Erigeron breviscapus chalcone synthase activity and / or selectivity enhanced mutant as well as screening method and application thereof

Abstract

The invention discloses an erigeron breviscapus chalcone synthase activity and / or selectivity enhanced mutant as well as a screening method and application thereof. The construction method comprises the following steps: constructing a recombinant plasmid for synthesizing phloretin by taking p-coumaric acid as a substrate, screening out sites related to the catalytic activity and selectivity of EbCHS through structural prediction and a catalytic mechanism, and carrying out experimental screening to obtain effective mutants and effective mutation sites; comparing the EbCHS with different CHS sequences, and carrying out screening experiments to obtain effective mutants of non-conservative amino acid sites adjacent to the effective mutation sites; combining the two obtained effective mutants in pairs to obtain related detection information; according to the experimental information library building, machine learning modeling prediction and experimental verification, the EbCHS with obviously enhanced activity and selectivity in the phloretin synthesis route is obtained, the phloretin can be efficiently synthesized by taking p-coumaric acid as a substrate, byproducts are reduced, and a technical support is provided for promoting the industrial production of the phloretin.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a mutant of Erigeron breviscapus with enhanced chalcone synthase activity and / or selectivity, and a screening method and application thereof. Background Art

[0002] Phloretin, a natural dihydrochalcone product, exists in apple plants and has anti-inflammatory, antioxidant, antibacterial and other physiological activities. It is widely used in food, medicine, cosmetics and other industries. p -coumaric acid as substrate, through p -Coumaroyl-CoA ligase ( p Phloretin is catalyzed by 4-coumaroyl-CoA ligase (4CL), double-bond reductase (DBR), and chalcone synthase (CHS). Currently, studies have been conducted on the synthesis of phloretin using microbial cell factories. However, since CHS is a common enzyme in the flavonoid biosynthesis pathway and has substrate promiscuity, it produces intermediates and by-products such as naringenin chalcone, naringenin, BNY, CTAL, 2H-BNY, and 2H-CTAL during the heterologous synthesis of phloretin. Furthermore, the catalytic activity of CHS used in current literature reports is relatively low. These issues all lead to low phloretin biosynthesis yields. Previous researchers have added phloreticaci as a precursor and used the 4CL and CHS genes to artificially synthesize phlorretin. However, compared with the heterologous synthesis of other flavonoids, the yield is still low. In addition, there are problems such as the expensive phloreticaci substrate, high production cost, and the inability to achieve de novo synthesis of phlorretin.

[0003] Current research has achieved some success in flavonoid synthesis by screening CHS genes from different sources, adjusting CHS promoter strength, employing protein fusion techniques, and increasing gene copy number. However, this still fails to effectively reduce or eliminate byproducts produced by the synthesis pathway. Therefore, modifying CHS to produce CHS with improved substrate specificity and enhanced catalytic activity is an important approach to increasing phloretin biosynthesis. Summary of the Invention

[0004] The primary purpose of the present invention is to provide a method for screening mutants with enhanced chalcone synthase activity and / or selectivity of Erigeron breviscapus.

[0005] Another object of the present invention is to provide a mutant of Erigeron breviscapus chalcone synthase with enhanced activity and / or selectivity.

[0006] Another object of the present invention is to provide the use of the mutant of the above-mentioned Erigeron breviscapus chalcone synthase with enhanced activity and / or selectivity.

[0007] The purpose of the present invention is achieved through the following technical solutions: A method for screening mutants of Erigeron breviscapus with enhanced chalcone synthase activity and / or selectivity, comprising the following steps: (1) Put the parsley p -Coumaryl-CoA ligase (Pc4CL) encoding gene, Saccharomyces cerevisiae endogenous double bond reductase (ScTSC13) encoding gene and Erigeron breviscapus chalcone synthase (EbCHS) encoding gene were constructed on the same vector backbone to obtain recombinant plasmid; (2) By predicting the three-dimensional structure of EbCHS and based on the CHS catalytic mechanism, we preliminarily screened out sites related to the catalytic activity and selectivity of EbCHS; (3) constructing a single site saturation mutation plasmid for the site selected in step (2), and then transforming it into the Saccharomyces cerevisiae chassis cells, culturing on uracil-deficient agar plates to obtain a recombinant strain; then p -coumaric acid fermentation medium, culturing the recombinant strain, measuring phloretin and by-products in the fermentation broth, screening for effective mutants related to EbCHS catalytic activity and selectivity, and obtaining effective mutation sites; (4) Align EbCHS with CHS sequences from different plant sources and screen for non-conserved amino acid sites adjacent to the effective mutation sites obtained in step (3); (5) constructing a single site saturation mutation plasmid for the site selected in step (4), and then transforming it into the Saccharomyces cerevisiae chassis cells, culturing it on a uracil-deficient agar plate to obtain a recombinant strain; p -coumaric acid fermentation medium, culturing the recombinant strain, measuring phloretin and by-products in the fermentation broth, and screening for effective mutants with non-conservative amino acids; (6) Based on the information of the effective mutants obtained in step (3) and the non-conservative amino acid effective mutants obtained in step (5), a sequence containing two site mutation combinations in each EbCHS mutant was designed; then a plasmid containing two site mutations was constructed, and then transformed into the Saccharomyces cerevisiae chassis cells, and cultured on uracil-deficient agar plates to obtain a recombinant strain; then, a plasmid containing two site mutations was constructed, and then ... p - Cultivate the recombinant strain in a fermentation medium containing coumaric acid, and measure the phloretin and by-products in the fermentation broth to obtain relevant information; (7) Performing single-site saturation mutagenesis on the effective mutation site obtained in step (3) and the non-conserved amino acid site obtained in step (4), respectively, and combining them with the two mutations obtained in step (6) to obtain a mutant sequence library; (8) Based on the test results of step (3), step (5) and step (6), the mutant sequence library obtained in step (7) is subjected to machine learning and prediction, and then a plasmid containing the mutation is constructed based on the mutant sequence predicted by EbCHS activity, and the plasmid is transferred into the Saccharomyces cerevisiae chassis cells and cultured on uracil-deficient agar plates to obtain a recombinant strain; p The recombinant strain is cultured in a fermentation medium containing 1,2-coumaric acid, phloretin and by-products in the fermentation broth are determined, and mutants with enhanced chalcone synthase activity and / or selectivity of Erigeron breviscapus are screened.

