Saccharomyces cerevisiae recombinant strain with high gene copy integration and high geraniol yield as well as construction method and application of saccharomyces cerevisiae recombinant strain

Abstract

The invention relates to the technical field of synthetic biology, in particular to a saccharomyces cerevisiae recombinant strain with high gene copy integration and high geraniol yield as well as a construction method and application of the saccharomyces cerevisiae recombinant strain. According to the method, the copy number of geraniol synthase genes is increased through a saccharomyces cerevisiae gene multi-copy integration technology, efficient and stable expression of the geraniol synthase genes is achieved, supply of geraniol precursors is improved by further combining the modes of blocking a precursor competitive pathway and the like, a saccharomyces cerevisiae recombinant strain capable of efficiently and stably synthesizing geraniol is constructed, and the method is suitable for large-scale production of geraniol. Restriction factors of synthesis of geraniol by saccharomyces cerevisiae are removed, and the recombinant strain has high genetic stability and good environmental protection property. The recombinant strain constructed by the invention can be used for efficiently synthesizing geraniol and derivatives of geraniol, and is applied to essence, fragrance and food industries.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of synthetic biology, and particularly relates to a Saccharomyces cerevisiae recombinant strain with high copy number of genes for high yield of geraniol and a construction method and application thereof. BACKGROUND

[0002] Geraniol, also known as citronellol, is an acyclic monoterpene alcohol, which exists in many plants such as roses, geraniums and lemongrass, and can be used for blending edible flavors, daily perfumes and the like, and is widely used in the perfume, cosmetic, pharmaceutical and food industries. At present, the production of geraniol mainly relies on plant extraction or chemical synthesis, which has problems such as high cost, low yield and serious environmental pollution. Synthetic biology can realize new functions by artificially designing, modifying and constructing new biological systems. With the help of synthetic biology technology, the production of target products has many advantages such as high efficiency, economy and environmental friendliness, which provides a new idea for the green and efficient production of natural products.

[0003] Saccharomyces cerevisiae itself cannot synthesize geraniol, and it has been reported that a recombinant Saccharomyces cerevisiae strain can synthesize geraniol, but the yield is low. The existence of competitive pathways of geraniol precursors in Saccharomyces cerevisiae and the low catalytic efficiency of geraniol synthase are the bottleneck problems leading to insufficient yield of geraniol. The plasmid expression system has problems such as easy loss and increase of metabolic burden of the host, and single copy gene integration often has insufficient expression, which is difficult to realize industrialization.

[0004] Patent application CN117624378A discloses a fusion protein, an expression vector, a construction method and application, specifically discloses cloning of Saccharomyces cerevisiae Erg20 gene; preparation of Erg20 F96W-N127W variant gene; synthesis of Diandongchun geraniol synthase gene; construction of GES-Erg20 F96W-N127W fusion protein; construction of geraniol synthase and geranyl pyrophosphate synthase GES-Erg20 F96W-N127W fusion expression vector, introduces GPP metabolic flow into the geraniol synthesis pathway, and improves the yield of geraniol biosynthesis, which can solve the problem of low geranyl pyrophosphate (GPP) level in Saccharomyces cerevisiae engineering bacteria for biosynthesis of monoterpenoids such as geraniol, resulting in low yield of geraniol. However, the strain still has problems such as insufficient modification of competitive pathways of geraniol precursors and insufficient expression of geraniol synthase gene with only one copy. SUMMARY

[0005] The present application provides a Saccharomyces cerevisiae recombinant strain with high copy number of genes for high yield of geraniol and a construction method and application thereof.

[0006] Specifically, the present application provides the technical solutions described below.

[0007] In a first aspect, the present application provides a recombinant Saccharomyces cerevisiae strain for geraniol production, wherein a plurality of copies of a geraniol synthase gene is integrated into the genome of the recombinant Saccharomyces cerevisiae strain; wherein the geraniol synthase is derived from Catharanthus roseus Catharanthus roseus or is a signal peptide-truncated variant thereof; the integration site of the geraniol synthase gene is a delta site of the Saccharomyces cerevisiae genome.

[0008] The integration efficiency and expression level of the integrated gene vary greatly at different integration sites, and the selection of the integration site is a key factor affecting the efficient and stable integration of the gene into the genome and the high-level expression. The delta sequence in the Saccharomyces cerevisiae genome is a high-copy repeat sequence. In the present application, the integration site of the plurality of copies of the geraniol synthase gene derived from Catharanthus roseus Catharanthus roseus is screened, and it is found that the delta sequence in the Saccharomyces cerevisiae genome can be used as an integration target to achieve the stable integration of more than 10 copies of the geraniol synthase gene per cell, thereby achieving the high-level expression of the geraniol synthase gene, promoting the synthesis of geraniol, and improving the genetic stability of the strain.

