A cyclodextrin glycosyltransferase mutant and its application in the synthesis of rebaudioside RI

Abstract

The present invention belongs to the field of genetic engineering and enzyme engineering technology, and specifically relates to a cyclodextrin glycosyltransferase mutant and its application in the synthesis of rebaudioside RI. Specifically, the present invention uses the theory of directed evolution to transform the enzyme molecule, thereby successfully obtaining a cyclodextrin glycosyltransferase mutant. At the same time, the cyclodextrin glycosyltransferase mutant is used to replace the expensive UDP-glucose with cheap and easily available cyclodextrin as a glycosyl donor, and the catalytic efficiency is greatly improved, with a conversion rate of 98.8%. This reaction has opened up a new way to prepare rebaudioside RI, the product yield of the reaction is high, the preparation process is simple, it is suitable for industrial production, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a cyclodextrin glycosyltransferase mutant and application thereof in synthesizing rebaudioside RI. Background Art

[0002] Stevioside is a diterpenoid compound with the diterpenoid steviol as its basic skeleton. Stevioside is primarily found in the leaves of the Stevia rebaudiana plant, which contain a variety of natural steviol glycosides, including stevioside, rebaudioside RA, rebaudioside RB, rebaudioside RD, rebaudioside RE, rebaudioside RI, and rebaudioside RM. Stevioside is widely used. Although this product is 300 to 600 times sweeter than sucrose, it still suffers from disadvantages such as a bitter aftertaste, and its taste needs improvement. Rebaudioside RI offers excellent sweetness and taste, along with low calories, making it a recognized ideal steviol glycoside product. However, the content of rebaudioside RI in the original plant is very low, and plant-based extraction and purification is extremely costly. Current production is far from meeting market demand.

[0003] Cyclodextrin glycosyltransferase (CGTase, EC2.4.1.19), a member of the α-cyclodextrinase family, has been shown to be a multifunctional enzyme capable of catalyzing four reactions: hydrolysis, disproportionation, cyclization, and coupling. CGTase is a typical transglycosidase, with a prominent transglycosylation reaction and a weak hydrolysis reaction. Disproportionation and coupling reactions are intermolecular transglycosylation reactions that transfer maltooligosaccharides and cyclodextrins to various acceptor molecules to produce glycosylated derivatives and modify the acceptor molecules. Currently, accepted acceptor molecules include alcohols (inositol, sorbitol, lactitol, xylitol, and maltitol), sugars (sucrose, fructose, and rhamnose), glycosides (rutin, salicin, and stevioside), flavonoids (rutin, hesperidin, naringin, and quercetin), and some other small molecules such as genistein, hydroquinone, and L-ascorbic acid. Rebaudioside RI is a polysaccharide formed by glycosylation of the aglycone steviol. Therefore, cyclodextrin glycosyltransferase can be applied to the synthesis of rebaudioside RI. However, natural cyclodextrin glycosyltransferases generally suffer from low enzymatic activity, instability, and poor substrate compatibility, making them difficult to meet production requirements. Summary of the Invention

[0004] To overcome the above-mentioned problems, the present invention provides a cyclodextrin glycosyltransferase mutant and its use in the synthesis of rebaudioside RI. The present invention utilizes directed evolution theory to modify the enzyme molecule, thereby obtaining a cyclodextrin glycosyltransferase mutant with higher catalytic efficiency. The present invention utilizes the cyclodextrin glycosyltransferase mutant to catalyze the one-pot conversion of stevioside ST to rebaudioside RI using stevioside ST as a substrate and cyclodextrin as a cosubstrate. Based on the above-mentioned research results, the present invention was completed.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a cyclodextrin glycosyltransferase mutant, which may be cyclodextrin glycosyltransferase mutant M1 and / or cyclodextrin glycosyltransferase mutant M2;

[0007] The cyclodextrin glycosyltransferase mutant M1 is obtained by mutating the lysine at position 74 to histidine, the proline at position 115 to asparagine, the tyrosine at position 179 to serine, and the glycine at position 397 to threonine of the wild-type cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO. 2;

[0008] The cyclodextrin glycosyltransferase mutant M2 is obtained by mutating the 128th aspartic acid to serine, the 130th serine to asparagine, and the 232nd aspartic acid to glycine of the wild-type cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO.4.

