Method for producing noottanone at high yield by using saccharomyces cerevisiae

Abstract

The invention discloses a method for producing noottanone with high yield by using saccharomyces cerevisiae, and belongs to the technical field of genetic engineering and bioengineering. The recombinant saccharomyces cerevisiae with improved noottanone production capacity is constructed by optimizing a noottanone metabolic pathway. When the saccharomyces cerevisiae is horizontally fermented for 96 hours in a shake flask, the yield of the nodulinone is 466.1 mg / L, and when the saccharomyces cerevisiae is fermented and optimized for 168 hours in a 5L fermentation tank, the yield of the nodulinone is 6.5 g / L, so that the industrial fermentation production of the nodulinone is favorably realized.

Description

Technical Field

[0001] The invention relates to a method for high-producing grapefruit ketone by utilizing brewer's yeast, and belongs to the technical field of genetic engineering and bioengineering. Background Art

[0002] Grapefruit ketone is a volatile bicyclic sesquiterpene ketone, which was originally extracted from the heartwood of Alaskan yellow cypress. It was later found to be present in trace amounts in the peels of citrus plants such as sweet oranges, grapefruits, and lemons. Because of its strong and lasting citrus fruit smell and low perception threshold, it is widely used in the food and beauty industries. In medical research, grapefruit ketone has anti-cancer, anti-platelet aggregation, antibacterial and anti-inflammatory effects. In addition, grapefruit ketone has good insect repellent and insecticidal effects in the agricultural field.

[0003] At present, plant extraction and chemical synthesis are the main methods for obtaining oleanone. However, these methods are limited by raw material instability, low titer and environmental pollution. Microbial fermentation is to use the endogenous metabolic pathway of microorganisms to heterologously synthesize oleanone. This method has the advantages of low energy consumption, low cost and high yield, and is more suitable for large-scale industrial production. However, the current microbial production of oleanone still has the problem of low conversion rate of the precursor valencene and low yield of the final product oleanone. Summary of the invention

[0004] The present invention provides a polynucleotide which is (a) or (b):

[0005] (a) With P GAL10 -ERG20-(GSG)2-CnVS shown in the structure of the polynucleotide; wherein the promoter P GAL10 The nucleotide sequence of is shown in SEQ ID NO.18; the nucleotide sequence of ERG20 is shown in SEQ ID NO.1; the nucleotide sequence of (GSG)2 is shown in SEQ ID NO.19; the nucleotide sequence of CnVS is shown in SEQ ID NO.2;

[0006] (b) The gene encoding valencene oxidase shown in SEQ ID NO.13.

[0007] The present invention also provides a recombinant microorganism expressing the polynucleotide.

[0008] In one embodiment, the recombinant microorganism includes but is not limited to Saccharomyces cerevisiae.

[0009] The present invention also provides an engineered strain of Saccharomyces cerevisiae with high production of naringenone, which has at least one of the following improvements on the basis of the starting strain:

[0010] (1) Expressed “P GAL10-ERG20-(GSG)2-CnVS” expression box;

[0011] (2) Expression of the truncated valerian oxidase gene tHPO V480A / V482A ;

[0012] (3) The farnesyl pyrophosphate synthase gene ERG20, valencene synthase gene CnVS, and valencene oxidase gene HPO were integrated and expressed in the genome. V480A / V482A , cytochrome P450 reductase gene AtCPR1, alcohol dehydrogenase gene ZSD1;

[0013] (3) replacing the endogenous ERG9 gene promoter sequence with the HXT1 gene promoter sequence;

[0014] (4) Reduce the expression of endogenous genes NEM1 and DPP1.

[0015] In one embodiment, the farnesyl pyrophosphate synthase gene ERG20, valencene synthase gene CnVS, valencene oxidase gene HPO are expressed at one or more sites in W17, W18, NEM1. V480A / V482A , cytochrome P450 reductase gene AtCPR1 or alcohol dehydrogenase gene ZSD1.