[0009] For the geraniol synthase, the present application preferably uses a signal peptide-truncated variant of the geraniol synthase derived from Catharanthus roseus Catharanthus roseus which lacks a signal peptide targeting chloroplast.

[0010] Preferably, the amino acid sequence of the geraniol synthase is shown in SEQ ID NO. 1.

[0011] Preferably, the geraniol synthase gene is a codon-optimized gene for the Saccharomyces cerevisiae host.

[0012] In some embodiments of the present application, the nucleotide sequence of the geraniol synthase gene is shown in SEQ ID NO. 2.

[0013] In the recombinant Saccharomyces cerevisiae strain described in the present application, the copy number of the geraniol synthase gene is not less than 5 copies per cell. Preferably, it is not less than 10 copies per cell.

[0014] In some embodiments of the present application, the copy number of the geraniol synthase gene is 5-20; preferably, 10-20; more preferably, 14-18.

[0015] To strengthen the upstream mevalonate (MVA) pathway to improve the supply of the geraniol precursor GPP, the MVA pathway is optimized in the present application.

[0016] Specifically, the recombinant Saccharomyces cerevisiae strain described above overexpresses a truncated HMG-CoA reductase gene tHMG1 and an isopentenyl diphosphate isomerase geneIDI1 .

[0017] Preferably, the truncated HMG-CoA reductase gene is overexpressed by replacing its promoter with a promoter with higher transcriptional activity. tHMG1 tHMG1 The expression cassette of the gene comprises a promoter P PGK1 , tHMG1 the gene and a terminator T CYC1 . Wherein, the P PGK1 , T CYC1 and HMG-CoA reductase are all endogenous sequences of Saccharomyces cerevisiae.

[0018] Preferably, the prenyl diphosphate isomerase gene is overexpressed by replacing its promoter with a promoter with higher transcriptional activity. IDI1 The promoter is preferably P ENO2 .

[0019] Preferably, the amino acid sequence of the truncated HMG-CoA reductase is as shown in SEQ ID NO. 3.

[0020] Preferably, the amino acid sequence of the prenyl diphosphate isomerase is as shown in SEQ ID NO. 4.

[0021] The recombinant Saccharomyces cerevisiae strain described above also expresses geranyl pyrophosphate synthase ERG20 F96W,N127W mutant.

[0022] Preferably, the endogenous ERG20 of Saccharomyces cerevisiae is mutated to express the ERG20 F96W,N127W mutant.

[0023] Preferably, the amino acid sequence of the ERG20 F96W,N127W mutant is as shown in SEQ ID NO. 5.

[0024] The recombinant Saccharomyces cerevisiae strain described above has reduced or inactivated activity of geraniol reductase OYE2.

[0025] Reducing or inactivating the activity of geraniol reductase OYE2 can eliminate the competitive pathway and prevent the degradation of geraniol.

[0026] Preferably, the geraniol reductase OYE2 of the recombinant Saccharomyces cerevisiae strain is inactivated.

[0027] Preferably, the geraniol reductase OYE2 is inactivated by knocking out the geraniol reductase gene OYE2 .

[0028] Preferably, the amino acid sequence of the geraniol reductase OYE2 is as shown in SEQ ID NO. 6.​

[0029] Secondly, the present invention provides a method for constructing the above-described geraniol-producing recombinant Saccharomyces cerevisiae strain, the method comprising: integrating the geraniol synthase gene into the delta site of the Saccharomyces cerevisiae genome using a CRISPR / Cas9-mediated homologous recombination system.

[0030] Preferably, the method further includes: introducing a yeast strain containing... tHMG1 The nucleic acid molecules of the gene expression cassette enable the... tHMG1 The gene expression cassette is integrated into the genome. The integration site is preferably... YJL064W .

[0031] Preferably, the method further includes: transferring the isopentenyl diphosphate isomerase gene. IDI1 The promoter is replaced with a promoter that has higher transcriptional activity. Preferably, the promoter is P. ENO2 .

[0032] Preferably, the method further includes: using endogenous brewer's yeast ERG20 Mutation to ERG20 F96W,N127W The gene encoding the mutant.

[0033] The above genetic modification is preferably performed using a CRISPR / Cas9-mediated homologous recombination system.

[0034] Thirdly, the present invention provides the application of the above-described recombinant brewer's yeast strain that produces geraniol in the fermentation production of geraniol.

[0035] Fourthly, the present invention provides the application of the above-described geraniol-producing recombinant Saccharomyces cerevisiae strain in constructing a strain for fermentation production of geraniol.

