Construction and preparation method of multifunctional fusion enzyme system constructed based on artificially designed substrate microchannel principle and substrate cyclic regeneration system

Abstract

The invention provides a method for preparing rebaudioside M by constructing a UGT91C1-UGT76G1-SuSy fusion enzyme on the basis of an artificially constructed substrate microchannel principle and using relatively cheap raw materials to efficiently catalyze a cascade reaction. EAAAK 3 and (GGGGS) 4 are used as connecting peptides, UGT76G1 is fused at the C end of UGT91C1, SuSy is fused at the C end of UGT76G1, mass transfer of an intermediate product is enhanced, and the intermediate product is prevented from being diffused to a reaction main body. The invention also discloses a construction method of the ugt91C1-C1-ugt76G1-suusy fusion gene, a construction method of a recombinant plasmid, a construction method of a recombinant strain, a protein expression and purification method and a method for catalytically preparing a product. The ugt91C1-C1-ugt76G1-suusy fusion gene has a very good application prospect in biological industrial catalytic production.

Description

Technical Field

[0001] The present invention relates to a UGT91C1-UGT76G1-SuSy fusion enzyme molecule rationally designed by computer-aided prediction of structure under the guidance of the principle of artificially designed substrate microchannels, a preparation method for highly expressing the same in Pichia pastoris, and its application in substrate cycle regeneration and cascade catalysis, belonging to the field of biocatalysis and biotransformation. Technical Background

[0002] At present, there is a lot of evidence indicating that excessive sugar intake can lead to diseases such as obesity, dental caries, hypertension, diabetes, etc. Therefore, finding a low-calorie and high-sweetness sugar substitute has become the focus of the food and beverage industry. Steviol glycosides are a class of substances present in Stevia rebaudiana. As a representative of natural sweeteners, they have a long history of use, high sweetness and low calories, and stable properties. Their sweetness is 250-450 times that of sucrose, but the calories are only 0.3% of it. In addition to being used in the food industry, they also have certain pharmacological and health care effects. Steviol glycosides are derivatives composed of the diterpene steviol backbone. The hydroxyl group at the C13 position and the carboxyl group at the C19 position of the steviol aglycone in the side chain can be glycosylated to connect different numbers of glucosyl groups. Among the components of steviol glycosides, stevioside (ST) and rebaudioside A (RA) account for 5%-10% and 2%-5% respectively, which are their main components. However, their bitter aftertaste limits their application. The proportion of rebaudioside D (RD) and rebaudioside M (RM) is extremely low, only 0.2%. However, because it has no bitter aftertaste, it is considered the best substitute for sweeteners.

[0003] The conversion of RA to RM is an enzymatic reaction catalyzed by the glycosyltransferases UGT91C1 and UGT76G1. First, RA forms a 1,2-β-D glycosidic bond at the C-13 position under the catalysis of the glycosyltransferase UGT9C1 to generate RD, and RD forms a 1,3-β-D glycosidic bond at the C-19 position under the catalysis of UGT76G1 to generate RM. Therefore, using the cheap and easily available RA to generate RM through the cascade catalysis of UGT91C1 and UGT76G1 is a green and feasible method. However, UGT91C1 and UGT76G1 are two UDPG-dependent glycosyltransferases, and their catalysis strictly depends on the second substrate UDPG as a glycosyl donor to proceed. However, the high price of UDPG limits its popularization and application. Therefore, how to reduce the production cost is an urgent problem to be solved for the industrialization of the enzymatic synthesis of rebaudioside M.

