Curcumin synthetase mutant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a curcumin synthetase mutant as well as a preparation method and application thereof, and the curcumin synthetase mutant is obtained by performing one or more of the following mutations on an amino acid sequence as shown in SEQ ID NO.1: phenylalanine at the 93rd site is mutated into tyrosine; phenylalanine at the 152nd site is mutated into leucine; phenylalanine at the 152nd site is mutated into serine; threonine at the 218th site is mutated into alanine; serine at the 259th site is mutated into glycine; valine at the 288 site is mutated into glycine; valine at the 288 site is mutated into glutamic acid; histidine at the 316th site is mutated into tyrosine; valine at the 343rd site is mutated into methionine. According to the invention, curcumin synthetase is subjected to directional modification, a basic group of a CUS gene is subjected to random mutation through error-prone PCR, and five CUS mutants with improved activity are screened. The curcumin synthesis capacities of the five CUS mutants are respectively 2.489 times, 3.887 times, 5.23 times, 6.439 times and 11.567 times of those of a wild type.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a curcumin synthase mutant, a preparation method thereof and an application thereof. Background Art

[0002] Curcuminoid synthase, with the English name curcuminoid synthase and abbreviated as CUS, is a key enzyme in the curcumin biosynthesis pathway. It belongs to type III polyketide synthase, abbreviated as PKSs, and is widely present in Zingiberaceae plants. CUS can catalyze the condensation of aromatic coenzyme A and malonyl coenzyme A to generate analogues such as demethoxycurcumin, bisdemethoxycurcumin and curcumin. When the aromatic coenzyme A is feruloyl coenzyme A, curcumin is generated. First, CUS catalyzes the decarboxylative condensation of malonyl coenzyme A onto the feruloyl coenzyme A thioester to form a diketone coenzyme A intermediate, then hydrolyzes to β-keto acid, and then continues to catalyze the decarboxylative condensation of β-keto acid onto the feruloyl coenzyme A thioester to form curcumin.

[0003] Curcumin, that is, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, with the English name curcumin, is an aromatic diphenylheptadione compound, mainly present in the tubers or rhizomes of Zingiberaceae and Araceae plants, and is considered to be its main active ingredient. The main chain of curcumin is an unsaturated aliphatic and aromatic group. Due to its special conjugated structure, including two methylated phenols, which are connected in the form of heptadiene-3,5-ene, it shows a bright yellow color. As a natural orange-yellow substance, curcumin is not only mainly used as a food colorant, but also used as an acid-base indicator in chemistry due to its red color when the pH value is greater than 8. In recent years, curcumin has also been proven to have many effects in reducing blood lipids, anti-tumor, antioxidant and other aspects.

[0004] The synthesis of curcumin uses ferulic acid and L-arabinose as substrates. Ferulic acid generates feruloyl coenzyme A under the action of p-coumaroyl coenzyme A ligase; L-arabinose generates malonyl coenzyme A through an enzymatic reaction; subsequently, CUS catalyzes 2 molecules of feruloyl coenzyme A and 1 molecule of malonyl coenzyme A to generate 1 molecule of curcumin. In this catalytic pathway, CUS is determined as the key rate-limiting enzyme. However, the efficiency of wild-type CUS in synthesizing curcumin is not high, which greatly limits the further application of curcumin. Therefore, there is an urgent need to provide a new strategy for efficiently synthesizing curcumin. Summary of the Invention

[0005] The object of the present invention is to provide a curcumin synthase mutant for preparing curcumin, which solves the problem of low efficiency of wild-type CUS in synthesizing curcumin in the prior art.

[0006] The technical solution adopted by the present invention is:

[0007] In the first aspect of the present invention, a curcumin synthase mutant is provided, and the curcumin synthase mutant is one or more of the following mutations to the amino acid sequence shown in SEQ ID NO.1: phenylalanine at position 93 is mutated to tyrosine; phenylalanine at position 152 is mutated to leucine; phenylalanine at position 152 is mutated to serine; threonine at position 218 is mutated to alanine; serine at position 259 is mutated to glycine; valine at position 288 is mutated to glycine; valine at position 288 is mutated to glutamic acid; histidine at position 316 is mutated to tyrosine; valine at position 343 is mutated to methionine.