[0036] Preferably, in the above applications, the recombinant Saccharomyces cerevisiae strain producing geraniol is used as the starting strain to construct a strain for fermentation production of geraniol. The construction method includes, but is not limited to, genetic engineering techniques.

[0037] Fifthly, the present invention provides a method for producing geraniol, the method comprising: culturing the above-described recombinant Saccharomyces cerevisiae strain that produces geraniol, and collecting geraniol from the culture medium.

[0038] Preferably, the culture temperature is 28-32℃.

[0039] The beneficial effects of this invention include at least the following: This invention increases the copy number of the geraniol synthase gene through multi-copy integration technology in *Saccharomyces cerevisiae*, achieving efficient and stable expression of the geraniol synthase gene. Furthermore, by combining this with methods such as blocking competitive pathways for precursors, the supply of geraniol precursors is increased, constructing a recombinant *Saccharomyces cerevisiae* strain capable of efficiently and stably synthesizing geraniol (yield reaching 776.86 mg / L after 5 days of shake-flask fermentation). This removes the limiting factors for geraniol synthesis in *Saccharomyces cerevisiae*, and the recombinant strain exhibits high genetic stability (geraniol yield remains >95% after 100 generations of continuous subculturing, eliminating the need for antibiotic selection). Simultaneously, this strain has good environmental friendliness (avoiding plasmid use and reducing the risk of environmental release of resistance genes). The recombinant strain constructed in this invention can be used for the efficient synthesis of geraniol and its derivatives, for use in the flavoring, fragrance, and food industries. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 This is a schematic diagram of gene multicopy integration in Saccharomyces cerevisiae in Example 6 of the present invention.

[0042] Figure 2 The following are gas chromatographic analysis results of the fermentation products of the recombinant strain in Example 7 of this invention. (A) Detection results of geraniol yield of the recombinant strain; (B) Standard curve plotted by gas chromatographic detection of geraniol standard; (C) Peak diagram of gas chromatographic detection of fermentation products of the recombinant strain.

[0043] Figure 3 For the determination of recombinant strains in Example 8 of the present invention tCrGES Gene copy number results.

[0044] Figure 4 This is a graph showing the genetic stability test results of the high-yielding geraniol recombinant strain in Example 9 of the present invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0046] Example 1: Overexpression in CEN.PK2-1D tHMG1 Gene Through CEN.PK2-1D YJL064W Site integration P PGK1 - tHMG1 -T CYC1 Expression box, overexpression tHMG1 Genes, the specific methods are as follows.

[0047] 1. Extraction of Saccharomyces cerevisiae genome CEN.PK2-1D glycerol tube strain stored at -80℃ was streaked onto SD agar plates and incubated at 30℃ for 48 h. Colonies of CEN.PK2-1D from the plates were picked and inoculated into 5 mL of liquid SD medium, and incubated overnight at 30℃ with a shaking incubator at 220 rpm. The cells were collected by centrifugation at 5000 rpm, and the genome was extracted (using the Beijing Coolplay Technology Co., Ltd. Fungal Genomic DNA Rapid Extraction Kit, catalog number: DE241, yeast genome extraction was performed according to the manufacturer's instructions).

[0048] 2. PCR amplification Using the CEN.PK2-1D genome as a template, the promoter P was amplified by PCR using the primers in Table 1. PGK1 Shorten genes tHMG1 Termination of sub-T CYC1 The nucleic acid fragments were recovered by gel extraction. The amplification system was as follows: 10 μL of 2 × Phanta Max Master Mix (Dye Plus) (Novizan Biotechnology Co., Ltd., catalog number: P525), 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 1 μL of template DNA, and 8 μL of ddH2O. The amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 56~72℃ annealing for 15 sec, 72℃ extension for 30 sec / kb (30 cycles); 72℃ extension for 5 min. Adjacent nucleic acid fragments contained 20 bp homologous arms. Phanta Max was obtained using fusion PCR technology. PGK1 - tHMG1 -T CYC1 Fragments with brewer's yeast at both ends YJL064WHomologous arms at the locus. The amplification systems are as follows: Step one system: 2 × Phanta Max Master Mix (DyePlus) 5 μL, fragment 1 0.5 μL, fragment 2 1 μL, fragment 3 0.5 μL, ddH2O 3 μL; Step two system: 2 × Phanta Max Master Mix (Dye Plus) 10 μL, upstream primer (10 μM) 0.5 μL, downstream primer (10 μM) 0.5 μL, step one system 1 μL, ddH2O 8 μL. The amplification program was as follows: Step one system: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 58℃ annealing for 15 sec, 72℃ extension for 30 sec / kb (15 cycles); 72℃ extension for 10 min; Step two system: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 56~72℃ annealing for 15 sec, 72℃ extension for 30 sec / kb (35 cycles); 72℃ extension for 10 min. The amplified product was recovered by gel cutting.

