A yarrowia lipolytica strain for synthesizing (2s)-naringenin and application thereof

Abstract

The application discloses a Yarrowia lipolytica strain for synthesizing (2S)-naringenin and application thereof, and belongs to the field of genetic engineering and bioengineering technology. The application improves the yield of (2S)-naringenin by 60% through semi-rational modification of a key enzyme CHS, and further integrates the modified CHS into the yeast genome. In addition, the application promotes the conversion of coumaric acid to (2S)-naringenin by strengthening the shikimic acid pathway and enhancing the supply of auxiliary factors, so that the yield of (2S)-naringenin reaches 776 mg / L after 96 hours of fermentation of the constructed strain in a 24-hole plate, and the yield reaches 8.65 g / L in a 5L fermenter. The application lays a foundation for subsequent biosynthesis of flavonoids, and has potential value and significance for the development of synthetic biology.

Description

Technical Field

[0001] This invention relates to a synthetic (2S)-naringenin-producing Yeast strain and its application, belonging to the fields of genetic engineering and bioengineering technology. Background Technology

[0002] (2S)-Narcissin is a natural flavonoid compound widely found in the peels and pulps of Rutaceae plants such as tomatoes, grapefruits, oranges, and tangerines. Due to its various pharmacological activities, including antibacterial, anti-inflammatory, antioxidant, anti-atherosclerotic, liver-protective, and lipid metabolism-regulating effects, it has been widely applied in the food, chemical, and pharmaceutical industries. (2S)-Narcissin is biosynthesized via the phenylpropane pathway. First, L-Phe / L-Tyr is deaminated by phenylalanine / tyrosine aminolyase (PAL / TAL) to generate p-coumaric acid. Then, 4-coumaryl-CoA is generated via 4-coumaryl-CoA ligase (4CL). Next, narcissin chalcone is synthesized by combining three molecules of malonyl-CoA with chalcone synthase (CHS). Finally, narcissin chalcone is catalyzed by chalcone isomerase (CHI) to generate (2S)-narcissin.

[0003] Currently, (2S)-naringenin is mainly extracted from plants, which is time-consuming, labor-intensive, and has low production efficiency. Chemical synthesis methods use toxic solvents and extreme chemical reaction conditions, are difficult to process, and are not suitable for large-scale production. In contrast, microbial methods have significant advantages such as being economical, environmentally friendly, and having low production costs, making them a promising alternative. Therefore, biosynthesis based on metabolic engineering and enzyme engineering can be considered for obtaining (2S)-naringenin. However, the current microbial production of (2S)-naringenin still suffers from low yields. Summary of the Invention

[0004] The present invention provides a chalcone synthase mutant, which is based on the parental form and has a mutation of one or more amino acids at positions 132, 196, and 208.

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

[0006] In one embodiment, the mutant is obtained by mutating threonine at position 132 to cysteine ​​and valine at position 196 to alanine, based on the parent, to obtain 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 present application also provides an expression cassette containing the gene.

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

[0011] (a) an expression cassette "P TEF -SjCHS V196A / T132C -T XPR2 -P TEF -MsCHI-T XPR2 ”

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

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

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

[0015] The present application also provides a Yarrowia lipolytica engineering strain with high yield of (2S)-naringenin, which expresses tyrosine ammonia-lyase FjTAL from Flavobacterium johnsoniae, 4-coumarate coenzyme ligase Pc4CL from Petroselinum crispum, chalcone synthase SjCHS from Thermus sp. or a mutant thereof, and chalcone isomerase MsCHI from Medicago sativa based on the starting strain.

[0016] In one embodiment, the engineering strain further overexpresses SjCHS or a mutant thereof; the overexpression includes, but is not limited to, integration of the coding gene of SjCHS or a mutant thereof at A3 site, AXP site and E3 site.

[0017] In an embodiment, the engineered bacteria further overexpress FjTAL, Pc4CL, SjCHS and MsCHI, and strains NAG04-NAG07 are constructed, and it is found that overexpression of SjCHS can increase the yield of (2S)-naringenin by 1.2 times.