[0008] In the second aspect of the present invention, a nucleotide sequence encoding the curcumin synthase mutant is provided.

[0009] In the third aspect of the present invention, a recombinant vector containing the nucleotide sequence is provided.

[0010] In the fourth aspect of the present invention, a genetically engineered bacterium containing the recombinant vector is provided.

[0011] In the fifth aspect of the present invention, an application of the curcumin synthase mutant, the nucleotide sequence, the recombinant vector or the genetically engineered bacterium is provided, and the application refers to being used for preparing curcumin.

[0012] Preferably, the substrate for preparing the curcumin is ferulic acid.

[0013] Preferably, the curcumin is used for preparing a food colorant.

[0014] Preferably, the curcumin is used for preparing an acid-base indicator.

[0015] Preferably, the curcumin is used for preparing an antioxidant.

[0016] Preferably, the curcumin is used for preparing a lipid-lowering drug.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The present invention provides a curcumin synthase mutant, which is one or more of the following mutations to the amino acid sequence shown in SEQ ID NO. 1: phenylalanine at position 93 is mutated to tyrosine; phenylalanine at position 152 is mutated to leucine; phenylalanine at position 152 is mutated to serine; threonine at position 218 is mutated to alanine; serine at position 259 is mutated to glycine; valine at position 288 is mutated to glycine; valine at position 288 is mutated to glutamate; histidine at position 316 is mutated to tyrosine; valine at position 343 is mutated to methionine. The present invention conducts directed evolution modification on curcumin synthase, makes the bases of the CUS gene randomly mutated by error-prone PCR, and establishes a CUS random mutant library. Through high-throughput screening method, 5 CUS mutants with improved activity are screened. The abilities of these 5 CUS mutants to synthesize curcumin are 2.489 times, 3.887 times, 5.23 times, 6.439 times and 11.567 times that of the wild type respectively.

[0019] In the present invention, compared with the wild type, the specific activity of the CUS mutant is improved, indicating that the increase in curcumin production is due to the increase in the protein expression level of curcumin synthase in the mutant.

[0020] The present invention uses the method of directed evolution to modify curcumin synthase and uses the high-throughput screening method. Compared with the growth screening and 96-well plate screening methods, the throughput of one round of screening is larger, the efficiency is higher, and the positive rate is also higher. Description of the Drawings

[0021] Figure 1 CUS catalyzes ferulic acid to synthesize curcumin via feruloyl-CoA.

[0022] Figure 2 Detection results of the abilities of 5 CUS mutants obtained by high-throughput screening to synthesize curcumin Detailed Embodiments

[0023] The following further illustrates the present invention through specific embodiments, but does not limit the scope of the present invention. Modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but these modifications or substitutions all fall within the protection scope of the present invention.

[0024] The inventive concept of the present invention is as follows:

[0025] Curcumin synthase is a key enzyme in the curcumin biosynthesis pathway. The synthesis of curcumin usually uses ferulic acid as a substrate. Ferulic acid is converted into feruloyl-CoA under the action of p-coumaroyl-CoA ligase. Subsequently, CUS catalyzes the reaction of 2 molecules of feruloyl-CoA and 1 molecule of malonyl-CoA to produce 1 molecule of curcumin. In this catalytic pathway, CUS is identified as the key rate-limiting enzyme. However, the efficiency of curcumin synthesis by wild-type CUS is not high, which greatly limits the further application of curcumin.

[0026] Based on this, the present invention provides a curcumin synthase mutant, which is one or more of the following mutations of the amino acid sequence shown in SEQ ID NO.1: phenylalanine at position 93 is mutated to tyrosine; phenylalanine at position 152 is mutated to leucine; phenylalanine at position 152 is mutated to serine; threonine at position 218 is mutated to alanine; serine at position 259 is mutated to glycine; valine at position 288 is mutated to glycine; valine at position 288 is mutated to glutamate; histidine at position 316 is mutated to tyrosine; valine at position 343 is mutated to methionine.