[0049] 3. p426- YJL064W -gRNA plasmid construction Using p426-gRNA plasmid as a template, linearized p426-gRNA was amplified using the primers in Table 1. YJL064W -gRNA plasmid. The amplification product was digested with DpnI enzyme to remove methylated template plasmid, and the digested product was then gel-cleaved and recovered. The recovered product was subjected to a recombination reaction to obtain circularized p426- YJL064W -gRNA plasmid was transformed into competent DH5α cells.

[0050] 4. Construction of Strain1 recombinant bacteria Nucleic acid fragment P PGK1 - tHMG1 -T CYC1 p426- YJL064W -gRNA and p414-CAS9 plasmid were co-transformed into CEN.PK2-1D competent cells. The transformation procedure was as follows: CEN.PK2-1D colonies were picked and inoculated into 5 mL of SD liquid medium and cultured overnight at 30°C and 220 rpm in a shaking incubator; the cells were collected by centrifugation at 5000 rpm, the cells were resuspended in 1 mL of sterile water and centrifuged to remove the supernatant, the cells were resuspended again in 1 mL of 1×TE / LiAC mixture and centrifuged to remove the supernatant; the cells were resuspended in 70 μL of 1×TE / LiAC mixture to prepare competent cells; 5 μL of salmon sperm DNA and 2 μL of p426- YJL064W -gRNA plasmid, 2 μL p414-CAS9 plasmid and 5 μL PPGK1 - tHMG1 -T CYC1 Fragments were transformed into 600 μL of yeast in a PEG / LiAC mixture and gently pipetted to mix. The transformation system was then heat-shocked at 42°C for 15 min. The mixture was centrifuged at 8,000 r / min for 1 min, the supernatant was removed, and the cell pellet was retained. The cells were resuspended in 100 μL of sterile water and plated on SD-URA-TRP agar plates. The plates were then incubated at 30°C for 48 h.

[0051] 5. PCR verification of transformant colonies Add 6 μL of sterile water to a 200 μL centrifuge tube, pick transformants from the transformation plate into the centrifuge tube, and mix by pipetting; take 3 μL of bacterial culture and inoculate it into a fresh SD-URA-TRP medium plate, add 17 μL of 0.02M NaOH solution to the remaining bacterial culture; perform alkaline lysis of the bacterial culture in the centrifuge tube, and heat-treat at 95℃ for 15 min; the supernatant obtained after heat treatment can be used as a template for PCR verification. Rapid amplification was performed using SapphireAmp® Fast PCR Master Mix (RR350B) from Takara Bio Inc. The amplification system is as follows: SapphireAmp ® The following reagents were used: Fast PCR Master Mix 5 μL, upstream primer (10 μM) 0.4 μL, downstream primer (10 μM) 0.4 μL, template 1 μL, and ddH2O 3.2 μL. The amplification program was as follows: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 sec, 56-72℃ annealing for 20 sec, 72℃ extension for 30 sec / kb (35 cycles); 72℃ extension for 5 min. After colony PCR, agarose gel electrophoresis and first-generation sequencing were performed. Sequencing results matching the expected sequence were identified as Strain1 (CEN.PK2-1D). YJL064W ::P PGK1 - tHMG1 -T CYC1 Recombinant strains, through continuous passage in SD medium to lose p426- YJL064W -gRNA and p414-CAS9 plasmid.

[0052] Example 2: Overexpression of Strain1 in recombinant strains IDI1 Gene In the Strain1 recombinant strain constructed in Example 1, by... IDI1 P of the gene IDI1 Replace the promoter with P ENO2 overexpression IDI1 Genes, the specific methods are as follows.

[0053] 1. PCR amplification Using the CEN.PK2-1D genome as a template, the promoter P was amplified by PCR using the primers in Table 1. ENO2 It has brewer's yeast promoter P at both ends IDI1 Homologous arms were extracted, and nucleic acid fragments were recovered by gel extraction. The amplification system was as follows: 10 μL of 2 × Phanta Max MasterMix (Dye Plus), 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 1 μL of template DNA, and 8 μL of ddH2O. The amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, annealing at 56–72℃ for 15 sec, extension at 72℃ for 30 sec / kb (30 cycles); and extension at 72℃ for 5 min.

[0054] 2. p426-P IDI1 -gRNA plasmid construction p426-P IDI1 The construction method of the -gRNA plasmid is described in Example 1, section 3, p426- YJL064W The method for constructing -gRNA plasmids uses the corresponding primers listed in Table 1.

