Saccharomyces cerevisiae from scratch synthesis of naringin engineering strain and its construction method and application

Abstract

The application discloses a Saccharomyces cerevisiae de novo synthesis of naringenin engineering strain and a construction method and application thereof, and the engineering strain is constructed by modifying a genome of the Saccharomyces cerevisiae CEN.PK2-1C as follows: substrate competition pathway genes are knocked out, anti-feedback inhibition genes are overexpressed, and the like, so as to improve a metabolic flux j of a precursor substance tyrosine; tyrosine deaminase, 4-coumarate:CoA-ligase, chalcone synthase and chalcone isomerase are expressed heterogeneously, so as to realize de novo synthesis of naringenin. Finally, the yield of naringenin is further improved by high-efficiency expression of Sc4CL and HaCHS at multiple copy sites.

Description

(I) Technical Field

[0001] This invention relates to a combined strategy that promotes efficient expression of heterologous genes by enhancing tyrosine metabolic flux and integrating delta multicopy sites in the genome, and particularly to an engineered Saccharomyces cerevisiae strain that produces naringenin, its construction method, and its application. (II) Background Technology

[0002] Naringenin is the core skeletal structure of flavonoids. As a platform compound, it can be used to derive a large number of flavonoids, possessing various physiological activities such as antiviral, antibacterial, antihypertensive, and antioxidant properties, and is widely used in the food, chemical, and pharmaceutical industries. Given the limitations of plant extraction and chemical synthesis, the biosynthesis of naringenin has become a research hotspot. Referring to plant metabolic pathways, two metabolic pathways for heterologous synthesis of naringenin have been reported: using tyrosine as a substrate, p-coumaric acid is generated under the catalysis of tyrosine ammonia-lyase (TAL), followed by the synthesis of p-coumaryl-CoA under the catalysis of 4-coumaric acid:coenzyme A ligase (4CL), and then the synthesis of naringenin chalcone and naringenin, respectively, under the catalysis of chalcone synthase (CHS) and chalcone isomerase (CHI); the pathway of synthesizing naringenin from tyrosine is currently a popular research approach. In another pathway, naringenin is synthesized from phenylalanine via a five-step enzymatic reaction: phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4CL, CHS, and CHI. The phenylalanine synthesis scheme involves C4H, a P450 enzyme that requires a P450 reductase to convert it to its redox form and needs to be anchored to the endoplasmic reticulum to complete the catalytic reaction, limiting the application of this pathway in prokaryotic expression systems.

[0003] Synthesizing naringenin via microbial fermentation is an ideal way to reduce the cost of flavonoids while also adhering to green and environmentally friendly principles. Saccharomyces cerevisiae, a GRAS (generally regarded as safe) organism, possesses a favorable genetic background, excellent stress resistance, superior fermentation characteristics, stable production performance, and high safety, making it an attractive microbial host for naringenin production. (III) Summary of the Invention

[0004] The first objective of this invention is to provide an engineered Saccharomyces cerevisiae strain that produces tyrosine. The engineered strain uses S. cerevisiae CEN.PK2-1C as the substrate bacteria and undergoes metabolic modification to obtain a high-yield Saccharomyces cerevisiae strain T01 that produces L-tyrosine.

[0005] The second objective of this invention is to provide a *Saccharomyces cerevisiae* engineered strain that enables and enhances de novo synthesis of naringenin. The engineered strain uses a high-tyrosine-producing strain T01 as a host, and overexpresses RgTAL and Sc4CL using a high-copy 2μ plasmid to construct a de novo naringenin synthesis strain (N01). Subsequently, by integrating the Sc4CL and HaCHS expression cassette into the delta site (this strain was named N02), naringenin synthesis was significantly promoted without a significant impact on biomass.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a recombinant expression plasmid containing a tyrosine ammonia-lyase gene (TAL) and a 4-coumaric acid:coenzyme A ligase gene (4CL).

[0008] In one embodiment of the present invention, the vector for the recombinant expression plasmid is pESC-His, and the tyrosine ammonia-lyase gene is linked to a TEF promoter for expression control. Additionally, for plasmid screening, the TEF promoter may also carry a screening gene, such as a hygromycin resistance gene.

[0009] In one embodiment of the present invention, the tyrosine ammonia-lyase gene encodes the amino acid sequence shown in SEQ ID NO:6, derived from *Rhodotorula glutinis*; the 4-coumaric acid:coenzyme A ligase gene encodes the amino acid sequence shown in SEQ ID NO:7, derived from *Streptomyces coelicolor*. The codon optimization required when inserting these genes into the host bacterium is common knowledge in the art.

[0010] In one embodiment of the present invention, the recombinant expression plasmid is named pESC-TAL-4CL-HygR plasmid.

