Yarrowia lipolytica strain for synthesizing (2S)-naringenin and application of Yarrowia lipolytica strain

Abstract

The invention discloses a Sarcabomyces lipolytica strain for synthesizing (2S)-naringenin and application of the Sarcabomyces lipolytica strain, and belongs to the technical field of engineering and bioengineering. According to the invention, a key enzyme CHS is subjected to semi-rational transformation, so that the yield of the strain (2S)-naringenin expressing the mutant V196A / T132C is increased by 60%. According to the method, the modified CHS is further integrated into a yeast genome, conversion of p-coumaric acid to (2S)-naringenin is promoted by strengthening a shikimic acid pathway and enhancing supply of cofactors, so that the yield of the (2S)-naringenin reaches 776mg / L after the constructed strain is fermented in a 24-pore plate for 96 hours, and the yield of the constructed strain can reach 8.65 g / L after the strain is fermented in a 5L fermentation tank; the method lays a foundation for the subsequent biosynthesis of flavonoid compounds, and has potential value and significance for the development of synthetic biology.

Description

Technical Field

[0001] The invention relates to a Yarrowia lipolytica strain for synthesizing (2S)-naringenin and application thereof, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] (2S)-naringenin is a natural flavonoid compound widely found in the peel and pulp of Rutaceae plants such as tomatoes, grapefruits, oranges, and tangerines. It has been widely used in food, chemical industry, and medicine due to its antibacterial, anti-inflammatory, antioxidant, anti-atherosclerotic, liver protection, and lipid metabolism regulation. (2S)-naringenin is biosynthesized through the phenylpropanoid pathway. First, L-Phe / L-Tyr is deaminated by phenylalanine / tyrosine ammonia lyase (PAL / TAL) to form p-coumaric acid, then 4-coumaroyl-CoA ligase (4CL) to form 4-coumaroyl-CoA, and then chalcone synthase (CHS) combines with three molecules of malonyl-CoA to synthesize naringenin chalcone. Finally, naringenin chalcone is catalyzed by chalcone isomerase (CHI) to generate (2S)-naringenin.

[0003] At present, (2S)-naringenin is mainly extracted from plants, which is time-consuming and labor-intensive, and has low production efficiency. The chemical synthesis method uses toxic solvents and extreme chemical reaction conditions, is difficult to handle, and is not suitable for large-scale production. In contrast, the microbial method has obvious advantages such as economy, environmental protection, and low production cost, and is a promising alternative. Therefore, it is possible to consider applying the biosynthesis method based on metabolic engineering and enzyme engineering to obtain (2S)-naringenin. However, the current microbial method for producing (2S)-naringenin still has the problem of low yield. Summary of the invention

[0004] The invention provides a chalcone synthase mutant, which has mutations of one or more amino acids at positions 132, 196 and 208 on the basis of the parent.

[0005] In one embodiment, the mutant is obtained by mutating the threonine at position 132 to cysteine ​​based on the parent SjCHS, thereby obtaining the mutant T132C.

[0006] In one embodiment, the mutant is obtained by mutating the threonine at position 132 to cysteine ​​and the valine at position 196 to alanine based on the parent, thereby obtaining the mutant V196A / T132C.

[0007] In one embodiment, the parent SjCHS has the amino acid sequence shown in SEQ ID NO.23.

[0008] The present invention also provides a gene encoding the mutant.

[0009] The invention also provides an expression cassette containing the gene.

[0010] In one embodiment, the expression cassette includes but is not limited to:

[0011] (a); Expression cassette "P" for expressing SjCHS and MsCHI TEF -SjCHS V196A / T132C -T XPR2 -P TEF -MsCHI-T XPR2 ”

[0012] (b) The expression cassette "P" for expressing FjTAL, SjCHS, and MsCHI TEF -FjTAL-T XPR2 -P TEF -SjCHS-T XPR2 -P TEF -MsCHI-T XPR2 ”;

[0013] (c) The expression cassette "P" for expressing FjTAL, Pc4CL, AtCHS, and MsCHI TEF -FjTAL-T XPR2 -P TEF -Pc4CL-T XPR2 -P TEF -AtCHS-T XPR2 -P TEF -MsCHI-T XPR2 ”.