[0055] 3. Construction of Strain2 recombinant bacteria The promoter segment P ENO2 p426-P IDI1 -gRNA and p414-CAS9 plasmid were co-transformed into Strain1 competent cells. The transformation procedure was as described in section 4 of Example 1.

[0056] 4. PCR verification of transformant colonies The PCR verification method for transformant colonies is the same as that described in section 5 of Example 1. The sequencing results are compared; if the sequence matches the expected sequence, it is Strain2 (Strain1, P...). IDI1 ::P ENO2 The recombinant strain was passaged sequentially in SD medium to lose p426-P. IDI1 -gRNA and p414-CAS9 plasmid.

[0057] Example 3 Expression in Strain2 recombinant strain ERG20 F96W,N127W Mutant genes Based on the Strain2 recombinant strain constructed in Example 2, it was... ERG20 Mutation ERG20 F96W,N127W The specific method is as follows.

[0058] 1. ERG20 Site-directed mutation of genes Using the CEN.PK2-1D genome as a template, PCR amplification was performed using the primers listed in Table 1. ERG20 The gene, with homologous arms at both ends near the NotI and SpeI restriction sites of the pSP-G1 plasmid, was excised and the nucleic acid fragment was recovered from the gel. The amplification system was as follows: 10 μL of 2 × Phanta MaxMaster Mix (Dye Plus), 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 1 μL of template DNA, and 8 μL of ddH2O. The amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, annealing at 56~72℃ for 15 sec, extension at 72℃ for 30 sec / kb (30 cycles); and extension at 72℃ for 5 min. The pSP-G1 plasmid was digested with NotI and SpeI, excised from the gel, and cloned using the ClonExpress one-step cloning kit from Novizan Biotechnology Co., Ltd. ® pSP-G1- was obtained through homologous recombination using the Ultra One Step Cloning Kit (item number: C115). ERG20 Plasmid.

[0059] pSP-G1- ERG20 Using the plasmid as a template, amplification was performed using primers listed in Table 1 (Novizan Biotechnology Co., Ltd. MutExpress II Fast Mutagenesis Kit, catalog number: C214). The primers contained double point mutations of F96W and N127W. The amplification system was as follows: 2 × Max Buffer 25 μL, dNTP Mix (10 mM each) 1 μL, template DNA 1 μL, primer 1 (10 μM) 2 μL, primer 2 (10 μM) 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, ddH2O 18 μL. The amplification program was as follows: 95℃ pre-denaturation for 30 sec; 95℃ denaturation for 15 sec, 60~72℃ annealing for 15 sec, 72℃ extension for 30 sec / kb (30 cycles); 72℃ extension for 5 min. The amplification product was digested with DpnI enzyme to remove methylated template plasmid, and the digested product was then gel-extracted. The recovered product was subjected to a recombination reaction to obtain cyclized pSP-G1- ERG20 F96W,N127W The plasmid was then transformed into competent DH5α cells.

[0060] 2. p426- ERG20 -gRNA plasmid construction Refer to page 426 of Example 1, section 3. YJL064W -gRNA plasmid construction method and p426- using the corresponding primers in Table 1-ERG20 Construction of -gRNA plasmid.

[0061] 3. Construction of Strain3 recombinant bacteria pSP-G1- ERG20 F96W,N127W Using plasmid as a template, amplification ERG20 F96W,N127W Gene fragments with brewer's yeast at both ends ERG20 The two homologous arms of the gene. ERG20 F96W,N127W Gene fragment, p426- ERG20 -gRNA and p414-CAS9 plasmid were co-transformed into Strain2 competent cells. The transformation procedure was as described in section 4 of Example 1.

[0062] 4. PCR verification of transformant colonies The colony PCR verification method is the same as that described in section 5 of Example 1. The sequencing results are compared; if the sequence matches the expected sequence, it is Strain3 (Strain2, ...). ERG20 :: ERG20 F96W,N127W Recombinant strain. Through sequential passage in SD medium to lose p426- ERG20 -gRNA and p414-CAS9 plasmid.

[0063] Example 4: Knockout in Strain3 recombinant strain OYE2 Gene Based on the Strain3 recombinant strain constructed in Example 3, the strain was knocked out. OYE2 Genes, the specific methods are as follows.