[0016] In one embodiment, the improvement includes: integrating the endogenous gene ERG20 of Saccharomyces cerevisiae and the naringenone pathway gene CnVS into the bidirectional terminator site W17 of the Saccharomyces cerevisiae genome, and V480A / V482A , AtCPR1, and ZSD1 were integrated into the bidirectional terminator site W18 of the Saccharomyces cerevisiae genome.

[0017] In one embodiment, the improvement includes: knocking out the endogenous ERG9 gene promoter sequence, and integrating the HXT1 gene promoter sequence.

[0018] In one embodiment, the improvement comprises: knocking out the endogenous gene ROX1, and replacing "P GAL10 -ERG20-(GSG)2-CnVS" expression cassette was integrated into the ROX1 locus.

[0019] In one embodiment, the improvement comprises: knocking out the endogenous gene NEM1 and integrating the gene tHPO V480A / V482A , AtCPR1, ZSD1.

[0020] In one embodiment, the engineered Saccharomyces cerevisiae strain also knocks out the endogenous gene DPP1.

[0021] In one embodiment, the engineered Saccharomyces cerevisiae strain also complements the amino acid tag sequences TRP1 and URA3 that were knocked out during the strain construction process.

[0022] In one embodiment, the starting strain is an improvement of Saccharomyces cerevisiae CENPK2-1D with "Ty1::ADH1t-tHMG1-GAL1,10p-IDI1-TDH3t-HIS3"; the strain α02 has been disclosed in the paper "Elucidation of the Biosynthesis of Asiaticoside and Its Reconstitution in Yeast" and is named Saccharomyces cerevisiae NO1 in this application.

[0023] In one embodiment, the nucleotide sequence of the farnesyl pyrophosphate synthase gene ERG20 is shown in SEQ ID NO.1; the nucleotide sequence of the valencene synthase gene CnVS is shown in SEQ ID NO.2; the valencene oxidase gene HPO V480A / V482A The nucleotide sequence of is shown in SEQ ID NO.3; the nucleotide sequence of cytochrome P450 reductase gene AtCPR1 is shown in SEQ ID NO.4; and the nucleotide sequence of alcohol dehydrogenase gene ZSD1 is shown in SEQ ID NO.5.

[0024] In one embodiment, the nucleotide sequence of site W17 is shown as SEQ ID NO.6; the nucleotide sequence of site W18 is shown as SEQ ID NO.7.

[0025] In one embodiment, the genes ERG20, CnVS, HPO V480A / V482A , AtCPR1, and ZSD1 were integrated into the bidirectional terminator sites W17 and W18 of the Saccharomyces cerevisiae genome.

[0026] In one embodiment, the promoter gene P ERG9 , P HXT1 And the nucleotide sequence of gene HIS3 is shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0027] In one embodiment, the gene ERG9 promoter P ERG9 Replaced with gene HXT1 promoter P HXT1 , and complement the amino acid tag HIS3.

[0028] In one embodiment, the nucleotide sequence of the gene ROX1 is shown as SEQ ID NO.11.

[0029] In one embodiment, the nucleotide sequences of the genes NEM1, LEU2 are shown as SEQ ID NO.12 and SEQ ID NO.14.

[0030] In one embodiment, after the gene NEM1 is knocked out, tHPO V480A / V482A , AtCPR1, ZSD1, and LEU2 were integrated into the NEM1 site.

[0031] In one embodiment, the nucleotide sequences of the genes DPP1, TRP1, and URA3 are shown as SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17.

[0032] In one embodiment, after the gene DPP1 is knocked out, TRP1 and URA3 are integrated into the DPP1 site.

[0033] In one embodiment, the promoter P TDH3 Initiate ERG20 gene expression.

[0034] In one embodiment, the promoter P TEF Initiate expression of the CnVS gene.

[0035] In one embodiment, the promoter P PGK1 Starting HPO V480A / V482A Gene or tHPO V480A / V482A Gene expression.

[0036] In one embodiment, the promoter P TDH3 Initiate the expression of AtCPR1 gene.

[0037] In one embodiment, the promoter P TEF Initiate ZSD1 gene expression.