[0009] The second aspect of the present invention provides a nucleic acid molecule encoding the cyclodextrin glycosyltransferase mutant described in the first aspect.

[0010] In the present invention, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0011] The third aspect of the present invention provides an expression cassette, which comprises the nucleic acid molecule according to the second aspect.

[0012] In the present invention, the expression cassette includes a promoter capable of initiating transcription of the nucleic acid molecule and the nucleic acid molecule, and may further include a termination sequence and an enhancer sequence.

[0013] The fourth aspect of the present invention provides a recombinant vector comprising the nucleic acid molecule described in the second aspect.

[0014] The recombinant vector of the present invention is obtained by connecting a vector to the nucleic acid molecule. The vector can be constructed using any method known in the art or can be purchased commercially. For example, in one or more embodiments of the present invention, the vector is plasmid pET22b, which can be expressed in Escherichia coli.

[0015] The fifth aspect of the present invention provides a recombinant microorganism comprising the nucleic acid molecule described in the second aspect or the recombinant vector described in the fourth aspect.

[0016] The recombinant microorganism of the present invention may be a recombinant bacterium, specifically a recombinant Escherichia coli.

[0017] The seventh aspect of the present invention provides the use of the cyclodextrin glycosyltransferase mutant described in the first aspect, the nucleic acid molecule described in the second aspect and / or the recombinant microorganism described in the fifth aspect in catalyzing the synthesis of rebaudioside RI.

[0018] An eighth aspect of the present invention provides a method for synthesizing rebaudioside RI, comprising:

[0019] Rebaudioside RI is synthesized in a liquid environment using wet cells obtained by induction culture of the recombinant microorganism described in the fifth aspect or a crude enzyme solution extracted by crushing the wet cells as a catalyst, stevioside ST as a substrate, and cyclodextrin as a co-substrate;

[0020] The liquid environment is preferably a phosphate buffer solution with a pH of 7-8; the reaction conditions include: reacting at 35-45°C (preferably 40°C) and 200-800 rpm (preferably 500 rpm) to accelerate the reaction progress.

[0021] The amount of the catalyst used is 5-25 g / L based on the total weight of the wet cells, the initial concentration of the substrate is 10-80 mM, and the amount of the auxiliary substrate added is 30-220 mM.

[0022] In the present invention, the wet bacterial cells can be prepared by the following method: inoculating the recombinant bacteria into LB liquid medium containing benzyl ampicillin resistance, culturing at 37°C and 200 rpm for 12 h, then inoculating the recombinant bacteria into fresh LB liquid medium containing 50 μg / mL ampicillin resistance at a volume concentration of 1%, culturing at 37°C and 200 rpm until the bacterial OD600 reaches 0.6-0.8, adding IPTG with a final concentration of 24 μg / mL, inducing the culture at 25°C for 16 h, centrifuging at 4°C and 8000 rpm for 20 min, discarding the supernatant, and collecting the precipitate to obtain wet bacterial cells;

[0023] Furthermore, the preparation method of the crude enzyme solution includes: adding the wet bacteria to a pH 7.5, 100 mM phosphate buffer, resuspending the wet bacteria, and ultrasonically disrupting them on an ice-water mixture for 5 minutes; wherein the ultrasonic disruption conditions include: ultrasonic power of 200 W, disruption for 1 s, and pause for 2 s.

[0024] Beneficial technical effects of the above technical solution:

[0025] (1) The above technical scheme successfully obtained cyclodextrin glycosyltransferase mutants M1 and M2 by site-directed mutagenesis of wild-type cyclodextrin glycosyltransferase. Using stevioside ST as a substrate, in the presence of the auxiliary substrate cyclodextrin, the two cyclodextrin glycosyltransferase mutants catalyzed stevioside ST to produce rebaudioside RI in one pot. The co-expression strain finally completely catalyzed 70 mM stevioside ST to produce rebaudioside RI in just 16 hours. This method has high raw material conversion rate, high yield, and short conversion time.