[0011] Secondly, the present invention provides a genetically engineered bacterium containing the above-mentioned recombinant expression plasmid, wherein the host bacterium is obtained from S. cerevisiae CEN.PK2-1C strain through the following modifications: the gal80 gene is replaced with an expression cassette A of the chalcone synthase gene, the aro10 gene is replaced with an expression cassette of the chalcone isomerase gene, the pdc5 gene and the gal2 gene are knocked out, and the PHA2 gene is replaced with aro7. G141S Gene expression cassette, TRP2 replaced with aro4 k229L Gene expression cassettes were used to obtain the host bacteria.

[0012] The aro7 G141SThe expression cassette of the gene encodes an amino acid sequence by mutating glycine at position 141 of the amino acid sequence encoded by the aro7 gene to serine. G141S The amino acid sequence encoded by the gene expression cassette is shown in SEQ ID NO:10; the aro7 G141S The promoter and terminator of the gene expression cassette are the PGK1 promoter and HXT7 terminator, respectively. The aro4 k229L The amino acid sequence encoded by the expression cassette of the aro4 gene is obtained by mutating lysine at position 229 to leucine. k229L The amino acid sequence encoded by the gene expression cassette is shown in SEQ ID NO:11, namely aro4. k229L The promoter and terminator of the gene expression cassette are the TEF1 promoter and the CYC1 terminator, respectively.

[0013] In one embodiment of the present invention, the promoter and terminator of the expression cassette A of the chalcone synthase (CHS) gene are the gal7 promoter and the CYC1 terminator, respectively, and the edited amino acid sequence of the expression cassette A of the chalcone synthase (CHS) gene is shown in SEQ ID NO:8; the promoter and terminator of the expression cassette of the chalcone isomerase (CHI) gene are the gal1 promoter and the ADH1 terminator, respectively, and the edited amino acid sequence of the expression cassette of the chalcone isomerase (CHI) gene is shown in SEQ ID NO:9.

[0014] Preferably, the genetically engineered bacteria are further modified as follows: the expression cassette B of the chalcone synthase gene and the expression cassette of the 4-coumaric acid:coenzyme A ligase gene are integrated together into the delta site of the genetically engineered bacteria to obtain a genetically engineered bacteria that produces high levels of naringenin.

[0015] Furthermore, all the above modifications were performed using the CRISPR-Cas9 single-plasmid gene editing system. In one embodiment of the present invention, the Cas9 expression cassette (P...) was first... TEF -spCas9-T ADH2This invention optimizes the CRISPR-Cas9 dual-plasmid gene editing system by integrating gRNA into the IX-1 site of the genome of *S. cerevisiae* CEN.PK2-1C strain. Using Di-CRISPR / Cas9 (delta-integration) technology, gRNA was designed to target delta sequences on the yeast genome, achieving simple and efficient integration of Sc4CL and HaCHS Donor DNA in the naringenin synthesis pathway at double-stranded breaks (DSBs). However, those skilled in the art should understand that similar or identical modifications performed using other gene editing methods are also within the scope of this invention.

[0016] In one embodiment of the present invention, the promoter and terminator of the expression cassette B of the chalcone synthase gene are the ERG20 promoter and the CYC1 terminator, respectively; the promoter and terminator of the expression cassette of the 4-coumaric acid:coenzyme A ligase gene are the SED1 promoter and the CYC1 terminator, respectively.

[0017] In embodiments of the present invention, the amino acid sequences edited in expression cassettes A and B of the chalcone synthase gene are the same, the only difference being the promoter.

[0018] In one embodiment of the present invention, the amino acid sequence edited by the expression cassette of the chalcone synthase gene is shown in SEQ ID NO:8; the amino acid sequence edited by the expression cassette of the 4-coumaric acid:coenzyme A ligase gene is shown in SEQ ID NO:7.

[0019] Thirdly, the present invention also provides the application of the above-mentioned genetically engineered bacteria in the fermentation production of naringenin.

[0020] Specifically, in one embodiment of the present invention, the application is as follows: the genetically engineered bacteria are inoculated into YPD liquid culture medium and cultured at 30°C and 180 rpm for 15 h to obtain seed liquid; the seed liquid is transferred to fresh YPD liquid culture medium at a volume inoculation rate of 2% and fermented at 30°C and 180-220 rpm for 48-96 h to obtain fermentation broth containing naringenin.

[0021] Specifically, the method for constructing the host bacterium includes the following steps:

[0022] (1) The S. cerevisiae CEN.PK2-1C genome was used as a template to amplify endogenous promoters, terminators, upstream and downstream homologous arms and endogenous genes; E. coli DH5α was used for plasmid amplification and preservation, etc.

[0023] (2) To facilitate gene manipulation, approximately 500 bp homologous arms upstream and downstream of the target locus IX1 were amplified using the genome as a template. A Cas9 expression cassette was amplified from the p42H-spCas9 plasmid, and the complete Cas9 expression cassette Donor DNA fragment was obtained by over-lap PCR. The above Donor DNA and gRNA-IX1 plasmid were transformed by LiAc / PEGylation. After colony PCR verification of the integration of the Cas9 expression cassette into the IX1 site, the gRNA-IX1 plasmid was lost by passage culture without antibiotic YPD. The resulting strain was C00(CEN.PK2-1C,IX1::P). TEF -spCas9-T ADH2 It serves as a host for subsequent DNA integration and biosynthesis pathways.