[0064] 1. Fragment amplification Using the CEN.PK2-1D genome as a template, PCR amplification was performed using the primers listed in Table 1. OYE2 The upstream and downstream homologous arm fragments of the gene were separated, and the nucleic acid fragments were recovered by gel extraction. The amplification system was as follows: 10 μL of 2 × PhantaMax Master Mix (Dye Plus), 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 1 μL of template DNA, and 8 μL of ddH2O. The amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, annealing at 56~72℃ for 15 sec, extension at 72℃ for 30 sec / kb (30 cycles); and extension at 72℃ for 5 min. The upstream-downstream fragment was obtained by fusion PCR and used to knock out Saccharomyces cerevisiae. OYE2Gene. The amplification system is as follows: Step one system: 5 μL of 2 × PhantaMax Master Mix (Dye Plus), 0.5 μL of fragment 1, 0.5 μL of fragment 2, and 3 μL of ddH2O; Step two system: 10 μL of 2 × PhantaMax Master Mix (Dye Plus), 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 1 μL of the step one system, and 8 μL of ddH2O. The amplification program is as follows: Step one system: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 58℃ annealing for 15 sec, 72℃ extension for 30 sec / kb (15 cycles); 72℃ extension for 10 min; Step two system: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 56~72℃ annealing for 15 sec, 72℃ extension for 30 sec / kb (35 cycles); 72℃ extension for 10 min. The amplified product was recovered by gel extraction.

[0065] 2. p426- OYE2 -gRNA plasmid construction Using the corresponding primers in Table 1, refer to p426- in Example 1, section 3. YJL064W -Construction method of gRNA plasmid p426- OYE2 -gRNA plasmid.

[0066] 3. Construction of Strain4 recombinant bacteria upstream-downstream nucleic acid fragments, p426- OYE2 -gRNA and p414-CAS9 plasmid were co-transformed into Strain3 competent cells. The transformation procedure was as described in section 4 of Example 1.

[0067] 4. PCR verification of transformant colonies Colony PCR verification was performed using the colony PCR method described in Example 1, paragraph 5. The sequencing results were compared, and those matching the expected sequence were identified as Strain4 (Strain3, oye2Δ A recombinant strain was developed that optimizes the mevalonic acid pathway for the efficient synthesis of geraniol precursor geraniyl pyrophosphate (GPP). This was achieved through sequential subculturing in SD medium to eliminate p426- OYE2 -gRNA and p414-CAS9 plasmid.

[0068] Example 5 Total Synthesis of Geranium Synthase Gene Genscript Biotechnology Co., Ltd. was commissioned to study the periwinkle ( Catharanthus roseus Geraniol synthase gene from CrGES(Genbank: JN882024.1) Codon optimization was performed on the Saccharomyces cerevisiae host, and a signal peptide lacking chloroplast targeting was synthesized. tCrGES Inserting it between the SmaI and SalI restriction sites of the pSP-G1 vector yields pSP-G1- tCrGES Plasmid.

[0069] Example 6 High-copy integration of geraniol synthase gene The expression cassette P of the geranium synthase gene synthesized in Example 5 was used. PGK1 - tCrGES -T CYC1 The delta sequence was integrated into the genome of the recombinant Strain4 bacteria using a multicopy integration strategy. The integration diagram is shown below. Figure 1 As shown, the specific method is as follows.

[0070] 1. Construction of p426-delta-gRNA plasmid Using the corresponding primers in Table 1, refer to p426- in Example 1, section 3. YJL064W The p426-delta-gRNA plasmid was constructed using the method described above.

[0071] 2. tCrGES PCR amplification of multi-copy integration fragments of genes Using the CEN.PK2-1D genome as a template, delta1 and delta2 fragments were amplified by PCR using primers listed in Table 1. pSP-G1- tCrGES Using plasmids as templates, PCR amplification was performed using primers listed in Table 1. tCrGES Expression box P PGK1 - tCrGES -T CYC1 The amplification system was as follows: 10 μL of 2 × Phanta Max Master Mix (Dye Plus) (Novizan Biotechnology Co., Ltd., catalog number: P525), 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 1 μL of template DNA, and 8 μL of ddH2O. The amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 56~72℃ annealing for 15 sec, 72℃ extension for 30 sec / kb (30 cycles); 72℃ extension for 5 min. delta1, P PGK1 - tCrGES -T CYC1 The delta2 nucleic acid fragment contains a 20bp homologous arm. Using fusion PCR technology, delta1-P was obtained. PGK1 - tCrGES -T CYC1 -delta2 fragment, used fortCrGES Multiple copy integration of genes.

[0072] 3. tCrGES Construction of multi-copy integration recombinant bacteria The nucleic acid fragment delta1-P PGK1 - tCrGES -T CYC1 Strain4 competent cells were co-transformed with p426-delta-gRNA and p414-CAS9 plasmid. The transformation procedure was as described in section 4 of Example 1.

