Saccharomyces cerevisiae engineering bacteria, preparation method and use thereof

Abstract

The application provides a saccharomyces cerevisiae engineering bacterium and a preparation method and application thereof. By modifying a saccharomyces cerevisiae strain, the content of santalene and santalol produced by fermentation can be significantly improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an engineered strain of Saccharomyces cerevisiae, its preparation method, and its uses. Background Technology

[0002] Currently, high-end natural fragrance oils, represented by sandalwood, are expensive and in high demand, largely monopolized by multinational corporations. Meanwhile, traditional chemical synthesis methods can only produce single fragrance molecules, resulting in meager profits and failing to reproduce the rich and subtle aromas of natural fragrances, making them difficult to substitute for them. Synthetic biology, with its ability to transfer functional elements and modules across species, is gradually becoming a primary means of solving this problem. Currently, based on genome sequencing, scientists are using synthetic biology methods to continuously increase the yield of heterologous biosynthesis of core components of natural sandalwood essential oil, such as santalene and santalol, and the proportion of terpenoid molecules in the metabolites of engineered bacteria is increasingly approaching the proportion found in natural sandalwood essential oil. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an engineered strain of *Saccharomyces cerevisiae*, its preparation method, and its uses, to solve the problem of low yields in the preparation of santalene and santalol using engineered strains in the prior art. This invention utilizes an endogenously loaded, highly efficient, and controllable exogenous gene expression system to overexpress the endogenous MVA pathway (a total of 7 enzymes: ERG12, mevalonate kinase; ERG8, valonate phosphate kinase; ERG19, mevalonate pyrophosphate decarboxylase; IDI1, dimethylallyl pyrophosphate isomerase; ERG10, acetyl-CoA acetyltransferase; ERG13, hydroxymethylglutaryl-CoA synthase; tHMGR1, truncated hydroxymethylglutaryl-CoA reductase) and ERG20 (farnesyl pyrophosphate synthase) in *Saccharomyces cerevisiae*, specifically the *Sansyn* strain derived from *Clausena lansium*. F441V Multiple copies of the mutant santalene synthase, CYP736A167 (cytochrome P450) and SaCPR2 (cytochrome P450 reductase) derived from sandalwood (Santalum album) were heterologously expressed. At the same time, ERG9 (squalene synthase), which competes with the pathway, was inhibited with low glucose. ATF1 and ATF2 (ethanol acetyltransferase), OYE2 and OYE3 (saturase), LPP1 (lipophosphophosphatase) and DPP1 (diacylglycerol pyrophosphatase) were knocked out to obtain the engineered yeast strain PCSan6-1.

[0004] One aspect of the present invention provides an engineered brewer's yeast strain, which has the following characteristics: a) Overexpression of mevalonate kinase, valonate kinase, mevalonate pyrophosphate decarboxylase, dimethylallyl pyrophosphate isomerase, acetyl-CoA acetyltransferase, hydroxymethylglutaryl-CoA synthase, truncated hydroxymethylglutaryl-CoA reductase and ERG20 in wild-type yeast engineered strains. b) Multiple copies of the mutant santalene synthase, cytochrome P450, and cytochrome P450 reductase were heterologously expressed. c) Ethanol acetyltransferase, senescent enzyme, lipophosphatase, and diacylglycerol pyrophosphatase are eliminated; d) ERG9 is suppressed.

[0005] Furthermore, the santalene synthase is derived from wampee, the cytochrome P450 is derived from sandalwood, and the cytochrome P450 reductase is derived from sandalwood. Another aspect of the present invention provides the use of the above-mentioned strains for preparing santalene and santalol.

[0006] Furthermore, ERG9 is suppressed by replacing its promoter with P. HXT1。

[0007] Another aspect of the present invention provides a method for constructing the above-mentioned engineered brewer's yeast, the method comprising the following steps: Using auxotrophic brewer's yeast as chassis cells, with P HXT1 The ERG9 promoter was replaced, and a yeast strain containing the overexpressed gene was constructed using gene editing technology; then, the target gene was knocked out using gene editing technology.

[0008] Furthermore, 12 yeast endogenous genes, namely LYS21, ILV5, HSP104, TDH1, RPS10A, PET9, TDH2, SOD1, RPS25A, CYS3, TEF1, and TDH3, were selected as genes in the santalol synthesis pathway, namely ERG8, ERG10, ERG12, ERG13, ERG19, IDI1, tHMGR1, ERG20, two SaCPR2 genes, and Sansyn. F441V The endogenous target genes of the CYP736A167 gene were linked using IGG6 elements, and a peroxisome localization signal MDH3 was added to the first 7 genes of the santalol synthesis pathway. The target genes were then transferred into yeast using CRISPR gene editing technology. The nucleotide sequences of the gRNA used in the gene editing technology are shown in Table 2, and the primer nucleotide sequences are shown in Table 3.

