Saccharomyces cerevisiae engineering bacteria for synthesizing beta-carotene as well as construction method and application thereof

Abstract

The invention provides a saccharomyces cerevisiae engineering bacterium for synthesizing beta-carotene. The saccharomyces cerevisiae engineering bacterium is characterized in that glycine at the 1154 site of an amino acid sequence of formyl glycine pyrimidine nucleotide synthetase ADE6 in saccharomyces cerevisiae is mutated into aspartic acid; or the expression quantity of formyl glycine pyrimidine nucleotide synthetase ADE6 in the saccharomyces cerevisiae is reduced. The invention also provides a construction method and application of the gene. The ADE6 mutant gene disclosed by the invention can be used for remarkably improving the yield of beta-carotene in saccharomyces cerevisiae. The discovery lays a foundation for further industrial production of biosynthesis of beta-carotene from saccharomyces cerevisiae, and provides a new strategy for biosynthesis of other tetraterpenoid natural products.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic biology applications, and relates to an engineering bacterium of saccharomyces cerevisiae for synthesizing beta-carotene, a construction method and an application thereof, wherein the ADE6 (formylglycine ribosylation synthetase) mutation site can significantly enhance the synthesis of beta-carotene by saccharomyces cerevisiae. Background Art

[0002] Saccharomyces cerevisiae has a clear genetic background and complete genetic manipulation and omics tools. It is an important microbial cell factory chassis cell. By utilizing its natural mevalonate pathway (MVA pathway) or performing metabolic modification, Saccharomyces cerevisiae can efficiently produce isoprenoid family products and a variety of high-value-added terpenoid compounds, such as geraniol and limonene of monoterpenes, patchouli alcohol and artemisinic acid of sesquiterpenes, sclareol and paclitaxel of diterpenes, ginsenosides and squalene of triterpenes, and β-carotene, astaxanthin and lycopene of tetraterpenes. Among them, β-carotene, as a natural pigment and nutrient, is widely used in pet food, healthcare and cosmetics industries. With the innovation of gene editing technology and the development of synthetic biology microbial cell factories, the synthesis of terpenoids by modifying the mevalonate pathway in Saccharomyces cerevisiae has become a hot topic in the metabolic process. The biosynthesis of β-carotene can be achieved by heterologously expressing CrtE, CrtI and CrtYB in Saccharomyces cerevisiae. However, the natural production of tetraterpenoids as secondary metabolites is usually low and currently cannot meet the needs of industrial scale-up. Therefore, an important demand at present is to develop new gene targets to further enhance the β-carotene biosynthesis capacity of Saccharomyces cerevisiae. Summary of the invention

[0003] In order to solve the problems existing in the prior art, the present invention provides an engineering yeast for synthesizing beta-carotene, a construction method and an application thereof.

[0004] In order to improve the ability of β-carotene biosynthesis of Saccharomyces cerevisiae, the present invention screened and characterized multiple mutant gene targets by isolating and characterizing mutant strains, high-throughput genome resequencing, reverse metabolic engineering and forward metabolic engineering, and proved that the mutation of Gly and Asp at position 1154 of the ADE6 amino acid sequence and the loss of ADE6 expression can significantly increase the β-carotene yield, increasing the β-carotene yield of the basic strain by 5.2 times and 6.1 times, respectively.

[0005] By using fluorescence activated cell sorting (FACS) to sort single cells of the Saccharomyces cerevisiae strains after subculture, the present invention separated and obtained two high-yield Saccharomyces cerevisiae mutant strains with darker single colony colors. High performance liquid chromatography (HPLC) quantitative analysis of β-carotene on the mutant strains found that the β-carotene production of the two strains increased by 4.4 times and 6.0 times, respectively. Subsequent high-throughput whole-genome sequencing and analysis of the two mutant strains showed that the amino acid sequence of acetyl-CoA synthetase (ERG10) of the first mutant strain mutated from phenylalanine (Phe) at position 51 in the N-terminus to serine (Ser); the second strain had two mutation sites, the amino acid sequence of formylglycine (FGAM) synthetase ADE6 mutated from glycine (Gly) at position 1154 in the N-terminus to aspartic acid (Asp), and the amino acid sequence of ribosomal quality control complex component RQC1 mutated from arginine (Arg) at position 385 in the N-terminus to threonine (Thr).