[0014] The present invention also provides a recombinant microorganism expressing the mutation.

[0015] The present invention also provides an engineered strain of Yarrowia lipolytica with high yield of (2S)-naringenin, which expresses tyrosine ammonia lyase FjTAL from Flavobacterium johnsonii, 4-coumarate coenzyme ligase Pc4CL from parsley, chalcone synthase SjCHS from Sophora japonica or its mutant, and chalcone isomerase MsCHI from alfalfa on the basis of a starting strain.

[0016] In one embodiment, the engineered bacteria further overexpress SjCHS or a mutant thereof; the overexpression includes but is not limited to integrating the coding gene of SjCHS or a mutant thereof into the A3 site, the AXP site and the E3 site.

[0017] In one embodiment, the engineered bacteria further overexpress FjTAL, Pc4CL, SjCHS and MsCHI to construct strains NAG04-NAG07. It was found that overexpression of SjCHS can increase the yield of (2S)-naringenin by 1.2 times.

[0018] In one embodiment, the engineered bacteria also enhance the expression of shikimate pathway genes; the shikimate pathway genes include but are not limited to genes YIARO1, YIARO2, and a highly active mutant YIARO3 that is feedback-resistant to aromatic amino acid regulation. K225L 、YlARO4 K225L 、YlARO7 G139S and EcAROG G146N One or more of .

[0019] In one embodiment, the engineered bacteria also enhances malonyl-CoA supply.

[0020] In one embodiment, the engineered bacteria also enhance the supply of NADPH.

[0021] In one embodiment, the starting strain is Yarrowia lipolytica ΔKu70; the Yarrowia lipolytica ΔKu70 is disclosed in the paper "Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica.".

[0022] In one embodiment, the strong promoter PTEF is used to drive gene expression.

[0023] In one embodiment, the nucleotide sequence of the tyrosine ammonia lyase of FjTAL derived from Flavobacterium johnsonii is shown as SEQ ID NO.1.

[0024] In one embodiment, the nucleotide sequence of the 4-coumarate coenzyme ligase Pc4CL derived from parsley is shown as SEQ ID NO.2.

[0025] In one embodiment, the nucleotide sequence of the chalcone synthase SjCHS derived from Epimedium is shown as SEQ ID NO.3.

[0026] In one embodiment, the nucleotide sequence of the chalcone isomerase MsCHI derived from alfalfa is shown as SEQ ID NO.4.

[0027] In one embodiment, the genes FjTAL, Pc4CL, SjCHS, and MsCHI are integrated together at the D17 site.

[0028] In one embodiment, the genes SjCHS and MsCHI are also integrated together at the AXP site.

[0029] In one embodiment, the promoter P TEF Expression of genes involved in the synthesis of (2S)-naringenin.

[0030] In one embodiment, the Yarrowia lipolytica engineered bacteria also overexpresses the Yarrowia lipolytica endogenous shikimic acid pathway genes YIARO1 and YIARO2 and a highly active mutant YIARO3 that is feedback-resistant to aromatic amino acid regulation. K225L 、YlARO4 K225L 、YlARO7 G139S and EcAROG G146N ; Genes YIARO1, YIARO2, YIARO3 K225L 、YlARO4 K225L 、YlARO7 G139S 、EcAROG G146N The nucleotide sequences are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.

[0031] In one embodiment, the engineered Yarrowia lipolytica bacteria also overexpresses a gene that enhances a cofactor.

[0032] In one embodiment, the genes that enhance the cofactors include one or more of the citrate synthase gene Y1CS, the α-ketoglutarate dehydrogenase gene Y10GDC, and the isocitrate dehydrogenase gene Y1IDH.

[0033] In one embodiment, the nucleotide sequence of gene YlCS is shown as SEQ ID NO.13; the nucleotide sequence of gene YlOGDC is shown as SEQ ID NO.14; and the nucleotide sequence of gene YlIDH is shown as SEQ ID NO.15.

[0034] The present invention also provides the use of the Yarrowia lipolytica engineered bacteria in producing (2S)-naringenin.

[0035] In one embodiment, the engineered Yarrowia lipolytica is inoculated into a YPD medium and fermented at 30° C. for 96 to 120 hours.