[0027] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below with reference to specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0028] The abbreviation list of the present invention is shown in Table 1.

[0029] Table 1 Abbreviation list

[0030] Abbreviation Full name CUS Curcumin synthase PKSs Polyketide synthases 4CL p-Coumaroyl-CoA ligase Feruloyl-CoA Feruloyl coenzyme A

[0031] Example 1

[0032] A curcumin synthase mutant is as follows:

[0033] (1) The CUS mutant plasmid library was constructed by error-prone PCR.

[0034] Using the wild-type CUS gene as a template for error-prone PCR, the PCR product was subjected to agarose gel electrophoresis and then recovered. Using the recovered PCR product as a megaprimer and the plasmid carrying the wild-type CUS gene as a template, MEGAWHOP PCR was carried out using Pyrobest DNA polymerase. The amino acid sequence encoded by the CUS gene is shown in SEQ ID NO.1.

[0035] The above-mentioned MEGAWHOP PCR products were digested with DpnⅠ overnight at 37°C for 14 h to eliminate the template plasmid. 1 μL of the digested product was electrotransformed into competent E. coli MC1061 cells. After recovery at 37°C for 1 h, it was evenly spread on a plate containing kanamycin resistance. After overnight culture at 37°C for 14 h, the monoclonal strains on the plate were scraped to extract plasmids, and the CUS mutant plasmid library was obtained.

[0036] (2) Screening of the CUS error-prone mutant library.

[0037] Principle: Utilizing the characteristic that curcumin is yellow, high-throughput screening was carried out on agar plates. Arabinose and ferulic acid were added to the agar plates, and the CUS error-prone mutant library was cultured on the agar plates respectively to enable the CUS mutants to complete the synthesis pathway from ferulic acid to curcumin. The medium changed from white to yellow, and the deeper the yellow color, the higher the curcumin content and the better the CUS activity. Finally, CUS mutants with enhanced CUS activity were obtained. The process of CUS catalyzing ferulic acid to synthesize curcumin via feruloyl coenzyme A is shown in Figure 1 .

[0038] The method is as follows: The CUS mutant plasmid library and the plasmid carrying the wild-type CUS gene were respectively electrotransformed into competent E. coli BW25113 cells. After recovery on a shaker at 37°C for 1 h, the recovered solutions of the wild-type and mutants were appropriately diluted and spread on YM9 solid medium supplemented with 8 mM ferulic acid, 1 mM L-arabinose, and 1‰ kanamycin for high-throughput screening. They were cultured in an incubator at 30°C for 24 h, and the changes and degrees of the colony colors on the plates were observed. Monoclonal spots with a deeper yellow color on the plate were picked and streaked onto a new YM9 solid medium for retesting. At the same time, wild-type strains were picked onto the same screening plate as a control. They were cultured at 30°C for 12 h, and the colors of the colonies on the plates were observed. Monoclonal strains that were still darker in color than the wild-type strains after retesting were picked into LB medium containing kanamycin and cultured on a shaker at 37°C for 14 h as the seed solution.

[0039] Add 1 μL of 1 M CaCl2 solution, 20 μL of 1 M MgSO4 solution and 1‰ kanamycin to 10 mL of YM9 liquid medium, and mix well. Inoculate the seed solutions of the wild-type and mutant strains screened in the previous step into 10 mL of YM9 liquid medium at a ratio of 1% respectively. After culturing at 37 °C until the OD600 is about 0.6, add 100 μL of 800 mM ferulic acid and 10 μL of 1 M L-arabinose, transfer to 30 °C and culture for 12 h, and detect the OD600 of the fermented bacterial liquid to judge the growth of the strains. Take 200 μL of the fermented liquid, add 800 μL of absolute ethanol, vortex for 2 min, centrifuge at 10000 g for 5 min, take the supernatant, filter it, and detect the filtrate by high performance liquid chromatography (HPLC), and the concentration of curcumin can be obtained by calculation.

