CN115851810A - Engineering strain for de novo synthesis of naringenin by saccharomyces cerevisiae and construction method and application thereof - Google Patents

Abstract

The invention discloses an engineering strain for de novo synthesis of naringenin by saccharomyces cerevisiae, a construction method and application thereof, wherein the engineering strain is used for reconstructing and constructing a saccharomyces cerevisiae S.cerevisiae CEN.PK2-1C genome according to the following method: knocking out substrate competition pathway genes, overexpressing anti-feedback inhibition genes and the like to improve the metabolic flux of precursor substance tyrosine, heterologously expressing tyrosine ammonia lyase and 4-coumaroyl: coA-ligase, chalcone synthase and chalcone isomerase effect de novo synthesis of naringenin. Finally, the Sc4CL and HaCHS are efficiently expressed through multiple copy sites, so that the yield of naringenin is further improved.

Description

Engineering strain for de novo synthesis of naringenin by saccharomyces cerevisiae and construction method and application thereof

(I) technical field

The invention relates to a combination strategy for promoting high-efficiency expression of heterologous genes by enhancing tyrosine metabolism flux and integrating delta multicopy sites of a genome, in particular to a saccharomyces cerevisiae engineering bacterium for producing naringenin and a construction method and application thereof.

(II) background of the invention

Naringenin is a core skeleton structure of a flavonoid compound, can be used as a platform compound to derive a large amount of flavonoid substances, has various physiological activities such as antivirus, antibiosis, hypertension resistance, antioxidation and the like, and is widely applied to the industries such as food, chemical industry, medicine and the like. In view of the limitations of plant extraction and chemical synthesis, naringenin biosynthesis has become a research hotspot. With plant metabolic pathways as references, there are 2 metabolic pathways reported for the heterologous synthesis of naringenin: tyrosine is taken as a substrate, p-coumaric acid is generated under the catalysis of Tyrosine Ammonia Lyase (TAL), then p-coumaroyl-CoA is synthesized under the catalysis of 4-coumaric acid-coenzyme A ligase (4 CL), and naringenin chalcone and naringenin are respectively synthesized under the catalysis of chalcone synthase (CHS) and chalcone isomerase (CHI); the pathway for naringenin synthesis from tyrosine is the hot protocol currently being investigated. In another approach, naringenin is synthesized from phenylalanine by a 5-step enzyme catalyzed reaction, which is: phenylalanine Ammonia Lyase (PAL), cinnamic acid-4-hydroxylase (C4H), 4CL, CHS and CHI. The phenylalanine synthesis scheme involves C4H, an enzyme belonging to the P450 class, which needs to be converted into a redox form by a P450 reductase and needs to be anchored on the endoplasmic reticulum to complete a catalytic reaction, limiting the utility of this pathway in prokaryotic expression systems.

The naringenin synthesized by the microbial fermentation method is an ideal source for reducing the cost of the flavonoid compound, and simultaneously meets the concept of environmental protection of green and resources. Saccharomyces cerevisiae, as a GRAS (genetically regulated as safe) organism, has good genetic background, excellent stress resistance, excellent fermentation characteristics, stable production performance and higher safety, and has become an attractive microorganism host for producing naringenin.

Disclosure of the invention

The first purpose of the invention is to provide a saccharomyces cerevisiae engineering bacterium for producing tyrosine, which takes S.cerevisiae CEN.PK2-1C as a substrate bacterium and obtains a saccharomyces cerevisiae T01 strain with high L-tyrosine yield through metabolic modification.

The second purpose of the invention is to provide a saccharomyces cerevisiae engineering bacterium for realizing and enhancing the de novo synthesis of naringenin. The engineering bacteria take a high-yield tyrosine T01 strain as a host, and overexpress RgTAL and Sc4CL by using a high-copy 2 mu plasmid to realize the construction of a first-synthesis strain (N01) of naringenin; subsequently, by integrating the Sc4CL and HaCHS expression cassettes into the delta site (this strain was named N02), naringenin synthesis was significantly promoted without significant impact on biomass.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a recombinant expression plasmid comprising a tyrosine ammonia lyase gene (TAL) and a 4-coumaric acid-CoA ligase gene (4 CL).

In one embodiment of the invention, the vector of the recombinant expression plasmid is pESC-His, and the tyrosine ammonia lyase gene is connected with a TEF promoter to control the expression. In addition, for plasmid selection, the TEF promoter may also carry a selection gene, such as a hygromycin resistance gene.

In one embodiment of the invention, the tyrosine ammonia lyase gene encodes an amino acid sequence shown as SEQ ID NO. 6, derived from Rhodotorula gluteninis; the 4-coumaric acid-coenzyme A ligase gene codes an amino acid sequence shown as SEQ ID NO. 7 and is derived from Streptomyces coelicolor. The genes need to be optimized correspondingly by codons when inserted into host bacteria, and the common knowledge in the field is provided.