[0006] By using reverse metabolic engineering, the present invention verifies the effects of these three mutation sites on the yield of β-carotene. First, single base mutations were performed on the basic bacteria to achieve mutation recovery at the three sites. Subsequent control experiments and yield tests showed that mutations in the two genes RQC1 and ERG10 had little effect on the yield of β-carotene, while mutations in the ADE6 gene increased the yield of β-carotene by 5.2 times. In order to further explore the effect of ADE6 on β-carotene production, the present invention further constructed ADE6 deletion and overexpression strains. The test results showed that the deletion of the ADE6 gene increased the yield of β-carotene by 6.1 times, while the ADE6 overexpression strain almost completely inhibited the production of β-carotene. This discovery proves the key regulatory role of the ADE6 gene in β-carotene synthesis, and the effect of this gene on β-carotene synthesis has never been reported.

[0007] The first object of the present invention is to provide an engineered yeast for synthesizing β-carotene, by mutating the 1154th position of the amino acid sequence of formylglycine nucleotide synthetase ADE6 in Saccharomyces cerevisiae from glycine to aspartic acid; or reducing the expression level of formylglycine nucleotide synthetase ADE6 in Saccharomyces cerevisiae.

[0008] Preferably, the Saccharomyces cerevisiae is a ZB01 recombinant yeast strain.

[0009] Preferably, the amino acid sequence of the formylglycine nucleotide synthetase ADE6 is SEQ ID NO.3 in the sequence listing.

[0010] The second object of the present invention is to provide a nucleotide sequence encoding the above-mentioned formylglycine nucleotide synthetase ADE6;

[0011] Preferably, the nucleotide sequence of the formylglycine nucleotide synthetase ADE6 is SEQ ID NO.4 in the sequence list;

[0012] Preferably, the nucleotide sequence of the mutant formylglycine nucleotide synthetase ADE6 is based on SEQ ID NO.2 in the sequence table, with the 3461st position mutated from G to A.

[0013] The third object of the present invention is to provide a method for constructing the above-mentioned β-carotene-synthesizing Saccharomyces cerevisiae engineered bacteria, using the CRISPR method to mutate the 1154th position of the ADE6 amino acid sequence; including the steps of designing and amplifying gRNA and donor fragments;

[0014] The gRNA sequence is: ATGGTGATGTCTTAGGTGCA.

[0015] The donor sequence is SEQ ID NO.5 in the sequence listing.

[0016] The fourth object of the present invention is to provide a method for constructing the above-mentioned β-carotene-synthesizing Saccharomyces cerevisiae engineered bacteria, using the CRISPR method to knock out the ADE6 gene in Saccharomyces cerevisiae to reduce the expression level of ADE6; including the steps of designing and amplifying gRNA and donor fragments;

[0017] The gRNA sequence is: ACAAGCTGGATTCAACTCAG.

[0018] The donor sequence is SEQ ID NO.6 in the sequence listing.

[0019] The fifth object of the present invention is to provide the use of the above-mentioned engineered yeast Saccharomyces cerevisiae in synthesizing β-carotene.

[0020] Preferably, the synthesis of β-carotene is carried out by inoculating the above-mentioned engineered yeast Saccharomyces cerevisiae into a YPD liquid culture medium for culturing; preferably, the culture conditions are: overnight culturing at 30° C. and 200 rpm in a shaking incubator.

[0021] The sixth object of the present invention is to provide a method for synthesizing β-carotene, wherein the above-mentioned engineered yeast Saccharomyces cerevisiae is inoculated into a YPD liquid culture medium for cultivation.

[0022] Preferably, the culture conditions are: overnight culture at 30°C and 200 rpm in a shaking incubator.

[0023] In summary, the ADE6 mutant gene of the present invention can significantly increase the yield of β-carotene in Saccharomyces cerevisiae. This discovery lays a foundation for further industrial production of β-carotene biosynthesis by Saccharomyces cerevisiae and provides a new strategy for the biosynthesis of other tetraterpenoid natural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0025] Figure 1 The graphs are growth diagrams and β-carotene production comparisons of mutant strains separated by FACS.

[0026] Figure 2 This is a comparison chart of β-carotene production between the point mutation strains of ADE6, RQC1, and ERG10 and the control strain.

[0027] Figure 3 This is a comparison chart of β-carotene production in ADE6 gene knockout, overexpression strains and control strain. DETAILED DESCRIPTION

[0028] In order to make the present invention easier to understand, the present invention will be described in detail below with reference to the accompanying drawings and examples. These examples are only for illustration and are not limited to the application scope of the present invention. Specific experimental methods not mentioned in the following examples are usually carried out according to conventional experimental methods.

[0029] I. Terminology

[0030] The term "nucleotide mutant" used in the present invention refers to the smallest unit in which a mutation can occur in the nucleotide sequence of a gene.