[0018] In an embodiment, the engineered bacteria further enhance the expression of shikimic acid pathway genes; the shikimic acid pathway genes include but are not limited to one or more of genes YlARO1, YlARO2, and high-activity mutants YlARO3, YlARO4, YlARO7, and EcAROG that are resistant to feedback regulation of aromatic amino acids. K225L K225L G139S G146N

[0019] In an embodiment, the engineered bacteria further enhance the supply of malonyl-CoA.

[0020] In an embodiment, the engineered bacteria further enhance the supply of NADPH.

[0021] In an embodiment, the starting strain is Yarrowia lipolytica △Ku70; the Yarrowia lipolytica △Ku70 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 an embodiment, the expression of the genes is initiated by the strong promoter PTEF.

[0023] In an embodiment, the tyrosine ammonia-lyase nucleotide sequence of FjTAL derived from Flavobacterium johnsoniae is shown in SEQ ID NO. 1.

[0024] In an embodiment, the nucleotide sequence of 4-coumarate coenzyme ligase Pc4CL derived from Petroselinum crispum is shown in SEQ ID NO. 2.

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

[0026] In an embodiment, the nucleotide sequence of chalcone isomerase MsCHI derived from Medicago sativa is shown in SEQ ID NO. 4.

[0027] ​​​​In an embodiment, the genes FjTAL, Pc4CL, SjCHS and MsCHI are co-integrated into the D17 locus.

[0028] In an embodiment, the genes SjCHS and MsCHI are co-integrated into the AXP locus.

[0029] In an embodiment, the promoter P TEF The genes of the synthetic (2S)-naringenin synthesis pathway are expressed.

[0030] In an embodiment, the Y. lipolytica engineering strain further overexpresses the endogenous shikimic acid pathway genes YlARO1, YlARO2 and the high-activity mutant YlARO3 K225L , YlARO4 K225L , YlARO7 G139S and EcAROG G146N that are resistant to feedback regulation of aromatic amino acids. K225L , YlARO4 K225L , YlARO7 G139S , EcAROG G146N The nucleotide sequences of the genes YlARO1, YlARO2, YlARO3 K225L , YlARO4 K225L , YlARO7 G139S , EcAROG G146N are shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, respectively.

[0031] In an embodiment, the Y. lipolytica engineering strain further overexpresses a gene that enhances cofactors.

[0032] In an embodiment, the gene that enhances cofactors comprises one or more of the genes YlCS, YlOGDC and YlIDH.

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

[0034] The present application also provides the use of the Y. lipolytica engineering strain in the production of (2S)-naringenin.

[0035] In an embodiment, the Y. lipolytica engineering strain is inoculated into YPD medium and fermented at 30°C for 96-120 h.

[0036] In an embodiment, the Yarrowia lipolytica engineering bacteria is inoculated into a fermentation medium, and is fermented at 28 DEG C for 156 h, with the dissolved oxygen level controlled at 10-20%, the pH controlled at 4.9 during the fermentation, and the glucose concentration controlled at 0.1-1 g / L by feeding glucose when the glucose concentration is lower than 1 g / L.

[0037] In an embodiment, the fermentation medium contains 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O

[0038] In an embodiment, the fermentation is performed for at least 156 h.

[0039] The application also claims the use of the Yarrowia lipolytica engineering bacteria in the production of flavonoid-containing products in the fields of food, medicine and chemical industry.

[0040] Advantages:

[0041] 1. The application screens for exogenous (2S)-naringenin synthesis genes in Yarrowia lipolytica and uses a strong promoter P TEF to start the genes, so as to obtain an optimal strain producing initial (2S)-naringenin.

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

[0043] 3. The application 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 co-factors, thereby further improving the yield of (2S)-naringenin, so that the yield of (2S)-naringenin of the constructed strain reaches 776 mg / L after 96 h of fermentation in a 24-well plate.

[0044] 4. The constructed strain can obtain 8.65 g / L of (2S)-naringenin after fermentation in a 5L fermenter. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 It is a metabolic schematic diagram of heterologous synthesis of (2S)-naringenin in Yarrowia lipolytica.

[0046] Figure 2 It is a (2S)-naringenin yield diagram of the engineering strains NAG01-NAG04 under YPD culture.