[0036] In one embodiment, the engineered bacteria of Yarrowia lipolytica are inoculated into a fermentation medium, fermented at 28° C. for 156 hours, and the dissolved oxygen level is controlled at 10-20%; the pH is controlled at 4.9 during the fermentation process; and glucose is fed when the glucose is lower than 1 g / L to keep the glucose concentration at 0.1 to 1 g / L.

[0037] In one embodiment, the fermentation medium contains: 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / LMgSO4·7H2O

[0038] In one embodiment, the fermentation is for at least 156 hours.

[0039] The present invention also seeks to protect the application of the engineered Yarrowia lipolytica in producing flavonoid-containing products in the fields of food, medicine and chemical industry.

[0040] Beneficial effects:

[0041] 1. The present invention screens exogenous (2S)-naringenin synthesis genes in lipolytic yeast and uses a strong promoter P TEF The genes were activated to obtain the best strain producing the initial (2S)-naringenin.

[0042] 2. The present invention increases the yield of (2S)-naringenin in the strain expressing the mutant V196A / T132C by 60% by semi-rational modification of the key enzyme CHS.

[0043] 3. The present invention further integrates the modified CHS into the yeast genome, and promotes the conversion of p-coumaric acid to (2S)-naringenin by strengthening the shikimic acid pathway and enhancing the supply of cofactors, thereby further increasing the yield of (2S)-naringenin. The yield of (2S)-naringenin in the constructed strain reaches 776 mg / L after 96 fermentations in a 24-well plate.

[0044] 4. The strain constructed by the present invention was fermented in a 5L fermenter to obtain 8.65 g / L of (2S)-naringenin. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the metabolism of heterologous synthesis of (2S)-naringenin in Yarrowia lipolytica.

[0046] Figure 2 This is a graph showing the (2S)-naringenin production of engineered strains NAG01 to NAG04 under YPD culture.

[0047] Figure 3 (2S)-naringenin production diagram to verify that CHS is the key enzyme.

[0048] Figure 4The (2S)-naringenin production map shows the semi-rational modification of the key enzyme CHS and site-specific integration of the modified key enzyme.

[0049] Figure 5 Diagram of (2S)-naringenin production for enhanced shikimate pathway flux.

[0050] Figure 6 The (2S)-naringenin production diagram is shown after the NCM pathway is introduced and TCA cycle-related genes are overexpressed.

[0051] Figure 7 The graph shows the (2S)-naringenin production in a fed-batch culture in a 5 L fermenter. DETAILED DESCRIPTION

[0052] (I) Culture medium

[0053] LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L. Add 20g / L agar powder to prepare LB solid medium.

[0054] 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).

[0055] YPD medium: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L.

[0056] (II) Preparation of competent cells of Y. lipolytica: The competent cells of Y. lipolytica were prepared using the Frozen-EZYeast Transformation II kit and cultured with 5 mL YPD medium at 30°C until the OD 600 =0.8-1.0). The following steps were carried out at room temperature.

[0057] 1. Centrifuge the cells at 3500 rpm for 5 min and remove the supernatant;

[0058] 2. Add 10 mL of EZ1 solution to wash the precipitate, centrifuge the precipitated cells again, and remove the supernatant;

[0059] 3. Add 1 mL of EZ2 solution to resuspend the pelleted cells.

[0060] (III) Transformation of Yarrowia lipolytica:

[0061] 1. Scrape the yeast colony from the YPD plate and inoculate it into YPD liquid medium, then place it in a constant temperature incubator at 30°C and grow for 16-22 hours;

[0062] 2. Prepare yeast transformation buffer solution (if multiple transformations are required, magnification × n): 50% PEG3350, added in a volume of 90 μL; 2M lithium acetate (M CHCOOLi 65.99), volume 5 μL; ssDNA, volume 5 μL, ssDNA needs to be boiled in water for 3 min before adding to the transformation buffer, and then put on ice for cooling;