[0031] Similarly, the term "amino acid mutant" used in the present invention refers to the smallest unit that can mutate in the amino acid sequence of a protein.

[0032] The mark "*" used in the present invention indicates the termination of the protein translation process.

[0033] II. Implementation Plan

[0034] The inventors isolated a mutant high-yield strain by fluorescence activated cell screening (FACS), confirmed the mutation by high-throughput sequencing, then used CRISPR technology for base mutation, and high performance liquid chromatography (HPLC) for β-carotene yield determination.

[0035] Specifically, the inventors found the ADE6 target and high-yield β-carotene strains through the following steps:

[0036] First, FACS is used to sort the culture liquid of the Saccharomyces cerevisiae strain after subculture into a 96-well culture plate for single cells. For strains with significantly darkened single colony colors, high-performance liquid chromatography (HPLC) is used for quantitative analysis of β-carotene. Subsequently, high-throughput whole-genome sequencing and analysis are performed for two mutant strains with significantly increased yields, and mutation targets are obtained. The present invention finds a mutation site in the first mutant strain, that is, the amino acid sequence of acetyl-CoA synthetase (ERG10) mutates from the 51st phenylalanine (Phe) in the N-terminal to C-terminal direction to serine (Ser); two mutation sites are found in the second strain, the amino acid sequence of formylglycine pyrimidine nucleotide (FGAM) synthetase ADE6 mutates from the 1154th glycine (Gly) in the N-terminal to C-terminal direction to aspartic acid (Asp), and the amino acid sequence of the ribosome quality control complex component RQC1 mutates from the 385th arginine (Arg) in the N-terminal to C-terminal direction to threonine (Thr).

[0037] Then, CRISPR technology was used to perform reverse mutation. For the strains verified to be correct by sequencing, HPLC was used to perform the next step of β-carotene production verification. Since the mutation of ADE6 significantly increased the β-carotene production, the present invention further used CRISPR technology to construct ADE6 deletion and overexpression strains, and performed strain production tests.

[0038] For more specific operations, please refer to Examples 1-6 of the present invention below. Examples 1-6 provide detailed descriptions of the process of obtaining mutant strains with high β-carotene production.

[0039] The detection method and instrument in the present invention:

[0040] (1) PCR amplification was performed using a T100 PCR instrument (Bio-Rad, USA).

[0041] (2) A MINI-SUB CELL GT POWER PAC Basic agarose gel electrophoresis instrument (Bio-Rad, USA) was used to detect and separate PCR products.

[0042] (3) The PCR products were detected and verified using a Gel Doc XR+ gel imager (Bio-Rad, USA).

[0043] (4) The absorbance was measured using a GENESYS 50 UV-visible spectrophotometer (Thermo Fisher Scientific, USA).

[0044] (5) The concentrations of DNA and RNA were determined using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA).

[0045] (6) Single cell sorting was performed using a BD Influx flow cytometer (BD Biosciences, USA).

[0046] (7) The β-carotene yield was determined using a Shimadzu LC-20A / SPD-20AV high performance liquid chromatography system (Shimadzu Corporation, Japan) and a SUPELCOSILTM LC-18 column (Merck, USA). The mobile phase was acetonitrile: methanol: isopropanol = 5:3:2, the flow rate was 1 ml / min, and the detection time was 20 min.

[0047] III. Examples

[0048] The present invention is described in detail below through specific examples. The experimental methods described below, unless otherwise specified, are all routine laboratory methods and use conventional experimental equipment. The experimental materials described below, unless otherwise specified, can all be obtained from commercial channels.

[0049] In the following examples, the experimental basic strain is the ZB01 recombinant yeast preserved in the laboratory, and its genotype is:

[0050] MATa MAL2-8c SUC2 ura3-52 leu2ΔhoΔ::ADH1t-crtYB-TDH3p-TEF1p-crtI-CYC1tHMG1::ADH1t-CRTE-TDH3p-TEF1p-tHMG1 -CYC1tXII-2::ERG20t-ERG20-TDH3p-TEF1p-tHMG1-CYC1tX-2::TEF1p-dCas9NG-VPR-ADH1t.