[0026] (2) The current bioenzymatic synthesis of rebaudioside RI usually requires the addition of expensive UDP-glucose as a substrate. UDP-glucosyltransferase is used to catalyze the production of rebaudioside RI using stevioside ST as a substrate and UDP-glucose as a glycosyl donor. The extremely high price of UDP-glucose almost completely limits the feasibility of industrial production of rebaudioside RI, resulting in poor economic efficiency and lack of market competitiveness. The above-mentioned technical solution utilizes two co-expressed cyclodextrin glycosyltransferases to catalyze the conversion of stevioside ST to rebaudioside RI in one pot. This solution uses inexpensive cyclodextrin as a glycosyl donor and has good economic benefits. Therefore, it has significant application value for the production of rebaudioside RI. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1 for E.coli Reaction route of the synthesis of rebaudioside RI from stevioside ST catalyzed by BL21(DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T(M1)-cgt3-D128S-S130N-D232G(M2).

[0029] Figure 2 for E.coliReaction process diagram of the synthesis of rebaudioside RI catalyzed by BL21(DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T(M1)-cgt3-D128S-S130N-D232G(M2). DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0032] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0033] The term "expression cassette" generally refers to a nucleic acid construct containing nucleic acid elements sufficient to express a gene of interest. A typical expression cassette includes a promoter, a multiple cloning site (MCS), and / or a terminator. An expression cassette may also include the gene of interest, marker genes (such as TK, DHFR, CAT, and NEO), ribosome recognition and binding sites (SDs), transcription factor binding sites (TFBSs), enhancers, silencers, repressors, introns, poly(A) signal sequences, and / or mRNA splicing signal sequences. The various elements in an expression cassette can be linked directly or indirectly via linkers.

[0034] The term "vector" generally refers to a vehicle capable of transporting exogenous DNA or a gene of interest into host cells for amplification and / or expression. Such a vector can be a cloning vector or an expression vector. A vector can be introduced into host cells through transformation, transduction, or transfection, enabling amplification and / or expression of the genetic material it carries. Those skilled in the art can select an appropriate vector based on the objectives of the genetic engineering project and the properties of the recipient cells. Such vectors include, but are not limited to, plasmids, bacteriophages (such as lambda phage or M13 phage), cosmids (cosmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), P1 artificial chromosomes (PACs), or Ti plasmid artificial chromosomes (TACs)), and viral vectors (such as baculovirus vectors, retroviruses, adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, papovaviruses, and herpesviruses). A vector may contain a variety of elements that control expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain a replication initiation site.

[0035] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, and the like. For example, the bacteria may be from the genera Corynebacterium, Brevibacterium, Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, and Bacillus. The viruses may include rotaviruses, baculoviruses, retroviruses (such as lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, influenza viruses, papillomaviruses (such as SV40), and herpesviruses (such as herpes simplex virus). The fungi may be from the genera Saccharomyces (such as Saccharomyces cerevisiae, Candida, Methanol yeast, Pichia pastoris), Fusarium, Rhizoctonia, Verticillium, Penicillium, Aspergillus, and Cephalosporium. The actinomycetes may be from the genus Streptomyces. The algae may be from the phylum Cyanobacteria, the genera Fucus, Angiospermum, Cocospermum, Diplophytum, Fibrophytum, Astrophytum and Golden Chromophytum, etc.

[0036] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0037] The term "recombinant microorganism" generally refers to a microorganism whose genes have been manipulated and modified to produce a functionally altered recombinant microorganism. This can be achieved by introducing an exogenous gene of interest or a recombinant vector into the microorganism, or by directly editing the endogenous genes of the microorganism.

[0038] The term "mutation" generally refers to changes in amino acid or nucleotide sequences, which may include changes in the base pair composition or arrangement order of the gene structure, such as point mutations caused by single base changes, or deletions, duplications and insertions of multiple bases, etc. It may also include replacements, deletions and insertions (additions) of one or more amino acid residues in a protein.