[0047] Figure 3 It is a (2S)-naringenin yield diagram for verifying that CHS is a key enzyme.

[0048] Figure 4(2S)-Naringenin production platform for semi-rational improvement of key enzyme CHS and site-directed integration of the improved key enzyme.

[0049] Figure 5 (2S)-Naringenin production platform for enhancing the flux of shikimate pathway.

[0050] Figure 6 (2S)-Naringenin production platform for introducing NCM pathway and overexpressing TCA cycle related genes.

[0051] Figure 7 (2S)-Naringenin production platform for fed-batch cultivation in 5L fermenter. DETAILED DESCRIPTION

[0052] (I) Culture medium

[0053] LB medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 10 g / L. Add 20 g / 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: Tryptone 20 g / L, Yeast extract 10 g / L, Glucose 20 g / L.

[0056] (II) Preparation of Yarrowia lipolytica competence: Yarrowia lipolytica competence was prepared using Frozen-EZ Yeast Transformation II reagent kit, 30°C, and 5 mL YPD medium was used to culture Yarrowia lipolytica bacteria to the middle order (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 aspirate the supernatant;

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

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

[0060] (III) Transformation of Yarrowia lipolytica:

[0061] 1. Scrape the yeast colonies from the YPD plate and inoculate them in YPD liquid medium, and place them in a constant temperature incubator at 30°C for 16-22 h;

[0062] 2. Prepare yeast transformation buffer solution (if multiple transformations, scale up x n): 50% PEG 3350, add 90 μL; 2 M Lithium Acetate (M CHCOOLi 65.99), 5 μL; ssDNA, 5 μL, which needs to be boiled in water for 3 min before adding to the transformation buffer, then put on ice to cool down;

[0063] 3. Take 500 μL of the bacterial solution with a pipette and add to a centrifuge tube, centrifuge the tube at an appropriate speed, and discard the supernatant to obtain the yeast cells. Transfer the yeast cells to a centrifuge tube containing 100 μL of the transformation buffer, mix slowly and evenly, add 0.25-0.5 μg of plasmid DNA or linear DNA, mix thoroughly and shake for 2 min (at least 0.25-0.5 μg of DNA is added for each plasmid);

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

[0065] 5. Transfer the centrifuge tube to a centrifuge and centrifuge at 8000 rpm for 2 min. Discard the supernatant in a sterile clean bench, resuspend the cells with an appropriate amount of sterile water, and add to the YPD solid medium. Uniformly spread on the YPD solid medium with a sterile spreader, and then place the medium in a constant temperature incubator at 30°C for culture.

[0066] (Four) HPLC determination of (2S)-naringenin: Shimadzu high performance liquid chromatography was used for determination. HPLC conditions: column oven temperature was set to 30°C, flow rate was 0.7 mL / min, and sample size was 5 μL. The mobile phase A was 10 mM ammonium formate (pH 3.0, adjusted by formic acid), and the mobile phase B was acetonitrile. The mobile phase was A:B = 95.0%:5.0% for the first 0.5 min. Then, the solvent composition was changed according to a linear gradient, A:B = 40.0%:60.0% at 7.0 min. The gradient was changed to the initial condition (A:B = 95.0%:5.0%) after 9.6 min, and remained unchanged until the end of the run at 12 min. The peak time of coumaric acid was at 277 nm. The peak time of naringenin was at 290 nm, with a retention time of 5.7 min.

[0067] (Five) Strain information is shown in Table 1.

[0068] Table 1 Strains involved in the present application

[0069]

[0070] (VI) Integration sites involved in the embodiments

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] Example 1: Construction of (2S)-Naringenin biosynthesis pathway in Yarrowia lipolytica

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

[0081] Firstly, we designed primers for heterologous genes using Snapgene software, and used primers D17-B-F / D17-B-R and AXP-B-F / AXP-B-R to PCR amplify the fragments formed by the homologous arms of D17 and AXP sites and the target genes, and add the PCR amplified fragments and the Cas9 plasmid of the corresponding site during the process of yeast transformation. Then the genes of (2S)-naringenin biosynthesis pathway were combined, firstly FjTAL and Pc4CL were integrated at D17 site, then AtCHS-MsCHI, AtCHS-AtCHI, SjCHS-MsCHI and SjCHS-AtCHI were integrated at AXP site respectively, and the constructed engineering bacteria were named as NAG01-NAG04.