[0009] Furthermore, the method for knocking out the target gene is as follows: using Sansyn F441VThe open reading frames (OPFs) of ATF1 and OYE2 were replaced with the open reading frames of the CYP736A167 gene; the open reading frames of ATF2 and OYE3 were replaced with the open reading frame of the CYP736A167 gene; and the CYP736A167 gene, SaCPR2 gene, and SanSyn gene were linked using IGG6. F441V Genes, with P TDH3 Using IGG6 as the promoter, a tricistronic expression cassette was constructed to replace the open reading frame of DPP1; the SaCPR2 gene and SanSyn were linked together. F441V Gene, CYP736A167 gene, with P FBA1 Using LPP1 as the promoter, a tricistronic expression cassette was constructed to replace the open reading frame of LPP1; the target gene was then transferred into yeast using CRISPR gene editing technology. The nucleotide sequences of the primers used in the CRISPR gene editing technology are shown in Tables 4 and 5.

[0010] Another aspect of the present invention provides a method for preparing santalene and santalol, wherein the method comprises fermenting the above-mentioned strain, collecting the fermentation product, and purifying it after collection.

[0011] As described above, the engineered brewer's yeast strain, its preparation method, and its uses of the present invention have the following beneficial effects: The engineered brewer's yeast provided in this application can effectively improve the yield of santalene and santalol, as detailed in the examples below. Attached Figure Description

[0012] Figure 1 The results show the detection of santalene and santalol in PCSan3. (A) GC-MS mass spectrometry chromatogram. (B) Chromatogram of the characteristic fragment ion extracted at m / z=94.0. (C) GC-MS mass spectrum of the fragment ion (mass spectrum of the fragment ion corresponding to the elution time at m / z=94.0). (D) Mass spectra of characteristic fragment ions of santalene and santalol from the database.

[0013] Figure 2 The results show the detection of santalene and santalol in PCSan6-1 and PCSan6-2. (A) GC-MS mass spectrometry chromatogram. (B) Chromatogram of the extracted characteristic fragment ion at m / z=94.0. Detailed Implementation

[0014] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0015] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0016] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

[0017] pJET1.2 / blunt Cloning Vector: purchased from Thermo Fisher Scientific.

[0018] Saccharomyces cerevisiae CEN.PK2-1D: MATα; ura3-52; trp1-289; leu2-3,112; his3Δ 1;MAL2-8C; SUC2.

[0019] Enzymes and kits: The high-fidelity DNA amplification MIX and seamless cloning kit were purchased from Novizan. DNA loading buffer and DNA marker were purchased from Kangwei Century Company; The DNA purification gel recovery kit was purchased from Thermo Fisher Scientific. Escherichia coli DH5α competent cells were purchased from Kangwei Century Company.

[0020] The solvent used in the experiment, dodecane, was produced by Beijing Chemical Reagent Factory and was of analytical grade. Chromatographically pure ethyl acetate was produced by Fisher Chemical Company.

[0021] Culture medium: LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0); SC-Ura Deficit Medium (2% glucose, 6.7% amino acid-free yeast nitrogen source, uracil-deficient amino acid mixture); YPD medium (2% yeast extract, 2% peptone, 2% glucose), If preparing a solid culture medium, add 2% agar powder.

[0022] Example 1: Reconstruction of the Santalol Synthesis Pathway 1.1. Experimental Objective Using auxotrophic brewer's yeast CEN.PK2-1D as the chassis cells, and P HXT1 The promoter of ERG9 was replaced. Twelve endogenous yeast genes (LYS21, ILV5, HSP104, TDH1, RPS10A, PET9, TDH2, SOD1, RPS25A, CYS3, TEF1, and TDH3) were selected as the santalol synthesis pathway genes (ERG8, ERG10, ERG12, ERG13, ERG19, IDI1, tHMGR1, ERG20, two SaCPR2 genes, and Sansyn) to represent the santalol synthesis pathway. F441V The target gene (CYP736A167 gene) was identified. It was linked using the IGG6 element 5'-CAATCAAAC-3' (SEQ ID NO.1), and a peroxisome localization signal MDH3 was added to the first seven genes in the santalol synthesis pathway. The target gene was transferred into yeast using CRISPR gene editing technology, and the production of santalene and santalol in positive transformants was detected by GC-MS.