[0024] (3) Using C00 as the chassis bacteria, firstly, knocking out gal80 can relieve the gal promoter's galactose-dependent expression, and at the same time, integrate the HaCHS expression cassette at this site (the chalcone synthase CHS is derived from Hypericum mandrosaemum); knocking out aro10 and integrating the MsCHI expression cassette can achieve CHI overexpression (the chalcone isomerase CHI is derived from alfalfa (Medicago sativa); knocking out pdc5 reduces the consumption of 4-HPP by competitive metabolic pathways; then, knock out gal2; next, knock out PHA2 and overexpress aro7. G141S and knocking out TRP2 and overexpressing aro4 k229L To prevent carbon flux from being directed towards the synthesis of phenylalanine and tryptophan, the carbon flux entering shikimic acid and cladonic acid metabolism can be increased, and the feedback inhibition of tyrosine can be relieved. The T01 strain exhibited the highest tyrosine yield at 840 mg / L after 96 hours of fermentation, representing an increase of approximately 494% compared to the starting strain.

[0025] Specifically, in the embodiments of the present invention, the genetically engineered bacteria and the high-yield naringenin genetically engineered bacteria are constructed according to the following method:

[0026] 1) The tyrosine ammonia-lyase TAL described in this invention is derived from *Rhodotorula glμtinis*, and the 4-coumaric acid:coenzyme A ligase 4CL is derived from *Streptomyces coelicolor*. First, the genes RgTAL and Sc4CL were cloned into the pESC-His vector after codon optimization. Subsequently, the His auxotrophic selection marker was replaced with a hygromycin resistance tag to obtain the pESC-TAL-4CL-HygR plasmid.

[0027] 2) When the pESC-TAL-4CL-HygR expression plasmid was overexpressed in the high-tyrosine-producing strain T01, strain N01 was obtained, with a tyrosine yield of 403 mg / L, a p-coumaric acid yield of 40.99 mg / L, and a naringenin yield of 81.45 mg / L.

[0028] 3) The method for integrating the Sc4CL and HaCHS expression cassettes at the delta site described in this invention is as follows: Using the S. cerevisiae CEN.PK2-1C genome as a template, the upstream homologous arm of the delta site (approximately 160 bp), the SED1 promoter, the ERG20 promoter, and the downstream homologous arm of the delta site (approximately 250 bp) are amplified. Using pESC-TAL-4CL-His and pESC-CHS-CHI-His plasmids as templates, 4CL and its ADH1 terminator and CHS and its CYC1 terminator are amplified, respectively. The above PCR products are purified or gel-recovered to obtain 6 DNA fragments. Multiple fusions of the two fragments yield delta-integrated Donor DNA, which is then integrated into the genome of strain N01 via LiAc / PEGylation to obtain a delta-integrated strain, named N02. N02 has a tyrosine yield of 344.19 mg / L, a coumaric acid yield of 67.96 mg / L, and a naringenin yield of 157.68 mg / L.

[0029] Those skilled in the art will know that completing genome modification before introducing recombinant plasmids should also fall within the scope of protection of this invention.

[0030] Compared with existing technologies, the main advantages of this invention are as follows: This invention integrates the Cas9 gene expression cassette into the yeast genome, optimizing the CRISPR / Cas9 dual-plasmid gene editing system, which can shorten the yeast gene editing cycle. The Di-CRISPR / Cas9 (delta-integration) platform can significantly improve the synthesis of target metabolites as an effective method. The engineered Saccharomyces cerevisiae strain constructed in this invention can achieve de novo synthesis of naringenin using glucose as a substrate via microbial methods, with the NO2 strain yielding 157.68 mg / L during shake-flask fermentation. (iv) Description of the attached drawings

[0031] Figure 1 Chemical structural formula of naringenin

[0032] Figure 2 Schematic diagram of the modified naringenin synthesis and metabolism pathway in brewer's yeast

[0033] Figure 3 Schematic diagram of the biomass of engineered strains of Saccharomyces cerevisiae

[0034] Figure 4 Schematic diagram of tyrosine production by engineered strains of Saccharomyces cerevisiae

[0035] Figure 5 Schematic diagram of naringenin production by engineered strains of brewing yeast

[0036] Figure 6 Schematic diagram of Sc4CL and HaCHS expression cassettes integrated into the delta site (V) Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be fully described below. However, what is described are only some embodiments of the present invention, not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0038] The culture media involved in the examples described in this invention are as follows:

[0039] LB medium: 0.5% yeast extract, 1% peptone, 1% sodium chloride, solvent: water, pH: natural;

[0040] YPD medium: 1% yeast extract, 2% peptone, 2% glucose, water as solvent, natural pH;

[0041] For solid culture media, 1.5% agar powder is added.