[0082] The constructed engineering bacteria NAG01, NAG02, NAG03 and NAG04 were respectively fermented in YPD medium at 30°C, 220 rpm for 96 h, and the fermentation broth was extracted and identified by liquid phase. The results are shown in Table 1, all the four engineering strains constructed in this embodiment realized the synthesis of (2S)-naringenin, and the combination of genes FjTAL-Pc4CL-SjCHS-MsCHI showed the highest yield of (2S)-naringenin, reaching 24.1 mg / L, as shown in Table 1. Figure 2 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 the reports of prior art, CHS is the rate-limiting enzyme of (2S)-naringenin biosynthesis. According to the method of Example 1, a gene integration expression frame was constructed, and FjTAL, Pc4CL, SjCHS and MsCHI were overexpressed in NAG03, respectively, to obtain strains NAG05-NAG08. Fermentation was carried out according to the method of Example 1, and 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 to (2S)-naringenin.

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

[0089] (1) In order to improve the activity of the key enzyme SjCHS and promote the effective conversion of p-coumaric acid to (2S)-naringenin, semi-rational modification was performed on the key enzyme CHS. Docking analysis was performed on the enzyme and the substrate in order to infer the interaction between the enzyme and the substrate. According to the obtained conformation, active sites such as 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 TEF -FjTAL-T XPR2 -P TEF -Pc4CL-T XPR2 -P TEF -AtCHS-T XPR2 was constructed, 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 mutation was performed, and isomeric residue substitution strategy was used to replace T132 and S133 with polar residues. On the basis of V196A, mutations were introduced into T132 and S133, respectively. The recombinant bacteria constructed were cultured in YPD medium according to the method of Example 1, and the results showed that CHS mutant V196A / T132C could further increase the yield to 38 mg / L, and the yield of (2S)-naringenin was increased by 60% compared with the control (NAG03) Figure 4 C).

[0092] (3) The expression cassette P containing mutant V196A / T132C and MsCHI was constructed according to the method of Example 1 TEF -SjCHS V196A / T132C -T XPR2- P TEF -MsCHI-T XPR2 , and the expression cassette was subjected to three rounds of site-specific integration at the A3 site, the AXP site and the E3 site of the strain NAG03, respectively, to obtain strains NA09-NAO11. The strains were fermented according to the method of Example 1, and the results showed that the precursor p-coumaric acid of the strain NAG11 still had 45 mg / L, and the yield of (2S)-naringenin reached 246.4 mg / L Figure 4 D).

[0093] Example 4: Strengthening of the shikimic acid pathway

[0094] To promote the supply of the precursor p-coumaric acid, the endogenous genes YlAROl (nucleotide sequence as shown in SEQ ID NO. 5), YlARO2 (nucleotide sequence as shown in SEQ ID NO. 6) and high-activity mutant YlARO3 K225L , YlARO4 K225L , YlARO7 G139S and EcAROG G146N (nucleotide sequences as shown in SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10) with feedback resistance to aromatic amino acid regulation were overexpressed to further improve the yield of (2S)-naringenin.

[0095] The specific steps are as follows: the primers YlAROl-F / YlAROl-R are used to amplify the genes YlAROl from the Y. lipolytica genome, the primers YlARO2-F / YlARO2-R are used to amplify the genes YlARO2 from the Y. lipolytica genome, the primers YlARO4-F / YlARO4-R are used to amplify the genes YlARO4 from the Y. lipolytica genome, the primers YlARO3-F / YlARO3-R are used to amplify the genes YlARO3 from the Y. lipolytica genome, the primers YlARO7-F / YlARO7-R are used to amplify the genes YlARO7 from the Y. lipolytica genome, the primers EcAROG-F / EcAROG-R are used to amplify the genes EcAROG from the Y. lipolytica genome, and the primers F1-3-B-F / F1-3-B-R are used to amplify the upper and lower homologous arms of the F1-3 site, respectively. The YlARO3 K225L fragment, the YlARO4 K225L fragment, the YlARO7 G139SFragment, EcAROG G146N Fragment, about 1 μg of integrated fragment and about 600 ng of sgRNA were transformed into Y. lipolytica engineering strain using the Frozen-EZ Yeast Transformation II kit, spread on the screening solid medium, and incubated at 30 °C for 3 days until colonies appeared. The obtained engineering strains NAG12-NAG18 were fermented in YPD medium at 30 °C, 220 rpm for 96 h, and the fermentation broth was extracted for HPLC analysis. The results showed that the yield of (2S)-naringenin of the recombinant engineering strain NAG18 was the highest, 478.4 mg / L( Figure 5 ).