[0073] 4. PCR verification of transformant colonies Colony PCR verification was performed using the method described in Example 1, paragraph 5. The sequencing results were compared, and if the sequence matched the expected sequence, it was considered a successfully integrated recombinant strain (Strain4, delta::P). PGK1 - tCrGES -T CYC1 The p426-delta-gRNA and p414-CAS9 plasmid were lost by continuous passage in SD medium.

[0074] Table 1 Primer sequences used in this invention Example 7: Shake-flask fermentation and detection of recombinant strains 1. Recombinant strain shake flask fermentation Twenty recombinant strains obtained in Example 6 (strains that successfully integrated the tCrGES gene and lost p426-IDI1-gRNA and p414-CAS9 plasmid through successive passages in SD medium, named Strain5-1 to 5-20 respectively) were randomly selected and inoculated into 5 mL of SD liquid medium. They were cultured overnight at 30°C and 220 rpm in a shaking incubator. The bacterial concentration of each fragment was measured using a spectrophotometer. Each fragment was then inoculated into 100 mL Erlenmeyer flasks (containing 20 mL of fresh SD liquid medium and 2 mL of dodecane (10%, v / v)). The initial bacterial concentration OD was measured. 600 The value was adjusted to 0.1, and fermentation was carried out in a shaker at 30℃ and 220r / min for 5 days.

[0075] 2. Geraniol gas chromatography detection The fermentation broth was centrifuged at 12000 r / min for 5 min, and the upper organic layer was collected for gas chromatography detection. The specific detection steps are as follows: (1) The fermentation organic layer was diluted 10 times with ethyl acetate, passed through a 0.22 μm organic nylon membrane, and detected by gas chromatography. Instrument model: Agilent 6890A gas chromatograph. (2) Geraniol standard was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (item number: 48798). (3) The gas chromatography detection procedure for the fermentation product is as follows: injection port temperature 240℃, injection volume 1 μL, splitless; chromatographic column: HP-5ms (30m*0.25mm*0.5μm); chromatographic conditions: 70℃, 4 min, 4℃ / min to 220℃, hold for 5 min.

[0076] 3. The shake-flask fermentation and gas-phase quantitative detection experiments were repeated three times. The results showed that among the 20 recombinant strains tested, the Strain5-11 recombinant strain had the highest geraniol yield after 5 days of fermentation. Figure 2 The concentration was 776.86 mg / L.

[0077] Example 8 Recombinant strains tCrGES Gene copy number detection This embodiment uses Hieff, a product of Yisheng Biotechnology Co., Ltd. ® qPCR was performed using SYBR Green MasterMix for real-time quantitative PCR to detect the recombinant strains Strain5-1 to Strain5-20 in Example 7. tCrGES Gene copy number.

[0078] 1. Real-time quantitative PCR The genome of the recombinant strain was extracted using the fungal genomic DNA rapid extraction kit from Beijing Coolplay Technology Co., Ltd. Real-time quantitative PCR was performed using the genome as a template. The reaction system was Hieff... ® qPCR reaction mixture: 10 μL SYBR Green MasterMix, 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM), 1 μL template, 8.2 μL ddH2O. Vortex the prepared reaction mixture and briefly centrifuge. Then, place it in a real-time quantitative PCR instrument and perform the following program: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 sec, 55℃ annealing for 20 sec, 72℃ extension for 20 sec (40 cycles).

[0079] 2. Draw the standard curve Build a single copy tCrGES Target gene and single-copy internal reference gene ALG9The recombinant plasmid pSPcopy1 was used as a reference template for the qPCR standard curve. The plasmid concentration was determined using an ultra-micro spectrophotometer, and the molecular copy number per unit volume of plasmid solution was calculated based on the plasmid molecular weight. A series of concentration standards were prepared using a 10-fold serial dilution method and used as templates for qPCR amplification to establish a standard curve between Ct values ​​and the logarithm of copy number. This standard curve will be used in subsequent recombinant yeast strains... tCrGES Precise quantitative analysis of gene copy number. Three technical replicates were set up during the experiment to ensure data reliability, and all operations were performed in a nuclease-free environment.

[0080] 3. tCrGES Gene copy number detection Real-time quantitative PCR was performed using 1 μL of recombinant bacterial genomic DNA as a template. Three technical replicates were set up for each sample, and three parallel experiments were conducted simultaneously to ensure data reliability. The obtained Ct values ​​were substituted into a pre-established standard curve to calculate the Ct values ​​for each recombinant strain. tCrGES Absolute copy number of genes. Results showed that in strain Strain5-11... tCrGES The gene copy number is the highest, at 16 copies. Figure 3 This aligns with the fact that it has the highest geraniol production.