[0038] In one embodiment, the endogenous promoter P of Saccharomyces cerevisiae is used. HIS3 Initiate expression of the HIS3 gene.

[0039] In one embodiment, the endogenous promoter P of Saccharomyces cerevisiae is used. LEU2 Initiate LEU2 gene expression.

[0040] In one embodiment, the endogenous promoter P of Saccharomyces cerevisiae is used. TRP1 Initiate TRP1 gene expression.

[0041] In one embodiment, the endogenous promoter P of Saccharomyces cerevisiae is used. URA3 Initiate expression of the URA3 gene.

[0042] The invention also provides a method for preparing grapefruit ketone, which comprises culturing the engineering yeast of saccharomyces cerevisiae in a fermentation medium at 28-30°C.

[0043] In one embodiment, the fermentation medium includes but is not limited to YPD medium.

[0044] In one embodiment, the engineered Saccharomyces cerevisiae is inoculated into a YPD medium and fermented at 28-30° C. for at least 96 hours.

[0045] In one embodiment, feeding is also performed during the fermentation process.

[0046] In one embodiment, the feed is supplemented with glucose and one or more inorganic salts including but not limited to potassium salt, sodium salt, and magnesium salt.

[0047] In one embodiment, the potassium salt includes but is not limited to KH2PO4, the magnesium salt includes but is not limited to MgSO47H2O, and the sodium salt includes but is not limited to Na2SO3.

[0048] The present invention also provides the application of the engineered yeast Saccharomyces cerevisiae in the fields of food, cosmetics, agriculture or medicine.

[0049] In one embodiment, the use is for preparing novolac ketone or a product containing novolac ketone.

[0050] Beneficial effects:

[0051] (1) The present invention realizes the de novo synthesis of oleic acid in Saccharomyces cerevisiae and strengthens the supply and conversion of oleic acid precursors. First, the endogenous gene ERG20 of Saccharomyces cerevisiae, the oleic acid pathway genes CnVS and HPO V480A / V482A , AtCPR1, and ZSD1 were integrated into the bidirectional terminator sites W17 and W18 of the Saccharomyces cerevisiae genome, realizing the de novo synthesis of oleic acid ketone in Saccharomyces cerevisiae. On this basis, the endogenous ERG9 gene promoter sequence was knocked out, and the HXT1 gene promoter sequence and gene HIS3 were integrated to achieve the inhibition of expression of the competition pathway.

[0052] (2) The present invention knocks out the endogenous gene ROX1, screens the connecting peptides and optimizes the promoters of the pathway genes ERG20 and CnVS, and selects the selected "P GAL10 -ERG20-(GSG)2-CnVS" gene expression cassette was integrated into the ROX1 locus to increase the supply of the precursor valencene.

[0053] (3) The present invention uses AlphaFold 2 to predict the HPO V480A / V482A A signal peptide was found at the N-terminus of HPO. V480A / V482A The terminal 24 amino acids and wild-type HPO V480A / V482A and tHPO V480A / V482AAs well as the precursor valencene, tHPO was subjected to 100 nanosecond molecular dynamics simulations. V480A / V482A The B-factor of the tHPO showed less fluctuation than that of the wild-type protein. By truncating the signal peptide, the distance between the C-2 position of the substrate valencene and the iron ion of the heme was reduced from 5.7 to 3.4. V480A / V482A The pocket is more tightly bound, which is more conducive to the subsequent catalytic reaction.

[0054] (4) The present invention knocks out the endogenous gene NEM1 and enhances the gene tHPO V480A / V482A , AtCPR1, ZSD1, and LEU2 to enhance the conversion rate of the precursor and further increase the accumulation of nopalxone.

[0055] (5) The present invention also improves the growth state of the strain by knocking out the endogenous gene DPP1 and supplementing the amino acid tags TRP1 and URA3.