[0040] By comparing the curcumin concentrations synthesized by the wild-type strain and the mutant strain, the N16 mutant with a higher curcumin synthesis ability than the wild-type strain was obtained. Extract the plasmid of the N16 mutant, sequence the CUS gene on the N16 mutant, and the amino acid sequence at the CUS gene locus in the N16 mutant is shown in SEQ ID NO.2.

[0041] Example 2

[0042] A curcumin synthase mutant is as follows:

[0043] In this example, error-prone PCR was carried out using the N16 mutant obtained in Example 1 as a template, and further high-throughput screening was carried out. The experimental method was the same as that in Example 1, and the S7 mutant was obtained. The amino acid sequence at the CUS gene locus in the S7 mutant is shown in SEQ ID NO.3.

[0044] Example 3

[0045] A curcumin synthase mutant is as follows:

[0046] In this example, error-prone PCR was carried out using the S7 mutant obtained in Example 2 as a template, and further high-throughput screening was carried out. The experimental method was the same as that in Example 1, and the R1 mutant was obtained. The amino acid sequence at the CUS gene locus in the R1 mutant is shown in SEQ ID NO.4.

[0047] Example 4

[0048] A curcumin synthase mutant is as follows:

[0049] In this example, error-prone PCR was carried out using the R1 mutant obtained in Example 3 as a template, and further high-throughput screening was carried out. The experimental method was the same as that in Example 1, and the P10 mutant was obtained. The amino acid sequence at the CUS gene locus in the P10 mutant is shown in SEQ ID NO.5.

[0050] Example 5

[0051] A curcumin synthase mutant is as follows:

[0052] In this example, error-prone PCR was performed using the P10 mutant obtained in Example 4 as a template, and further high-throughput screening was carried out. The experimental method was the same as that in Example 1, and the P11 mutant was obtained. The amino acid sequence at the CUS gene locus in the P11 mutant is shown in SEQ ID NO.6.

[0053] The activities of several CUS mutants obtained in Examples 1 to 5 were compared:

[0054] The mutants described in Examples 1 to 5 were respectively cultured in YM9 medium containing 15 mM ferulic acid and 1 mM L-arabinose at 30 °C for 14 h. Take 200 μL of the fermentation sample, add 800 μL of ethanol and mix and shake. Filter the sample with a 0.2 μm filter membrane and detect by HPLC on a Shimadzu LC-20A system equipped with a photodiode array detector.

[0055] The HPLC separation conditions were as follows: at 35 °C, a Waters Symmetry C18 chromatographic column was used with 20% acetonitrile containing 0.1% formic acid as the mobile phase, the flow rate was 0.48 mL / min, and curcumin was monitored at 420 nm. A standard curve was drawn with commercially available analytical pure curcumin standard to calculate the curcumin concentration of the sample.

[0056] After detection and statistics, the results are shown in Figure 2 , the abilities of the five mutants, namely the N16 mutant, S7 mutant, R1 mutant, P10 mutant, and P11 mutant, to synthesize curcumin were 2.489 times, 3.887 times, 5.23 times, 6.439 times, and 11.567 times that of the wild-type CUS, respectively. Among them, the P11 mutant had the best effect, and the concentration of curcumin synthesized was 7.44 mM.

[0057] The amino acid sequence of wild-type CUS, SEQ ID NO.1:

[0058]

[0059] The amino acid sequence of the N16 mutant, SEQ ID NO.2:

[0060]

[0061] The amino acid sequence of the S7 mutant, SEQ ID NO.3:

[0062]

[0063]

[0064] Amino acid sequence of the R1 mutant, SEQ ID NO.4:

[0065]

[0066] Amino acid sequence of the P10 mutant, SEQ ID NO.5:

[0067]

[0068] Amino acid sequence of the P11 mutant, SEQ ID NO.6:

[0069]

[0070]

[0071] The summary of CUS mutant sites is shown in Table 2.