[0042] Tyrosine and naringenin were detected by high performance liquid chromatography (HPLC).

[0043] The specific conditions for HPLC detection of tyrosine were as follows: Welchrom C18 column (250 mm × 4.6 mm, 5 μm), mobile phase of 0.1 mol / L sodium acetate solution (pH = 4.0): methanol = 90:10 (v / v), flow rate of 1 mL / min, detection wavelength of UV detector of 280 nm, injection volume of 10 μL, and column temperature of 30 ℃.

[0044] The specific conditions for HPLC detection of naringenin were as follows: Welchrom C18 column (250 mm × 4.6 mm, 5 μm) was used; mobile phase A was pure methanol (containing 0.2% acetic acid); mobile phase B was an aqueous solution containing 0.2% acetic acid; the flow rate was 1 mL / min; gradient elution of the mobile phase is shown in the table below; the detection wavelength for coumaric acid using a UV detector was 308 nm; the detection wavelength for naringenin was 288 nm; the injection volume was 10 μL; and the column temperature was 30 °C.

[0045]

[0046] Example 1

[0047] 1. Construction of engineered tyrosine-producing Saccharomyces cerevisiae T01

[0048] First, the Cas9 expression box (P TEF -spCas9-T ADH2 ) was integrated into the S. cerevisiae CEN.PK2-1C genome at site IX-1 to optimize the CRISPR-Cas9 dual plasmid gene editing system; subsequently, gal80 was replaced with P gal7 -CHS-T cyc1 The galactose-dependent expression of the gal promoter was deactivated and CHS was overexpressed; then, aro10 was replaced with P gal1 -CHI-T ADH1 Subsequently, pdc5 and gal2 were knocked out in sequence, and finally, PHA2 was replaced with P. PGK1 -aro7 G141S -T HXT7 TRP2 is replaced with P TEF1 -aro4 k229L -T cyc1 To achieve overexpression of the tyrosine-resistant feedback inhibition gene and reduce the metabolic flux of the phenylalanine and tryptophan competitive pathways, strain T01 was obtained.

[0049] 2. Integration of Cas9 expression cassette into the S. cerevisiae CEN.PK2-1C genome

[0050] Construct the gRNA-IX1 plasmid (the gRNA plasmid required for subsequent gene editing is constructed using this example).

[0051] First, the plasmid pKan100-LEU2-gRNA (CN112322512A) was used as a template for reverse amplification (primers: gRNA-F and gRNA-R). After digestion with Dpn I for 2.5 h, it was transformed into E. coli DH5α. After overnight culture on Amp plates, single colonies were picked and inoculated into LB broth containing Amp (the working concentration of ampicillin solution described in this article is 100 mg / mL) and cultured for 12-15 h. The plasmid was extracted, and sequencing was performed using the universal primer M13R to verify whether the LEU2 tag had been knocked out. If the knockout was successful, the pKan100 plasmid was obtained. Then, using pKan100 plasmid as a template, the pKan100-IX1 DNA fragment was obtained by reverse amplification using primers (gRNA-IX1-F and gRNA-IX1-R) designed for the first 20 bp of the PAM site of the IX1 target site. The remaining steps were the same as those for pKan100 plasmid construction. After extracting the plasmid, sequencing was performed using the universal vector primer M13R to verify whether the first 20 bp of the IX1 target site (i.e., N20) was successfully mutated. If the N20 mutation was successful, the gRNA-IX1 plasmid was obtained.

[0052] Using plasmid pH-spCas9 (CN112322512A) as a template, spCas9 was amplified (primers: spCAS9-p42H-F and spCAS9-p42H-R). Then, using plasmid pRS42H-iCas9 (CN112322512A) as a template, it was reverse-amplified (primers: p42H-spCAS9-F and p42H-spCAS9-R). Complete recombinant linearized plasmid fragments were obtained via overlap PCR (primers: spCAS9-p42H-F and p42H-spCAS9-R; the molar ratio of the two DNA fragments was 1:3). After ligation, transformation was performed, and colony PCR was used for verification (primers: spCAS9-CHECK-F1 and spCAS9-CHECK-R1). Positive transformants were screened, and plasmids were extracted and sequenced to verify their sequence correctness. Correct sequencing yielded the p42H-spCas9 plasmid. Then, using the S. cerevisiae CEN.PK2-1C genome as a template, approximately 500 bp homologous arms upstream and downstream of the target locus IX1 were amplified (primers: UPIX1-F, UP IX1-R, DOWN IX1-F, and DOWN IX1-R). The Cas9 expression cassette was amplified from the p42H-spCas9 plasmid (primers: SpCas9-F and SpCas9-R), and the complete Donor DNA fragment was obtained by overlap PCR (primers: UP IX1-F and DOWN IX1-R; the three DNA fragments were used in a molar ratio of 1:3:1). First, the p42H-spCas9 plasmid was transformed into S. cerevisiae CEN.PK2-1C; subsequently, the Donor DNA containing the Cas9 expression cassette and the gRNA-IX1 plasmid were co-transformed into S. cerevisiae CEN.PK2-1C. After verification by colony PCR (primers: IX1-CHECK-F1 and IX1-CHECK-R1) and sequencing, strain C00 (CEN.PK2-1C, IX1::P) was obtained. TEF -spCas9-T ADH2 It is used as a host for subsequent DNA integration and biosynthesis pathways.