[0051] The construction method of the ZB01 strain is as follows: based on the Saccharomyces cerevisiae strain YZ03 reported in the literature (DOI: 10.1002 / bit.27717), the following genetic engineering modifications were performed, and the genomic integration sites used were reported in the literature (DOI: 10.1016 / j.ymben.2012.01.006): the open reading frame of the LEU2 gene was knocked out; the gene expression cassette TEF1p-dCas9NG-VPR-ADH1t was inserted into the X-2 site of the genome, and the dCas9NG-VPR gene has been codon-optimized for Saccharomyces cerevisiae, and the sequence is SEQ ID NO.1 in the sequence table; the gene expression cassette ERG20t-ERG20-TDH3p was inserted into the XII-2 site of the genome, and the gene sequence was from the Saccharomyces Genome Database website (Saccharomyces Genome Database|SGD); the gene expression cassette TEF1p-tHMG1-CYC1t was inserted into the XII-2 site of the genome, and the gene sequence is SEQ ID NO.2 in the sequence table.

[0052] dCas9NG-VPR gene sequence optimized for Saccharomyces cerevisiae codons (SEQ ID NO.1 in the sequence list):

[0053]

[0054]

[0055]

[0056] tHMG1 gene sequence (SEQ ID NO.2):

[0057]

[0058] Example 1 Isolation of mutant bacteria using flow cytometry screening technology

[0059] (1) Take out the recombinant yeast ZB01 from the -80°C ultra-low temperature freezer, activate it on a YPD plate, and culture it in a 30°C incubator for 48 hours;

[0060] (2) Pick a single clone of ZB01 strain and culture it in 5 mL YPD medium overnight. Transfer the culture medium to 5 mL of new complete synthetic medium (SC) and control the light absorption (OD 600 ) was 0.1, cultured for 72 hours, culture temperature was 30°C, and shaking speed was 200 rpm;

[0061] (3) Dilute the bacterial solution to OD in a 2 mL flow cytometer using sterile water. 600 The vertical axis of the sorting channel is 460 / 50-[405 / 100], and the horizontal axis is 530 / 40-[488 / 200]. The single cells with the top 5% of the horizontal axis intensity are sorted to 50 96-well SC solid culture plates. Then, they are cultured in a 30°C incubator for 3 to 5 days, and the color difference of the single clones is observed, and the dark clones are separated for yield verification.

[0062] Example 2 Verification of β-carotene yield of mutant strain by shake flask fermentation

[0063] (1) Pick the dark clones from the above SC solid culture plate and place them in a 50 mL shake tube containing 5 mL YPD liquid culture medium. Culture them overnight at 30°C and 200 rpm in a shaker.

[0064] (2) Measure the absorbance OD of overnight culture solution 600 , transferred to a 100 mL shake flask containing 20 mL YPD liquid medium, with an initial OD 600The concentration of 0.1 was used and the cells were cultured at 30°C and 200 rpm in a shaking incubator for 72 hours.

[0065] (3) Take 200 μL of the fermentation broth, centrifuge and remove the supernatant, add acid-washed glass beads (425-600 μm) for grinding and shake to mix, then add 1 mL of acetone and continue shaking for 20 minutes. After centrifugation, take the supernatant and filter it through a microporous nylon filter membrane and add it to a 2 mL brown injection bottle.

[0066] (4) The β-carotene yield of the above sample was determined by high performance liquid chromatography using a SUPELCOSILTM LC-18 column. The mobile phase was acetonitrile: methanol: isopropanol = 5:3:2, the flow rate was 1 ml / min, and the detection time was 20 minutes.

[0067] (5) HPLC results showed that the β-carotene production of the two dark clones increased by 4.4 and 6.0 times, respectively, compared with the original strain. Figure 1 ). The carotene concentrations of the control strain ZB01, the mutant strain 14 and the mutant strain 16 were 8.2 mg / L, 35.9 mg / L and 49.4 mg / L respectively. Next, high-throughput whole genome sequencing and analysis were performed on these two mutant strains.

[0068] Figure 1 The graphs are growth diagrams and β-carotene production comparisons of mutant strains separated by FACS.

[0069] Example 3 Genome sequencing and analysis of mutant strains

[0070] (1) The above-mentioned ZB01 recombinant yeast was selected as a genome sequencing control template, and the genome sequencing and comparative analysis of the high-yield mutant bacteria were performed.

[0071] (2) After analyzing the sequencing results, the present invention found that the first mutant strain ( Figure 1 A mutation site was found in the mutant No. 14 in the acetyl-CoA synthetase (ERG10), that is, the 51st phenylalanine (Phe) in the amino acid sequence from the N-terminus to the C-terminus was mutated to serine (Ser), and correspondingly, the 152nd position in the nucleotide sequence from the 5' end to the 3' end was mutated from T to C; in the second mutant strain ( Figure 1Two mutation sites were found in mutant No. 16 in the strain, the amino acid sequence of formylglycine (Gly) synthetase ADE6 mutated from glycine (Gly) at position 1154 to aspartic acid (Asp) from N-terminus to C-terminus, and the amino acid sequence of ribosomal quality control complex component RQC1 mutated from arginine (Arg) at position 385 to threonine (Thr) from N-terminus to C-terminus; accordingly, the nucleotide sequence of ADE6 mutated from G to A at position 3461 from 5' to 3' end; the nucleotide sequence of RQC1 mutated from G to C at position 1154 from 5' to 3' end.