[0056] The strain constructed by the present invention has a 466.1 mg / L tautomerone production when the shake flask fermentation is optimized for 96 hours in a YPD medium, and a 6.5 g / L tautomerone production when the fermentation is optimized for 168 hours in a 5L fermenter. The present invention significantly improves the tautomerone production by heterologously introducing a tautomerone pathway synthesis gene and regulating an endogenous metabolic pathway of saccharomyces cerevisiae, thereby providing a reference for the subsequent expansion of tautomerone production. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the metabolism of nootoketone in Saccharomyces cerevisiae.

[0058] Figure 2 This is the gas chromatography-mass spectrometry (GC-MS) graph of oleic acid ketone in the engineered strain cultured in YPD.

[0059] Figure 3 These are the results of screening valencene oxidase truncations; A is the AlphaFold 2 prediction map; B and C are 100 nanosecond molecular dynamics simulations; and D is the distance map between the C-2 position of the substrate valencene and the iron ion of heme.

[0060] Figure 4 This is a graph showing the production of naringenone by the engineered strain under YPD culture.

[0061] Figure 5 This is a graph showing the production of naringenone by the engineered strain in a 5L fermenter using YPD culture. DETAILED DESCRIPTION

[0062] The terms "comprising", "including", "having", and "containing" used in the present invention mean "including but not limited to", "including but not limited to", "having but not limited to", and "containing but not limited to", and can be used interchangeably with corresponding phrases. Unless the context clearly indicates otherwise, the term "or" used in this article is used in this article to mean the term "and / or" and can be used interchangeably with it. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, the examples of suitable methods and materials described below are illustrative only and do not limit the present invention in any way.

[0063] (I) Culture medium

[0064] LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L. Add 20g / L agar powder to prepare LB solid medium.

[0065] YNB medium: Yeast Nutrition Base 67.4 g / L, glucose 20 g / L, amino acids (5 g / L uracil, 10 g / L tryptophan, 10 g / L leucine, 10 g / L histidine, and appropriate deletion of corresponding amino acids as needed).

[0066] YPD medium: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L.

[0067] Feed medium: glucose 800 g / L, KH2PO4 18 g / L, MgSO4 7H2O 10.24 g / L, K2SO4 7 g / L and Na2SO3 0.56 g / L.

[0068] (II) Preparation of competent cells of Saccharomyces cerevisiae: The competent cells of Saccharomyces cerevisiae were prepared using the Frozen-EZ Yeast Transformation II kit at 30°C with 10 mL of YPD medium to a medium magnitude (OD 600 =0.8-1.0). The following steps were carried out at room temperature.

[0069] 1. Centrifuge the cells at 3500 rpm for 5 min and remove the supernatant;

[0070] 2. Add 10 mL of EZ1 solution to wash the precipitate, centrifuge the precipitated cells again, and remove the supernatant;

[0071] 3. Add 1 mL of EZ2 solution to resuspend the pelleted cells.

[0072] (III) Transformation of Saccharomyces cerevisiae:

[0073] 1. Mix 50 μL competent cells with 0.2-1 μg DNA (volume less than 5 μL); add 500 μL EZ3 solution and mix thoroughly;

[0074] 2. Incubate at 30℃ for 45min. During the incubation, flick the tube or vortex 2-3 times to mix well.

[0075] 3. Transfer 50-150 μL of the transformation mixture to appropriate auxotrophic plates.

[0076] 4. Incubate the plates at 30°C for 3-5 days to grow transformants.

[0077] (IV) Determination of valeriane and viniferone: SH-Rxi-5ms column was used for gas phase detection. The detection conditions of viniferone were as follows: the sample volume was 1 L, the initial temperature was maintained at 40°C for 1 minute, then the temperature was increased to 160°C at a rate of 10°C / min, and then increased to 250°C at a rate of 15°C / min, and the temperature was maintained for 8 minutes.

[0078] (V) The strain information is shown in Table 1.

[0079] Table 1 Strains involved in the present invention

[0080]

[0081] Note: Saccharomyces cerevisiae NO1 has been published in the paper "Elucidation of the Biosynthesis of Asiaticoside and Its Reconstitution in Yeast", corresponding to the strain α02 in the paper (see Table S1).