[0053] 3. CRISPR / Cas9 gene replacement procedures, taking gal80Δ::Pgal7-CHS-Tcyc1 as an example.

[0054] 1) Construct the gRNA-gal80 plasmid. The operation is the same as that for the gRNA-IX1 plasmid. The only difference is that the first 20bp of the PAM site of the target gene gal80 is different. That is, the amplification primers are gRNA-gal80-F and gRNA-gal80-R.

[0055] 2) Preparation of Donor DNA

[0056] Amplification of upstream and downstream homologous arms (50 μL system): Using 1 μL of S. cerevisiae CEN.PK2-1C genome as a template (the genome concentration used in this study was 100 ng / μL), 25 μL of 2×Phanta Max Master Mix (Vazyme, Nanjing) high-fidelity DNA polymerase, 1 μL each of primers UP gal80-F and UP gal80-R (upstream homologous arm primers) or DOWN gal80-F and DOWN gal80-R (downstream homologous arm primers), and ddH2O to a final volume of 50 μL. PCR reaction conditions were: pre-denaturation at 98℃ for 8 min, followed by temperature cycling at 98℃ for 10 sec; 58℃ for 15 sec; 72℃ for 30 sec; for a total of 35 cycles, ending at 16℃. After PCR amplification, the correct PCR products were verified by 1% agarose gel electrophoresis. PCRPurification Kit (TransGen, Beijing) is used to purify upstream or downstream homologous arm fragments.

[0057] Amplification of the CHS expression cassette (Pgal7-CHS-Tcyc1): Using 1 μL of pESC-CHS+CHI-His plasmid (synthesized by Hangzhou Tsingke Biotechnology Co., Ltd., nucleotide sequence of which is shown in SEQ ID NO:1) at a concentration of 100 ng / μL as a template, 25 μL of 2×Phanta Max Master Mix (Vazyme, Nanjing) high-fidelity DNA polymerase, 1 μL each of primers Pgal7-CHS-TF and Pgal7-CHS-TR, and ddH2O to a final volume of 50 μL. PCR reaction conditions were: pre-denaturation at 98℃ for 5 min, followed by temperature cycling at 98℃ for 10 sec; 58℃ for 15 sec; 72℃ for 1 min; for a total of 35 cycles, ending at 16℃. After amplification, the correct PCR product was verified by 1% agarose gel electrophoresis. The CHS expression cassette was purified using a PCR Purification Kit.

[0058] Donor DNA was obtained using overlap PCR. Using DNA fragments from the upstream homologous arm of gal80, the CHS expression cassette, and the downstream homologous arm of gal80 as templates (added at a fragment molar ratio of 1:3:1 based on the recovered fragment concentrations), 25 μL of 2×PhantaMax Master Mix (Vazyme, Nanjing) high-fidelity DNA polymerase, 1 μL each of primers UP gal80-F and DOWN gal80-R, and ddH2O were added to a final volume of 50 μL. The PCR reaction conditions were: pre-denaturation at 98℃ for 5 min, followed by temperature cycling at 98℃ for 10 sec; 58℃ for 15 sec; 72℃ for 1 min; for a total of 35 cycles, ending at 16℃. After amplification, the PCR product obtained through purification or gel recovery was sequenced correctly to obtain the Donor DNA.

[0059] 3) Preparation of S. cerevisiae CO00 competent cells (using this as an example for the preparation of Saccharomyces cerevisiae competent cells required for subsequent gene editing).

[0060] (1) Take out the preserved bacterial strain C00, streak it on YPD solid plate, and incubate it in a constant temperature incubator at 30℃ for 2-3 days.

[0061] (2) Pick a fresh single colony and inoculate it into 5 mL of YPD liquid medium. Incubate at 30°C and 200 rpm for 15-20 h with shaking.

[0062] (3) Take the above bacterial solution and inoculate it into 20 mL of fresh YPD medium at a 2% inoculation rate. Incubate at 30°C and 200 rpm with shaking until OD600 = 0.5-0.7.

[0063] (4) Transfer 20 mL of bacterial culture to a 50 mL sterile centrifuge tube, centrifuge at 4000 rpm and 4℃ for 5 min, and collect the cells.

[0064] (5) Use a pipette to add 900 μL of pre-cooled sterile water and 100 μL of 10×LiAc solution to the above 50 mL centrifuge tube, gently resuspend the cells, transfer the bacterial solution to a 2 mL sterile EP tube, centrifuge at 4000 rpm and 4℃ for 1 min, and collect the cells.