[0072] The amino acid sequence of ADE6 of recombinant yeast ZB01 (SEQ ID NO.3 in the sequence listing):

[0073]

[0074]

[0075] The nucleotide sequence of ADE6 of recombinant yeast ZB01 (SEQ ID NO.4 in the sequence listing):

[0076]

[0077]

[0078] Example 4 Reversion using CRISPR technology

[0079] (1) Design gRNA and donor fragments

[0080] According to the results of whole genome sequencing, the two mutant strains had three amino acid mutations, which were located in the three genes ADE6, RQC1, and ERG10, so the three target sites were reversed by mutation, and the mutation method adopted the reported GTR 2.0 point mutation strategy. This method is based on the CRISPR system of NGN PAM and uses homologous recombination experimental mutation. The pre-experimental design method includes gRNA design for targeting and DNA donor design for mutation.

[0081] a. Design of gRNA

[0082] First, the original genome sequence of the mutant gene was obtained by searching the website https: / / yeastgenome.org (SGD). According to the results of whole-genome sequencing, the mutation position was found. If it was a CG mutation, it was used as the PAM sequence. If it was an AT mutation, NGN was found as the PAM sequence nearby in the downstream, and gRNA primers were designed using Snapgene software.

[0083] The PAM sequence of ADE6 is: GG.

[0084] The ADE6 forward primer sequence is:

[0085]

[0086] The sequence of the ADE6 reverse primer is:

[0087]

[0088] The PAM sequence of RQC1 is: AG.

[0089] The RQC1 forward primer sequence is:

[0090]

[0091]

[0092] The sequence of the RQC1 reverse primer is:

[0093]

[0094] The PAM sequence of ERG10 is: TG.

[0095] The ERG10 forward primer sequence is:

[0096]

[0097] The sequence of the ERG10 reverse primer is:

[0098]

[0099] Among them, the underlined part is the gRNA sequence.

[0100] b.donor's design

[0101] Design primers as upstream homology arms and downstream homology arms 500 bp upstream and downstream of the gRNA recognition site, respectively, and control Tm at 59°C, about 30 bp.

[0102] The upstream primers for ADE6 are:

[0103]

[0104] The downstream primers for ADE6 are:

[0105]

[0106] The upstream primers for RQC1 are:

[0107]

[0108] The downstream primers for RQC1 are:

[0109]

[0110] The upstream primers for ERG10 are:

[0111]

[0112] The downstream primers for ERG10 are:

[0113]

[0114] (2) Obtaining gRNA and donor fragments

[0115] a. Amplification of gRNA and donor gene fragments

[0116] The gRNA and donor fragments were amplified using a PCR instrument using 2×Phanta Flash Master Mix. The PCR reaction system is shown in Tables 1 and 2, the PCR reaction program is shown in Table 3, and the total volume is 50 μL.

[0117] Table 1 PCR reaction system of gRNA

[0118]

[0119] Table 2 PCR reaction system of donor

[0120]

[0121]

[0122] Table 3 2×Phanta Flash Master Mix PCR program

[0123]

[0124] After the PCR products were separated and tested by agarose gel electrophoresis, they were purified by column using the Novagen FastPure Gel DNA Extraction Mini Kit to obtain pure gRNA and donor fragments, and the recovered DNA concentration and quality were detected using a Nanodrop ultra-micro spectrophotometer.

[0125] The nucleotide sequence of ADE6 point mutation donor (SEQ ID NO.5 in the sequence listing):

[0126]

[0127] b. Golden Gate method to construct recombinant plasmid

[0128] The reported vector plasmid pCas9NG-lacZ plasmid was assembled with gRNA and donor fragment using the Golden Gate method. The Golden Gate reaction system is shown in Table 4. The total volume is 20 μL. The Golden Gate reaction procedure is shown in Table 5.

