Chalcone synthase mutants and uses thereof

Abstract

The application discloses chalcone synthase mutants and application thereof, and belongs to the technical field of biology.The application obtains multiple CHS mutants, which are 1.1-2.96 times of the yield of wild-type synthesized naringenin, wherein the best mutant is CHS-3-15, and the yield of naringenin is 1.85 mM.Through in-vitro characterization and pure-enzyme activity comparison, the specific activity of CHS-2-7, CHS-2-12, CHS-3-15 and CHS-3-27 is 1.11-2.30 times of that of the wild type.To improve the synthesis of naringenin and reduce the synthesis of by-product CTAL, a specific chassis is constructed, and the CHIL protein capable of reducing by-products is co-expressed, the CHIL expression amount is optimized by optimizing the ribosome binding site sequence, and finally the yield of naringenin reaches 3.98 mM in a shake flask.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to chalcone synthase mutants and their applications. Background Technology

[0002] Flavonoids are a class of polyphenolic plant secondary metabolites, widely distributed in the plant kingdom, consisting of two phenolic hydroxyl benzene rings linked by a central three-carbon bond. Based on a C6-C3-C6 structure, these compounds are classified into several categories according to the degree of oxidation and conformation of the three-carbon bond (C3): flavones, flavonols, flavanones (dihydroflavones), flavanone alcohols (dihydroflavonols), isoflavones, isoflavones (dihydroisoflavones), chalcones, dihydrochalcones, flavans, flavanols, and other flavonoids. Flavonoids play a crucial role in plant growth and development and possess pharmacological activities such as antibacterial, antioxidant, and hepatoprotective effects. Therefore, flavonoids have been a research hotspot in the biomedical field both domestically and internationally. Naringenin is an important platform compound in their synthesis and is key to the synthesis of other flavonoids. In plants and bacteria, p-coumaric acid (p-CA) is generally used as a precursor, and naringenin chalcone is generated by the catalysis of p-coumaryl-CoA ligase (4CL) and type III polyketide synthase chalcone synthase (CHS). Naringenin is then generated by the catalysis of chalcone isomerase or spontaneous isomerization.

[0003] In this catalytic pathway, chalcone synthase catalyzes the conversion of 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA into 1 molecule of chalcone naringenin; this step has been identified as the critical rate-limiting step. Simultaneously, CHS catalysis may generate the byproduct CTAL (see...). Figure 1 Previous studies have primarily focused on increasing the copy number or expression intensity of chalcone synthase to enhance naringenin production. However, this can negatively impact metabolic flux, burdening the strain and hindering the synthesis of the final product. Weijia Cao et al. improved the synthesis of pinocembrin, a downstream product, through site-directed mutagenesis of chalcone synthase ("Enhanced pinocembrin production in escherichia coli by regulating cinnamic acid metabolism"). However, studies on improving naringenin production and reducing byproduct synthesis through directed evolution of chalcone synthase have yet to be reported. Summary of the Invention

[0004] One objective of this invention is to provide a chalcone synthase mutant, which is obtained by mutating SEQ ID NO.1 with amino acids, wherein the amino acid mutation is selected from any of the following:

[0005]

[0006]

[0007] A second objective of this invention is to provide a DNA molecule that encodes the aforementioned chalcone synthase mutant.

[0008] A third objective of this invention is to provide a recombinant plasmid, wherein the recombinant plasmid is linked to the aforementioned DNA molecule.

[0009] Preferably, the recombinant plasmid is pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+). +), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE3 2. pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX- 6p2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 or pUC-19.

[0010] The fourth objective of this invention is to provide a host cell containing the aforementioned recombinant plasmid, wherein the host cell is a non-plant cell.

[0011] Preferably, the host cell is a prokaryotic cell.

[0012] More preferably, the prokaryotic cell is Escherichia coli BW25113.

[0013] More preferably, the *Escherichia coli* is BW25113ΔpoxB::acsΔadhEΔfabFΔtesBΔyigLΔyciAΔfadMΔpaaI.

[0014] The fifth objective of this invention is to provide a method for producing naringin, the method comprising the following steps:

[0015] (1) Obtain a DNA molecule encoding the amino acid sequence of the mutant of claim 1;

[0016] (2) Construct a recombinant expression vector using the DNA molecule obtained in step (1), and then transform it into a host cell;

[0017] (3) Induce host cells containing recombinant expression vectors and isolate and purify naringenin.

[0018] The sixth objective of this invention is to provide the application of the above-mentioned chalcone synthase mutant in the production of naringenin.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention utilizes a growth-coupled high-throughput screening method to screen mutants with enhanced CHS activity. Simultaneously, this method is used to screen genes within the *E. coli* host genome that can influence naringenin synthesis. This study aims to enhance CHS activity, reduce CTAL byproduct synthesis, and ultimately achieve high naringenin production. Attached Figure Description

[0021] Figure 1 This diagram shows the CHS-catalyzed synthesis pathway of naringenin from coumaroyltriacetica acid lactone (CTAL), with CTAL being the main byproduct.