[0065] (6) Resuspend the bacterial cells again in 900 μL of pre-cooled sterile water and 100 μL of 10×LiAc solution, and centrifuge. Repeat the above steps three times.

[0066] (7) Finally, add 900 μL of pre-cooled sterile water and 100 μL of 10×LiAc solution to the EP tube to resuspend the cells. Let stand for 5 min, centrifuge at 4000 rpm and 4℃ for 1 min, discard 900 μL of supernatant, and resuspend the cells in the remaining supernatant to obtain CO0 competent cells.

[0067] 4) PEG / LiAc transformation of Saccharomyces cerevisiae (using this as an example for subsequent gene editing transformation)

[0068] (1) Add the conversion system to the sterilized EP tubes. The conversion system is shown in the table below:

[0069]

[0070] (2) Add 25 μL of the prepared competent cells to the above transformation system and mix using a vortex mixer for 10 s. Place the EP tube in a 30℃ incubator for 30 min.

[0071] (3) Add 36 μL of dimethyl sulfoxide and mix in a vortex mixer for 10 s. Heat shock in a 42℃ water bath for 15 min.

[0072] (4) Centrifuge at 4000 rpm and 4℃ for 1 min to collect cells. Discard the supernatant, add 400 μL of 5 mM CaCl2 solution to resuspend the cells, and let stand for 15 min.

[0073] (5) Collect cells by centrifugation. Discard the supernatant, add 1 mL of sterile YPD medium, gently resuspend the cells, and incubate at 30°C and 200 rpm for 1 h with shaking.

[0074] (6) Collect cells by centrifugation. Discard the supernatant, add 1 mL of sterile water to wash the cells, and centrifuge as above.

[0075] (7) Discard 700-800 μL of YPD medium, gently resuspend the bacterial cells in the remaining medium, and spread them on YPD+G418 (in this article, the final concentration of G418 used is 200 mg / L) solid plates. Incubate at 30℃ for 2-3 days.

[0076] 5) Identification of Saccharomyces cerevisiae transformants (using this as an example for subsequent colony PCR during gene editing).

[0077] MightyPrep reagent for DNA (Takara, Dalian) was used to obtain colony PCR templates from *Saccharomyces cerevisiae* cells through cell lysis. Using 1 μL of supernatant cell lysis buffer as template, 25 μL of Green Taq Mix (Vazyme, Nanjing), 1 μL each of primers gal80-CHECK-F1 and gal80-CHECK-R1, and ddH2O to a final volume of 50 μL were added. The PCR reaction conditions were: pre-denaturation at 98℃ for 8 min, followed by temperature cycling at 98℃ for 15 sec; 58℃ for 15 sec; 72℃ for 30 sec; for a total of 35 cycles, ending at 16℃. Once the PCR product was correctly sequenced, gene editing was considered complete.

[0078] 6) gRNA-gal80 plasmid elimination (using this as an example for gRNA plasmid elimination during subsequent gene editing)

[0079] (1) Inoculate the transformant with the correct genotype into 3 mL YPD and culture for 12 h; (2) Take 100 μL to inoculate with new 3 mL LYPD and passage 3 times consecutively; (3) Pour 5 μL onto a YPD solid plate and culture for 2-3 days; (4) Pick the same single colony on a YPD-free solid plate and a YPD+G418 resistant plate; if a single colony can grow on the YPD-free solid plate but not on the G418 plate, then the engineered bacteria with the gRNA-gal80 plasmid lost is obtained.

[0080] 7) If subsequent gene editing involves genome replacement, refer to the gal80Δ::Pgal7-CHS-Tcyc1 procedure.

[0081] 4. CRISPR / Cas9 gene knockout procedure, taking the pdc5 gene as an example.

[0082] The difference between gene knockout and replacement is that gene knockout only requires the direct fusion of upstream and downstream homologous arm fragments during the Donor DNA construction step.

[0083] Amplification of upstream and downstream homologous arms: Using 1 μL of 100 ng / μL *Saccharomyces cerevisiae* CEN.PK2-1C genome as a template, 25 μL of 2×PhantaMax Master Mix high-fidelity DNA polymerase, 1 μL each of primers UPPdc5-F and UPPdc5-R or DOWNPdc5-F and DOWNPdc5-R, and ddH2O to a final volume of 50 μL. PCR reaction conditions were: pre-denaturation at 98℃ for 8 min, followed by temperature cycling at 98℃ for 10 sec; 58℃ for 15 sec; 72℃ for 15 sec; for a total of 35 cycles, ending at 16℃. After PCR amplification, the correct PCR products were verified by 1% agarose gel electrophoresis. The PCR Purification Kit was used to purify upstream or downstream homologous arm fragments.

[0084] Overlap PCR was performed to obtain Donor DNA. Using fragments recovered from the upstream and downstream homologous arms of pdc5 as templates (fragment molar ratio 1:1), 25 μL of 2×PhantaMax Master Mix high-fidelity DNA polymerase, 1 μL each of primers UPPdc5-F and DOWNPdc5-R, and ddH2O were added to a final volume of 50 μL. After amplification, the PCR product was purified or recovered via gel extraction, and after successful sequencing, the Donor DNA was obtained.