[0072] Table 2 Summary of CUS mutant sites

[0073]

[0074] Curcumin synthase plays a core role in the synthesis of curcumin and is of crucial significance for the biosynthesis of curcumin, plant growth and development, medical research, industrial production, etc.

[0075] Key link in biosynthesis: Curcumin synthase is a key enzyme in the curcumin biosynthesis pathway and directly participates in the catalytic synthesis reaction. In plants, it can specifically recognize and bind to related substrates, promoting the chemical reaction to proceed in the direction of curcumin production. For example, it can catalyze the condensation reaction between feruloyl coenzyme A and malonyl coenzyme A, and finally produce curcumin through a series of complex biochemical processes. Without curcumin synthase, the curcumin biosynthesis pathway will be blocked and plants cannot synthesize curcumin normally.

[0076] Influence on plant physiological functions: Curcumin has antioxidant, antibacterial and other properties, which are of great significance for the growth, development and defense mechanisms of plants themselves. Curcumin synthase indirectly affects plant physiological processes by regulating the synthesis amount of curcumin. When facing the invasion of external pathogens, plants will increase the synthesis of curcumin, the activity of curcumin synthase is enhanced, and more curcumin is synthesized to resist pathogens and protect plants from damage, which helps plants survive and reproduce in the complex natural environment.

[0077] Key Targets in Medical Research: Curcumin has a wide range of applications in the medical field and possesses various potential medicinal values such as anti-inflammatory, anti-cancer, and antioxidant properties. As a key regulatory factor in curcumin synthesis, curcumin synthase has become an important target in medical research. Researchers can study the structure and function of this enzyme to gain a deeper understanding of the curcumin synthesis mechanism, thereby developing more effective drugs or treatment methods. For example, by regulating the activity of curcumin synthase, it is expected to achieve precise control over curcumin production, providing theoretical support for the production of high-purity and high-activity curcumin-based drugs.

[0078] Important Basis for Industrial Production: In the industrial production of curcumin, the characteristics and activity of curcumin synthase are important bases for optimizing the production process. Understanding information such as the catalytic characteristics and optimal reaction conditions of this enzyme helps improve the production efficiency and yield of curcumin. Through genetic engineering techniques, the gene encoding curcumin synthase is introduced into a suitable expression system to achieve high-level expression of this enzyme, thereby enhancing the efficiency of microbial fermentation for curcumin production; or by using protein engineering means to modify curcumin synthase to enhance its stability and catalytic activity, improving the industrial production level of curcumin.

[0079] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as falling within the scope described in this specification.

[0080] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A curcumin synthase mutant, characterized in that: The curcumin synthase mutant is one or more of the following mutations to the amino acid sequence shown in SEQ ID NO.1: The phenylalanine at position 93 was mutated to tyrosine; The phenylalanine at position 152 was mutated to leucine; The phenylalanine at position 152 was mutated to serine; The threonine at position 218 was mutated to alanine; The serine at position 259 was mutated to glycine; The valine at position 288 was mutated to glycine; The valine at position 288 was mutated to glutamic acid; The histidine at position 316 was mutated to tyrosine; The valine at position 343 was mutated to methionine.

2. A nucleotide sequence encoding the curcumin synthase mutant according to claim 1.

3. A recombinant vector comprising the nucleotide sequence of claim 2.

4. A genetically engineered bacterium comprising the recombinant vector according to claim 3.

5. Use of the curcumin synthase mutant according to claim 1, the nucleotide sequence according to claim 2, the recombinant vector according to claim 3 or the genetically engineered bacteria according to claim 4, characterized in that: The application refers to the preparation of curcumin.

6. The use according to claim 5, characterized in that The substrate for preparing the curcumin is ferulic acid.

7. The use according to claim 5, characterized in that The curcumin is used for preparing food coloring agent.

8. The use according to claim 5, characterized in that The curcumin is used for preparing an acid-base indicator.

9. The use according to claim 5, characterized in that The curcumin is used for preparing antioxidant.

10. The use according to claim 5, characterized in that The curcumin is used for preparing blood lipid lowering medicine.

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