost. This shows that the method of the present application can enable Saccharomyces cerevisiae to be stably inherited for more than 125 generations without screening pressure.

[0098] PCR identification results Fig.15As shown in the figure, two sets of primers were used to detect the POT-CK-HIS-GFP-TPI1 plasmid of the strain after passage without screening pressure. PCR was able to amplify the target band, and the size was consistent with the expectation, indicating that the plasmid was not lost and could be stably inherited. The results of the fluorescent reporter gene are shown in Fig.16 As shown, the 125th generation strain still has fluorescence without selection pressure, indicating that the system can stably inherit the plasmid without selection pressure.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for stable plasmid inheritance in Saccharomyces cerevisiae without selection pressure, characterized in that: The method comprises the following steps: S1: constructing a first plasmid and a second plasmid carrying essential genes of Saccharomyces cerevisiae, wherein the first plasmid comprises the LEU2 gene and the second plasmid comprises the URA3 gene; S2: using the POT2-ccdB plasmid as a template to amplify the URA3 fragment containing the homology arm, the URA3 fragment and the first plasmid of S1 are co-transfected into Saccharomyces cerevisiae, and the essential genes of Saccharomyces cerevisiae in the genome of Saccharomyces cerevisiae are knocked out using a plate lacking both uracil and leucine to obtain recombinant bacteria 1; S3: amplifying the upstream and downstream homologous arm sequences of the essential gene of Saccharomyces cerevisiae in the genome of Saccharomyces cerevisiae, fusing the fragment 1 by fusion PCR, transferring the fragment 1 into the recombinant bacteria 1 described in S2, and screening using a plate containing 5FOA to obtain the recombinant bacteria 2 with the URA3 gene removed; S4: The second plasmid of S1 is transferred into the recombinant bacteria 2, and screened using a plate lacking uracil to obtain the recombinant bacteria 3; S5: construct a third plasmid carrying the target gene, transfer it into the recombinant bacteria 3, and screen it using a plate lacking uracil to obtain the recombinant bacteria 4; S6: Subculture the recombinant bacteria 4 described in S5 without selection pressure.

2. The method according to claim 1, characterized in that The essential gene of Saccharomyces cerevisiae is TPI1 gene, PGI1 gene, CDC4 gene, CDC9 gene or CDC28 gene.

3. The method according to claim 1, characterized in that S1 The first plasmid is obtained by connecting a plasmid containing an open reading frame of an essential gene of Saccharomyces cerevisiae, a promoter plasmid and a terminator plasmid with a POT5-LEU2-ccdB vector.

4. The method according to claim 1, characterized in that: S1 The second plasmid is obtained by connecting a plasmid containing an open reading frame of an essential gene of Saccharomyces cerevisiae, a promoter plasmid and a terminator plasmid to a POT3-URA3-RFP vector.

5. The method according to claim 1, characterized in that The third plasmid described in S5 is obtained by connecting a plasmid containing an open reading frame of an essential gene of Saccharomyces cerevisiae, a target gene plasmid, a promoter plasmid and a terminator plasmid with a POT3-URA3-RFP vector.

6. The method according to any one of claims 3 to 5, characterized in that: The connection uses Golden Gate technology.

7. The method according to claim 3, characterized in that The nucleotide sequence of the POT3-URA3-RFP vector is shown in SEQ ID NO.

5.

8. The method according to claim 4, characterized in that The nucleotide sequence of the POT5-LEU2-ccdB vector is shown in SEQ ID NO.

6.

9. The method according to claim 1, characterized in that: The nucleotide sequence of fragment 1 described in S3 is shown in SEQ ID NO.

17.

10. Use of the method according to any one of claims 1 to 9 in metabolic engineering production or biosynthesis.

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