[0004] Sucrose synthase (SuSy) is one of the key enzymes involved in sucrose metabolism in plants. It can catalyze the reversible conversion of sucrose and uridine diphosphate (UDP) into fructose and UDPG. The catalytic reaction of SuSy avoids the complex de novo synthesis pathway of UDPG. With only the relatively inexpensive UDP and sucrose, it can achieve the efficient one-step synthesis of UDPG, reducing the acquisition cost of the sugar donor UDPG. Currently, some application methods for synthesizing RM based on UGT91C1 and UGT76G1 have been developed. However, there are no patent and literature reports on the regeneration of the sugar donor UDPG and its participation in the combined cascade catalytic reaction, especially the construction and preparation of the UGT91C1-UGT76G1-SuSy trifunctional fusion enzyme based on the principle of artificially constructed substrate microchannels. The UGT91C1-UGT76G1-SuSy trifunctional fusion enzyme catalyzes the cascade reaction, which can proceed with only the relatively inexpensive UDP and sucrose added, and realizes the recycling of substrates. It can also strengthen the mass transfer process of intermediate products and substrates through the artificially designed substrate microchannels, avoid the diffusion of intermediate products and substrates into the bulk phase, increase the local concentration of intermediate products and substrates near the catalytic center. At the same time, the trifunctional enzyme fusion strategy makes the protein expression and purification more convenient and the reaction system simpler, showing important prospects for biocatalytic industrial production. Summary of the Invention

[0005] The object of the present invention is to provide a method for constructing and recombinantly preparing a UGT91C1-UGT76G1-SsSy fusion enzyme molecule with high catalytic performance and capable of realizing substrate recycling. This fusion system can be used for biosynthesis with relatively inexpensive substrates, and can achieve efficient mass transfer of substrates through the artificially designed and constructed substrate microchannels, avoiding the diffusion of intermediate products and substrates into the reaction system, thereby realizing the low-cost and high-efficiency synthesis of rebaudioside M.

[0006] To achieve the above object, the main implementation steps involved in the present invention include:

[0007] (1) Based on the principle of artificially designed substrate microchannels, design the sequence of the linker peptide so that UGT76G1 is fused to the C-terminus of UGT91C1D, and SuSy is fused to the C-terminus of UGT76G1.

[0008] (2) Through the linker peptide database and computer-aided design, it is determined that UGT91C1 and UGT76G1 are connected by (EAAAK)3, and UGT76G1 and SuSy are connected by (GGGGS)4.

[0009] (3) Construction of the UGT91C1-UGT76G1-SsSy fusion enzyme gene and expression vector;

[0010] (4) Transformation of the UGT91C1-UGT76G1-SsSy fusion enzyme recombinant plasmid and its expression and purification in Pichia pastoris;

[0011] (5) Characterization of the catalytic cascade reaction of the UGT91C1-UGT76G1-SsSy fusion enzyme.

[0012] The gene sequence of the UGT91C1-UGT76G1-SuSy fusion enzyme constructed in the present invention is Seq ID NO.1, and the corresponding gene is named ugt76G1-ugt91C1-susy.

[0013] The protein and sequence of the constructed fusion enzyme are Seq ID NO.2

[0014] UGT91C1 and UGT76G1 in step (1) involved in the present invention are mutant sequences integrating mutant sites with better catalytic performance. Among them, UGT91C1 has mutated F208M, and UGT76G1 has mutated M88L, L200A, T284S, but it can also be a wild-type sequence or other mutants.

[0015] The linker peptides in step (2) involved in the present invention are EAAAK and GGGGS, but are not limited to these two.

[0016] The plasmid used in step (3) involved in the present invention is pPICZA, but is not limited to this one.

[0017] The recombinant expression systems used in step (4) involved in the present invention are Pichia pastoris and Escherichia coli, but are not limited to these two.

[0018] In step (5) involved in the present invention, high performance liquid chromatography is used to detect the enzyme activity, but is not limited to this one.

[0019] Compared with the existing technologies, the beneficial effects of the present invention include: (1) Based on the principle of artificially designed substrate microchannels, screening of the linker peptide database, and computer-aided design, the fusion sequence of the fusion enzyme and the linker peptide are rationally designed; (2) Compared with the free enzyme mixture system expressing UGT91C1, UGT76G1, and SuSy separately, the expression and purification of the fusion enzyme are more convenient and more conducive to industrial production; (3) SuSy can use relatively inexpensive UDP and sucrose to synthesize UDPG and use it for the cascade reaction catalyzed by UGT91C1 and UGT76G1, greatly reducing the production cost; (4) The cascade reaction catalyzed by the fusion enzyme can timely convert the intermediate product by means of the artificially designed substrate microchannel, enhance the mass transfer efficiency, avoid the accumulation of the intermediate product and its diffusion to the reaction main body, and improve the catalytic efficiency; (5) It provides a green and economic solution for other similar reactions that require expensive UDPG as a glycosyl donor; (6) It provides a reference for the construction and modification of other fusion enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. Figure 1 is the structural diagram of the UGT91C1-UGT76G1-SuSy fusion enzyme designed by computer-aided design