[0008] The nucleotide sequence of the Pc4CL encoding gene described in step (1) is preferably as shown in SEQ ID NO.1.

[0009] The nucleotide sequence of the ScTSC13 encoding gene described in step (1) is preferably as shown in SEQ ID NO.3.

[0010] The nucleotide sequence of the EbCHS encoding gene described in step (1) is preferably as shown in SEQ ID NO.2.

[0011] The Pc4CL encoding gene and the ScTSC13 encoding gene in step (1) are respectively set in independent expression cassettes, or connected to form a fusion gene in the same expression cassette through a linker encoding gene.

[0012] The basic components of the expression cassette are a promoter, a coding gene and a terminator.

[0013] The linker is preferably (GGGGS)3.

[0014] The nucleic acid sequence of the Linker encoding gene is preferably as shown in SEQ ID NO.4.

[0015] The EbCHS encoding gene described in step (1) is placed in a separate expression cassette.

[0016] The recombinant plasmid described in step (1) preferably contains a gene fragment with the following structure: promoter- Pc4CL -( GGGGS )3- ScTSC13 -Double terminator- EbCHS -promoter.

[0017] The promoters are the same or different; preferably different, wherein, Pc4CL The promoter is preferably the SED1 promoter, EbCHS The promoter is preferably the TDH1 promoter.

[0018] The double terminator is preferably a ter22 double terminator.

[0019] Genes are indicated in italics.

[0020] The prediction software described in step (2) is preferably AlphaFold3.

[0021] The CHS catalytic mechanism described in step (2) is preferably the catalytic mechanism of MsCHS.

[0022] The sites described in step (2) are preferably K58, R61, K65, A311, S136, E195, T197, T200, S341T135, M140, I257, G259, F268, P378, R11, I46, P84, K107, F168, S211, V264, D64 and L217.

[0023] The saturation mutation described in step (3) refers to mutation to 19 amino acids other than the wild type.

[0024] The codon preference sequence of Saccharomyces cerevisiae in the saturation mutation is: A (GCT), G (GGT), V (GTT), L (TTG), I (ATT), E (GAA), Q (CAA), D (GAT), N (AAT), M (ATG), S (TCT), T (ACT), F (TTT), W (TGG), Y (TAT), R (AGA), H (CAT), C (TGT), P (CCA), K (AAA).

[0025] The Saccharomyces cerevisiae chassis cells described in step (3), step (5), step (6) and step (8) are preferably Saccharomyces cerevisiae CEN.PK series strains; more preferably Saccharomyces cerevisiae CEN.PK2-1C.

[0026] The transformation steps described in step (3), step (5), step (6) and step (8) are preferably as follows: the Saccharomyces cerevisiae chassis cells, plasmid, transformation liquid and ssDNA are mixed evenly to obtain a transformation system; the transformation system is incubated in a 37±1°C water bath and then centrifuged to discard the supernatant; the obtained cells are resuspended in water to obtain a transformation bacterial solution.

[0027] The composition of the transformation system is preferably as follows: 2×10 7 ~1×10 8 cells, 100 μL transformation medium, 1 μg plasmid, and 3 μL ssDNA at a concentration of 10 mg / mL.

[0028] The transformation solution consists of 800 μL of 500 g / L PEG3350 solution, 200 μL of 2 M LiAc solution, and 7.5 μL of β-mercaptoethanol.

[0029] The incubation time is preferably 20 to 40 minutes, more preferably 30 minutes.

[0030] The centrifugation condition is preferably 4000-8000 g for 1-5 min; more preferably 6000 g for 3 min.

[0031] The water is preferably deionized water.

[0032] Steps (3), (5), (6) and (8) contain p -Coumaric acid fermentation medium is based on p -coumaric acid as the substrate, and its composition is preferably as follows: p -Coumaric acid 0.3-0.5 g / L, tryptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L, and the solvent is deionized water.

[0033] Steps (3), (5), (6) and (8) are described in p The recombinant strain is cultured in a fermentation medium containing -coumaric acid by shaking culture. The culture conditions are preferably 30±1°C, 150-250 rpm for 3-4 days; more preferably 30±1°C, 200-250 rpm for 3-4 days.

[0034] The by-products in step (3), step (5), step (6) and step (8) are naringenin and 2H-BHY.

[0035] The phloretin and by-products described in step (3), step (5), step (6) and step (8) are determined by HPLC.