[0023] 1.2. Experimental Methods: (1) Obtaining plasmids and donor DNA for CRISPR gene editing Genes for the exogenous santalol synthesis pathway (CYP736A167, SaCPR2, SanSyn) were obtained through gene synthesis. F441V cDNA positive clones. Based on endogenous target gene or sequence, target gene or P HXT1 Primers were designed based on the sequences of IGG elements and MDH3. A homologous fragment of the target gene and / or no IGG sequence was added to the 5' end of the forward primer, and a homologous fragment of the target gene and / or no MDH3 sequence was added to the 5' end of the reverse primer. The target gene was then targeted using the genome of *Saccharomyces cerevisiae* or exogenous santalol synthesis pathway genes (CYP736A167, SaCPR2, SanSyn). F441V Using a cDNA-positive clone as a template, the donor DNA was amplified by PCR.

[0024] Table 1

[0025] Primers were designed targeting the gRNAs of the LYS21, ILV5, HSP104, TDH1, RPS10A, PET9, TDH2, SOD1, RPS25A, CYS3, TEF1, and TDH3 genes to construct plasmids pCAS-sgRNA ERG9, pCAS-sgRNA ILV5+TDH1+TDH2+HSP104, pCAS-sgRNA SOD1+LYS21+PET9, pCAS-sgRNA RPS10A+CYS3+RPS25A, pCAS-sgRNA TEF1, and pCAS-sgRNA TDH3.

[0026] Table 2 .

[0027] (2) Yeast transformation Yeast electroporation and plasmid elimination were performed. First, pCAS-sgRNA ERG9 and its corresponding donor DNA T were... PTH1 -P HXT1 -ERG9 was transformed into CEN.PK2-1D, and positive transformants CEN-1 were obtained by colony PCR screening. After plasmid elimination, pCAS-sgRNA ILV5+TDH1+TDH2+HSP104 and its corresponding donor DNA ILV5-IGG6-ERG10-MDH3, TDH1-IGG6-ERG13-MDH3, TDH2-IGG6-tHMGR1-MDH3 and HSP104-IGG6-ERG12-MDH3 were transformed into pCAS-sgRNA SOD1+LYS21+PET9 and its corresponding donor DNA were obtained by colony PCR screening. After plasmid elimination, pCAS-sgRNA SOD1+LYS21+PET9 and its corresponding donor DNA were transformed into pCAS-sgRNA SOD1+LYS21+PET9 and its corresponding donor DNA were obtained by colony PCR screening. SOD1-IGG6-ERG20, LYS21-IGG6-ERG8-MDH3, and PET9-IGG6-IDI1-MDH3 were used to select positive transformants P1 by colony PCR. After plasmid removal, pCAS-sgRNA RPS10A+CYS3+RPS25A and its corresponding donor DNA RPS10A-IGG6-ERG19-MDH3, RPS25A-IGG6-SaCPR2, and CYS3-IGG6-SaCPR2 were used as the host to select positive transformants PCSan1 by colony PCR. After plasmid removal, pCAS-sgRNA TEF1 and its corresponding donor DNA TEF1-IGG6-Sansyn were used as the host to select positive transformants PCSan1. F441VPositive transformants PCSan2 were screened by colony PCR. After eliminating the plasmid, pCAS-sgRNA TDH3 and its corresponding donor DNA TDH3-IGG6-CYP736A167 were introduced into the plasmid as the host, and positive transformants PCSan3 were screened by colony PCR.

[0028] Table 3 .

[0029] (3) Fermentation culture First, an appropriate amount of PCSan3 yeast transformants were inoculated into 3 mL of SC-Ura liquid deficient medium and cultured at 30℃ and 200 rpm for about 16 h to obtain the seed culture. An appropriate amount of the seed culture was then inoculated into 20 mL of YPD medium, with an initial cell OD value of approximately 0.1. After culturing at 30℃ and 200 rpm for 1 day, 2 mL of filtered and sterilized n-dodecane was added, and the culture was continued for 4 days. The culture was then centrifuged at 4000 rpm for 10 minutes, and the n-dodecane phase was collected.

[0030] (4) GC-MS detection of santalene and santalol Take 100 µL of n-dodecane phase, dilute it 10 times with ethyl acetate, filter it through a 0.22 µm filter membrane, and then detect it by GC-MS.