In one embodiment of the invention, the recombinant expression plasmid is designated as pESC-TAL-4CL-HygR plasmid.

In a second aspect, the invention provides a genetically engineered bacterium containing the recombinant expression plasmid, wherein a host bacterium of the genetically engineered bacterium is obtained by modifying a S.cerevisiae CEN.PK2-1C strain as follows: the gal80 gene was replaced with the expression cassette A of the chalcone synthase gene, the aro10 gene was replaced with the expression cassette of the chalcone isomerase gene, the pdc5 gene and gal2 gene were knocked out, and the PHA2 gene was replaced with aro7 ^G141S Expression cassette of Gene, TRP2 to aro4 ^k229L And (3) an expression cassette of the gene to obtain the host bacterium.

The aro7 ^G141S The amino acid sequence coded by the expression cassette of the gene is obtained by mutating glycine 141 of the amino acid sequence coded by the aro7 gene into serine, wherein aro7 ^G141S The amino acid sequence coded by the expression cassette of the gene is shown as SEQ ID NO. 10; the aro7 ^G141S The promoter and terminator of the expression cassette of the gene are the PGK1 promoter and the HXT7 terminator, respectively. The aro4 ^k229L The amino acid sequence coded by the expression cassette of the gene is obtained by mutating the 229-site lysine of the amino acid sequence coded by the expression cassette of the aro4 gene into leucine, wherein the aro4 gene is a mutant of the amino acid sequence ^k229L The amino acid sequence of the gene expression cassette code is shown as SEQ ID NO. 11, and the aro4 ^k229L The promoter and terminator of the expression cassette of the gene are the TEF1 promoter and CYC1 terminator, respectively.

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

Preferably, the genetically engineered bacterium is further modified as follows: integrating an expression cassette B of a chalcone synthase gene and an expression cassette of a 4-coumaric acid-coenzyme A ligase gene into a delta site of the genetic engineering bacteria together to obtain the genetic engineering bacteria with high naringenin yield.

Further, the transformation is completed by a CRISPR-Cas9 single plasmid gene editing system. In one embodiment of the invention, the Cas9 expression cassette (P) is engineered first _TEF -spCas9-T _ADH2 ) Integrate into IX-1 site of the genome of s.cerevisiae cen.pk2-1C strain to optimize the CRISPR-Cas9 dual plasmid gene editing system. Di-CRISPR/Cas9 (delta-integration) technology is adopted to design a delta sequence on a gRNA targeting yeast genome, and simple and efficient integration of Sc4CL and HaCHS Donor DNA in an naringenin synthesis pathway is realized at a double-strand break (DSB). However, it will be appreciated by those skilled in the art that the same or similar modifications using other methods of gene editing are also within the scope of the present invention.

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; 4-Coumaric acid-coenzyme A ligase Gene the promoter and terminator of the expression cassette are the SED1 promoter and CYC1 terminator, respectively.

In the examples of the present invention, the amino acid sequences edited by the expression cassettes A and B of the chalcone synthase gene are the same, and differ only in the promoter.

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

In a third aspect, the invention also provides application of the genetic engineering bacteria in fermentation production of naringenin.

Specifically, in one embodiment of the present invention, the application is: inoculating the genetic engineering bacteria to an YPD liquid culture medium, and culturing at 30 ℃ and 180rpm for 15h to obtain a seed solution; transferring the seed liquid to a fresh YPD liquid culture medium by the volume inoculation amount of 2%, and fermenting for 48-96h at 30 ℃ and 180-220 rpm to obtain naringenin-containing fermentation liquid.

Specifically, the construction method of the host bacterium comprises the following steps:

(1) Genome of Cerevisiae CEN. PK2-1C is used as template to amplify endogenous promoter, terminator, upstream and downstream homology arms and endogenous gene; coli DH5 α was used for plasmid amplification and preservation, etc.

(2) For facilitating gene manipulation, a genome is taken as a template, and about 500bp homologous arms of the upstream and downstream of a locus of a target site IX1 are amplified; and amplifying a Cas9 expression cassette from a p42H-spCas9 plasmid, and obtaining a complete Cas9 expression cassette Donor DNA fragment by over-lap PCR. The Donor DNA and the gRNA-IX1 plasmids are transformed by a LiAc/PEG transformation method, the integration of a Cas9 expression cassette to an IX1 site is verified by colony PCR, and then the gRNA-IX1 plasmids are subcultured by non-resistance YPD to obtain a strain C00 (CEN. PK2-1C, IX1:: P) _TEF -spCas9-T _ADH2 ) Used as hosts for subsequent DNA integration and biosynthetic pathways.