[0036] The effective mutant described in step (3) is a mutant with enhanced chalcone synthase activity and / or selectivity of Erigeron breviscapus.

[0037] The effective mutant described in step (3) is preferably EbCHS F168Y 、EbCHS P84S 、EbCHS T200C 、EbCHS S341A 、EbCHS S211G .

[0038] The EbCHS F168Y The 168th amino acid of EbCHS is mutated from phenylalanine to tyrosine. P84S The 84th amino acid of EbCHS is mutated from proline to serine. T200CThe 200th amino acid of EbCHS is mutated from threonine to cysteine. S341A The amino acid at position 341 of EbCHS is mutated from serine to alanine. S211G It refers to the mutation of amino acid 211 of EbCHS from serine to glycine.

[0039] The different plant-derived CHSs described in step (4) include PhCHS 、 SjCHS1 、 HvCHS1 、 HvCHS2 、 PmCHS 、 SbCHS2 、 CsC 、 VaCHS 、 FqV 、 HaCHS 、 PcCHS 、 MdCHS1 、 MdCHS2 .

[0040] The non-conserved amino acid sites described in step (4) are preferably N83, S85, A169, G171, V199, L212 and C344.

[0041] The effective mutant described in step (5) is a mutant with enhanced chalcone synthase activity and / or selectivity of Erigeron breviscapus.

[0042] The non-conservative amino acid effective mutant described in step (5) is preferably EbCHS C344S .

[0043] The EbCHS C344S It refers to the mutation of amino acid 344 of EbCHS from cysteine to serine.

[0044] The combination described in step (6) is preferably as follows: T200C, F168Y, T200C, P84S, T200C, S341A, T200C, S211G, F168Y, P84S, F168Y, S341A, F168Y, S211G, P84S, S341A, P84S, S211G, S341A, S211G, C344S, T200C, C344S, F168Y, C344S, P84S, C344S, S341A, C344S, S211G.

[0045] The learning and prediction described in step (8) are preferably performed by an XGBoost model.

[0046] The specific operation of constructing a plasmid containing a mutation based on the mutant sequence predicted by EbCHS activity in step (8) is preferably as follows: the mutant sequences predicted by EbCHS activity are sorted from high to low according to activity, and the top 30 mutant sequences are selected as the basis for constructing a plasmid containing a mutation; preferably, the top 20 mutant sequences are selected as the basis for constructing a plasmid containing a mutation; more preferably, the top 10 mutant sequences are selected as the basis for constructing a plasmid containing a mutation.

[0047] The mutant with enhanced activity and / or selectivity of the chalcone synthase of Erigeron breviscapus described in step (8) is preferably EbCHS F168Y, S211G, C344A 、EbCHS F168Y, S211G, C344S or EbCHS F168Y, V199I, S211G, C344A .

[0048] The EbCHS F168Y, S211G, C344A It refers to the mutation of the 168th amino acid of EbCHS from phenylalanine to tyrosine, the 211th amino acid of EbCHS from serine to glycine, and the 344th amino acid of EbCHS from cysteine to alanine.

[0049] The mutant with enhanced activity and / or selectivity of the breviscapine chalcone synthase refers to a mutant with increased phloretin accumulation relative to the wild-type breviscapine chalcone synthase, or a mutant with increased phloretin accumulation and reduced by-product accumulation, or a mutant with a greater reduction in by-product accumulation than in phloretin accumulation.

[0050] A mutant of Erigeron breviscapus chalcone synthase with enhanced activity and / or selectivity is obtained by the above screening method, comprising: The mutant of EbCHS with enhanced activity is mutant EbCHS F168Y 、EbCHS P84S ; EbCHS activity-enhanced and selectivity-enhanced mutants are EbCHS S211G 、EbCHS F168Y,S211G 、EbCHSC3 F168Y, C344S 、EbCHSC3 P84S, C344S 、EbCHSC3 S211G, C344S 、EbCHS F168Y, S211G, C344A 、EbCHS F168Y , S211G, C344S 、EbCHS F168Y, V199I, S211G, C344A ; The mutant of EbCHS selectively enhanced is mutant EbCHS T200C 、EbCHS S341A 、EbCHS C344A ; Preferably EbCHS F168Y 、EbCHSF168Y, S211G, C344A .

[0051] Application of the above-mentioned mutant with enhanced activity and / or selectivity of Erigeron breviscapus chalcone synthase in phloretin synthesis.

[0052] The present invention has the following advantages and effects compared to the prior art: (1) The screening method provided by the present invention can quickly screen out the Erigeron breviscapus chalcone synthase that is beneficial to the synthesis of phloretin.

[0053] (2) The present invention can screen out mutants that have advantages in increasing phloretin production, such as EbCHS F168Y 、EbCHS P84S wait.

[0054] (3) The present invention can screen out mutants that have advantages in reducing by-products in the process of phloretin synthesis, such as EbCHS T200C 、EbCHS S341A 、EbCHS C344S .

[0055] (4) The present invention can screen out mutants that have advantages in increasing phloretin production and reducing by-product production, such as EbCHS S211G 、EbCHS F168Y, S211G, C344A wait. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram of plasmid p416-Pc4CL-(GGGGS)3-ScTSC13-EbCHS in Example 1.

[0057] Figure 2 The standard curves of phloretin and naringenin are shown in Figure 2.