[0063] 3. Use a pipette to draw 500 μL of bacterial solution into a centrifuge tube, centrifuge the tube at an appropriate speed, and discard the supernatant to obtain yeast cells. Transfer the yeast to a centrifuge tube containing 100 μL of transformation buffer, slowly blow and mix, add 0.25-0.5 μg of plasmid DNA or linear DNA, mix thoroughly and oscillate for 2 minutes (add at least 0.25-0.5 μg of DNA to each plasmid);

[0064] 4. Incubate the centrifuge tube containing the transformation mixture in a metal bath at 30°C for 30-45 min, oscillate the mixture for 15 s every 10 min, and then heat shock at 39°C for an additional 10 min to improve transformation efficiency;

[0065] 5. Transfer the centrifuge tube to a centrifuge and centrifuge at 8000 rpm for 2 minutes. Pour out the supernatant in a sterile clean bench, add an appropriate amount of sterile water to resuspend the bacteria, and add it to the YPD solid medium. Use a sterile coating rod to evenly spread on the YPD solid medium, and then place the medium in a constant temperature incubator at 30°C for cultivation.

[0066] (iv) HPLC determination of (2S)-naringenin: Shimadzu high performance liquid chromatography was used for determination. HPLC conditions: the column box temperature was set at 30°C, the flow rate was 0.7 mL / min, and the injection volume was 5 μL. Mobile phase A was 10 mM ammonium formate (pH 3.0, adjusted by formic acid), and mobile phase B was acetonitrile. The mobile phase for the first 0.5 minutes was A:B = 95.0%:5.0%. Then, the solvent composition was changed according to a linear gradient, and at the 7th minute it was A:B = 40.0%:60.0%. After 9.6 minutes, the gradient returned to the initial conditions (A:B = 95.0%:5.0%) and remained unchanged until 12 minutes after the end of the run. The peak time of p-coumaric acid at 277 nm was 4.7 minutes. The peak of naringenin at 290 nm has a retention time of 5.7 minutes.

[0067] (V) The strain information is shown in Table 1.

[0068] Table 1 Strains involved in the present invention

[0069]

[0070] (VI) Integration sites involved in specific implementations:

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] Example 1: Construction of the (2S)-naringenin synthesis pathway in Yarrowia lipolytica

[0080] In order to synthesize (2S)-naringenin, AtCHS and AtCHI in Arabidopsis thaliana, SjCHS in Sophora japonica and MsCHI in alfalfa were co-expressed with FjTAL and Pc4CL in different combinations in Saccharomyces lipolytica. All heterologous genes were synthesized by the company to obtain a plasmid template containing the target gene, wherein the nucleotide sequence of the gene AtCHS is shown in SEQ ID NO.21; the nucleotide sequence of the gene AtCHI is shown in SEQ ID NO.22; the nucleotide sequence of the gene SjCHS is shown in SEQ ID NO.3; the nucleotide sequence of the gene MsCHI is shown in SEQ ID NO.4; the nucleotide sequence of the gene FjTAL is shown in SEQ ID NO.1; and the nucleotide sequence of the gene Pc4CL is shown in SEQ ID NO.2.

[0081] First, we used Snapgene software to design primers for heterologous genes, and used primers D17-BF / D17-BR and AXP-BF / AXP-BR to PCR amplify the homology arms of the D17 site and the AXP site and the fragment formed by the target gene. During the yeast transformation process, the PCR amplified fragments and the Cas9 plasmid of the corresponding site were added. Then, the genes of the (2S)-naringenin biosynthesis pathway were combined, first integrating FjTAL and Pc4CL at the D17 site, and then integrating AtCHS-MsCHI, AtCHS-AtCHI, SjCHS-MsCHI and SjCHS-AtCHI at the AXP site, respectively. The constructed engineered bacteria were named NAG01-NAG04.

[0082] The engineered bacteria NAG01, NAG02, NAG03, and NAG04 were fermented in YPD medium at 30°C and 220 rpm for 96 h, and the fermentation broth was extracted and identified by liquid phase. Figure 2 As shown in FIG. 1 , the four engineered strains constructed in this example all achieved the synthesis of (2S)-naringenin, and the gene combination FjTAL-Pc4CL-SjCHS-MsCHI showed the highest (2S)-naringenin production, reaching 24.1 mg / L. Figure 2 .