[0031] GC-MS analysis was performed using an 8890-5977B gas chromatograph-mass spectrometer (Agilent Technologies, USA). A 1 µL sample was taken and a DB-5MS UI capillary column (30 m × 0.25 mm, 0.25 µm; Agilent Technologies, USA) was used with high-purity helium as the carrier gas. The fragmentation voltage was set to 70 electron volts (EI), the ion source temperature to 230 °C, and the transfer line temperature to 250 °C. The injection port temperature was 250 °C, the pressure to 9.181 psi, splitless operation, and the column flow rate to 1.0 mL / min (constant flow). The column temperature was set as follows: 80 °C initially held for 1 min, then increased at 10 °C / min to 120 °C; then increased at 3 °C / min to 160 °C; and finally increased at 10 °C / min to 270 °C, held for 3 min. Data acquisition was performed in full scan mode (m / z 35-450).

[0032] 1.3. Experimental Results and Analysis The contents of santalene and santalol in the sample were detected by GC-MS, and 1.15 ± 0.23 mg L was successfully detected in PCSan3. -1 Santalene, 0.62 ± 0.11 mg L -1 Santalol, of which α-santalol yielded 0.54 ± 0.08 mg / L. -1 .

[0033] Example 2: Optimization and Enhancement of the Santalol Synthesis Pathway 2.1. Experimental Objective Using PCSan3 as the chassis cell, and Sansyn F441V The open reading frames (OPFs) of ATF1 and OYE2 were replaced with the open reading frames of the CYP736A167 gene; the OPFs of ATF2 and OYE3 were replaced with the open reading frame of the CYP736A167 gene. The CYP736A167 gene, SaCPR2 gene, and SanSyn gene were linked by IGG6 (5'-CAATCAAAC-3') (SEQ ID NO.74). F441V Genes, with P TDH3 Using IGG6 (5'-CAATCAAAC-3') (SEQ ID NO.75) as the promoter, a tricistronic expression cassette was constructed to replace the open reading frame of DPP1; the SaCPR2 gene and SanSyn were linked together. F441V Gene, CYP736A167 gene, with P FBA1 Using LPP1 as the promoter, a tricistronic expression cassette was constructed to replace the open reading frame of LPP1. The target gene was transferred into yeast using CRISPR gene editing technology, and santalene and santalol produced in positive transformants were detected by GC-MS.

[0034] 2.2. Experimental Methods: (1) Obtaining plasmids and donor DNA for CRISPR gene editing Primers are designed based on the sequences of endogenous target genes, target genes, promoters, or IGG elements. Homologous fragments or IGG sequences are added to the 5' end of the corresponding primers as needed, targeting the genome of *Saccharomyces cerevisiae* or exogenous santalol synthesis pathway genes (CYP736A167, SaCPR2, SanSyn). F441V Using a cDNA-positive clone as a template, the donor DNA was amplified by PCR.

[0035] Table 4

[0036] EuPaGDT was used to predict and design primers for the gRNAs of ATF1, ATF2, OYE2, OYE3, LPP1, and DPP1 genes, and plasmids pCAS-sgRNA OYE2+OYE3, pCAS-sgRNA ATF1+ATF2, and pCAS-sgRNA DPP1+LPP1 were constructed.

[0037] Table 5 .

[0038] (2) Yeast transformation Yeast electroporation and plasmid removal were performed. First, PCSan3 was deplasmidized and then used as a host to transform pCAS-sgRNA ATF1+ATF2 and its corresponding donor DNA uATF1-Sansyn. F441V -dATF1 and uATF2-CYP736A167-dATF2 were used to screen for positive transformants PCSan4 via colony PCR. After plasmid elimination, PCSan4 was used as a host to transform pCAS-sgRNA OYE2+OYE3 and its corresponding donor DNA uOYE2-Sansyn. F441V -dOYE2 and uOYE3-CYP736A167-dOYE3 were used to screen for positive transformants PCSan5 via colony PCR. After plasmid removal, PCSan5 was used as a host to transform pCAS-sgRNA DPP1+LPP1 and its corresponding donor DNA uDPP1-P TDH3 -CYP736A167-IGG6-SaCPR2-IGG6-SanSyn F441V -dDPP1, uLPP1-P FBA1 -SaCPR2-IGG6-SanSyn F441V The positive transformant PCSan6-1 was obtained by screening the bacterial colony PCR strain IGG6-CYP736A167-dLPP1. After plasmid removal, the positive transformant PCSan6-2 was obtained.

[0039] (3) Fermentation culture Same as before.

[0040] (4) GC-MS detection of santalene and santalol Same as before.