[0058] Figure 3 This is the three-dimensional structure diagram of EbCHS predicted by AlphaFold3 in Example 2; wherein a represents the 24 screened mutation sites, and b represents the 7 non-conserved amino acids adjacent to the 5 effective sites.

[0059] Figure 4 Schematic diagram of the K58 site of the plasmid p416-Pc4CL-(GGGGS)3-ScTSC13-EbCHS in Example 3.

[0060] Figure 5 This is the sequencing result of the plasmid p416-Pc4CL-(GGGGS)3-ScTSC13-EbCHS after K58A mutation in Example 3.

[0061] Figure 6 This is the liquid phase peak diagram of the product and standard in Example 4.

[0062] Figure 7 This is the LC-MS mass spectrum identification diagram of 2H-BNY in Example 4.

[0063] Figure 8 This is a graph showing the results of enhanced activity or selectivity of the EbCHS mutant in the phloretin biosynthesis pathway detected by high performance liquid chromatography in Examples 4 and 5.

[0064] Figure 9 This is a diagram showing the results of two-point combination mutations at the six effective sites in Example 6.

[0065] Figure 10 This is a graph showing the results of the machine learning modeling prediction and verification in Example 6. DETAILED DESCRIPTION

[0066] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0067] The experimental process of the present invention is summarized as follows: CHS is a III polyketide synthase (PKS). Based on the crystal structure of alfalfa CHS (MsCHS) (1CGK), the catalytic cavity of MsCHS consists of the catalytic active center, the malonyl-CoA binding channel, the cyclization pocket, p -Coumaryl-CoA binding pocket. The amino acid residues that make up these structures are believed to play a decisive role in the specificity of the starting substrate and product, and are important targets for the directional modification of CHS. Therefore, the present invention selected Erigeron breviscapus CHS (EbCHS) with high specificity for phloretin synthesis as the wild type, used AlphaFold3 to predict its three-dimensional structure, and screened the CoA-binding channel sites (K58, R61, K65, A311). p -Coumaryl-CoA binding pocket sites (S136, E195, T197, T200, S341), cyclization pocket sites (T135, M140, I257, G259, F268, P378) and other activity-related sites reported in the literature (R11, I46, P84, K107, F168, S211, V264, D64, L217) are numbered according to the EbCHS amino acid sequence. Unless otherwise specified, the following numbering is used. A total of 24 sites were saturated mutated. pUsing 4-coumaric acid as a substrate for phloretin synthesis, high-performance liquid chromatography (HPLC) analysis identified five effective single-point mutations in EbCHS that resulted in high phloretin yield and low byproduct production. Sequence alignment of CHSs from different plant sources with EbCHS revealed saturation mutagenesis of seven non-conserved amino acids adjacent to the five effective sites (N83, S85, A169, G171, V199, L212, and C344), resulting in one effective single-point mutation. Pairwise combination mutagenesis of the six effective single-point sites yielded 15 two-point combination mutation sequences. A library of 243 EbCHS mutation sequences was constructed based on the effective sites, their adjacent non-conserved amino acid sequences, and the two-point mutation sequences for machine learning modeling. Ten EbCHS mutation sequences with predicted high activity were selected for experimental validation, ultimately yielding an evolved EbCHS enzyme with significantly enhanced activity and selectivity in the phloretin biosynthesis pathway.

[0068] The PCR amplification in the present invention includes conventional denaturation, annealing and extension steps; wherein the annealing temperature is based on the temperature recommended when the primers are designed, generally 57° C., and the extension time of the fragment is determined according to the instructions of the enzyme used.

[0069] The culture medium used in the present invention is as follows: The composition of YPD medium is as follows: 20 g / L tryptone, 10 g / L yeast powder, 20 g / L glucose, and the solvent is deionized water.

[0070] The composition of the uracil-deficient agar plate is as follows: 1.74 g / L yeast nitrogen base, 5 g / L ammonium sulfate, 20 g / L glucose, 50 mg / L histidine, 50 mg / L leucine, 50 mg / L tryptophan, 1.5% agar powder, and deionized water as the solvent.

[0071] The composition of the transformation solution is as follows: 800 μL of 500 g / L PEG3350 solution, 200 μL of 2 M LiAc solution, and 7.5 μL of β-mercaptoethanol.

[0072] Determination of phloretin, naringenin, and 2H-BNY (Dihydro-bisnoryangonin) accumulation: Equal volumes of fermentation broth and HPLC-grade methanol were placed in a crushed tube. Extraction was performed using a Bioprep-24R homogenizer, followed by centrifugation at 12,000 g for 10 minutes. The supernatant was filtered through a 0.22 μm membrane filter and analyzed by high-performance liquid chromatography (HPLC) using an LC-16 (Shimadzu Corporation, Japan) equipped with an SPD-16 dual-wavelength UV detector and a C18 column (250 × 4.6 mm, 5 μm). Gradient elution, phase A: 0.1% (v / v) formic acid aqueous solution, phase C: acetonitrile, elution conditions: 0-10 min 10-40% v / v phase C, 10-20 min 40-60% v / v phase C, 20-23 min 60% v / v phase C, 23-25 min 60-10% v / v phase C, 25-28 min 10% v / v phase C, flow rate: 1 mL / min, injection volume: 10 μL, detection wavelength: 285 nm. Phloretin and naringenin standards with concentration gradients of 200 mg / L, 100 mg / L, 50 mg / L, 25 mg / L, 12.5 mg / L, 5 mg / L, 2.5 mg / L, and 1 mg / L were prepared, and the HPLC detection was used to establish a standard curve and calculate the yields of phloretin and naringenin in the fermentation broth. There is no commercial standard for 2H-BNY, and the accumulation amount was calculated based on the liquid phase peak.