[0083] Table 2 Primers and sequences

[0084]

[0085]

[0086] Example 2: Screening of key enzymes affecting the conversion of p-coumaric acid to (2S)-naringenin

[0087] According to prior art reports, CHS is the rate-limiting enzyme in the biosynthesis of (2S)-naringenin. A gene integration expression cassette was constructed by referring to the method of Example 1, and FjTAL, Pc4CL, SjCHS and MsCHI were overexpressed in NAG03, respectively, to construct strains NAG05-NAG08. Fermentation was performed according to the method of Example 1. The results showed that overexpression of SjCHS could increase the yield of (2S)-naringenin by 1.2 times ( Figure 3 ). Therefore, it is crucial to improve the activity of the key enzyme CHS to effectively convert p-coumaric acid into (2S)-naringenin.

[0088] Example 3: Semi-rational transformation of the key enzyme SjCHS

[0089] (1) In order to improve the activity of the key enzyme SjCHS and promote the efficient conversion of p-coumaric acid to (2S)-naringenin, the key enzyme CHS was semi-rationally modified. In order to infer the interaction between the enzyme and the substrate, the enzyme and the substrate were docked and analyzed. Based on the obtained conformation, the active sites Thr132, Gly218 and Phe373 were selected as potential mutation sites, and alanine mutation was performed.

[0090] According to the method of Example 1, a strain D17::P that does not produce (2S)-naringenin was constructed. TEF -FjTAL-T XPR2 -P TEF -Pc4CL-T XPR2 -P TEF -AtCHS-T XPR2 , and on this basis, a fragment containing the SjCHS mutant gene was integrated at the A3 site, and the strain expressing the mutant was fermented according to the method of Example 1. The results showed that the expression of CHS mutant V196A could increase the yield of (2S)-naringenin from 24 mg / L to 29 mg / L ( Figure 4 B).

[0091] (2) Based on V196A, iterative mutations were performed, and the polar residues of T132 and S133 were replaced by the same residue replacement strategy. On the basis of V196A, mutations were introduced into T132 and S133, respectively. The constructed recombinant bacteria were cultured in YPD medium according to the method of Example 1. The results showed that the CHS mutant V196A / T132C could further increase the yield to 38 mg / L. Compared with the control (NAG03), the yield of (2S)-naringenin was increased by 60% ( Figure 4 C).

[0092] (3) Construct an expression cassette P containing the mutant V196A / T132C and MsCHI according to the method of Example 1 TEF -SjCHS V196A / T132C -T XPR2- P TEF -MsCHI-T XPR2 The expression cassette was site-specifically integrated at the A3 site, AXP site and E3 site of strain NAG03 for three rounds to obtain strains NA09 to NAG11. The strains were fermented according to the method of Example 1. The results showed that the precursor p-coumaric acid of strain NAG11 was 45 mg / L, and the (2S)-naringenin production reached 246.4 mg / L ( Figure 4 D).

[0093] Example 4: Enhancement of the Shikimate Pathway

[0094] To promote the supply of precursors to coumaric acid, the endogenous genes YIARO1 (nucleotide sequence shown in SEQ ID NO.5), YIARO2 (nucleotide sequence shown in SEQ ID NO.6) of Y. lipolytica and the highly active mutant YIARO3 with feedback resistance to aromatic amino acid regulation were overexpressed. K225L 、YlARO4 K225L 、YlARO7 G139S and EcAROG G146N (The nucleotide sequences are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10) further improve the production of (2S)-naringenin.