[0022] Figure 2a The results show the comparison of naringenin production yields of the CHS mutant obtained through three rounds of high-throughput screening in Example 1. Dark gray indicates naringenin production, and light gray indicates the production of the byproduct CTAL.

[0023] Figure 2b The results of in vitro testing of enzyme specific activities after purifying the enzyme protein from the CHS mutant in Example 1 are shown.

[0024] Figure 3 The results of the effect of overexpression of several thioesterases in Escherichia coli on the synthesis of naringenin and its byproducts in Example 2 are shown.

[0025] Figure 4 The results of optimizing naringenin synthesis yield were obtained by co-expressing the CHS mutant and CHIL in Example 2. Detailed Implementation

[0026] Example 1

[0027] Using p-coumaric acid (p-CA) as a precursor, p-coumaric acid-CoA is generated via p-coumaric acid-CoA ligase (4CL), which inhibits the growth of host bacterial cells. Therefore, increasing CHS enzyme activity helps to eliminate the accumulation of toxic intermediates, meaning that strains with enhanced CHS activity can achieve faster growth. CHS mutant libraries were screened using a growth assay. After several rounds of growth and enrichment following the addition of p-CA, the substrate required for naringenin synthesis, CHS mutants with enhanced activity were finally obtained.

[0028] Following the above high-throughput screening and three rounds of growth high-throughput screening, seven CHS activity-enhancing mutants, namely CHS-1-1, CHS-1-2, and CHS-1-7, were obtained (see Table 1 for details). Naringin was synthesized via fermentation, with the byproduct CTAL being generated. The yields were 0.623 (0.207), 0.712 (0.358), 0.588 (0.452), 0.208 (0.387), 0.624 (0.356), 0.924 (0.526), ​​1.09 (0.556), 0.95 (0.49), 1.70 (0.52), 1.85 (0.59), and 1.53 (0.57) mM, respectively (the yield of the byproduct CTAL is in parentheses). Figure 2a As shown, the naringenin production was 1.1-2.96 times that of the wild-type (CHS-WT) mutant, with the best-performing mutant being CHS-3-15, which produced 1.85 mM of naringenin.

[0029] In vitro characterization and comparison of pure enzyme activities showed that CHS-2-7, CHS-2-12, CHS-3-15, and CHS-3-27 had 1.11-2.30 times higher specific activities than the wild type. The specific activities of the wild type, CHS-2-7, CHS-2-12, CHS-3-15, and CHS-3-27 were 3.28, 4.97, 4.58, 7.88, and 7.85 U / mg, respectively.

[0030] The specific experimental plan is as follows:

[0031] Naringin was synthesized by fermentation: pGAP-CHS-4CL plasmids carrying wild-type CHS and mutants (Reference: Xiong, D., Lu, S., Wu, J., Liang, C., Wang, W., Wang, W., Jin, J.-M., Tang, S.-Y. 2017. Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor. Metabolic Engineering, 40, 115-123) were transformed into Escherichia coli BW25113ΔpoxB::acsΔadhEΔfabF (Wu, J. et al. Metabolic Engineering for Improved Curcumin Biosynthesis in Escherichia coli. J Agric Food Chem 68, 10772-10779 (2020)). Single plasmids were picked, cultured overnight in LB medium at 37°C in a shaker, transferred to YM9 medium, and p-CA 4.5 mM substrate was added. After culturing at 30°C for 48 hours, samples were taken for analysis.

[0032] In vitro characterization of enzyme activity: pET28a-CHS wild-type and mutant plasmids were constructed and transformed into Escherichia coli BL21(DE3) host bacteria, respectively, and plated on kanamycin-resistant plates. Single colonies were picked and cultured in LB medium with the corresponding antibiotics at 37°C for 14 hours. This culture was then used as a seed culture for inoculation into LB medium with the corresponding antibiotics at a 1% (v / v) inoculation rate. When the cell concentration reached approximately 0.6, 0.4 mM IPTG inducer was added, and the cells were cultured for another 12 hours at 37°C. The cells were then collected, sonicated to disrupt the cell walls, and purified using nickel column affinity chromatography to purify the recombinant protein CHS enzyme. The pure enzyme reaction system consisted of HEPES-NaOH buffer (100mM, pH 7.5), 50μM p-CA, 150μM malonyl-CoA, and an appropriate amount of CHS pure enzyme. After incubating at 30℃ for 1 hour, 50μL of pure enzyme was added to terminate the reaction. Quantification was performed by HPLC. The unit enzyme activity was defined as the amount of pure CHS enzyme required to generate 1 nmol of naringenin per minute.

[0033] HPLC detection conditions were as follows: Shimadzu LC-20AT system (Shimadzu Corporation, Kyoto, Japan), Waters Symmetry C18 column (5 μm, 250 mm × 4.6 mm), column temperature 35℃. Mobile phase A was 0.1‰ formic acid, and mobile phase B was acetonitrile. Elution conditions were gradient elution with mobile phase B (5–30 min, 30–50%) at a flow rate of 0.5 mL / min. The detection wavelengths for naringenin, CTAL, and p-CA were 287 nm, 320 nm, and 305 nm, respectively. The concentrations of the compounds were quantified using their respective standard curves.