(3) Taking C00 as a basidiomycete, firstly, knocking out gal80 can relieve gal promoter galactose-dependent expression, and simultaneously integrating a HaCHS expression cassette at the site (the chalcone synthase CHS is derived from hypericum baccatum (Hyperic μm androsaem μm), knocking out aro10 and integrating with an MsCHI expression cassette to realize overexpression of CHI (chalcone isomerase CHI is derived from Medicago sativa; knocking out pdc5 reduces consumption of 4-HPP by a competitive metabolic branch), then knocking out gal2, knocking out PHA2 and overexpressing aro7 ^G141S And knock-out TRP2 and overexpress aro4 ^k229L Avoiding the carbon flux from tending to synthesize phenylalanine and tryptophan can increase the carbon flux entering shikimic acid and chorismate metabolism and relieve the feedback inhibition of tyrosine. The tyrosine yield of the T01 strain is the highest when the T01 strain is fermented for 96 hours, is 840mg/L, and is improved by about 494 percent compared with the original strain.

Specifically, the genetically engineered bacteria and the genetically engineered bacteria for high yield of naringenin in the embodiment of the invention are constructed according to the following method:

1) The tyrosine ammonia lyase TAL is derived from Rhodotorula glutinis (Rhodotorula gla) and the 4-coumaric acid coenzyme A ligase 4CL is derived from Streptomyces coelicolor. Firstly, genes RgTAL and Sc4CL are cloned to a pESC-His vector after codon optimization; subsequently, the His auxotrophic selection marker was replaced with a Hygromycin (Hygromycin) resistance tag to obtain the pESC-TAL-4CL-HygR plasmid.

2) When the pESC-TAL-4CL-HygR expression plasmid is over-expressed in a high-yield tyrosine T01 strain, a strain N01 is obtained, the tyrosine yield is 403mg/L, the p-coumaric acid yield is 40.99mg/L, and the naringenin yield is 81.45mg/L.

3) The method for integrating the Sc4CL and HaCHS expression cassettes at the delta sites comprises the following steps: and (2) respectively amplifying an upstream homology arm of the delta site, about 160bp, an SED1 promoter, an ERG20 promoter, a downstream homology arm of the delta site and about 250bp by using the S.cerevisiae CEN.PK2-1C genome as a template. pESC-TAL-4CL-His and pESC-CHS-CHI-His plasmids are taken as templates to respectively amplify 4CL and ADH1 terminator thereof and CHS and CYC1 terminator thereof. The PCR product is purified or gel recovered to obtain 6 DNA fragments. Fusing the two fragments for multiple times to obtain delta integrated Donor DNA, and integrating the delta integrated Donor DNA to the genome of the N01 strain by a LiAc/PEG conversion method to obtain a delta integrated strain named as N02. The yield of N02 tyrosine is 344.19mg/L, the yield of p-coumaric acid is 67.96mg/L, and the yield of naringenin is 157.68mg/L.

It is understood by those skilled in the art that the genome modification and the introduction of the recombinant plasmid may be performed before the introduction of the recombinant plasmid, and are within the scope of the present invention.

Compared with the prior art, the invention has the following beneficial effects: according to the invention, the Cas9 gene expression cassette is integrated into the yeast genome, the CRISPR/Cas9 dual-plasmid gene editing system is optimized, and the yeast gene editing period can be shortened. The Di-CRISPR/Cas9 (delta-integration) platform can significantly improve the synthesis of metabolites of interest as an efficient way. The saccharomyces cerevisiae engineering bacteria constructed by the invention can realize the de novo synthesis of naringenin by a microbiological method by taking glucose as a substrate, and the yield of shake flask fermentation of the N02 strain is 157.68mg/L.

(IV) description of the drawings

FIG. 1 chemical structural formula of naringenin

FIG. 2 schematic diagram of naringenin anabolism pathway modification in Saccharomyces cerevisiae

FIG. 3 schematic diagram of engineered strain biomass of Saccharomyces cerevisiae

FIG. 4 is a diagram showing tyrosine production of engineered Saccharomyces cerevisiae strains

FIG. 5 schematic diagram of naringenin yield of Saccharomyces cerevisiae engineering strain

FIG. 6 schematic representation of integration of Sc4CL and HaCHS expression cassettes into the delta site

(V) detailed description of the preferred embodiments

In order to make the objects, 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 has been described is one embodiment of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without inventive step, are within the scope of protection of the invention.

The media involved in the examples of the invention are as follows:

LB culture medium: 0.5% of yeast powder, 1% of peptone, 1% of sodium chloride and water as a solvent, wherein the pH value is natural;

YPD medium: 1% of yeast powder, 2% of peptone and 2% of glucose, wherein the solvent is water, and the pH value is natural;

1.5% agar powder was added to the solid medium.

Tyrosine and naringenin are detected by High Performance Liquid Chromatography (HPLC).

The specific conditions for the tyrosine HPLC detection are as follows: a Welchrom C18 column (250 mm. Times.4.6 mm,5 μm) was used, the mobile phase was 0.1mol/L sodium acetate solution (pH = 4.0), methanol =90 (v/v), the flow rate was 1mL/min, the detection wavelength of the UV detector was 280nm, the sample was taken 10 μ L, and the column temperature was 30 ℃.