[0095] The specific steps are as follows: using primers YlARO1-F / YlARO1-R to amplify gene YlARO1 from the Y. lipolytica genome, using primers YlARO2-F / YlARO2-R to amplify gene YlARO2 from the Y. lipolytica genome, using primers YlARO4-F / YlARO4-R to amplify gene YlARO4 from the Y. lipolytica genome, using primers YlARO3-F / YlARO4-R to amplify gene YlARO4 from the Y. lipolytica genome, ARO3-R was used to amplify the gene YlARO3 from the Yarrowia lipolytica genome, and the primers YlARO7-F / YlARO7-R were used to amplify the gene YlARO7 from the Yarrowia lipolytica genome, and the primers EcAROG-F / EcAROG-R were used to amplify the gene EcAROG from the Yarrowia lipolytica genome, and the primers F1-3-BF / F1-3-BR were used to amplify the upstream and downstream homology arms of the F1-3 site. K225L Fragment, YlARO4 K225L Fragment, YlARO7 G139SFragment, EcAROG G146N The fragment, about 1 μg of the integrated fragment and about 600 ng of sgRNA were transformed into the Yeast lipolytica engineered strain using the yeast transformation kit Frozen-EZ Yeast Transformation II, spread on the screening solid medium, and cultured at 30°C for 3 days until colonies appeared. The obtained engineered strains NAG12 to NAG18 were fermented in YPD medium at 30°C and 220 rpm for 96 hours, and the fermentation broth was extracted and analyzed by HPLC. The results showed that the recombinant engineered strain NAG18 had the highest yield of (2S)-naringenin, which was 478.4 mg / L ( Figure 5 ).

[0096] Table 3 Primer sequences

[0097]

[0098] Example 5: Strengthening the supply of malonyl-CoA to increase (2S)-naringenin production

[0099] In the synthesis process of (2S)-naringenin, the catalysis of the key enzyme CHS requires the supply of malonyl-CoA. Therefore, increasing the content of malonyl-CoA is a feasible strategy to increase the titer of (2S)-naringenin. The NCM pathway MCR-C-BauA (the nucleotide sequence of MCR-C is shown in SEQ ID NO.11; the nucleotide sequence of BauA is shown in SEQ ID NO.12) was introduced into the strain NAG18 constructed in Example 4, and the above genes were amplified using primers MCR-CF / and MCR-CR, BauA-F and BauA-R, respectively, and integrated at the C7 site to obtain strain NAG19. The constructed strain NAG19 was fermented in YPD medium at 30°C and 220rpm for 96h. The results showed that the yield of (2S)-naringenin was increased from 478.4mg / L to 513.5mg / L (compared with NAG18). Figure 6 B).

[0100] Based on strain NAG18, the key genes of TCA cycle CS (nucleotide sequence as shown in SEQ ID NO.13), OGDC (nucleotide sequence as shown in SEQ ID NO.14) and IDH (nucleotide sequence as shown in SEQ ID NO.15) and ScACS1 (nucleotide sequence as shown in SEQ ID NO.16), ScACS2 (nucleotide sequence as shown in SEQ ID NO.17) and YlACS (nucleotide sequence as shown in SEQ ID NO.18) that decompose pyruvate into acetyl-CoA were overexpressed to further increase the content of malonyl-CoA. The specific steps are: using primers YlCS-F / YlCS-R, YlOGDC-F / YlOGDC-R, YlIDH-F / YlIDH-R, ScACS1-F / ScACS1-R, ScACS2-F / ScACS2-R, YlACS-F / YlACS-R, respectively, amplify the above genes from the genome, integrate them at the C7 site, and construct strains named NAG20-NAG25. The strains were fermented in a 24-well plate containing YPD medium at 30°C for 96 h. The results showed that the (2S)-naringenin content in the fermentation broth of strains NAG20-NAG22 increased significantly, while the (2S)-naringenin level in the fermentation broth of strains NAG23 and NAG24 overexpressing ACS did not change significantly ( Figure 6 B).

[0101] According to the same method as above, the genes CS, OGDC and IDH were co-expressed in strain NAG19 to construct strain NAG26. Strain NAG26 was fermented in a 24-well plate containing YPD medium at 30°C and 220rpm for 96h. The results showed that the yield of (2S)-naringenin could reach 582.5mg / L ( Figure 6 B).

[0102] Example 6: Strengthening NADPH supply to increase (2S)-naringenin production

[0103] Establish an NADPH regeneration system to promote the synthesis of (2S)-naringenin, that is, overexpress ZWF1 (nucleotide sequence as shown in SEQ ID NO.19) and POS5 (nucleotide sequence as shown in SEQ ID NO.20) in strain NAG26 to increase the regeneration of NADPH. The specific steps are: amplify the above genes from the genome with primers YlZWF1-F / YlZWF1-R and YlPOS5-F / YlPOS5-R, respectively, integrate them at the B6 site, and construct strain NAG27. The constructed strain NAG27 was fermented in YPD medium at 30°C and 220rpm for 96h. The results showed that ( Figure 6B), the (2S)-naringenin production in the fermentation broth of strain NAG27 reached 615.0 mg / L, but a certain amount of p-coumaric acid (194.2 mg / L) was still accumulated at this time.