[0096] Table 3 Primer sequences

[0097]

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

[0099] In the synthesis 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 improve the titer of (2S)-naringenin. The NCM pathway MCR-C-BauA (the nucleotide sequence of MCR-C is shown as SEQ ID NO. 11; the nucleotide sequence of BauA is shown as SEQ ID NO. 12) was introduced into the strain NAG18 constructed in Example 4, and the above-mentioned genes were amplified using primers MCR-C-F / and MCR-C-R, 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, 220 rpm for 96 h, and the results showed that the yield of (2S)-naringenin was increased from 478.4 mg / L to 513.5 mg / L compared with NAG18( Figure 6 B).

[0100] On the basis of strain NAG18, the key genes 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) of TCA cycle 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) which decompose pyruvic acid into acetyl coenzyme A were overexpressed to further increase the content of malonyl coenzyme A. The specific steps were as follows: the above-mentioned genes were amplified from the genome by using primers YlCS-F / YlCS-R, YlOGDC-F / YlOGDC-R, YlIDH-F / YlIDH-R, ScACS1-F / ScACS1-R, ScACS2-F / ScACS2-R and YlACS-F / YlACS-R, respectively, and were integrated into C7 site to obtain the strains NAG20-NAG25. The strains were fermented in YPD medium in 24-well plates at 30°C for 96h, and the results showed that the content of (2S)-naringenin in the fermentation broth of strains NAG20-NAG22 was significantly increased, while the level of (2S)-naringenin in the fermentation broth of strains NAG23 and NAG24 overexpressing ACS did not change significantly Figure 6 B).

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

[0102] Example 6: Strengthening NADPH supply to improve the yield of (2S)-naringenin

[0103] A NADPH regeneration system was established to promote the synthesis of (2S)-naringenin, that is, ZWF1 (nucleotide sequence as shown in SEQ ID NO. 19) and POS5 (nucleotide sequence as shown in SEQ ID NO. 20) were overexpressed in strain NAG26 to increase the regeneration of NADPH. The specific steps were as follows: the above-mentioned genes were amplified from the genome by using primers YlZWF1-F / YlZWF1-R and YlPOS5-F / YlPOS5-R, respectively, and were integrated into B6 site to construct strain NAG27. The constructed strain NAG27 was fermented in YPD medium at 30°C and 220 rpm for 96h, and the results showed that Figure 6B), the yield of (2S)-naringenin reached 615.0 mg / L in the fermentation broth of strain NAG27, but a certain amount of p-coumaric acid (194.2 mg / L) was still accumulated.

[0104] Considering that p-coumaric acid could not be completely converted into (2S)-naringenin, the subsequent goal was to improve the downstream metabolic pathway by using the high-copy sites identified in Y. lipolytica. Therefore, a Pc4CL-SjCHS-MsCHI fusion expression frame (P TEF -FjTAL-T XPR2 -P TEF -SjCHS-T XPR2 -P TEF -MsCHI-T XPR2 ) carrying a URA3 marker was constructed and expressed in strain NAG27 and fermented in a 24-well plate to obtain strains NAG28-NAG38. The strains were fermented in YPD medium at 30°C and 220 rpm for 96 h, and the yield reached 583-705.5 mg / L. The (2S)-naringenin titer in the fermentation broth of strain NAG38 was 705.5 mg / L, and the yield of p-coumaric acid decreased to 86 mg / L, with a conversion rate of 60%.