[0082] (vi) Promoter sequence.

[0083] Table 2 Promoter sequences involved in the present invention

[0084]

[0085]

[0086]

[0087]

[0088] (VII) Connecting peptide sequence.

[0089] Table 3 Connecting peptide sequences involved in the present invention

[0090]

[0091] Example 1: ERG20, CnVS, HPO V480A / V482A Integration of AtCPR1 and ZSD1

[0092] Using NO1 as the starting strain, the endogenous gene ERG20 (nucleotide sequence as shown in SEQ ID NO.1) and the oleic acid pathway genes CnVS (nucleotide sequence as shown in SEQ ID NO.2), HPO V480A / V482A (nucleotide sequence as shown in SEQ ID NO.3), AtCPR1 (nucleotide sequence as shown in SEQ ID NO.4), and ZSD1 (nucleotide sequence as shown in SEQ ID NO.5) were integrated into the bidirectional terminator sites W17 (nucleotide sequence as shown in SEQ ID NO.6) and W18 (nucleotide sequence as shown in SEQ ID NO.7) of the Saccharomyces cerevisiae genome to obtain strain NO2.

[0093] The upstream and downstream homology arms of the W17 locus were amplified using primers W17-F-UP, W17-R-UP; W17-F-DOWN, W17-R-DOWN. The upstream and downstream homology arms of the W18 locus were amplified using primers W18-F-UP, W18-R-UP; W18-F-DOWN, W18-R-DOWN. The upstream and downstream homology arms of the W18 locus were amplified using primers ERG20-F, ERG20-R; CnVS-F, CnVS-R; HPO V480A / V482A -F, HPO V480A / V482A -F; AtCPR1-F, AtCPR1-R; ZSD1-F, ZSD1-R amplify ERG20, CnVS, HPO V480A / V482A , AtCPR1, and ZSD1 target gene fragments. The above fragments were fused by fusion PCR to obtain the expression cassette P TEF -CnVS-T TER22 -ERG20-P TDH3 , P TEF -ZSD1-T TEF -P PGK1 -HPO V480A；V482A -T TER22 -AtCPR1-P TDH3 , and were integrated into the bidirectional terminator sites W17 and W18 of the Saccharomyces cerevisiae NO1 genome by the PEG / LiAc method to construct the engineered strain NO2.

[0094] The obtained engineered strain NO2 was cultured in YPD medium at 30°C and 220rpm for 96 hours, 10% (v / v) n-dodecane was added as an extractant before the start of fermentation, and the content of oleic acid in the fermentation broth was detected after the fermentation. The gas chromatography verification results showed that the engineered strain NO2 can produce oleic acid, and the yield of oleic acid is 19.2 mg / L after 96 hours of fermentation.

[0095] Table 4 Primer sequences

[0096]

[0097] Example 2: P HXT1 Replace P ERG9 To suppress the competition path

[0098] Based on the yeast Saccharomyces cerevisiae NO2 constructed in Example 1, the ERG9 promoter P was transformed into ERG9 (nucleotide sequence as shown in SEQ ID NO.8) replaced with the promoter P of gene HXT1 HXT1 (the nucleotide sequence is shown in SEQ ID NO.9), and the amino acid tag HIS3 (the nucleotide sequence is shown in SEQ ID NO.10) was complemented to obtain strain NO3.

[0099] With primer P ERG9 -F-UP, P ERG9 -R-UP;P ERG9 -F-DOWN, P ERG9 -R-DOWN amplifies P ERG9 The upstream and downstream homology arms of the site were amplified with primer P HXT1 -F, P HXT1 -R; HIS3-F, HIS3-R amplification P HXT1 The promoter sequence and HIS3 target gene sequence were synthesized by fusion PCR to obtain the expression cassette P HXT1 -HIS3, integrated into the ERG9 promoter of Saccharomyces cerevisiae NO2 by PEG / LiAc method ERG9 The engineered strain NO3 with the promoter sequence replaced was constructed.