[0073] All primers used for introducing mutations in the present invention were designed based on the EbCHS nucleic acid shown in SEQ ID NO. 2. The primers used for the K58 site saturation mutagenesis are shown in Table 1. The primers for the saturation mutagenesis primers are 46 bp upstream and 51 bp downstream of the mutation site. The K58-F primer has 21 bp complementary to the upstream mutation site of the saturation mutagenesis primer, and the downstream primer carries the mutation site, allowing the mutation site to be introduced into the expression vector via PCR amplification. The primer designs for the remaining mutants (R61, K65, A311, S136, E195, T197, T200, S341, T135, M140, I257, G259, F268, P378, R11, I46, P84, K107, F168, S211, V264, D64, L217) are similar to those for the K58 site saturation mutagenesis primers. The codons for the saturation mutagenesis are the same as those used for the K58 site mutations in Table 1. The codon preference sequence of Saccharomyces cerevisiae for saturated mutant amino acids is: A (GCT), G (GGT), V (GTT), L (TTG), I (ATT), E (GAA), Q (CAA), D (GAT), N (AAT), M (ATG), S (TCT), T (ACT), F (TTT), W (TGG), Y (TAT), R (AGA), H (CAT), C (TGT), P (CCA), K (AAA).

[0074] Table 1 Primers

[0075] Example 1: Constructing p -Coumaric acid-based phloretin biosynthesis pathway Codon optimization of the Pc4CL and EbCHS coding genes was performed using the online tool GenSmart Optimization (GenScript Biotech Co., Ltd.). Shanghai Sangon Biotech (Shanghai) Co., Ltd. synthesized the EbCHS coding gene with the nucleotide sequence shown in SEQ ID NO.2, the Pc4CL coding gene with the nucleotide sequence shown in SEQ ID NO.1 (excluding the stop codon taa), the three tandem GGGGS coding genes with the nucleotide sequence shown in SEQ ID NO.4, and the ScTSC13 coding gene with the nucleotide sequence shown in SEQ ID NO.3 to form the Pc4CL-(GGGGS)3-ScTSC13 gene, and the Ter22 double terminator with the nucleotide sequence shown in SEQ ID NO.5. pY26-GPD-TEF (also known as pY26TEF-GPD, BioVector) was used to synthesize the EbCHS coding gene with the nucleotide sequence shown in SEQ ID NO.1 (excluding the stop codon taa), the Pc4CL-(GGGGS)3-ScTSC13 gene, and the Ter22 double terminator with the nucleotide sequence shown in SEQ ID NO.5 were synthesized. The plasmid vector was obtained from the NTCC Plasmid Vector Culture Cell Gene Collection Center) as a template, and the plasmid framework was amplified with primers pY26-1F and pY26-1R; the EbCHS coding gene was used as a template, and the EbCHS gene fragment was amplified with primers EbCHS-F and EbCHS-R; the Pc4CL-(GGGGS)3-ScTSC13 gene was used as a template, and the primers Pc4CL-(GGGGS)3-ScTSC13-F and Pc4CL-(GGGGS)3-ScTSC13-F were used to amplify the EbCHS gene fragment. The Pc4CL-(GGGGS)3-ScTSC13 gene fragment was amplified using primers Ter22-F and Ter22-R, using the Ter22 double terminator as a template. The SED1p promoter gene fragment (nucleotide sequence shown in SEQ ID NO. 6) and the TDH1p promoter gene fragment (nucleotide sequence shown in SEQ ID NO. 7) were amplified using primers SED-F / SED-R and TDH-F / TDH-R, respectively, using Saccharomyces cerevisiae CEN.PK2-1C as a template. After the product was recovered, the pY26-GPD-TEF plasmid framework, EbCHS gene fragment, Pc4CL-(GGGGS)3-ScTSC13 gene fragment, Ter22 double terminator fragment, SED1p promoter gene fragment and TDH1p promoter gene fragment were assembled into a plasmid using Gibson assembly technology to obtain the pY26-SED1p-Pc4CL-(GGGGS)3-ScTSC13-Ter22-EbCHS-TDH1p (pY26-Pc4CL-(GGGGS)3-ScTSC13-EbCHS is abbreviated) plasmid, as shown in FIG. Figure 1 shown.

[0076] In 1×10 8In a Saccharomyces cerevisiae chassis cell CEN.PK2-1C (purchased from Shanghai Lianzu Biotechnology Co., Ltd.), 1 μg of plasmid pY26-Pc4CL-(GGGGS)3-ScTSC13-EbCHS, 100 μL of transformation solution, and 3 μL of 10 mg / mL salmon sperm DNA (ssDNA) were mixed evenly to obtain a transformation system. The transformation system was incubated in a 37°C water bath for 30 minutes, centrifuged at 6000 g for 3 minutes, and the supernatant was discarded. The resulting cells were resuspended in sterile deionized water to obtain a transformation solution, which was then plated on a uracil-deficient YNB agar plate. Colonies were selected on a plate supplemented with 400 g / L p The culture was shaken in 25 mL YPD medium containing 1-coumaric acid and cultured at 220 rpm and 30°C for 72 h. The accumulation of phloretin and the byproduct naringenin was determined by HPLC. The results showed that 43.83 mg / L of phloretin and 26.77 mg / L of naringenin were synthesized, and the phloretin / naringenin ratio was 1.64. The standard curves of phloretin and naringenin were as follows: Figure 2 shown.