[0039] The term "site-directed mutagenesis" generally refers to the alteration of one or more bases in a gene through site-directed mutagenesis. This includes base additions, deletions, and point mutations, resulting in changes in the amino acid composition of the corresponding protein. Site-directed mutagenesis methods include oligonucleotide primer-mediated mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis.

[0040] The term "homologous recombination" generally refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. For example, a site-directed mutagenesis strategy based on homologous recombination can be achieved by ligating the ends of a nucleic acid molecule encoding the cyclodextrin glycosyltransferase mutant described herein to homology arms of a wild-type cyclodextrin glycosyltransferase gene, followed by introduction into a recipient bacterium to replace the wild-type cyclodextrin glycosyltransferase gene, thereby achieving site-directed mutagenesis.

[0041] The term "fermentation" generally refers to a biological reaction process that produces and accumulates desired products through the growth, reproduction and metabolic activities of organisms, including microbial fermentation.

[0042] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0043] The culture medium formulations and HPLC detection methods used in the following examples are as follows:

[0044] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, solvent: water, pH 7.4.

[0045] LB plate: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar, solvent is water, pH 7.4.

[0046] The concentration of rebaudioside RI was determined by high-performance liquid chromatography (HPLC) using a QS-C18 column, 5 μm, 4.6 × 250 mm. The mobile phase consisted of a 68:32 ratio of A (water):B (acetonitrile), with an injection volume of 10 μL, a detection wavelength of 210 nm, an 18-min detection time, and a flow rate of 0.5 mL / min. The column temperature was 40°C.

[0047] Sample treatment: Take 20 μL of the sample after the reaction is completed, dilute it 10 times, add 16 μL of 2M H2SO4 solution and 160 μL of 60% methanol solution to terminate the reaction, filter it through a 0.22 μm filter membrane, and perform HPLC detection.

[0048] Example 1: Construction of expression vector and engineered bacteria

[0049] Two genes were mined from the NCBI database. Thermoanaerobacterium saccharolyticum and Clostridium sp. The cyclodextrin glycosyltransferase of USBA 49 has NCBI accession numbers WP_045409091.1 and WP_078681113.1, and the nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively, and the amino acid sequences are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively.

[0050] Primers F1 and R1 were designed based on the nucleotide sequence shown in SEQ ID NO.1, and NdeI and NotI restriction enzyme sites were introduced into the primers respectively:

[0051] F1: 5'-CATATGATGAAAAAAACGTTTAAATTGATATTG-3';

[0052] R1: 5'-GCGGCCGCCTACTGTTGCCAGTTTACAATTA-3';

[0053] Primers F2 and R2 were designed based on the nucleotide sequence shown in SEQ ID NO.3, and NdeI and NotI restriction enzyme sites were introduced into the primers respectively:

[0054] F2: 5'-CATATGATGCGTAAATTTAATGCCTTAATAA-3';

[0055] R2: 5'-GCGGCCGCTTATTGTTGCCAATTAACAGTTAT-3';

[0056] Using pET-22b plasmid as the expression vector, E. coli BL21 (DE3) / pET22b-cgt2 and E. coli BL21 (DE3) / pET22b-cgt3 were constructed.

[0057] Construction of expression plasmids: Under the priming of the above primers, the target gene was used as a template and amplified using high-fidelity Pfu DNA polymerase to obtain the cyclodextrin glycosyltransferase gene sequence. After sequencing, the amplified fragment was treated with NdeI and NotI restriction endonucleases (TaKaRa) and ligated with the vector pET-22b treated with the same restriction endonucleases using T4 DNA ligase (TaKaRa) to construct the expression vectors pET22b-cgt2 and pET22b-cgt3.