[0021] FIG. Figure 2 is the schematic diagram of the cascade reaction catalyzed by the fusion enzyme UGT91C1-UGT76G1-SuSy

[0022] FIG. Figure 3 is the schematic diagram of the recombinant plasmid structure of the UGT91C1-UGT76G1-SuSy fusion enzyme

[0023] FIG. Figure 4 is the influence of sucrose concentration on the cascade reaction catalyzed by the fusion enzyme

[0024] FIG. Figure 5 is the influence of UDP concentration on the cascade reaction catalyzed by the fusion enzyme DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will be further described below through specific examples and the accompanying drawings of the specification. However, these examples should only be understood as being used to illustrate the present invention in detail and should not be used to limit the protection scope of the present invention.

[0026] Example 1:

[0027] (1) Design of the fusion enzyme UGT91C1-UGT76G1-SuSy

[0028] Based on the principle of artificially designed microchannels, by screening the LINKER database, the sequence of the linker peptide can be designed according to the length of the required linker, the potential sites of proteases or restriction endonucleases to be avoided, and the preferred composition of amino acids. The UGT76G1 from Stevia rebaudiana is fused to the C-terminus of UGT91C1 from rice through the linker peptide, and the SuSy from mung bean is fused to the C-terminus of UGT76G1 from Stevia rebaudiana; the designed amino acid sequence of the recombinant target gene is imported into AlphaFold2, and the three-dimensional structure of the fusion enzyme UGT91C1-UGT76G1-SuSy is generated through the computational simulation of AlphaFold2. According to this, it is preliminarily judged whether the fusion enzyme can be successfully folded and whether there will be interference between the functional proteins. By comparing different types and lengths of linker peptides, it is determined that three segments of EAAAK are used as the linker peptide between UGT91C1 and UGT76G1, and four segments of GGGGS are used as the linker peptide between UGT76G1 and SuSy, which is beneficial to the cascade reaction and there will be no interference between the functional proteins.

[0029] (2) Synthesis of ugt91C1-ugt76G1-susy gene and construction of recombinant plasmid

[0030] According to the recombinant plasmid map designed on SnapGene, the forward and reverse primers for each gene segment are designed, and the sequence of the linker peptide is added by the reverse primer of the previous gene segment, namely 91C1-R and 76G1-R; it is required that there is a 15-20 bp overlapping sequence between 76G1-F and 91C1-R, and between SuSy-F and 76G1-R, so as to fuse and connect these three gene segments by the method of overlap extension in the follow-up; in addition, it is also required that the primers 91C1-F and SuSy-R carry the restriction enzyme cleavage sites of EcoRⅠ and ApaⅠ respectively, so as to carry out restriction enzyme digestion and ligation operations for the subsequent fusion of the target gene and the vector. Since the GC content and annealing temperature of the overlapping sequences at the junctions of the three gene segments are different, the success rate of connecting the three gene segments by the one-pot method is very low, so the pairwise connection method is adopted.