[0077] Example 2: Semi-rational design screening of EbCHS saturation mutation sites The catalytic active cavity of EbCHS consists of the catalytic active center, malonyl-CoA binding channel, cyclization pocket, p -Coumaryl-CoA binding pocket. The amino acid residues that make up these structures are believed to play a decisive role in the specificity of the starting substrate and product, and are important targets for the directed modification of CHS molecules. Among them, the catalytic active center residues are highly conserved, and mutations have a greater impact on their activity, so they are selected as candidate mutation sites. Screening CoA-binding channel sites (K58, R61, K65, A311), p -Coumaryl-CoA binding pocket sites (S136, E195, T197, T200, S341), cyclization pocket sites (T135, M140, I257, G259, F268, P378) and other activity-related sites reported in the literature (R11, I46, P84, K107, F168, S211, V264, D64, L217) are numbered according to the EbCHS amino acid sequence. Unless otherwise specified, the following numbering is used. A total of 24 sites were saturated mutagenesis, such as Figure 3 As shown in a in .

[0078] Example 3: Construction of EbCHS saturation mutation site plasmid The primer sequences were synthesized by the company and the plasmid pY26-Pc4CL-(GGGGS)3-ScTSC13-EbCHS ( Figure 4 ), obtaining pY26-EbCHSK58A After verification by nucleic acid gel electrophoresis, the p-loop fragment was recovered and transformed into E. coli DH5α and cultured for 12 hours. A single colony was randomly selected and cultured for 12 hours, and the plasmid was extracted and sent for sequencing verification. Figure 5 As shown, plasmid pY26-Pc4CL-(GGGGS)3-ScTSC13-EbCHS was obtained. K58A The other mutants are the same as pY26-Pc4CL-(GGGGS)3-ScTSC13-EbCHS K58A Construction method.

[0079] Example 4: Screening for EbCHS single-point effective mutation sites with enhanced activity and selectivity 456 plasmids (24 mutation sites × 19 = 456) were transformed into the Saccharomyces cerevisiae chassis cell CEN.PK2-1C, spread on uracil-deficient agar plates, and cultured in a 30°C incubator for four days. The colonies were selected and grown on a plate supplemented with 400 g / L p -coumaric acid in 2 mL YPD medium, and the accumulation of phloretin, the by-products naringenin and 2H-BNY were measured after culturing at 220 rpm and 30 °C for 72 h. The liquid phase peak diagram is shown in Figure 2. Figure 6 As shown, the LC-MS mass spectrum identification of 2H-BNY is shown in Figure 7 The phloretin production of the five effective mutation sites was enhanced or the by-product production was decreased compared with the EbCHS wild type, respectively. F168Y (phloretin, 1.69-fold; naringenin, 1.15-fold; 2H-BNY, 1.16-fold), EbCHS P84S (phloretin, 1.41 times; naringenin, 1.29 times; 2H-BNY, 1.21 times), EbCHS T200C (phloretin, 0.98-fold; naringenin, 0.43-fold; 2H-BNY, 0.43-fold), EbCHS S341A (phloretin, 0.81 times; naringenin, 0.28 times; 2H-BNY, 0.42 times), EbCHS S211G (phloretin, 1.05 times; naringenin, 0.91 times; 2H-BNY, 0.85 times), such as Figure 8 shown.

[0080] Example 5: Screening for non-conserved amino acids adjacent to effective mutation sites Choose from 13 different plant-derived CHS ( PhCHS 、 SjCHS1 、 HvCHS1 、 HvCHS2 、 PmCHS 、 SbCHS2 、 CsC 、 VaCHS 、 FqV 、 HaCHS 、 PcCHS 、 MdCHS1 、 MdCHS2 ) for sequence alignment, where PhCHS The amino acid sequence is shown in Genbank AAF60297.1, amino acids 1-389; SjCHS1 The nucleotide sequence of the gene is shown in SEQ ID NO.8; HvCHS1 The amino acid sequence is shown in Genbank P26018.1, amino acids 1-398; HvCHS2 The amino acid sequence is shown in Genbank Q96562.1, amino acids 1-399; PmCHS The nucleotide sequence of the gene is shown in Genbank MK058493.1, bases 1-1194; SbCHS2 The amino acid sequence is shown in Genbank AMW91736.1, amino acids 1-390; CsC The amino acid sequence is shown in Genbank AAO13091.1, amino acids 1-389; VaCHS The amino acid sequence is shown in Genbank BAO58434.1, amino acids 1-389; FqV The amino acid sequence is shown in Genbank ACZ51475.1, amino acids 1-395; HaCHS The amino acid sequence is shown in Genbank Q9FUB7.1, amino acids 1-390; PcCHS The amino acid sequence is shown in Genbank P16107.1, amino acids 1-398; MdCHS1 The amino acid sequence is shown in Genbank NP_001306186.1, amino acids 1-391; MdCHS2 The amino acid sequence is shown in Genbank NP_001306181.1, amino acids 1-389.