[0058] Preparation of competent cells: from -80℃ freezer E. coli The BL21 (DE3) strain preserved in a glycerol tube was obtained, streaked on an antibiotic-free LB plate, cultured at 37°C for 10 h, and a single colony was obtained; a single colony on the LB plate was picked and inoculated into a test tube containing 5 mL of LB medium, and cultured at 37°C and 180 rpm for 9 h; 200 μL of bacterial solution was taken from the test tube and inoculated into 50 mL of LB medium, and cultured at 37°C and 180 rpm until OD600 reached 0.4-0.6; the bacterial solution was pre-cooled on ice, transferred to a sterilized centrifuge tube, placed on ice for 10 min, and centrifuged at 4°C and 5000 rpm for 10 min; the supernatant was poured out, paying attention to prevent contamination, and the precipitated cells were resuspended with pre-cooled 0.1 mol / L CaCl2 aqueous solution and placed on ice for 30 min; centrifuged at 4°C and 5000 rpm for 10 min, the supernatant was discarded, and the precipitated cells were resuspended with pre-cooled 0.1 mol / L CaCl2 aqueous solution and placed on ice for 10 min. Resuspend the precipitated cells in 10 mol / L CaCl2 aqueous solution, take 100 μL of the resuspended cells and dispense them into sterile 1.5 mL centrifuge tubes, store them in a -80℃ refrigerator, and take them out when needed.

[0059] Construction of recombinant Escherichia coli: First, the competent Escherichia coli BL21 (DE3) (Invitrogen) cells stored at -80°C were placed on ice at 0°C for 10 min. Then, 5 μL of the ligation product was added in a clean bench, ice-bathed at 0°C for 30 min, heat-shocked in a 42°C water bath for 90 s, and ice-bathed at 0°C for 2 min. 600 μL of LB medium was added and cultured at 37°C and 200 rpm for 1 h. The cells were spread on LB plates containing 50 μg / ml ampicillin resistance and cultured at 37°C for 8-12 h. Clones were randomly picked to extract plasmids for sequencing and identification, and recombinant Escherichia coli containing the recombinant plasmid were screened. E.coli BL21(DE3) / pET22b-cgt2 andE.coli BL21(DE3) / pET22b-cgt3.

[0060] Example 2: Inducible expression of cyclodextrin glycosyltransferase

[0061] Wet cells containing cyclodextrin glycosyltransferase gene: the recombinant Escherichia coli obtained in Example 1 E.coli BL21(DE3) / pET22b-cgt2 and E.coli BL21(DE3) / pET22b-cgt3 were inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance, cultured at 37°C and 200 rpm for 12 h, and then inoculated into fresh LB liquid medium containing 50 μg / mL ampicillin resistance at a 1% (v / v) inoculum. The cells were cultured at 37°C and 200 rpm until the OD600 of the cells reached 0.6-0.8. IPTG was added at a final concentration of 0.1 mM, and the cells were induced at 25°C for 16 h. The cells were centrifuged at 4°C and 8000 rpm for 20 min, the supernatant was discarded, the precipitate was collected, and the cells were washed twice with pH 7.5, 20 mM phosphate buffer to obtain recombinant bacteria containing cyclodextrin glycosyltransferase. E.coli BL21(DE3) / pET22b-cgt2 and E.coli Wet cells of BL21(DE3) / pET22b-cgt3 were resuspended in 100 mM phosphate buffer (pH 7.5) and disrupted by sonication over ice-water for 5 minutes at 200 W for 1 second and a 2-second pause to obtain a crude enzyme solution.

[0062] Example 3: Establishment of a cyclodextrin glycosyltransferase gene mutation library

[0063] 1. Site-directed mutagenesis

[0064] Constructed with Example 1 E.coli BL21(DE3) / pET22b-cgt2 and E.coli BL21(DE3) / pET22b-cgt3 was the starting strain.

[0065] Directed evolution was used to develop a complex model based on the crystal structure of cyclodextrin glycosyltransferase obtained through homology modeling and docking. Amino acid residues surrounding the substrate stevioside ST in the cgt2 structure were analyzed, and sites 74, 114, 115, 116, 197, and 397 were selected for site-directed mutagenesis. Primer designs are shown in Table 1.

[0066] Directed evolution was used to develop a complex model based on the crystal structure of cyclodextrin glycosyltransferase obtained through homology modeling and docking. Amino acid residues surrounding the substrate rebaudioside RA in the cgt3 structure were analyzed, and sites 126, 128, 130, 231, and 232 were selected for site-directed mutagenesis. Primers were designed as shown in Table 2.