[0085] The remaining steps are the same as those for gal80Δ::Pgal7-CHS-Tcyc1 gene replacement.

[0086] 5. CRISPR / Cas9 anti-feedback suppression point mutation operation, using PHA2Δ::P PGK1 -aro7 G141S -T HXT7 For example

[0087] The release of aro7 anti-feedback inhibition and its overexpression are gene substitutions. The difference between this and the substitutions described above is that point mutations are required at key sites of the aro7 gene to achieve the purpose of anti-feedback inhibition.

[0088] The glycine residue at position 141 of the aro7-encoded protein was mutated to a serine residue. First, aro7... G141S The expression cassette was constructed using a full plasmid amplification method, i.e., primer P. PGK1 -aro7 m -T HXT7 -F and P PGK1 -aro7 m -T HXT7 -R with p-aro7 m The plasmid (preserved in the laboratory, with the nucleotide sequence of the plasmid as shown in SEQ ID NO:2) was used as a template for reverse amplification to obtain P. PGK1 -aro7 m -T HXT7 First, the upstream and downstream homologous arms are amplified using the same method as described above (primers for amplifying the upstream homologous arm are: UP PHA2-F and UP PHA2-R; primers for amplifying the downstream homologous arm are: Down PHA2-F and Down PHA2-R); finally, the three fragments are fused using primers UP PHA2-F and Down PHA2-R via overlap PCR to obtain Donor DNA.

[0089] The remaining steps refer to the gal80Δ::Pgal7-CHS-Tcyc1 operation procedure. TRP2Δ::P TEF1 -aro4 k229L -T cyc1 Reference PHA2Δ::P PGK1 -aro7 G141S -T HXT7 Among them, p-aro4 m The plasmid was preserved in the laboratory, and its nucleotide sequence is shown in SEQ ID NO:3.

[0090] 6. Construction of recombinant plasmid pESC-TAL-4CL-HygR

[0091] The RgTAL and Sc4CL genes were synthesized by Hangzhou Tsingke Biotechnology Co., Ltd. using codon optimization of Saccharomyces cerevisiae and multiple cloning sites of Sac I and Xho I, and were placed in the pESC-His vector. The nucleotide sequence of the resulting pESC-TAL-4CL-His plasmid is shown in SEQ ID NO:4.

[0092] Using pESC-TAL-4CL-His plasmid as a template, the pESC-TAL-4CL vector backbone (with the His3 expression cassette removed) was amplified in reverse. Using p-gRNA-delta plasmid (a kind gift from Professor Hu Zhongce's laboratory at Zhejiang University of Technology, the nucleotide sequence of which is shown in SEQ ID NO:5) as a template, the expression cassette of the hygromycin resistance gene (P) was amplified using primers HygR-F and HygR-R. TEF The pESC-TAL+4CL and HygR expression cassettes were cloned in one step using the ClonExpress II One Step Cloning Kit and then transformed into E. coli DH5α. Colony PCR (primers: +HygR-CHECK-F1 and +HygR-CHECK-R1) and sequencing results showed that the pESC-TAL-4CL-HygR plasmid was successfully constructed.

[0093] 7. Construction of Saccharomyces cerevisiae N01

[0094] First, T01 competent cells were prepared. Second, the pESC-TAL-4CL-HygR plasmid was transformed into the T01 competent cells using LiAc / PEGylation and plated on YPD+Hyg (Hyg working concentration 200 mg / L) plates, and cultured at 30℃ for 2-3 days until transformants appeared. Finally, colony PCR was performed using primers +HygR-CHECK-F1 and +HygR-CHECK-R1 to verify the colony. The correctly verified strain was preserved in glycerol tubes to obtain the engineered Saccharomyces cerevisiae strain N01.

[0095] 8. Construction of Saccharomyces cerevisiae NO2

[0096] Construction of Donor DNA: Using the S. cerevisiae CEN.PK2-1C genome as a template, up-delta-F and up-delta-R primers amplified the upstream homologous arm of the delta site, approximately 160 bp; pSED1-F1 and pSED1-R1 primers amplified the SED1 promoter; pERG20-F1 and pERG20-R1 primers amplified the ERG20 promoter; and down-delta-F and down-delta-R primers amplified the downstream homologous arm of the delta site, approximately 250 bp. Using pESC-TAL-4CL-His and pESC-CHS-CHI-His plasmids as templates (synthesized by Hangzhou Tsingke Biotechnology Co., Ltd., the nucleotide sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:4, respectively), 4CL-F and 4CL-R, and CHS-F and CHS-R primers amplified 4CL and its ADH1 terminator, and CHS and its CYC1 terminator, respectively. The PCR products were purified or gel-recovered to obtain six DNA fragments. Overlap PCR yielded delta-integrated Donor DNA. A schematic diagram of the integration of Sc4CL and HaCHS expression cassettes into the delta site is shown below. Figure 6 .