[0081] Screening of 7 non-conserved sites (N83, S85, A169, G171, V199, L212, C344) adjacent to the effective sites F168, P84, T200, S341, S211, such as Figure 3 As shown in b. Saturation mutation was performed on 7 non-conserved sites to construct 133 mutant plasmids, which were then transformed into Saccharomyces cerevisiae chassis cells CEN.PK2-1C for screening (transformation and culture steps were the same as in Example 4), and one effective mutation site EbCHS was obtained. C344S (phloretin, 0.99 times; naringenin, 0.34 times; 2H-BNY, 0.77 times), such as Figure 8 shown.

[0082] Example 6: Machine learning modeling prediction and verification to obtain efficient EbCHS combination mutation sites Six effective single-point mutations EbCHS F168Y 、EbCHS P84S 、EbCHS T200C 、EbCHS S341A 、EbCHS S211G 、EbCHS C344S The transformation and culture steps of the plasmids containing these 15 mutations were the same as those in Example 4. The recombinant yeast was cultured at 30°C and 200 rpm for 72 h. The test results were as follows: Figure 9 shown.

[0083] The saturated mutation sequences of the effective mutation sites (F168, P84, T200, S341, S211, C344) and their adjacent non-conserved amino acid sites (N83, S85, A169, G171, V199, L212) and 15 sequences of two-point combination mutations of these 6 effective sites were used to establish a mutation sequence library with a total of 243 sequences (12×19+15=243). Based on the XGBoost model, the detection data of Example 4, Example 5 and the above-mentioned two-point mutations were subjected to machine learning modeling and prediction. The top 10 mutation sequences with the predicted value of EbCHS activity are shown in Table 2, which are 6 three-point combinations (P84Q, F168H, C344S; P84Q, V199I, C344S; F168Y, S211G, C344A; F168H, S211G, C344 4S; F168G, S211G, C344S; F168Y, S211G, C344S), and four combinations of four points (F168Y, V199I, S211G, C344A; N83F, F168Y, S211G, C344S; F168Y, V199I, S211G, C344S; F168Y, A169G, S211G, C344S) were experimentally verified.

[0084] Table 2 Predicted values of EbCHS activity

[0085] According to Example 4, the mutant plasmid was transformed into the Saccharomyces cerevisiae chassis cell CEN.PK2-1C, and the plasmid was spread on a uracil-deficient agar plate. After culturing in a 30°C incubator for four days, colonies were selected and grown on a plate supplemented with 400 g / L p-coumaric acid in 2 mL YPD medium, and the accumulation of phloretin, by-products naringenin and 2H-BNY were measured after culturing at 220 rpm and 30 °C for 72 h. Figure 10 As shown) shows that EbCHS F168Y, S211G, C344A (phloretin 93.64 mg / L, 2.14-fold; naringenin 5.62 mg / L, 0.21-fold; 2H-BNY, 0.56-fold) showed the highest phloretin production and lower naringenin production, EbCHS F168Y, S211G, C344S (Phloretin, 2.01-fold; Naringenin, 0.17-fold; 2H-BNY, 0.55-fold), EbCHS F168Y, V199I, S211G, C344A (Phloretin, 1.79-fold; Naringenin, 0.31-fold; 2H-BNY, 0.50-fold) also had higher phloretin production and lower naringenin production.

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for screening mutants with enhanced chalcone synthase activity and / or selectivity of Erigeron breviscapus, characterized in that The steps include: (1) The Pc4CL encoding gene, ScTSC13 encoding gene, and EbCHS encoding gene were constructed on the same vector backbone to obtain a recombinant plasmid; (2) By predicting the three-dimensional structure of EbCHS and based on the CHS catalytic mechanism, we preliminarily screened out sites related to the catalytic activity and selectivity of EbCHS; (3) constructing a single site saturation mutation plasmid for the site selected in step (2), and then transforming it into the Saccharomyces cerevisiae chassis cells, culturing on uracil-deficient agar plates to obtain a recombinant strain; then p -coumaric acid fermentation medium, culturing the recombinant strain, measuring phloretin and by-products in the fermentation broth, screening for effective mutants related to EbCHS catalytic activity and selectivity, and obtaining effective mutation sites; (4) Align EbCHS with CHS sequences from different plant sources and screen for non-conserved amino acid sites adjacent to the effective mutation sites obtained in step (3); (5) constructing a single site saturation mutation plasmid for the site selected in step (4), and then transforming it into the Saccharomyces cerevisiae chassis cells, culturing it on a uracil-deficient agar plate to obtain a recombinant strain; p -coumaric acid fermentation medium, culturing the recombinant strain, measuring phloretin and by-products in the fermentation broth, and screening for effective mutants with non-conservative amino acids; (6) Based on the information of the effective mutants obtained in step (3) and the non-conservative amino acid effective mutants obtained in step (5), a sequence containing two site mutation combinations in each EbCHS mutant was designed; then a plasmid containing two site mutations was constructed, and then transformed into the Saccharomyces cerevisiae chassis cells, and cultured on uracil-deficient agar plates to obtain a recombinant strain; then, a plasmid containing two site mutations was constructed, and then ... p - Cultivate the recombinant strain in a fermentation medium containing coumaric acid, and measure the phloretin and by-products in the fermentation broth to obtain relevant information; (7) Performing single-site saturation mutagenesis on the effective mutation site obtained in step (3) and the non-conserved amino acid site obtained in step (4), respectively, and combining them with the two mutations obtained in step (6) to obtain a mutant sequence library; (8) Based on the test results of step (3), step (5) and step (6), the mutant sequence library obtained in step (7) is subjected to machine learning and prediction, and then a plasmid containing the mutation is constructed based on the mutant sequence predicted by EbCHS activity, and the plasmid is transferred into the Saccharomyces cerevisiae chassis cells and cultured on uracil-deficient agar plates to obtain a recombinant strain; p The recombinant strain is cultured in a fermentation medium containing 1,2-coumaric acid, phloretin and by-products in the fermentation broth are determined, and mutants with enhanced chalcone synthase activity and / or selectivity of Erigeron breviscapus are screened.