[0067] The mutation PCR system (100 μL) consisted of 25 μL of 2*Phanta Max buffer, 1 μL of dNTPs, 1 μL of each upstream and downstream mutation primer, 1 μL of template (starting strain), 0.5 μL of Pfu DNA polymerase, and ddH2O supplemented to 50 μL. PCR conditions were: initial denaturation at 95°C for 3 min, 30 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 7 min 20 s, followed by a final extension at 72°C for 10 min. PCR results were verified by DNA agarose gel electrophoresis. The PCR product was digested with DpnI, inactivated at 37°C for 1 hour, and incubated at 200 rpm for 1 minute at 65°C. The PCR product was heat-shock transformed into Escherichia coli. E. coli BL21 (DE3) was activated and cultured at 37°C, 200 rpm for 1 hour. The cells were then spread on LB plates containing 50 μg / mL ampicillin resistance and cultured in an inverted manner at 37°C overnight.

[0068] Table 1 Primer design for site-directed mutagenesis of cyclodextrin glycosyltransferase cgt2

[0069]

[0070] Table 2 Primer design for site-directed mutagenesis of cyclodextrin glycosyltransferase cgt3

[0071]

[0072] Example 4: Screening of a cyclodextrin glycosyltransferase gene mutation library

[0073] A single clone was picked from the plate obtained in Example 3 and inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance, cultured at 37°C and 200 rpm for 12 h, the strain was preserved and sent to a sequencing company for sequencing verification. After sequencing verification was correct, the preserved strain was inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance at a 0.2% inoculum size and cultured at 37°C and 200 rpm for 12 h. Then, it was inoculated into fresh LB liquid medium containing 50 μg / mL ampicillin resistance at a 1% (v / v) inoculum size and cultured at 37°C and 200 rpm until the bacterial OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 25°C for 16 h. After centrifugation at 4°C and 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected to obtain wet bacteria containing the cyclodextrin glycosyltransferase cgt2 and cgt3 gene mutant library. The wet cells were resuspended in 100 mM phosphate buffer at pH 7.5 and ultrasonically disrupted for 5 min on an ice-water mixture. The ultrasonic disruption conditions were as follows: power 200 W, disruption for 1 s, and pause for 2 s to obtain the crude enzyme solution.

[0074] 1. CGT2 initial screening:

[0075] The reaction solution (200 μL) was prepared with the following: substrate stevioside ST at a final concentration of 20 mM, cyclodextrin at a final concentration of 30 mM, and a catalyst dosage of 5 g / L based on the total weight of wet cells before disruption. The reaction medium was phosphate buffer at pH 7.5. Reaction conditions: 15 hours of reaction in a reactor at 40°C and 500 rpm. After the reaction, a 20 μL sample was taken, diluted 10-fold, and terminated with 16 μL of 2 M H₂SO₄ solution and 160 μL of 60% methanol. The sample was filtered through a 0.22 μm filter and analyzed by HPLC. The results are shown in Table 3.

[0076] Table 3 Preliminary screening reaction results

[0077]

[0078] 2. CGT3 initial screening:

[0079] The reaction solution (200 μL) was prepared with the following: substrate rebaudioside RA at a final concentration of 10 mM, cyclodextrin at a final concentration of 30 mM, and a catalyst dosage of 5 g / L based on the total weight of wet cells before disruption. The reaction medium was phosphate buffer at pH 7.5. Reaction conditions: 16 hours of reaction in a reactor at 40°C and 500 rpm. After the reaction, a 20 μL sample was taken, diluted 10-fold, and terminated with 16 μL of 2 M H₂SO₄ solution and 160 μL of 60% methanol. The sample was filtered through a 0.22 μm filter and analyzed by HPLC. The results are shown in Table 4.