[0129] Table 4 Golden Gate reaction system

[0130]

[0131]

[0132] The volume v of gRNA and donor DNA in the system is calculated according to the following formula:

[0133]

[0134] Table 5 Golden Gate reaction procedure

[0135]

[0136] (3) Screening and verification of point mutation plasmids

[0137] Take 50 μL of E. coli competent cells melted in an ice bath, add 5 μL of the above Golden Gate reaction system, mix gently, and place in an ice bath for 30 minutes. Accurately heat shock in a 42°C water bath for 30 seconds, then quickly transfer the tube to an ice bath for 2 minutes. Add 500 μL of sterile SOC medium (without antibiotics) to each centrifuge tube, mix well and place in a 37°C, 200 rpm shaker for 1 hour. Pipette 100 μL of bacterial solution and spread it on an LB solid culture plate containing ampicillin resistance, and culture in a 37°C constant temperature incubator until a single clone is visible to the naked eye. Pick the above single clone into a 12 mL shake tube containing 4 mL LB culture medium (containing 1 / 1000 ampicillin), and culture overnight at 37°C, 200 rpm shaker. Use the Novozyme FastPure Plasmid Mini Kit plasmid extraction kit to extract the plasmid and perform gene sequencing. And save the gene point mutation plasmids of ADE6, RQC1 and ERG10 that have been sequenced correctly.

[0138] (4) Point mutation plasmid transformation

[0139] a. Preparation of electroporated Saccharomyces cerevisiae

[0140] ZB01 bacteria were revived by drawing lines on YPD plates and cultured at 30°C until a single colony was visible to the naked eye. A single colony was picked and placed in a 50 mL permeable shake tube containing 5 mL YPD and cultured in a shaker at 30°C and 200 rpm until OD 600 About 2. Press the initial OD 600 =0.3 inoculated into a 250 mL shake flask containing 50 mL YPD medium and cultured until 1.2 <OD 600 <1.6. Collect the culture medium in a sterile 50mL centrifuge tube and centrifuge at 3000 rpm for 3 minutes at 4℃. Pour off the supernatant in the clean bench. Add 20mL of 4℃ sorbitol and resuspend, and repeat the centrifugation step. Pour off the supernatant in the clean bench and add 16mL of 1M sorbitol, 2mL of 10×TE solution and 2mL of 1M LiAc solution to resuspend the bacteria. Incubate on a shaker at 30℃ and 200 rpm for 30 minutes. Add 200μL of 1M DTT solution and continue to incubate for 15 minutes. Centrifuge at 3000 rpm for 3 minutes at 4℃, and pour off the supernatant in the clean bench. Mix with 20mL of sorbitol and centrifuge at 3000 rpm for 3 minutes at 4℃. Repeat this step. Use a pipette to remove as much remaining liquid as possible. Resuspend the cells with 200μL of sorbitol and dispense into sterile tubes in 50μL portions.

[0141] b. Electrotransformation of Saccharomyces cerevisiae

[0142] Add the constructed point mutation plasmid (500ng) to 50μL competent cells prepared in step a and mix well. Use a 1mL pipette to blow and beat the mixture evenly. Use a pipette to quickly transfer it to a pre-cooled electroporation cup. Electric shock at 1500v, quickly add 1mL pre-cooled sorbitol after electric shock and transfer to a 50mL shake tube, then take 1mL of sorbitol and add it to the electroporation cup, and transfer the liquid to a 50mL shake tube. At the same time, add 2mL YPD culture medium to the tube. After 3-5 hours of recovery, centrifuge at 3000 rpm for 3min and discard the supernatant. Wash with 4mL sterile water and centrifuge, add 4mL of SC-ura liquid, and culture at 30℃, 200 rpm for 24 hours. Take 400μL of bacterial liquid and add it to 3600μL SC-ura liquid, and culture at 30℃, 200 rpm for 24 hours. Take 100 μL of bacterial solution and spread it evenly on the SC-Ura screening plate and culture it at 30°C for 2 to 3 days.

[0143] (5) Verification of point mutation strains

[0144] Single colonies were picked from the above SC-Ura screening plates for gene sequencing, and the successfully mutated strains were streaked onto 5-Foa solid culture plates and cultured at 30°C for 2 to 3 days.

[0145] Example 5: Verification of the yield of the reverse mutant bacteria

[0146] A single clone of the strain verified correctly in Example 4 was picked and placed in a 50 mL shaking tube containing 5 mL of YPD liquid medium, and cultured overnight in a shaking incubator at 30° C. and 200 rpm.