[0100] The obtained engineered strain NO3 was fermented in YPD medium at 30°C and 220rpm for 96 hours. 10% (v / v) n-dodecane was added as an extractant before the start of fermentation. After the fermentation, the content of oleic acid in the fermentation liquid was detected, and the yield of oleic acid was 54.5 mg / L.

[0101] Table 5 Primer sequences

[0102]

[0103]

[0104] Example 3: Integration of the ERG20-(GSG)2-CnVS expression cassette at the ROX1 locus to enhance precursor supply

[0105] In order to promote the supply of the precursor valencene, based on the Saccharomyces cerevisiae NO3 constructed in Example 2, the endogenous gene ROX1 (nucleotide sequence as SEQ ID NO.11) of the Saccharomyces cerevisiae strain NO3 was knocked out using multi-gene editing technology. The pathway genes ERG20 (nucleotide sequence as shown in SEQ ID NO.1) and CnVS (nucleotide sequence as shown in SEQ ID NO.2) were screened for connecting peptides and promoters were optimized. The screening types are shown in Table 4. GAL10 -ERG20-(GSG)2-CnVS was used as an example. The specific steps were as follows: primers ERG20-F, ERG20-R; CnVS-F, CnVS-R were used to amplify the ERG20 and CnVS target gene sequences, and Gibson assembly was used to construct a P GAL10 -ERG20-(GSG)2-CnVS expression cassette plasmid to obtain PY26-P GAL10 -ERG20-(GSG)2-CnVS, PY26-P GAL10 -ERG20-(GSG)2-CnVS was expressed in the NO1 strain and fermented in YNB medium at 30°C and 220rpm for 96 hours. 10% (v / v) n-dodecane was added as an extractant before the start of fermentation. After the fermentation, the valencene content in the fermentation broth was detected and the yield was 29.67 mg / L.

[0106] Primers ROX1-F-UP, ROX1-R-UP; ROX1-F-DOWN, ROX1-R-DOWN were used to amplify the upstream and downstream homology arms of the ROX1 locus, and primers ERG20-F, ERG20-R; CnVS-F, CnVS-R were used to amplify the ERG20 and CnVS target gene sequences. The above fragments were fused by fusion PCR to obtain the expression cassette P GAL10 -ERG20-(GSG)2-CnVS was integrated into the ROX1 site of Saccharomyces cerevisiae NO3 by the PEG / LiAc method to construct the engineered strain NO4.

[0107] The obtained engineered strain NO4 was fermented in YPD medium at 30°C and 220rpm for 96 hours. 10% (v / v) n-dodecane was added as an extractant before the start of fermentation. After the fermentation, the content of oleic acid in the fermentation liquid was detected, and the yield of oleic acid was 124.2 mg / L.

[0108] Table 6 Connector peptide screening and promoter optimization types

[0109]

[0110]

[0111] Table 7 Primer sequences

[0112]

[0113] Example 4: tHPO V480A / V482A Signal peptide truncation

[0114] By using AlphaFold 2 prediction, in HPO V480A / V482A A signal peptide was found at the N-terminus of HPO V480A / V482A The N-terminal 24 amino acids. V480A / V482A (coding sequence as shown in SEQ ID NO.3) and tHPO V480A / V482A (coding sequence as shown in SEQ ID NO.13) and the precursor valencene were subjected to 100 nanosecond molecular dynamics simulations, tHPO V480A / V482A The B-factor of the tHPO showed less fluctuation than that of the wild-type protein. By truncating the signal peptide, the distance between the C-2 position of the substrate valencene and the iron ion of the heme was reduced from 5.7 to 3.4. V480A / V482A The pocket is more tightly bound, which is more conducive to the subsequent catalytic reaction.