[0041] 2.3. Experimental Results and Analysis The contents of santalene and santalol in the sample were detected by GC-MS, and 98.62 ± 0.71 mg L was successfully detected in PCSan6-1. -1 Santalene, 40.62 ± 4.35 mg L -1 α-Santalol; 46.57 ± 1.80 mg L was detected in PCSan6-2 after plasmid removal. -1 Santalene, 25.62±2.89 mg L -1 α-Santalol ( Figure 2This indicates that optimizing and enhancing the santalol synthesis pathway can increase the yield of santalene and santalol. Simultaneously, we also measured the biomass of the yeast transformants and found that the biomass of PCSan6-1 was significantly higher than that of PCSan6-2. The eliminated plasmid contained a highly expressed URA3 gene; after plasmid removal, PCSan6-2 reverted to an auxotrophic yeast strain where the URA3 gene could not be expressed normally.

[0042] Table 6

Claims

1. An engineered brewing yeast strain, characterized in that, The engineered brewer's yeast has the following characteristics: a) Overexpression of mevalonate kinase, valonate kinase, mevalonate pyrophosphate decarboxylase, dimethylallyl pyrophosphate isomerase, acetyl-CoA acetyltransferase, hydroxymethylglutaryl-CoA synthase, truncated hydroxymethylglutaryl-CoA reductase, and farnesyl pyrophosphate synthase in wild-type engineered yeast. b) Multiple copies of santalene synthase, cytochrome P450, and cytochrome P450 reductase were heterologously expressed. c) Ethanol acetyltransferase, senescent enzyme, lipophosphatase, and diacylglycerol pyrophosphatase are eliminated; d) ERG9 is suppressed.

2. The engineered bacteria according to claim 1, characterized in that, The santalene synthase is derived from yellow peel, the cytochrome P450 is derived from sandalwood, and the cytochrome P450 reductase is derived from sandalwood.

3. The engineered bacteria according to claim 1, characterized in that, ERG9 is suppressed by replacing its promoter with P. HXT1 .

4. The use of the engineered Saccharomyces cerevisiae as described in any one of claims 1-3 for the preparation of santalene and santalol.

5. A method for constructing the engineered Saccharomyces cerevisiae as described in claim 1, the method comprising: Using auxotrophic brewer's yeast as chassis cells, with P HXT1 The ERG9 promoter was replaced, and a yeast strain containing the overexpressed gene was constructed using gene editing technology; then, the target gene was knocked out using gene editing technology.

6. The method according to claim 5, characterized in that, The method for constructing overexpression yeast is as follows: Twelve yeast endogenous genes, namely LYS21, ILV5, HSP104, TDH1, RPS10A, PET9, TDH2, SOD1, RPS25A, CYS3, TEF1, and TDH3, were selected as genes in the santalol synthesis pathway, namely ERG8, ERG10, ERG12, ERG13, ERG19, IDI1, tHMGR1, ERG20, two SaCPR2 genes, and Sansyn. F441V The endogenous target genes of the CYP736A167 gene were linked using the IGG6 element, and a peroxisome localization signal MDH3 was added to the first 7 genes of the santalol synthesis pathway. The target genes were then transferred into yeast using CRISPR gene editing technology.

7. The method according to claim 5, characterized in that, The method for knocking out the target gene is: using Sansyn F441V The open reading frames (OPFs) of ATF1 and OYE2 were replaced with the open reading frames of the CYP736A167 gene; the open reading frames of ATF2 and OYE3 were replaced with the open reading frame of the CYP736A167 gene; and the CYP736A167 gene, SaCPR2 gene, and SanSyn gene were linked using IGG6. F441V Genes, with P TDH3 Using IGG6 as the promoter, a tricistronic expression cassette was constructed to replace the open reading frame of DPP1; the SaCPR2 gene and SanSyn were linked together. F441V Gene, CYP736A167 gene, with P FBA1 Using LPP1 as the promoter, a tricistronic expression cassette was constructed to replace the open reading frame of LPP1; the target gene was then transferred into yeast using CRISPR gene editing technology.

8. The method according to claim 6, characterized in that: The nucleotide sequences of the gRNA used in the gene editing technology are shown in Table 2, and the primer nucleotide sequences are shown in Table 3.

9. The method according to claim 7, characterized in that: The nucleotide sequences of the primers used in the gene editing technology are shown in Tables 4 and 5.

10. A method for preparing santalene and santalol, the method comprising fermenting with the strain described in any one of claims 1-3, collecting the fermentation product, and purifying the product after collection.

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