[0104] Considering the accumulation of p-coumaric acid, which cannot be completely converted into (2S)-naringenin, the subsequent goal is to use the high-copy sites identified in Saccharomyces lipolytica to improve the downstream metabolic pathway. Therefore, the Pc4CL-SjCHS-MsCHI fusion expression cassette (P TEF -FjTAL-T XPR2 -P TEF -SjCHS-T XPR2 -P TEF -MsCHI-T XPR2 ) and carried the URA3 tag, which was expressed in strain NAG27 and fermented in 24-well plates to obtain strains NAG28-NAG38, respectively. The strains were fermented in YPD medium at 30°C and 220rpm for 96h, and the yields reached 583-705.5mg / L, among which the (2S)-naringenin titer in the fermentation broth of strain NAG38 was 705.5mg / L, the p-coumaric acid yield was reduced to 86mg / L, and the conversion rate reached 60%.

[0105] In order to further convert p-coumaric acid into (2S)-naringenin, the fusion expression cassette carrying Pc4CL-SjCHS-MsCHI (P TEF -FjTAL-T XPR2 -P TEF -SjCHS-T XPR2 -P TEF -MsCHI-T XPR2 ) was integrated into the ZETA multi-copy site, and the LEU tag was complemented to obtain strains NAG39-NAG49, respectively. The strains were fermented in YPD medium at 30°C and 220rpm for 96h, and the yields reached 738-776mg / L. The naringenin yield of strain NAG49 reached the highest 776mg / L, while p-coumaric acid was almost consumed ( Figure 6 C, 6D).

[0106] Table 4 Primers and sequences

[0107]

[0108]

[0109] Example 7: Fermentation of strains in a 5L fermenter

[0110] In order to further increase the yield of (2S)-naringenin in lipolytic yeast, the strain NAG49 constructed in Example 5 with the LEU and URA tags complemented was scaled up and cultured in a 5L bioreactor. During fermentation, the seed liquid was inoculated into the fermentation medium at an inoculum of 5%, and the dissolved oxygen level was controlled at 10-20% by stirring and dissolved oxygen association. As the fermentation proceeded, the pH value gradually decreased. Therefore, the pH value of the fermentation broth was maintained at 4.9 by automatic flow addition of NH3·H2O. Feeding was started when glucose was lower than 1g / L, and the fermentation was continued until OD 600 Feed was performed when the fermentation temperature reached 6-8 and the glucose concentration in the fermentation environment was controlled to be in the range of 0.1-1 g / L.

[0111] Until the end of fermentation. After 156h of fermentation, OD 600 Reach 240, (2S)-naringenin production can reach 8.65g / L ( Figure 7 ), which is the highest level of (2S)-naringenin synthesized de novo by microorganisms reported so far. In the future, we will focus on further optimizing the fermentation conditions to enhance the conversion of p-coumaric acid to (2S)-naringenin, thereby obtaining higher (2S)-naringenin yields.

[0112] Comparative Example:

[0113] The specific implementation method is the same as Example 2, except that mutants F215I, F215G, F215L, F215V, F265I, F265G, F265A, and F265G were also constructed, and the results showed that these mutants all lost their enzyme activity. Analysis found that both F215 and F265 are aromatic hydrophobic amino acids, which, although not electron-donating groups, can form intermolecular forces with substrates through π-π stacking.

[0114] 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 chalcone synthase mutant, characterized in that Relative to the parent, the threonine at position 132 is mutated to cysteine, and / or the valine at position 196 is mutated to alanine; the parent has the amino acid sequence shown in SEQ ID NO.

23.