[0080] Table 4 Results of initial screening reactions

[0081]

[0082] 3. cgt2 rescreening:

[0083] The strains obtained in the initial screening were rescreened, and the rescreened combined mutants were sent to a sequencing company for sequencing verification. After the sequencing verification was correct, the activity was verified. The rescreening reaction solution (10 mL) was configured: the final concentration of the substrate stevioside ST was 80 mM, the final concentration of cyclodextrin was 120 mM, the amount of catalyst was 20 g / L based on the total weight of the wet bacteria before crushing, and the reaction solution was composed of a phosphate buffer of pH 7.5 as the reaction medium. Reaction conditions: After reacting in a reactor at 40°C and 500 rpm for 15 hours, 20 μL of the reaction-completed sample was taken after the reaction was completed, diluted 10 times, and 16 μL of 2M H2SO4 solution and 160 μL of 60% methanol solution were added to terminate the reaction. The sample was filtered through a 0.22 μm filter membrane and tested by HPLC. The test results are shown in Table 5, and the strain with the highest activity was obtained. E.coli BL21(DE3) / pET22b-cgt2-K74H-P115N-Y179S-G397T(M1).

[0084] Table 5 Rescreening reaction results

[0085]

[0086] 4. cgt3 rescreening:

[0087] The strains obtained in the initial screening were rescreened, and the rescreened combined mutants were sent to a sequencing company for sequencing verification. After the sequencing verification was correct, the activity was verified. The rescreening reaction solution (10 mL) was configured: the final concentration of the substrate rebaudioside RA was 70 mM, the final concentration of cyclodextrin was 200 mM, the amount of catalyst was 20 g / L based on the total weight of the wet bacteria before crushing, and the reaction solution was composed of a phosphate buffer of pH 7.5 as the reaction medium. Reaction conditions: After reacting in a reactor at 40°C and 500 rpm for 16 hours, 20 μL of the reaction sample was taken after the reaction was completed, diluted 10 times, and 16 μL of 2M H2SO4 solution and 160 μL of 60% methanol solution were added to terminate the reaction. The sample was filtered through a 0.22 μm filter membrane and tested by HPLC. The test results are shown in Table 6, and the strain with the highest activity was obtained. E.coli BL21(DE3) / pET22b-cgt3-D128S-S130N-D232G(M2).

[0088] Table 6 Rescreening reaction results

[0089]

[0090] Example 5: Construction of a strain co-expressing cyclodextrin glycosyltransferase cgt2 and cyclodextrin glycosyltransferase cgt3

[0091] A co-expression strain was constructed by constructing the recombinant cyclodextrin glycosyltransferase cgt2 with the highest activity obtained in Example 4 and the cyclodextrin glycosyltransferase cgt3, cgt2 was cloned into the first multiple cloning site of the pETduet plasmid, and cgt3 was cloned into the second multiple cloning site of the pETduet plasmid to obtain the co-expression strain E. coli BL21 (DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T-cgt3-D128S-S130N-D232G, which can use stevioside ST as a substrate to catalyze the synthesis of rebaudioside RI.

[0092] Primers were designed based on the nucleotide sequences of cgt2-K74H-P115N-Y179S-G397T and cgt3-D128S-S130N-D232G and the pET-Duet vector sequence. Homologous recombination was used to construct cgt2-K74H-P115N-Y179S-G397T into the first cloning site of the pET-Duet vector between the NcoI-NotI restriction sites, and cgt3-D128S-S130N-D232G into the second cloning site of the pET-Duet vector between the NdeI-XhoI restriction sites. Construction of expression plasmids: Under the priming of primers F3 / R3, the target gene was used as a template and high-fidelity Pfu was used to construct the expression plasmid. DNA polymerase was used for amplification to obtain the cgt2-K74H-P115N-Y179S-G397T gene sequence with homology arms. Under the priming of primers F4 / R4, the pETduet plasmid was used as a template and amplified using high-fidelity Pfu DNA polymerase to obtain a linearized vector sequence. The target gene and the linearized vector were homologously recombined using a homologous recombinase. The transformation process of the recombinant product was referred to Example 1. After sequencing verification, the plasmid pETduet-cgt2-K74H-P115N-Y179S-G397T was obtained. Then, under the priming of primers F5 / R5, the target gene was used as a template and amplified using high-fidelity Pfu DNA polymerase to obtain the cgt3-D128S-S130N-D232G gene sequence with homology arms. Under the priming of primers F6 / R6, the pETduet-cgt2-K74H-P115N-Y179S-G397T plasmid was used as a template and amplified using high-fidelity Pfu DNA polymerase to obtain a linearized vector sequence. The target gene and the linearized vector were homologously recombined using a homologous recombinase. The transformation process of the recombinant product was referred to Example 1, and the co-expression strain was obtained after sequencing verification. E.coli BL21(DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T-cgt3-D128S-S130N-D232G.