The specific conditions of naringenin HPLC detection are as follows: a Welchrom C18 chromatographic column (250 mm. Times.4.6 mm,5 μm) was used, the mobile phase A was pure methanol (containing 0.2% acetic acid), the mobile phase B was an aqueous solution containing 0.2% acetic acid, the flow rate was 1mL/min, the mobile phase was eluted with a gradient as shown in the following Table, the detection wavelength for coumaric acid UV detector was 308nm, the naringenin detection wavelength was 288nm, the sample injection amount was 10 μ L, and the column temperature was 30 ℃.

Example 1

1. Construction of engineering bacterium T01 for producing tyrosine saccharomyces cerevisiae

First, the Cas9 expression cassette (P) _TEF -spCas9-T _ADH2 ) Integrate into the s.cerevisiae cen.pk2-1C genome IX-1 site to optimize the CRISPR-Cas9 dual plasmid gene editing system; subsequently, gal80 is replaced with P _gal7 -CHS-T _cyc1 Relieving galactose-dependent expression of the gal-type promoter and overexpressing CHS; next, aro10 was replaced with P _gal1 -CHI-T _ADH1 (ii) a Subsequently, pdc5 and gal2 were sequentially knocked out, and finally PHA2 was replaced with P _PGK1 -aro7 ^G141S -T _HXT7 Replacement of TRP2 by P _TEF1 -aro4 ^k229L -T _cyc1 So as to realize the over-expression of tyrosine anti-feedback inhibition genes and weaken the metabolic flux of phenylalanine and tryptophan competing branches to obtain the T01 strain.

2. Integrating a Cas9 expression cassette into S.cerevisiae CEN.PK2-1C genome

A gRNA-IX1 plasmid was constructed (a gRNA plasmid required for subsequent gene editing was constructed by way of example).

Firstly, plasmid pKan100-LEU2-gRNA (CN 112322512A) is used as a template for reverse amplification (primers are gRNA-F and gRNA-R), and single enzyme digestion is carried out for 2.5h by Dpn I, and transformation is carried out to E.coli DH5 alpha; after the Amp plate is cultured overnight, selecting a single colony, inoculating LB containing Amp (the working concentration of the ampicillin solution is 100 mg/mL) to culture for 12-15h, upgrading the plasmid, verifying whether the LEU2 label is knocked out by sequencing with a carrier universal primer M13R, and obtaining the pKan100 plasmid after successful knocking out. Then, taking a pKan100 plasmid as a template, carrying out reverse amplification by designing primers (gRNA-IX 1-F and gRNA-IX 1-R) of the first 20bp of the PAM site of the IX1 target site to obtain a pKan100-IX1 DNA fragment, constructing the rest steps by referring to the pKan100 plasmid, extracting the plasmid, and carrying out sequencing by using a vector universal primer M13R to verify whether the mutation of the first 20bp (namely, N20) of the IX1 target site is successful; the gRNA-IX1 plasmid is obtained after N20 mutation.

The method comprises the steps of amplifying spCas9 (primers are: spCAS9-p42H-F and spCAS9-p 42H-R) by taking plasmid pH-spCas9 (CN 112322512A) as a template, reversely amplifying by taking plasmid pRS42H-iCas9 (CN 112322512A) as a template (primers are: p42H-spCAS9-F and p42H-spCAS 9-R), obtaining complete recombinant linearized plasmid fragments (primers are: spCAS9-p42H-F and p42H-spCAS9-R; the usage amount of the two DNA fragments is 1 molar ratio. Then, using S.cerevisiae CEN.PK2-1C genome as template to amplify homologous arms (primer: UP IX1-F, UP IX1-R, DOWN IX1-F and DOWN IX 1-R) of about 500bp respectively at the upstream and downstream of the target site IX1 locus; the Cas9 expression cassette (primers: spCas9-F and SpCas 9-R) was amplified from the p42H-spCas9 plasmid, and the complete Donor DNA fragment was obtained by over-lap PCR (primers: UP IX1-F and DOWN IX1-R; three DNA fragments were used in a molar ratio of 1. First, plasmid p42H-spCas9 was transferred to s.cerevisiae cen.pk2-1C; subsequently, donor DNA with Cas9 expression cassette and gRNA-IX1 plasmid were co-transformed to s.cerevisiae cen.pk2-1C. The strain C00 (CEN. PK2-1C, IX1:: P) was obtained by colony PCR (primers: IX1-CHECK-F1 and IX 1-CHECK-R1) and sequencing verification _TEF -spCas9-T _ADH2 ) And are used as hosts for subsequent DNA integration and biosynthetic pathways.

3.CRISPR/Cas9 gene replacement operation, taking gal80 delta as an example, pgal7-CHS-Tcyc1

1) Constructing gRNA-gal80 plasmid, and operating the same as the construction of gRNA-IX1 plasmid, wherein the difference is only that the sequences of 20bp in front of a PAM locus of target gene gal80 are different, namely constructing an amplification primer as follows: gRNA-gal80-F and gRNA-gal80-R.