2. A gene encoding the chalcone synthase mutant according to claim 1.

3. An expression cassette containing the gene according to claim 2.

4. The expression frame according to claim 3, characterized in that: The expression frame includes but is not limited to: (a) Expression cassette for expressing SjCHS mutant and MsCHI TEF -SjCHS V196A / T132C -T XPR2 -P TEF -MsCHI-T XPR2 ” (b) Expression cassette for expressing FjTAL, SjCHS, MsCHI TEF -FjTAL-T XPR2 -P TEF -SjCHS-T XPR2 -P TEF -MsCHI-T XPR2 ”; (c) Expression cassette for FjTAL, Pc4CL, AtCHS, MsCHI TEF -FjTAL-T XPR2 -P TEF -Pc4CL-T XPR2 -P TEF -AtCHS-T XPR2 -P TEF -MsCHI-T XPR2 ”.

5. A recombinant microorganism expressing the chalcone synthase mutant according to claim 1, or carrying the gene according to claim 2, or carrying the expression cassette according to any one of claims 3 to 4.

6. An engineered strain of Yarrowia lipolytica producing (2S)-naringenin, characterized in that: On the basis of the starting strain, tyrosine ammonia lyase FjTAL from Flavobacterium johnsonii, 4-coumarate coenzyme ligase Pc4CL from parsley, chalcone synthase SjCHS or its mutant from Sophora japonica, and chalcone isomerase MsCHI from alfalfa are expressed; the mutant is based on the sequence shown in SEQ ID NO.23, and the threonine at position 132 is mutated to cysteine, and / or the valine at position 196 is mutated to alanine.

7. The engineered yeast Yarrowia lipolytica according to claim 6, characterized in that The coding gene of SjCHS or its mutant is integrated at the A3 site, the AXP site and / or the E3 site.

8. The engineered yeast Yarrowia lipolytica according to claim 6 or 7, characterized in that The engineered bacteria also enhance the expression of shikimic acid pathway genes; the shikimic acid pathway genes include but are not limited to genes YIARO1, YIARO2 and a highly active mutant YIARO3 that is feedback-resistant to aromatic amino acid regulation. K225L 、YlARO4 K225L 、YlARO7 G139S and EcAROG G146N One or more of .

9. The engineered yeast Yarrowia lipolytica according to any one of claims 6 to 8, characterized in that Genes that enhance cofactors are also overexpressed; the genes that enhance cofactors include one or more of the citrate synthase gene YlCS, the α-ketoglutarate dehydrogenase gene YlOGDC, and the isocitrate dehydrogenase gene YlIDH.

10. The engineered Yarrowia lipolytica according to any one of claims 6 to 9, characterized in that: The starting strain is Yarrowia lipolytica ΔKu70.

11. A method for increasing the yield of (2S)-naringenin, characterized in that: The Yarrowia lipolytica is subjected to at least one of the following improvements: (1) expressing tyrosine ammonia lyase FjTAL from Flavobacterium johnsonii, 4-coumarate coenzyme ligase Pc4CL from parsley, chalcone synthase SjCHS from Sophora japonica or its mutant, and expressing chalcone isomerase MsCHI from alfalfa; the mutant is a mutant of SEQ ID NO. 23, wherein the threonine at position 132 is mutated to cysteine, and / or the valine at position 196 is mutated to alanine; (2) enhancing the expression of genes in the shikimic acid pathway; the genes in the shikimic acid pathway include but are not limited to genes YIARO1, YIARO2, and a highly active mutant YIARO3 that is resistant to feedback regulation by aromatic amino acids. K225L 、YlARO4 K225L 、YlARO7 G139S and EcAROG G146N One or more of; (3) Overexpression of genes that enhance cofactors, including one or more of the citrate synthase gene YlCS, α-ketoglutarate dehydrogenase gene YlOGDC, and isocitrate dehydrogenase gene YlIDH.

12. A method for preparing (2S)-naringenin, characterized in that: The engineered Yarrowia lipolytica according to any one of claims 6 to 10 is fermented in a culture medium at 28 to 30° C. for 96 to 120 hours.

13. The method according to claim 12, characterized in that Glucose was also fed during the fermentation process.

14. Use of the engineered Yarrowia lipolytica according to any one of claims 6 to 10, or the method according to any one of claims 11 to 13, in the production of flavonoid-containing products in the fields of food, medicine, and chemical industry.

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