[0115] Example 5: NEM1 site tHPO V480A / V482A , AtCPR1, and ZSD1 integration enhances pathway gene expression

[0116] In order to improve the conversion of the precursor valencene and promote the accumulation of the final product naringenone, based on the Saccharomyces cerevisiae NO4 constructed in Example 3, the endogenous gene NEM1 (nucleotide sequence such as SEQ ID NO.12) of the Saccharomyces cerevisiae strain NO4 was knocked out by homologous recombination technology. The truncated naringenone pathway gene tHPO was overexpressed at the NEM1 site. V480A / V482A (nucleotide sequence shown in SEQ ID NO.13), AtCPR1 (nucleotide sequence shown in SEQ ID NO.4), ZSD1 (nucleotide sequence shown in SEQ ID NO.5), and back-complemented LEU2 amino acid tag (nucleotide sequence shown in SEQ ID NO.14) to obtain strain NO5.

[0117] The specific steps are: use primers NEM1-F-UP, NEM1-R-UP; NEM1-F-DOWN, NEM1-R-DOWN to amplify the upstream and downstream homology arms of the NEM1 site. V480A / V482A -F, tHPO V480A / V482A -F; AtCPR1-F, AtCPR1-R; ZSD1-F, ZSD1-R; LEU2-F, LEU2-R amplify tHPO V480A / V482A , AtCPR1, ZSD1, LEU2 target gene fragments. The above fragments were fused by fusion PCR to obtain the expression cassette P TEF -ZSD1-T TEF -P PGK1 -tHPO V480A；V482A -T TER22 -AtCPR1-P TDH3 -LEU2 was integrated into the NEM1 site of Saccharomyces cerevisiae strain NO4 by the PEG / LiAc method to construct the engineered strain NO5.

[0118] The obtained engineered strain NO5 was fermented in YPD medium at 30°C and 220rpm for 96 hours. 10% (v / v) n-dodecane was added as an extractant before the start of fermentation. After the fermentation, the content of oleic acid in the fermentation liquid was detected, and the yield of oleic acid was 466.1 mg / L.

[0119] Table 8 Primer sequences

[0120]

[0121]

[0122] Example 6: Replenishing TRP1 and URA3 amino acid tags to improve bacterial growth

[0123] In order to improve bacterial growth, based on the Saccharomyces cerevisiae NO5 constructed in Example 5, the endogenous gene DPP1 (nucleotide sequence as shown in SEQ ID NO.15) of the Saccharomyces cerevisiae strain NO5 was knocked out by multi-gene editing technology, and the defective amino acids TRP1 (nucleotide sequence as shown in SEQ ID NO.16) and URA3 (nucleotide sequence as shown in SEQ ID NO.17) of the Saccharomyces cerevisiae were complemented, and the upstream and downstream homologous arms of the DPP1 site were amplified using primers DPP1-F-UP, DPP1-R-UP; DPP1-F-DOWN, DPP1-R-DOWN. The target sequence genes of TRP1 and URA3 were amplified using primers TRP1-F, TRP1-R; URA3-F, URA3-R. The above fragments were fused by the fusion PCR method to obtain the expression cassette TRP1-URA3, which was integrated into the DPP1 site of Saccharomyces cerevisiae NO5 by the PEG / LiAc method to construct the engineered strain NO6.

[0124] The obtained engineered strain NO6 was fermented in YPD medium at 30°C and 220rpm for 96 hours, and 10% (v / v) n-dodecane was added as an extractant before the start of fermentation. The yield of oleic acid in the fermentation broth and the OD value of the bacteria were detected at the end of fermentation. The results showed that the growth of the bacteria was slightly improved, and the yield of oleic acid remained unchanged.

[0125] Table 9 Primer sequences

[0126]

[0127] Example 7: Preparation of grapefruit ketone by fermentation of strain NO6 in a fermenter

[0128] The strain NO6 constructed by Example 6 was fermented in a 5-L fermentation tank. Three or four fresh single colonies were selected and inoculated into 25mL YPD medium. After culturing for 17-20 hours at 30°C and 220rpm, 10% of the seed liquid was transferred to 200mL YPD medium and then cultivated for 17-20 hours. Based on the above work, at the beginning of fermentation, 200mL of seed liquid was added to a 5L bioreactor containing 2.3L YPD medium, 250μL defoamer (0.01%, v / v) and 250mL n-dodecane (10%, v / v). Dissolved oxygen was controlled to 30%, pH was controlled to 5.4, and temperature was controlled to 30°C. During the fermentation, 5M NaOH was used to adjust the pH value. Fermented at 30°C, the feed medium was added when the initial medium glucose was exhausted, so that the concentration of glucose in the fermentation system tended to 0g / L and the ethanol concentration was ≤10g / L. Regular sampling was performed during the fermentation to detect the content of grapefruit ketone. The results showed that the yield of grapefruit ketone was 6.5 g / L after 168 h of fermentation.