2) Preparation of Donor DNA

Amplification of upstream and downstream homology arms (50 μ L system): 1 uL of S.cerevisiae CEN.PK2-1C genome is used as a template (the concentration of the genome used in the present specification is 100 ng/. Mu.L), 25 uL of 2 x Phanta Max Master Mix (Vazyme, nanjing) high fidelity DNA polymerase, and primers UP gal80-F and UP gal80-R (upstream homology arm introduction)Substance) or DOWNgal80-F and DOWNgal80-R (downstream homology arm primers) each 1. Mu.L, ddH ₂ O make up to 50. Mu.L. The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 8min, and then entering temperature circulation at 98 ℃ for 10sec;58 ℃,15sec;72 ℃ for 30sec; for a total of 35 cycles, the end temperature was 16 ℃. After the PCR amplification is finished, the correct PCR product is verified by 1% agarose gel electrophoresis, and the PCR product is utilized

The upstream or downstream homology arm fragment was obtained by purifying the PCR Purification Kit (TransGen, beijing). />

Amplifying the CHS expression cassette (Pgal 7-CHS-Tcyc 1): mu.L of pESC-CHS + CHI-His plasmid (synthesized by Hangzhou tsingke Biometrics Ltd., nucleotide sequence of the synthesized plasmid is shown as SEQ ID NO: 1) with concentration of 100 ng/mu.L as a template, 25 mu.L of 2X Phanta Max Master Mix (Vazyme, nanjing) high fidelity DNA polymerase, 1 mu.L each of primers Pgal7-CHS-T-F and Pgal7-CHS-T-R, ddH ₂ O make up to 50. Mu.L. The PCR reaction conditions are as follows: performing pre-denaturation at 98 ℃ for 5min, and then performing temperature circulation at 98 ℃ for 10sec;58 ℃,15sec;72 ℃ for 1min; for a total of 35 cycles, the end temperature was 16 ℃. After amplification, the correct PCR product was verified by 1% agarose gel electrophoresis using

The CHS expression cassette was obtained by purifying the PCR Purification Kit.

Donor DNA was obtained by Overlap PCR. Using the DNA fragments of the gal80 upstream homology arm, CHS expression cassette and gal80 downstream homology arm as templates (added at a molar ratio of fragments of 1:3 according to the recovery concentration of each fragment), 2 XPphanta Max Master Mix (Vazyme, nanjing) high fidelity DNA polymerase 25. Mu.L, primers UP gal80-F and DOWNgal80-R each 1. Mu.L, ddH ₂ O make up to 50. Mu.L. The PCR reaction conditions are as follows: performing pre-denaturation at 98 ℃ for 5min, and then performing temperature circulation at 98 ℃ for 10sec;58 ℃,15sec;72 ℃ for 1min; for a total of 35 cycles, the end temperature was 16 ℃. After amplification is finished, the PCR product obtained by purification or gel recovery is sequenced correctly to obtain the Donor DNA.

3) Cerevisiae C00 transformation competent preparation (Saccharomyces cerevisiae competent preparation for subsequent Gene editing is exemplified here)

(1) Taking out the preserved strain C00, streaking on YPD solid plate, and culturing in 30 deg.C incubator for 2-3 days.

(2) Fresh single colonies were picked and inoculated into 5mL YPD liquid medium, and cultured at 30 ℃ for 15-20h with shaking at 200 rpm.

(3) The bacterial liquid is inoculated into a fresh 20mL YPD culture medium according to the inoculation amount of 2 percent, and the YPD culture medium is subjected to shaking culture at 30 ℃ and 200rpm until the OD600=0.5-0.7.

(4) 20mL of the bacterial solution was transferred to a 50mL sterilized centrifuge tube, centrifuged at 4000rpm and 4 ℃ for 5min, and the cells were collected.

(5) Adding 900 μ L of pre-cooled sterile water and 100 μ L of 10 XLiAc solution into the above 50mL centrifuge tube by using a pipette gun, gently resuspending the cells, transferring the bacterial solution into a 2mL sterilized EP tube, centrifuging at 4000rpm and 4 ℃ for 1min, and collecting the cells.

(6) The cells were resuspended in 900. Mu.L of precooled sterile water and 100. Mu.L of 10 XLiAc solution, and centrifuged. The above steps were repeated three times.

(7) Finally, 900 μ L of precooled sterile water and 100 μ L of 10 XLiAc solution are added into an EP tube to resuspend the thalli, the thalli is kept stand for 5min,4000rpm and 4 ℃, the centrifugation is carried out for 1min, 900 μ L of supernatant is discarded, and the residual supernatant is used for resuspending cells, thus obtaining the C00 competence.

4) PEG/LiAc Saccharomyces cerevisiae transformation (for example in the case of subsequent Gene editing)

(1) The transformation system was added to a sterile EP tube. The transformation system is shown in the following table:

(2) And adding 25 mu L of prepared competent cells into the transformation system, and uniformly mixing for 10s by using a vortex oscillator. The EP tube was placed in a 30 ℃ incubator for 30min.