[0081] Example 9: Detection of genetic stability of high-geraniol-producing recombinant strains This embodiment tests the genetic stability of the recombinant strain Strain5-11. The specific methods and results are described below.

[0082] 1. The constructed high-geraniol-producing recombinant strain Strain5-11 was inoculated into 5 mL of SD liquid medium and cultured overnight at 30°C and 220 r / min in a shaker. 2. Detection of bacterial culture OD 600 The value was then transferred to 5 mL of fresh SD liquid medium to allow OD to rise. 600 =0.1, and cultured in a shaker at 30℃ and 220r / min for 24h; 3. Take 1% of the bacterial culture from the cultured culture for 24 hours and transfer it to 5 mL of fresh SD liquid medium. Continue to culture for 24 hours. Repeat the above steps. According to the generation time of Saccharomyces cerevisiae (90 min), collect the bacterial culture from the 0th, 50th, and 100th generations, respectively. After appropriate dilution, spread it on SD plate medium and incubate in a 30℃ incubator. 4. Pick recombinant strains from passages 0, 50, and 100 from the plate medium and inoculate them into 5 mL of SD liquid medium. Incubate overnight at 30°C and 220 rpm in a shaker. 5. Detect the OD of bacterial solution 600The value was transferred to a 100mL Erlenmeyer flask containing 20mL of fresh SD liquid culture medium to allow OD to develop. 600 =0.1, fermented in a shaker at 30℃ and 220r / min for 5 days, and the geraniol yield was detected to reflect the genetic stability of the high-geraniol-producing recombinant strain.

[0083] The results showed that after 100 generations of subculturing, the recombinant strain Strain5-11 could still maintain a geraniol yield of 739.5 mg / L. Figure 4 This data indicates that the high-copy integration strategy employed has excellent genetic stability, with geraniol production retention exceeding 95% during long-term passaging.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant Saccharomyces cerevisiae strain that produces geraniol, characterized in that, The recombinant Saccharomyces cerevisiae strain has multiple copies of the geraniol synthase gene integrated into its genome. The geraniol synthase is derived from periwinkle ( Catharanthus roseus (or a truncated variant of its signal peptide;) The integration site of the geranium synthase gene is the delta site in the Saccharomyces cerevisiae genome.

2. The geraniol-producing recombinant Saccharomyces cerevisiae strain according to claim 1, characterized in that, The amino acid sequence of the geranium synthase is shown in SEQ ID NO.

1.

3. The geraniol-producing recombinant Saccharomyces cerevisiae strain according to claim 1, characterized in that, The number of copies is not less than 5 copies / cell, preferably not less than 10 copies / cell.

4. The geraniol-producing recombinant Saccharomyces cerevisiae strain according to any one of claims 1 to 3, characterized in that, The recombinant Saccharomyces cerevisiae strain overexpressed a truncated HMG-CoA reductase gene. tHMG1 and isopentenyl diphosphate isomerase gene IDI1 .

5. The geraniol-producing recombinant Saccharomyces cerevisiae strain according to any one of claims 1 to 3, characterized in that, The recombinant Saccharomyces cerevisiae strain expresses gerany pyrophosphate synthase ERG2. 0 F96W,N127W Mutant.

6. The recombinant Saccharomyces cerevisiae strain producing geraniol according to any one of claims 1 to 3, characterized in that, The activity of geraniol reductase OYE2 in the recombinant Saccharomyces cerevisiae strain was reduced or inactivated.

7. A method for constructing a recombinant Saccharomyces cerevisiae strain that produces geraniol, characterized in that, The geraniol-producing recombinant Saccharomyces cerevisiae strain is as described in any one of claims 1 to 6, wherein the method comprises: integrating the geraniol synthase gene into the delta site of the Saccharomyces cerevisiae genome using a CRISPR / Cas9-mediated homologous recombination system.

8. The application of a geraniol-producing recombinant Saccharomyces cerevisiae strain in the fermentation production of geraniol, wherein the geraniol-producing recombinant Saccharomyces cerevisiae strain is the geraniol-producing recombinant Saccharomyces cerevisiae strain according to any one of claims 1 to 6.

9. The application of a geraniol-producing recombinant Saccharomyces cerevisiae strain in constructing a strain for fermentation production of geraniol, wherein the geraniol-producing recombinant Saccharomyces cerevisiae strain is the geraniol-producing recombinant Saccharomyces cerevisiae strain according to any one of claims 1 to 6.

10. A method for producing geraniol, characterized in that, The method includes: culturing the recombinant Saccharomyces cerevisiae strain that produces geraniol according to any one of claims 1 to 6, and collecting geraniol from the culture medium.

Images