2. The method for screening mutants of Erigeron breviscapus with enhanced chalcone synthase activity and / or selectivity according to claim 1, characterized in that: The nucleotide sequence of the Pc4CL encoding gene described in step (1) is shown in SEQ ID NO.1; The nucleotide sequence of the ScTSC13 encoding gene described in step (1) is shown in SEQ ID NO. 3; The nucleotide sequence of the EbCHS encoding gene described in step (1) is shown in SEQ ID NO.2; The prediction software described in step (2) is AlphaFold3; The CHS catalytic mechanism described in step (2) is the catalytic mechanism of MsCHS; The learning and prediction described in step (8) are performed by the XGBoost model.

3. The method for screening mutants with enhanced chalcone synthase activity and / or selectivity of Erigeron breviscapus according to claim 2, characterized in that: The Pc4CL encoding gene and the ScTSC13 encoding gene in step (1) are connected to form a fusion gene in the same expression cassette through a linker encoding gene.

4. The method for screening mutants of Erigeron breviscapus with enhanced chalcone synthase activity and / or selectivity according to claim 3, characterized in that: The recombinant plasmid described in step (1) contains the gene fragments of the following structure: promoter- Pc4CL -( GGGGS )3- ScTSC13 -Double terminator- EbCHS -promoter.

5. The method for screening mutants with enhanced chalcone synthase activity and / or selectivity of Erigeron breviscapus according to claim 4, characterized in that: The Pc4CL The promoter is SED1 promoter, and the EbCHS The promoter is TDH1 promoter; The double terminator is ter22 double terminator; The nucleic acid sequence of the Linker encoding gene is shown in SEQ ID NO.

4.

6. The method for screening mutants of Erigeron breviscapus with enhanced chalcone synthase activity and / or selectivity according to claim 1, characterized in that: The Saccharomyces cerevisiae chassis cells described in step (3), step (5), step (6) and step (8) are Saccharomyces cerevisiae CEN.PK series strains; The transformation steps described in step (3), step (5), step (6) and step (8) are as follows: Saccharomyces cerevisiae chassis cells, plasmid, transformation solution and ssDNA are mixed evenly to obtain a transformation system; the transformation system is incubated in a 37±1°C water bath and then centrifuged to discard the supernatant; the obtained cells are resuspended in water to obtain a transformation bacterial solution; The transformation system is composed of 2×10 7 ~1×10 8 cells, 100 μL transformation medium, 1 μg plasmid, and 3 μL ssDNA at a concentration of 10 mg / mL; The transformation solution consists of 800 μL of 500 g / L PEG3350 solution, 200 μL of 2 M LiAc solution, and 7.5 μL of β-mercaptoethanol; The steps (3), (5), (6) and (8) described in p The composition of the fermentation medium for coumaric acid is as follows: p -Coumaric acid 0.3-0.5 g / L, tryptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L, the solvent is deionized water; Steps (3), (5), (6) and (8) are described in p The culture of the recombinant strain in the fermentation medium of -coumaric acid is a shaking culture; The by-products in step (3), step (5), step (6) and step (8) are naringenin and 2H-BHY; The phloretin and by-products described in step (3), step (5), step (6) and step (8) are determined by HPLC.

7. The method for screening mutants of Erigeron breviscapus with enhanced chalcone synthase activity and / or selectivity according to claim 6, characterized in that: The incubation time is 20 to 40 minutes; The centrifugal conditions are 4000-8000 g for 1-5 min; The shaking culture conditions are 30±1° C. and 150-250 rpm for 3-4 days.

8. The method for screening mutants of Erigeron breviscapus with enhanced chalcone synthase activity and / or selectivity according to claim 1, characterized in that: The different plant-derived CHSs described in step (4) include PhCHS 、 SjCHS1 、 HvCHS1 、 HvCHS2 、 PmCHS 、 SbCHS2 、 CsC 、 VaCHS 、 FqV 、 HaCHS 、 PcCHS 、 MdCHS1 、 MdCHS2 .

9. A mutant of Erigeron breviscapus with enhanced chalcone synthase activity and / or selectivity, characterized in that: Obtained by the screening method according to any one of claims 1 to 8, which is EbCHS F168Y 、EbCHS P84S 、EbCHS S211G 、EbCHS F168Y, S211G 、EbCHSC3 F168Y, C344S 、EbCHSC3 P84S, C344S 、EbCHSC3 S211G, C344S 、EbCHS F168Y, S211G, C344A 、EbCHS F168Y , S211G, C344S 、EbCHS F168Y, V199I, S211G, C344A 、EbCHS T200C 、EbCHS S341A or EbCHS C344A .

10. Use of the screening method according to any one of claims 1 to 8 or the mutant of Erigeron breviscapus with enhanced chalcone synthase activity and / or selectivity according to claim 9 in phloretin synthesis.

Images