(3) Add 36. Mu.L of dimethyl sulfoxide and mix well on a vortex shaker for 10s. Heat shock was performed in a water bath at 42 ℃ for 15min.

(4) The cells were collected by centrifugation at 4000rpm at 4 ℃ for 1 min. The supernatant was discarded, and 400. Mu.L of 5mM CaCl was added ₂ The solution was resuspended in cells and left for 15min.

(5) Cells were collected by centrifugation. The supernatant was discarded, 1mL of sterilized YPD medium was added, the cells were gently resuspended, and cultured at 30 ℃ for 1 hour with shaking at 200 rpm.

(6) Cells were collected by centrifugation. The supernatant was discarded, and 1mL of sterile water was added to clean the cells, followed by centrifugation.

(7) 700-800. Mu.L of YPD medium was discarded, and the cells were gently resuspended in the remaining medium and plated onto YPD + G418 (200 mg/L final concentration of G418 was used herein) solid plates. Culturing in 30 deg.C constant temperature incubator for 2-3 days.

5) Saccharomyces cerevisiae transformant identification (colony PCR for example in subsequent Gene editing)

MightyPrep reagent for DNA (Takara, dalian) was used for cell lysis of s.cerevisiae to obtain a template for colony PCR. Using 1. Mu.L of supernatant cell lysate as a template, 25. Mu.L of Green Taq Mix (Vazyme, nanjing), 1. Mu.L each of primers gal80-CHECK-F1 and gal80-CHECK-R1, ddH ₂ O make up to 50. Mu.L. The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 8min, and then entering temperature circulation at 98 ℃ for 15sec;58 ℃,15sec;72 ℃ for 30sec; for a total of 35 cycles, the end temperature was 16 ℃. And finishing gene editing after the PCR product is sequenced correctly.

6) gRNA-gal80 plasmid elimination (gRNA plasmid elimination during subsequent gene editing for example)

(1) Inoculating the transformant with the correct genotype into 3mL YPD for 12h; (2) taking 100 mu L of the mixture, transferring the mixture to new 3mL of YPD, and continuously carrying out passage for 3 times; (3) Drawing 5 μ L to line on YPD solid plate, and culturing for 2-3 days; (4) Picking the same single colony on a non-resistance YPD solid plate and a resistance YPD + G418 plate; if no resistance YPD solid plate can grow single colony, G418 plate can not grow single colony, and engineering bacteria with lost gRNA-gal80 plasmid can be obtained.

7) The subsequent gene editing refers to the gal80 delta & lt: & gt Pgal7-CHS-Tcyc1 operation flow if the gene editing is genome replacement.

Operation of CRISPR/Cas9 Gene knockout, exemplified by pdc5 Gene

The gene knockout is different from the substitution in that only the upstream and downstream homologous arm fragments are directly fused in the construction step of the Donor DNA.

Amplification of upstream and downstream homology arms: 1 muL of saccharomyces cerevisiae CEN. PK2-1C genome with the concentration of 100 ng/muL is taken as a template, 25 muL of 2 x Phanta Max Master Mix high-fidelity DNA polymerase, 1 muL of each of primers UPPdc5-F and UPPdc5-R or DOWNPdc5-F and DOWNPdc5-R, ddH ₂ O make up to 50. Mu.L. The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 8min, and then entering temperature circulation at 98 ℃ for 10sec;58 ℃,15sec;72 ℃,15sec; for a total of 35 cycles, the end temperature was 16 ℃. After the PCR amplification is finished, the correct PCR product is verified by 1% agarose gel electrophoresis, and the PCR product is utilized

The PCR Purification Kit was purified to obtain the upstream or downstream homology arm fragments.

And performing Overlap PCR to obtain Donor DNA. Using the fragments recovered from the pdc5 upstream homology arm and pdc5 downstream homology arm as templates (molar ratio of fragments: 1), 25. Mu.L of 2X Phanta Max Master Mix high fidelity DNA polymerase, 1. Mu.L each of primers UPPdc5-F and DOWNPdc5-R, ddH ₂ O make up to 50. Mu.L. After amplification is finished, the PCR product obtained by purification or gel recovery is sequenced correctly to obtain the Donor DNA.

The remaining steps refer to gal 80. Delta. Pgal7-CHS-Tcyc1 gene replacement.

5.CRISPR/Cas9 anti-feedback inhibition point mutation operation with PHA2 delta: P _PGK1 -aro7 ^G141S -T _HXT7 As an example

The release of aro7 anti-feedback inhibition and overexpression belong to gene replacement, and compared with the replacement described in the invention, the difference is that point mutation needs to be carried out on key sites of the aro7 gene to achieve the aim of anti-feedback inhibition.