[0147] The extraction and quantitative analysis of β-carotene were carried out according to the method in steps (2), (3) and (4) of Example 2. The results are shown in FIG. Figure 2 The yield verification results showed that the mutations of the two genes RQC1 and ERG10 had little effect on the yield of β-carotene, while the mutation of the ADE6 gene increased the yield of β-carotene by 5.2 times. The β-carotene yield of the control strain ZB01 was 6.5 mg / L, and the ADE6 gene point mutation strain, namely ZB01-ADE6 G1154D The β-carotene production of the strain with RQC1 gene point mutation was 46.3 mg / L, that of the strain with ERG10 gene point mutation was 5.7 mg / L, and that of the strain with ERG10 gene point mutation was 7.4 mg / L.

[0148] Figure 2 This is a comparison chart of β-carotene production between the point mutation strains of ADE6, RQC1, and ERG10 and the control strain.

[0149] Example 6 Construction of ADE6 deletion and overexpression strains and yield verification

[0150] (1) Design of gRNA and DNA donors for CRISPR systems for deletion and overexpression

[0151] a. ADE6 knockout gRNA and DNA donor

[0152] gRNA: Find an NGG site in the center of the open reading frame of the ADE6 gene as the PAM sequence, and use Snapgene software to design gRNA primers.

[0153] The PAM sequence of ADE6 is: GGG.

[0154] The forward primer sequence of ADE6 is:

[0155]

[0156] The reverse primer sequence of ADE6 is:

[0157]

[0158] Among them, the underlined part is the gRNA sequence.

[0159] DNA donor: Design primers as the upstream homology arm and downstream homology arm at 50bp upstream and downstream of the open reading frame (the whole donor is 100bp spliced ​​from the two), and control Tm at 59℃, about 30bp.

[0160] The upstream primer sequence of donor is:

[0161]

[0162] The downstream primer sequence of the donor is:

[0163]

[0164] b. ADE6 overexpression gRNA and donor

[0165] gRNA: Find an NGG integration site in the yeast gene (the reported X-3 site was used in this experiment). This integration site is a site reported to not affect the normal gene expression of yeast. This site is used as the PAM sequence, and the gRNA primers are designed using Snapgene software.

[0166] The PAM sequence is: AGG.

[0167] The forward primer sequence is:

[0168]

[0169] The reverse primer sequence is:

[0170]

[0171] Among them, the underlined part is the gRNA sequence.

[0172] DNA donor: Design primers as upstream homology arms and downstream homology arms at 50 bp before and after the integration site (donor is the whole spliced ​​by adding ADE6 gene in the middle of the 50 bp before and after), and control Tm at 59℃. The upstream primer sequence of donor is:

[0173]

[0174] The downstream primer sequence of the donor is:

[0175]

[0176] (2) Obtaining gRNA and donor for ADE6 overexpression and knockout

[0177] a. Obtaining gRNA fragments is the same as step (2) of Example 4.

[0178] b. Knockout donor was amplified by PCR using KOD-Plus-Neo. The PCR reaction system was shown in Table 6. The total volume was 50 μL.

[0179] Table 6 PCR reaction system

[0180] Reagents Volume / μL <![CDATA[ddH2O (double distilled water)]]> 15 10×PCR Buffer for KOD-Plus-Neo 5 KOD-Plus-Neo 2 2mM dNTPs 5 <![CDATA[25mMMgSO4]]> 3 F primer (10 μM) 10 R primer (10 μM) 10

[0181] Table 7 KOD-Plus-Neo PCR program

[0182]

[0183] Transfer all PCR products to a 1.5mL sterilized centrifuge tube with a pipette, add 3M NaAC (add 2.5μL of sodium acetate for every 50μL reaction solution), and use a pipette to gently blow and mix. Then add 100% ethanol (add 233μL 100% ethanol for every 100μL reaction solution), use a pipette to gently blow and mix, so that the DNA precipitates, and the final concentration of ethanol after addition is 70% (v / v). Transfer the centrifuge tube to a -80℃ refrigerator and freeze for 20 minutes. After taking it out, centrifuge it at the highest speed for 10 minutes, remove the supernatant, and leave the precipitate at the bottom of the tube. Wash the precipitate with 70% (v / v) ethanol, centrifuge it at high speed for 5 minutes, remove the supernatant, and repeat this step twice. Use a pipette to remove the liquid in the tube as much as possible, evaporate the precipitate at the bottom of the tube in a 55℃ metal bath, and add 20μLddH2O to resuspend the precipitate. Centrifuge at the highest speed for 1 minute, transfer the supernatant containing DNA to a sterilized centrifuge tube, and use a Nanodrop ultra-micro spectrophotometer to detect the concentration and quality of the recovered DNA.