[0034] Table 1

[0035] CHS mutant mutation site CHS-1-1* K57R, M159L, K281E CHS-1-2 V98A,I279V CHS-1-3 T131S, C341S CHS-1-5 K62N, T194S, A195A (synonymous mutation), V232V CHS-1-7 K62N, M159L CHS-2-7* K57R, M159L, K281E, K9R, V100D, G334G (synonymous mutation) CHS-2-12 K57R,M159L,K281E,L147Q,G200G,V261F CHS-3-3 K57R, M159L, K281E, K9R, V100D, G334G, I309I (synonymous mutation) CHS-3-15 K57R,M159L,K281E,K9R,V100D,G334G,L358Q CHS-3-27 K57R, M159L, K281E, K9R, V100D, G334G (synonymous mutation), S297N

[0036] The wild-type CHS sequence SEQ ID NO.1 is as follows:

[0037] MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTYPDYYFRITNSEHKTDLKEKFKRMCEKSMIKKRYMHLTEEILKENPSMCEYMAPSLDARQDIVVVEVPKLGKEAAQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNKGARVLVVCSEITA VTFRGPNDTHLDSLVGQALFGDGAGAIIIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLEEAFRPLSISDWNSLFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNYGNMSSACVLFILDEMRKASAKEGLGTTGEGLEWGVLFGFGPGLTVETVVLHSVAT*.

[0038] Example 2

[0039] Using the high-throughput screening method described above, the screening of overexpression libraries of the E. coli genome (i.e., mixed libraries that overexpress individual genes in the E. coli genome) showed that overexpression of thioesterase II (TesB) significantly increased the yield of the byproduct CTAL, reaching 1.69 mM, which is about 7 times that of the control group. Other thioesterases, such as YigI, FadM, YciA, and PaaI, can also increase the synthesis of byproducts to varying degrees.

[0040] Therefore, to improve naringenin synthesis and reduce the synthesis of the byproduct CTAL, several thioesterases were knocked out in the basal bacteria that biosynthesize naringenin, and a basal bacteria was constructed: BW25113ΔpoxB::acsΔadhEΔfabFΔtesBΔyigLΔyciAΔfadMΔpaaI. This was co-expressed with the previously reported CHIL protein, which can reduce byproducts (Reference: Role of achalcone isomerase-like protein in flavonoid biosynthesis in Arabidopsisthaliana). Plasmids pYB1k-CHS3-15-CHIL (regulated by the arabinose promoter, containing a kanamycin resistance gene, see Wu, J. et al. Metabolic Engineering for Improved Curcumin Biosynthesis in Escherichia coli. J Agric Food Chem 68, 10772-10779 (2020)) and pTrc99a-4CL (regulated by the Trc promoter, containing an ampicillin resistance gene, see Li, S. et al. De Novobiosynthesis of chlorogenic acid using an artificial microbial community. J Agric Food Chem 69, 2816-2825 (2021)) were constructed. Both plasmids were simultaneously transformed into the aforementioned substrate bacteria and spread on LB agar plates containing the two antibiotics. Single colonies were picked and inoculated into LB medium, cultured at 37°C in a shaker for 12 hours, and then transferred to YM9 medium (1×M9 salts, 10 g·L⁻¹). -1 yeast extract,3%glycerol,and 42g·L -1 MOPS [3-(N-morpholino)propanesulfonic acid] were incubated at 30°C in a shaker after adding 1 mM arabinose, 0.4 mM IPTG, and 4.5 mM p-CA substrate as inducers. Samples were then taken for analysis. Results are shown in [link to results]. Figure 4 After 72 hours of incubation, naringenin levels reached 3.98 mM.

[0041] Figure 44CL2M is a 4CL2 mutant derived from Arabidopsis thaliana (References: Rodrigues, JL; Prather, KLJ; Kluskens, LD; Rodrigues, LR, Heterologous production of curcuminoids. Micribiol. Mol. Biol. R 2015, 79(1), 66239-60.).

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A chalcone synthase mutant, characterized in that, The chalcone synthase mutant was obtained by mutating amino acids in SEQ ID NO.1, and the amino acid mutation sites are: K57R, M159L, K281E, K9R, V100D, G334G and L358Q.

2. A DNA molecule, characterized in that, The DNA molecule encodes the chalcone synthase mutant of claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid is ligated with the DNA molecule as described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmid is pET-28a(+).

5. A host cell, characterized in that, The host cell contains the recombinant plasmid as described in claim 3 or 4, and the host cell is a non-plant cell.

6. The host cell according to claim 5, characterized in that, The host cell is a prokaryotic cell.

7. The host cell according to claim 6, characterized in that, The prokaryotic cells were Escherichia coli BW25113.

8. A method for producing naringin, characterized in that, The production method includes the following steps: (1) Obtain a DNA molecule encoding the amino acid sequence of the mutant of claim 1; (2) Construct a recombinant expression vector using the DNA molecule obtained in step (1), and then transform it into a host cell; (3) Induce host cells containing recombinant expression vectors and isolate and purify naringenin.

9. The application of the chalcone synthase mutant according to claim 1 in the production of naringenin.

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