Glycine 141 of the aro7 encoded protein was mutated to serine. First, aro7 ^G141S The expression cassette is constructed in a full plasmid amplification manner, i.e.primer P _PGK1 -aro7 ^m -T _HXT7 -F and P _PGK1 -aro7 ^m -T _HXT7 -R is p-aro7 ^m Plasmid (stored in laboratory, nucleotide sequence of the plasmid is shown as SEQ ID NO: 2) is used as a template for reverse amplification to obtain P _PGK1 -aro7 ^m -T _HXT7 A fragment; second, it is used forAmplifying the upstream and downstream homologous arms by the method (the primers of the upstream homologous arms for amplification are UP PHA2-F and UP PHA2-R, and the primers of the downstream homologous arms for amplification are Down PHA2-F and Down PHA 2-R); finally, the above three fragments were fused by Overlap PCR using the primers UP PHA2-F and Down PHA2-R to obtain Donor DNA.

The rest steps refer to gal80 delta, pgal7-CHS-Tcyc1 operation flow. TRP 2. Delta. P _TEF1 -aro4 ^k229L -T _cyc1 Reference PHA2 Δ:: P _PGK1 -aro7 ^G141S -T _HXT7 . Wherein p-aro4 ^m The plasmid is preserved in the laboratory, and the nucleotide sequence of the plasmid is shown as SEQ ID NO. 3)

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

RgTAL and Sc4CL genes are subjected to saccharomyces cerevisiae codon optimization by Hangzhou tsingke biology Limited, and multiple cloning sites are as follows: sac I \ Xho I, synthesized on pESC-His vector, and the nucleotide sequence of pESC-TAL-4CL-His plasmid is shown as SEQ ID NO:4.

pESC-TAL-4CL-His plasmid is taken as a template, and pESC-TAL-4CL (His 3 expression frame is removed) vector skeleton is obtained by reverse amplification; using P-gRNA-delta plasmid (provided by Zhe Gong Huloy teacher's laboratory, nucleotide sequence of the plasmid is shown as SEQ ID NO: 5) as template, and amplifying primers HygR-F and HygR to obtain expression frame (P) of hygromycin resistance gene _TEF -HygR). The expression cassettes pESC-TAL +4CL and HygR were cloned in One Step by using Cloneexpress II One Step Cloning Kit and then transformed into E.coli DH5 alpha. Colony PCR (primers are + HygR-CHECK-F1 and + HygR-CHECK-R1) and sequencing results show that the pESC-TAL-4CL-HygR plasmid is successfully constructed.

7. Construction of Saccharomyces cerevisiae N01

Firstly, preparing T01 competence; next, the pESC-TAL-4CL-HygR plasmid was transferred into T01 competent cells by LiAc/PEG transformation, spread on YPD + Hyg (Hyg working concentration 200 mg/L) plates, and cultured at 30 ℃ for 2-3 days until transformants grew out. And finally, performing colony PCR verification by using the primers + HygR-CHECK-F1 and + HygR-CHECK-R1, and performing glycerin tube preservation on the strains with correct verification to obtain the saccharomyces cerevisiae engineering bacteria N01.

8. Construction of Saccharomyces cerevisiae N02

Construction of Donor DNA: using S.cerevisiae CEN.PK2-1C genome as template, amplifying upstream homology arm of delta site with up-delta-F and up-delta-R primers, about 160bp; the SED1 promoter is amplified by pSED1-F1 and pSED1-R1 primers; the ERG20 promoter is amplified by the pERG20-F1 and pERG20-R1 primers; the down-delta-F and down-delta-R primers amplify the homology arms downstream of the delta site, about 250bp. pESC-TAL-4CL-His, pESC-CHS-CHI-His plasmids are used as templates (synthesized by Hangzhou tsingke biology, inc., the nucleotide sequences of the plasmids are respectively shown in SEQ ID NO:1 and SEQ ID NO: 4), 4CL-F and 4CL-R, CHS-F and CHS-R primers respectively amplify 4CL and ADH1 terminator thereof, CHS and CYC1 terminator thereof. The PCR product is purified or recovered by gel to obtain 6 DNA fragments. Overlap PCR resulted in delta-integrated Donor DNA. A schematic of the integration of the Sc4CL and HaCHS expression cassettes into the delta site is shown in FIG. 6.

Preparing saccharomyces cerevisiae N01 competence, and referring to gal80 delta for the rest steps, namely an operation flow of Pgal7-CHS-Tcyc 1. And (5) verifying that the correct strain is preserved in glycerin tube to obtain the saccharomyces cerevisiae engineering bacteria N02.

Example 2: shake flask fermentation for producing tyrosine and naringenin

(1) The engineered Saccharomyces cerevisiae strains T01, N01 and N02 obtained in example 1 were inoculated into 20mL of YPD liquid medium, and cultured at 30 ℃ and 180rpm for 15 hours to obtain a seed solution.

(2) Inoculating the seed solution obtained in the step (1) into 30mL YPD liquid culture medium with the inoculation amount of 2%, and fermenting at 30 ℃ and 180rpm for 96h.