[0031] PCR was performed on pPICZA-ugt91C1, pPICZA-ugt76G1, and pPICZA-susy using the designed 91C1-F and 91C1-R, 76G1-F and 76G1-R, and SuSy-F and SuSy-R, respectively. The specific reaction conditions for ugt91C1 and ugt76G1 were: 98°C for 5 min; 30 cycles (98°C for 30 s, 60°C for 50 s, 72°C for 2 min); 72°C for 10 min. The specific reaction conditions for susy were: 98°C for 5 min; 30 cycles (98°C for 30 s, 60°C for 50 s, 72°C for 3 min); 72°C for 10 min. The PCR products were verified by 0.8% agarose gel electrophoresis. First, overlap extension PCR was performed on ugt91C1 and ugt76G1. The specific reaction conditions were: 98°C for 5 min; 10 cycles (98°C for 30 s, 55°C for 50 s, 72°C for 2 min); 72°C for 10 min. After 10 cycles of reaction, 91C1-F and 76G1-R were added, and the specific reaction conditions were: 98°C for 5 min; 20 cycles (98°C for 30 s, 60°C for 50 s, 72°C for 4 min); 72°C for 10 min. The overlap extension products were verified by 0.8% agarose gel electrophoresis. Then, overlap extension was performed on ugt91C1-ugt76G1 and susy. The specific reaction conditions were: 98°C for 5 min; 10 cycles (98°C for 30 s, 55°C for 50 s, 72°C for 4 min); 72°C for 10 min. After 10 cycles of reaction, 91C1-F and SuSy-R were added, and the specific reaction conditions were: 98°C for 5 min; 20 cycles (98°C for 30 s, 60°C for 50 s, 72°C for 6 min); 72°C for 10 min. The overlap extension products were verified by 0.8% agarose gel electrophoresis. The overlap extension products were purified using the Cycl Pure Kit. The purified products and the pPICZA empty plasmid were double digested with EcoRⅠ and ApaⅠ. The digested fusion target gene and the empty plasmid were verified by 0.8% agarose gel electrophoresis, respectively. The GelExtraction Kit was used to recover and purify the fusion target gene band and the linearized vector band from the gel. The gel-recovered and purified fusion target gene and the linearized vector were ligated using T4 DNA ligase. The ligation products were transformed into Escherichia coli Top 10 competent cells, and single colonies were picked and sent for sequencing. The correct recombinant plasmid pPICZA-ugt91C1-ugt76G1-susy was obtained by sequencing.

[0032] (3) Obtaining of recombinant yeast strains and expression and purification of fusion enzymes

[0033] The correctly sequenced recombinant plasmid pPICZA-ugt91C1-ugt76G1-susy was linearized using SacⅠ, and the linearized product was verified by 0.8% agarose gel electrophoresis; Pichia pastoris competent cells were prepared, and the linearized recombinant plasmid was electrotransformed into Pichia pastoris competent cells using a 7500 Real Time PCR System electroporator, and screened using a plate containing zeocin resistance. Single colonies were picked and cultured in YPD. Yeast genomic DNA was extracted using a yeast genomic DNA extraction kit, and the genome of the transformants was PCR amplified using AOX1 primers. The PCR products were verified by 0.8% agarose gel electrophoresis; after the positive transformants were activated in YPD, expanded in BMGY, and induced in BMMY for 5 days, the cells were collected by centrifugation, resuspended, and disrupted by high-pressure homogenization. After centrifugation, the supernatant was taken and filtered through a 0.45 μm filter head, and then purified by AKTA primer plus system in combination with HiTrap TM Chelating HP nickel column affinity chromatography and HiTrap TM Desalting gel filtration chromatography purification. The purified product was detected by SDS-PAGE, and the band was single and clear, with a molecular weight of 198KDa.

[0034] (4) Characterization of the catalytic performance of UGT91C1-UGT76G1-SuSy

[0035] The reaction system was a total of 200 μL, including: potassium phosphate buffer (a certain proportion of dipotassium hydrogen phosphate and potassium dihydrogen phosphate were mixed and adjusted to pH 7.5), 5 mM magnesium chloride, fusion enzyme, rebaudioside A, UDP, sucrose, added to a 2 mL round-bottom centrifuge tube, reacted in a shaker at 37 °C and 170 rpm. Immediately after the reaction ended, an equal volume of methanol was added to terminate the reaction. The reaction product was filtered through a 0.22 μm organic phase filter membrane and detected by high performance liquid chromatography. The detection conditions were: chromatographic column (Silgreen ODS C18 (5 μM, )), column temperature 30 °C, flow rate 1 mL / min, injection volume 20 μL, detection wavelength 210 nm, mobile phase (A: acetonitrile, B: water, A:B = 3:7). The results of the characterization are shown in the appendix Figure 3 as follows.