[0097] Prepare competent cells of *Saccharomyces cerevisiae* N01, following the same procedure as gal80Δ::Pgal7-CHS-Tcyc1. Verify the correct strain and preserve it in glycerol tubes to obtain engineered *Saccharomyces cerevisiae* strain N02.

[0098] Example 2: Production of tyrosine and naringenin by shake-flask fermentation

[0099] (1) The engineered Saccharomyces cerevisiae T01, N01 and N02 obtained in Example 1 were inoculated into 20 mL of YPD liquid medium and cultured at 30 °C and 180 rpm for 15 h to obtain seed liquid.

[0100] (2) The seed liquid obtained in step (1) was transferred to 30 mL of YPD liquid medium at an inoculation rate of 2%, and fermented at 30 °C and 180 rpm for 96 h.

[0101] (3) Samples were taken at 48h, 72h, and 96h to detect the biomass, L-tyrosine, and naringenin production of each engineered strain. The results are as follows: Figure 3 , 4As shown in Figure 5. The sample for detecting extracellular naringenin production was prepared as follows: 800 μL of fermentation broth was added to an equal volume of ethyl acetate and vortexed for 30 min. The supernatant was aspirated into an EP tube, and another 400 μL of ethyl acetate was added and vortexed for 30 min. The supernatants were collected twice, and the sample was concentrated by centrifugation at 50 °C for 40 min using a vacuum concentrator. An equal volume of chromatographic grade methanol was added to dissolve the concentrated and dried precipitate, and the sample was filtered through a 0.2 μm organic filter membrane to obtain the fermentation sample to be tested.

[0102] Table 1. Strains and plasmids involved in this invention.

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[0105] Table 2 Primers used in this invention

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[0109]

Claims

1. A high-yield brewer's yeast strain of naringenin, characterized in that, The engineered bacteria used the high-tyrosine-producing Saccharomyces cerevisiae T01 strain as the host, and were overexpressed using a high-copy 2μ plasmid. RgTAL and Sc4CL The strain obtained by integrating the expression cassette B of the chalcone synthase gene and the expression cassette of the 4-coumaric acid:coenzyme A ligase gene into the delta site of the genome of the engineered bacteria was named N02; wherein, the T01 strain was... S. cerevisiae CEN.PK2-1C is a chassis bacterium, obtained through the following modification: gal80 Gene replacement with expression cassette A of the chalcone synthase gene; aro10 Gene replacement with chalcone isomerase gene expression cassette; knockout pdc5 Gene; knockout gal2 Genes; will PHA2 Gene replacement aro7 G141S Gene expression cassettes; TRP2 Gene replacement aro4 K229L The gene expression cassette; the promoter and terminator of the expression cassette B of the chalcone synthase gene are the ERG20 promoter and the CYC1 terminator, respectively; the promoter and terminator of the expression cassette of the 4-coumaric acid:coenzyme A ligase gene are the SED1 promoter and the CYC1 terminator, respectively. The promoter and terminator of the expression cassette A of the chalcone synthase gene are respectively... gal7 promoters and CYC1 The terminator, encoding the amino acid sequence shown in SEQ ID NO:8; the promoter and terminator of the expression cassette of the chalcone isomerase gene are respectively... gal1 promoters and ADH1 The terminator encodes an amino acid sequence as shown in SEQ ID NO:

9.

2. The engineered brewer's yeast strain as described in claim 1, characterized in that: The RgTAL Source Rhodotorula glutinis, The amino acid sequence encoding the tyrosine ammonia-lyase shown in SEQ ID NO:6; Sc4CL Source Streptomyces coelicolor, The amino acid sequence of the 4-coumaric acid:coenzyme A ligase is encoded as shown in SEQ ID NO:

7.

3. The engineered brewing yeast strain as described in claim 1, characterized in that: The aro7 G141S The amino acid sequence encoded by the gene expression cassette is... aro7 The amino acid sequence encoded by the gene has a glycine residue at position 141 that is mutated to a serine residue, as shown in SEQ ID NO:

10. Its promoter and terminator are respectively... PGK1 promoters and HXT7 Terminator; the aro4 K229L The amino acid sequence encoded by the gene expression cassette is... aro4 The lysine at position 229 of the amino acid sequence encoded by the gene is mutated to leucine, as shown in SEQ ID NO:

11. Its promoter and terminator are respectively... TEF1 promoters and CYC1 Termination of contract.

4. The application of the engineered brewing yeast as described in claim 1 in the fermentation production of naringenin.

5. The application as described in claim 4, characterized in that: The engineered Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 30°C and 180 rpm for 15 h to obtain a seed culture. The seed culture was then transferred to fresh YPD liquid medium at an inoculation rate of 2% and fermented at 30°C and 180-220 rpm for 48-96 h to obtain a fermentation broth containing naringenin.

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