[0105] To further convert p-coumaric acid into (2S)-naringenin, a Pc4CL-SjCHS-MsCHI fusion expression frame (P TEF -FjTAL-T XPR2 -P TEF -SjCHS-T XPR2 -P TEF -MsCHI-T XPR2 ) was integrated into the ZETA multi-copy site while complementing the LEU marker, and strains NAG39-NAG49 were obtained. The strains were fermented in YPD medium at 30°C and 220 rpm for 96 h, and the yield reached 738-776 mg / L. The naringenin yield of strain NAG49 was as high as 776 mg / L, and p-coumaric acid was almost completely consumed. Figure 6 C、6D).

[0106] Table 4 Primers and sequences

[0107]

[0108]

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

[0110] To further improve the production of (2S)-naringenin in Y. lipolytica, the strain NAG49, which was constructed in Example 5 and complemented with LEU and URA markers, was scaled up in a 5L bioreactor. During the fermentation, the seed culture was inoculated into the fermentation medium at an inoculation amount of 5%, and the dissolved oxygen level was controlled at 10-20% by agitation and dissolved oxygen correlation. As the fermentation proceeded, the pH value gradually decreased, therefore, the pH value of the fermentation broth was maintained at 4.9 by automatically feeding NH3H2O. The feeding was started when the glucose was lower than 1g / L, and the fermentation was carried out until OD 600 6-8, the glucose concentration in the fermentation environment was controlled in the range of 0.1-1g / L.

[0111] until the end of the fermentation. After 156h of fermentation, OD 600 240, the production of (2S)-naringenin could reach 8.65g / L Figure 7 , which is the highest (2S)-naringenin level reported so far by microorganisms from scratch. In the future, we will strive to further optimize the fermentation conditions to enhance the conversion of coumaric acid to (2S)-naringenin, so as to obtain higher (2S)-naringenin production.

[0112] Comparative Example:

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

[0114] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore, the protection scope of the present application should be defined by the claims.

Claims

1. A chalcone synthase mutant, characterized in that, Relative to the parent amino sequence shown in SEQ ID NO.23, threonine at position 132 was mutated to cysteine, and valine at position 196 was mutated to alanine.

2. The gene encoding the chalcone synthase mutant of claim 1.

3. An expression cassette containing the gene of claim 2.

4. The expression box according to claim 3, characterized in that, The expression box is (a) or (b): (a) Expression SjCHS mutants and MsCHI Expression box "P TEF -SjCHS V196A / T132C -T XPR2 -P TEF -MsCHI -T XPR2 ” ; (b) Expression FjTAL、SjCHS mutants and MsCHI Expression box P TEF -FjTAL -T XPR2 - P TEF -SjCHS V196A / T132C -T XPR2 - P TEF -MsCHI -T XPR2 ".

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

6. Production (2) S The engineered strain of *Aristolochic acid* containing naringenin is characterized by, Based on the starting strain, the tyrosine ammonia-lyase FjTAL derived from Flavobacterium johnsonii, the 4-coumarate-coenzyme ligase Pc4CL derived from parsley, the chalcone synthase mutant of claim 1, and the chalcone isomerase derived from alfalfa are expressed. MsCHI The chalcone synthase mutant is based on the sequence shown in SEQ ID NO. 23, with threonine at position 132 mutated to cysteine ​​and valine at position 196 mutated to alanine; the nucleotide sequence encoding the tyrosine ammonia-lyase FjTAL is shown in SEQ ID NO. 1; the nucleotide sequence encoding the 4-coumarate coenzyme ligase Pc4CL is shown in SEQ ID NO. 2; and the nucleotide sequence encoding the chalcone isomerase... MsCHI The nucleotide sequence is shown in SEQ ID NO.

4.

7. The engineered *Yamylostella lipolytica* strain according to claim 6, characterized in that, The coding gene of the chalcone synthase mutant is integrated at the A3 site, AXP site, and / or E3 site.

8. The engineered *Yamylostella lipolytica* strain according to claim 6, characterized in that, The engineered bacteria also enhance the expression of genes related to the shikimic acid pathway; the genes for the shikimic acid pathway are genes. YlARO1 , YlARO2 And highly active mutants with feedback resistance to aromatic amino acid regulation. YlARO3 K225L YlARO4 K225L YlARO7 G139S and EcAROG G146N One or more of the following; genes YlARO1 , YlARO2 , YlARO3 K225L YlARO4 K225L YlARO7 G139S , EcAROG G146N The nucleotide sequences are shown in 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.