[0129] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A polynucleotide, characterized in that For (a) or (b): (a) With P GAL10 -ERG20-(GSG)2-CnVS shown in the structure of the polynucleotide; wherein the promoter P GAL10 The nucleotide sequence of is shown in SEQ ID NO.18; the nucleotide sequence of ERG20 is shown in SEQ ID NO.1; the nucleotide sequence of (GSG)2 is shown in SEQ ID NO.19; the nucleotide sequence of CnVS is shown in SEQ ID NO.2; (b) The gene encoding valencene oxidase shown in SEQ ID NO.

13.

2. A recombinant microorganism expressing the polynucleotide of claim 1.

3. An engineered strain of Saccharomyces cerevisiae producing naringenone, characterized in that: Based on the starting strain, there is at least one of the following improvements: (1) expressing the polynucleotide according to claim 1; (2) The farnesyl pyrophosphate synthase gene ERG20, valencene synthase gene CnVS, and valencene oxidase gene HPO were integrated and expressed in the genome. V480A / V482A , cytochrome P450 reductase gene AtCPR1, alcohol dehydrogenase gene ZSD1; (3) replacing the endogenous ERG9 gene promoter sequence with the HXT1 gene promoter sequence; (4) Reduce the expression of endogenous genes NEM1 and DPP1.

4. The engineered yeast of claim 3, characterized in that: The farnesyl pyrophosphate synthase gene ERG20, valencene synthase gene CnVS, valencene oxidase gene HPO are expressed at one or more sites in W17, W18, and NEM1. V480A / V482A , cytochrome P450 reductase gene AtCPR1 or alcohol dehydrogenase gene ZSD1.

5. The engineered yeast of claim 3, characterized in that: The endogenous gene ERG20 of Saccharomyces cerevisiae and the oleic acid pathway gene CnVS were integrated into the bidirectional terminator site W17 of the Saccharomyces cerevisiae genome, and the gene HPO V480A / V482A , AtCPR1, and ZSD1 were integrated into the bidirectional terminator site W18 of the Saccharomyces cerevisiae genome.

6. The engineered yeast of claim 3 or 5, characterized in that: The endogenous ERG9 gene promoter sequence of the starting strain was knocked out, and the HXT1 gene promoter sequence was integrated.

7. The engineered yeast of any one of claims 3 to 6, characterized in that: "P GAL10 -ERG20-(GSG)2-CnVS" expression cassette was integrated into the ROX1 locus.

8. The engineered yeast of any one of claims 3 to 7, characterized in that: Knock out the endogenous gene NEM1 and integrate the truncated valencene oxidase gene tHPO at the modified site V480A / V482A , cytochrome P450 reductase gene AtCPR1, alcohol dehydrogenase gene ZSD1.

9. A method for preparing grapefruit ketone, characterized in that: The fermentation is carried out using the engineered yeast of any one of claims 3 to 8.

10. The method according to claim 9, characterized in that The engineered yeast of Saccharomyces cerevisiae is fermented in a fermentation medium at 28-30° C. for at least 96 hours.

11. The method according to claim 9 or 10, characterized in that: Feeding is performed during the fermentation process; the feeding includes but is not limited to adding glucose.

12. The method according to claim 11, characterized in that The feed is supplemented with glucose and one or more inorganic salts including but not limited to potassium salt, sodium salt and magnesium salt.

13. Use of the engineered yeast of Saccharomyces cerevisiae according to any one of claims 3 to 8, or the method according to any one of claims 9 to 12 in the preparation of nopalxone and / or products containing nopalxone.

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