(3) Samples were taken at 48h, 72h and 96h to test the biomass of each engineering bacterium, the yield of L-tyrosine and naringenin, and the results are shown in the following figures 3, 4 and 5. The detection sample for the yield of extracellular naringenin is prepared by the following method: adding 800 mu L of fermentation liquor into equal volume of ethyl acetate, performing vortex oscillation for 30min, sucking supernatant into an EP tube, adding 400 mu L of ethyl acetate again, performing vortex oscillation for 30min, collecting supernatant twice, performing centrifugal concentration on the sample for 40min at 50 ℃ by using a vacuum concentrator, adding equal volume of chromatographic grade methanol to dissolve and concentrate the dried precipitate, and filtering by using a 0.2 mu m organic system filter membrane to obtain a fermentation sample to be detected.

TABLE 1 strains and plasmids according to the invention

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TABLE 2 primers used in the present invention

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Claims (10)

1. A recombinant expression plasmid characterized by: the recombinant expression plasmid contains tyrosine ammonia lyase gene and 4-coumaric acid-coenzyme A ligase gene.

2. The recombinant expression plasmid of claim 1, wherein: the vector of the recombinant expression plasmid is pESC-His, and the tyrosine ammonia lyase gene is connected with a TEF promoter to control expression.

3. The recombinant expression plasmid of claim 1, wherein: the tyrosine ammonia lyase gene codes an amino acid sequence shown as SEQ ID NO. 8; the 4-coumaric acid-coenzyme A ligase gene code is shown as an amino acid sequence in SEQ ID NO. 9.

4. Including the following claimsThe genetically engineered bacterium of a recombinant expression plasmid according to claim 1, characterized in that: the host strain of the genetic engineering strain is obtained by modifying an S.cerevisiae CEN.PK2-1C strain as follows: the gal80 gene was replaced with the expression cassette A of the chalcone synthase gene, the aro10 gene was replaced with the expression cassette of the chalcone isomerase gene, the pdc5 gene and gal2 gene were knocked out, and the PHA2 gene was replaced with aro7 ^G141S Expression cassette of Gene, TRP2 to aro4 ^k229L And (3) an expression cassette of the gene to obtain the host bacterium.

5. The genetically engineered bacterium of claim 4, wherein: the aro7 ^G141S The amino acid sequence coded by the expression cassette of the gene is obtained by mutating glycine 141 of the amino acid sequence coded by the aro7 gene into serine, wherein the aro7 gene is mutated into serine ^G141S The amino acid sequence coded by the expression cassette of the gene is shown as SEQ ID NO. 12; the aro7 ^G141S The promoter and terminator of the expression cassette of the gene are the PGK1 promoter and the HXT7 terminator, respectively; the aro4 ^k229L The amino acid sequence coded by the expression cassette of the gene is obtained by mutating the 229-site lysine of the amino acid sequence coded by the expression cassette of the aro4 gene into leucine, wherein the aro4 gene is a mutant of the amino acid sequence ^k229L The amino acid sequence coded by the expression cassette of the gene is shown as SEQ ID NO. 13; the aro4 ^k229L The promoter and terminator of the expression cassette of the gene are the TEF1 promoter and CYC1 terminator, respectively.

6. The genetically engineered bacterium of claim 4, wherein: the promoter and the terminator of the expression cassette A of the chalcone synthase gene are a gal7 promoter and a CYC1 terminator respectively, and the amino acid sequence edited by the expression cassette A of the chalcone synthase gene is shown in SEQ ID NO. 10; the promoter and the terminator of the expression cassette of the chalcone isomerase gene are a gal1 promoter and an ADH1 terminator respectively, and the amino acid sequence edited by the expression cassette of the chalcone isomerase gene is shown in SEQ ID NO. 11.

7. The genetically engineered bacterium of claim 4, wherein: the genetic engineering bacteria are further modified as follows: integrating an expression box B of a chalcone synthase gene and an expression box of a 4-coumaric acid-coenzyme A ligase gene to a delta site of the genetic engineering bacteria together to obtain the genetic engineering bacteria with high yield of naringenin.

8. The genetically engineered bacterium of claim 4, wherein: the promoter and terminator of the expression cassette B of the chalcone synthase gene are ERG20 promoter and CYC1 terminator respectively; 4-Coumaric acid-coenzyme A ligase Gene the promoter and terminator of the expression cassette are the SED1 promoter and CYC1 terminator, respectively.

9. The use of the genetically engineered bacterium of claim 4 in the fermentative production of naringenin.

10. The use according to claim 9, characterized in that the use is: inoculating the genetic engineering bacteria to an YPD liquid culture medium, and culturing at 30 ℃ and 180rpm for 15h to obtain a seed solution; transferring the seed liquid to a fresh YPD liquid culture medium according to the volume inoculation amount of 2%, and fermenting at 30 ℃ and 180-220 rpm for 48-96h to obtain fermentation liquid containing naringenin.

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