9. The engineered *Yamylostella lipolytica* strain according to claim 7, characterized in that, The engineered bacteria also enhance the expression of genes related to the shikimic acid pathway; the genes for the shikimic acid pathway are genes. YlARO1 , YlARO2 And highly active mutants with feedback resistance to aromatic amino acid regulation. YlARO3 K225L YlARO4 K225L YlARO7 G139S and EcAROG G146N One or more of the following; genes YlARO1 , YlARO2 , YlARO3 K225L YlARO4 K225L YlARO7 G139S , EcAROG G146N The nucleotide sequences are shown in 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.

10. The engineered *Yamylostella lipolytica* strain according to any one of claims 6 to 9, characterized in that, It also overexpressed a gene that enhances the cofactor; the gene that enhances the cofactor is the citrate synthase gene. YlCS α-Ketoglutarate dehydrogenase gene YlOGDC, Isocitrate dehydrogenase gene YlIDH One or more of the following; genes YlCS The nucleotide sequence is shown in SEQ ID NO. 13; gene YlOGDC The nucleotide sequence is shown in SEQ ID NO.14; gene YlIDH The nucleotide sequence is shown in SEQ ID NO.

15.

11. The engineered *Yamylostella lipolytica* strain according to any one of claims 6 to 9, characterized in that, The starting strain was *Yamylostella lipolytica* △Ku70.

12. The engineered *Yamylostella lipolytica* strain according to claim 10, characterized in that, The starting strain was *Yamylostella lipolytica* △Ku70.

13. An improvement (2) S A method for producing naringin, characterized in that, The following improvements were made to the lipophilic yeast: The enzyme expresses tyrosine ammonia-lyase FjTAL derived from Flavobacterium johnsonii, 4-coumarate-coenzyme ligase Pc4CL derived from parsley, the chalcone synthase mutant of claim 1, and chalcone isomerase derived from alfalfa. MsCHI The mutant is based on the sequence shown in SEQ ID NO. 23, with threonine at position 132 mutated to cysteine ​​and valine at position 196 mutated to alanine; the nucleotide sequence encoding the tyrosine ammonia-lyase FjTAL is shown in SEQ ID NO. 1; the nucleotide sequence encoding the 4-coumarate coenzyme ligase Pc4CL is shown in SEQ ID NO. 2; and the nucleotide sequence encoding the chalcone isomerase... MsCHI The nucleotide sequence is shown in SEQ ID NO.

4.

14. The method according to claim 13, characterized in that, It also enhances the expression of genes involved in the shikimic acid pathway; these genes include, but are not limited to, genes involved in the shikimic acid pathway. YlARO1 , YlARO2 And highly active mutants with feedback resistance to aromatic amino acid regulation. YlARO3 K225L YlARO4 K225L YlARO7 G139S and EcAROG G146N One or more of the following; genes YlARO1 , YlARO2 , YlARO3 K225L YlARO4 K225L YlARO7 G139S , EcAROG G146N The nucleotide sequences are shown in 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.

15. The method according to claim 14, characterized in that, It also overexpresses genes that enhance cofactors, including: citrate synthase gene. YlCS α-Ketoglutarate dehydrogenase gene YlOGDC, Isocitrate dehydrogenase gene YlIDH One or more of the following; genes YlCS The nucleotide sequence is shown in SEQ ID NO.13; gene YlOGDC The nucleotide sequence is shown in SEQ ID NO. 14; gene YlIDH The nucleotide sequence is shown in SEQ ID NO.

15.

16. A method for preparing (2 S The method for naringin is characterized by, The engineered *Yamylostella lipolytica* strain according to any one of claims 6 to 12 is fermented in a culture medium at 28 to 30°C for 96 to 120 h.

17. The method according to claim 16, characterized in that, Glucose is also added during the fermentation process.

18. The engineered *Yamylostella lipolytica* strain according to any one of claims 6-12, or the method according to any one of claims 13-17, for producing products containing (2...) in the food, pharmaceutical, or chemical industries. S Applications of naringin in products.

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