[0036] Example 2:

[0037] In this example, two segments of GGGGS in the polypeptide sequence were used as the linker peptide between UGT91C1 and UGT76G1, and four segments of A(EAAAK)A were used as the linker peptide between UGT76G1 and SuSy. Except for this, it was the same as step (1) of Example 1.

[0038] Example 3:

[0039] In this example, the expression vector in step (2) of Example 1 was replaced with PET-28a, and the expression host in step (3) was changed to Escherichia coli, and the remaining steps were the same.

[0040] Tests showed that except for a certain difference in the enzyme expression level between the fusion enzymes constructed in Example 2 and Example 3 and that in Example 1, other properties were close to those in Example 1.

Claims

1. A method for constructing and preparing a multifunctional fusion enzyme system based on the principle of artificially designed substrate microchannels and substrate recycling and regeneration system, characterized in that: The specific steps include: (1) Based on the principle of artificially designed substrate microchannels, the sequence of the connecting peptide was designed so that UGT76G1 was fused to the C-terminus of UGT91C1D and SuSy was fused to the C-terminus of UGT76G1. (2) Through the connection peptide database and computer-aided design, it was determined that UGT91C1 and UGT76G1 are connected through (EAAAK)3, and UGT76G1 and SuSy are connected through (GGGGS)4. (3) Construction of UGT91C1-UGT76G1-SsSy fusion enzyme gene and expression vector; (4) Transformation of UGT91C1-UGT76G1-SsSy fusion enzyme recombinant plasmid and its expression and purification in Pichia pastoris; (5) Characterization of the UGT91C1-UGT76G1-SsSy melting enzyme catalyzed cascade reaction.

2. The method for constructing and preparing a multifunctional fusion enzyme system based on the artificially designed substrate microchannel principle and substrate recycling system according to claim 1, characterized in that: Its gene sequence and protein primary structure are: SeqID NO.1 and Seq ID NO.2 respectively.

3. The method for constructing and preparing a multifunctional fusion enzyme system based on the artificially designed substrate microchannel principle and substrate recycling system according to claim 2, characterized in that: UGT91C1 and UGT76G1 in step 1 are mutant sequences that integrate mutation sites with better catalytic performance, wherein UGT91C1 mutates F208M, and UGT76G1 mutates M88L, L200A, and T284S, but they can also be wild-type sequences or other mutants.

4. The method for constructing and preparing a multifunctional fusion enzyme system based on the artificially designed substrate microchannel principle and substrate recycling system construction according to claim 3, characterized in that: The connecting peptides in step 2 are EAAAK and GGGGS, but are not limited to these two.

5. The method for constructing and preparing a multifunctional fusion enzyme system based on the artificially designed substrate microchannel principle and substrate recycling system construction according to claim 4, characterized in that: The plasmid used in step 3 is pPICZA, but is not limited to this one.

6. The method for constructing and preparing a multifunctional fusion enzyme system based on the artificially designed substrate microchannel principle and substrate recycling system construction according to claim 5, characterized in that: The recombinant expression system used in step 4 is Pichia pastoris and Escherichia coli, but is not limited to these two.

7. The method for constructing and preparing a multifunctional fusion enzyme system based on the artificially designed substrate microchannel principle and substrate recycling system construction according to claim 6, characterized in that: In step 5, high performance liquid chromatography is used to detect the enzyme activity, but it is not limited to this method.

8. The method for constructing and preparing a multifunctional fusion enzyme system based on the artificially designed substrate microchannel principle and substrate recycling system according to claim 7, characterized in that: The use of artificially designed substrate microchannel principles and the construction of a substrate recycling and regeneration system for the green, economical and efficient preparation of stevioside can not only enhance the mass transfer process of intermediates and substrates, avoid the diffusion of intermediates and substrates into the main phase, and accelerate the cascade reaction phase in the substrate direction, but also utilize relatively cheap raw materials for catalysis, which has important prospects for biocatalytic industrial production.

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