[0184] Nucleotide sequence of ADE6 knockout donor (SEQ ID NO.6 in the sequence listing):

[0185]

[0186]

[0187] c. ADE6 overexpression donor was amplified and recovered by 2×PhantaFlash MasterMix PCR according to the method in step (2) a of Example 4, and the template was the purified genome of ZB01 strain.

[0188] d. According to step (2) b of Example 4, the gRNA fragment was assembled into the reported vector plasmid pCas-lacZ-Brex27 (DOI: 10.1021 / acssynbio.2c00591) preserved in the laboratory using the Golden Gate method to construct the ADE6 deletion and overexpression plasmids.

[0189] (3) Screen and verify ADE6 deletion and overexpression plasmids according to the method in step (3) of Example 4.

[0190] (4) According to the method in step (4) of Example 4, the deletion and overexpression plasmids and the corresponding donor were transformed into ZB01 to construct the ADE6 deletion and overexpression strains.

[0191] (5) The ADE6 deletion and overexpression strains were verified according to the method in step (5) of Example 4.

[0192] (6) The yield of ADE6 deletion and overexpression strains was verified according to the method in Example 5. The results are shown in Figure 3 .

[0193] The results showed that the ADE6 gene deletion increased the production of β-carotene by 6.1 times, while the ADE6 overexpression strain almost completely inhibited the production of β-carotene. The β-carotene concentration of the control strain ZB01 was 7.2 mg / L, the 16 mutant was 45.5 mg / L, the β-carotene concentration of the ADE6 deletion strain ZB01-ade6Δ was 45.5 mg / L, and the β-carotene concentration of the ADE6 overexpression strain was 0.6 mg / L.

[0194] Figure 3 This is a comparison chart of β-carotene production in ADE6 gene knockout, overexpression strains and control strain.

[0195] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An engineered strain of Saccharomyces cerevisiae for synthesizing β-carotene, characterized in that: The method comprises mutating the 1154th position of the amino acid sequence of the formylglycine nucleotide synthetase ADE6 in the cerevisiae from glycine to aspartic acid; or reducing the expression level of the formylglycine nucleotide synthetase ADE6 in the cerevisiae.

2. The β-carotene-synthesizing yeast of claim 1, characterized in that: The Saccharomyces cerevisiae is a ZB01 recombinant yeast strain.

3. The β-carotene synthesizing yeast of claim 1, characterized in that: The amino acid sequence of the formylglycine nucleotide synthetase ADE6 is SEQ ID NO.3 in the sequence list.

4. A nucleotide sequence encoding the formylglycine nucleotide synthetase ADE6 and its mutants as described in any one of claims 1 to 3; Preferably, the nucleotide sequence of the formylglycine nucleotide synthetase ADE6 is SEQ ID NO.4 in the sequence list; Preferably, the nucleotide sequence of the mutant formylglycine nucleotide synthetase ADE6 is based on SEQ ID NO.2 in the sequence table, with the 3461st position mutated from G to A.

5. The method for constructing an engineered strain of Saccharomyces cerevisiae for synthesizing β-carotene according to any one of claims 1 to 3, characterized in that: The CRISPR method was used to mutate the 1154th position of the ADE6 amino acid sequence; including the steps of designing and amplifying the gRNA and donor fragments; The gRNA sequence is: ATGGTGATGTCTTAGGTGCA. The donor sequence is SEQ ID NO.5 in the sequence listing.

6. The method for constructing an engineered strain of Saccharomyces cerevisiae for synthesizing β-carotene according to any one of claims 1 to 3, characterized in that: The CRISPR method was used to knock out the ADE6 gene in Saccharomyces cerevisiae to reduce the expression of ADE6; Includes steps for designing and amplifying gRNA and donor fragments; The gRNA sequence is: ACAAGCTGGATTCAACTCAG. The donor sequence is SEQ ID NO.6 in the sequence listing.

7. Use of the engineered yeast Saccharomyces cerevisiae according to any one of claims 1 to 3 in synthesizing β-carotene.

8. The use according to claim 7, characterized in that: The synthesis of β-carotene is carried out by inoculating the engineered yeast of any one of claims 1 to 3 into a YPD liquid culture medium for culturing; preferably, the culture conditions are: overnight culturing at 30° C. and 200 rpm in a shaking incubator.

9. A method for synthesizing β-carotene, characterized in that: The engineered yeast of any one of claims 1 to 3 is inoculated into a YPD liquid culture medium for culture.

10. A method for synthesizing β-carotene according to claim 9, characterized in that: The culture conditions are: overnight culture at 30° C. and 200 rpm in a shaking incubator.

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