[0093] Table 7 Primer design

[0094]

[0095] Example 6: Application of co-expression strains in catalytic synthesis of rebaudioside RI

[0096] The recombinant cyclodextrin glycosyltransferase mutant with the highest activity obtained in Example 5 E.coliBL21 (DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T-cgt3-D128S-S130N-D232G. Inoculate into LB liquid medium containing a final concentration of 50 μg / mL ampicillin, culture at 37 ° C for 9 hours, and use it as a seed liquid. Inoculate it at a volume concentration of 3.5% in a 5 L fermenter containing 3 L fermentation medium for culture. Culture at 37 ° C, 500 rpm for about 3-4 hours. After the bacterial density OD reaches 6-8, it meets the requirements. After the fermenter temperature is lowered to 25 ° C, lactose with a final concentration of 5 g / L is added as an inducer, and then cultured at 25 ° C, 500 rpm for 12 hours. Centrifuge the fermented broth at 8000 rpm for 10 minutes to obtain a strain containing two cyclodextrin glycosyltransferase mutants co-expressed. E.coli The wet cells of BL21(DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T-cgt3-D128S-S130N-D232G were broken using a high-pressure homogenizer.

[0097] Among them, the fermentation tank culture medium composition: 45g tryptone, 36g yeast extract, 30g sodium chloride, 4.08g potassium dihydrogen phosphate, 45g glycerol, 6.84g dipotassium hydrogen phosphate trihydrate, 15g ammonium sulfate, 1.125g magnesium sulfate, 4g defoaming agent, add distilled water to 3 L for dissolution.

[0098] Catalyst dosage: The total weight of wet cells before high-pressure homogenization is 20 g / L, the final concentration of substrate stevioside ST is 70 mM, the final concentration of cyclodextrin is 200 mM, and the total volume of the reaction solution is 1 L using pH 7.5 phosphate buffer as the reaction medium. Reaction conditions: 40°C, 500 rpm for 16 hours. After the reaction, 20 μL of the reaction sample was taken, diluted 10-fold, and the reaction was terminated by adding 16 μL of 2M H2SO4 solution and 160 μL of 60% methanol solution. The sample was filtered through a 0.22 μm filter membrane and detected by HPLC. The reaction progress curve is shown in Figure 2. Figure 2 As shown in the figure, after the reaction, the concentration of rebaudioside RI reached 69.2 mM and the conversion rate reached 98.8%.

[0099] Nucleotide and amino acid sequence information used in the present invention

[0100] Thermoanaerobacterium saccharolyticum

[0101] Thermoanaerobacterium saccharolyticum Amino acid sequence of cyclodextrin glycosyltransferase from the source: MKKTFKLILVLMLSLTLVFGLTAPIQAASDTAVSNVVNYSTDVIYQIVTDRFVDGNTSNNPTGDLYDPTHTSLKKYFGGDWQGIINKINDGYLTGMGVTAIWISQPVENIYAVLPDSTFGGSTSYHGYWARDFKRTNPYFGSFTDFQNLINTAHAHNIKVIIDFAPNHTSPASETDPTYAENGRLYDNGTLLGGYTNDTNGYFHHYGGTDFSSYEDGIYRNLFDLADLNQQNSTIDSYLKSAIKVWLDMGIDGIRLDAVKHMPFGWQKNFMDSILSYRPVFTFGEWFLGTNEIDANNTYFANESGMSLLDFRFSQKVRQVFRDNTDTMYGLDSMIQSTASDYNFINDMVTFIDNHDMDRFYNGGSTRPVEQALAFTLTSRGVPAIYYGTEQYMTGNGDPYNRAMMTSFNTATTAYNVIKKLAPLRKSNPAIAYGTTQQRWINNDVYIYERKFENNVALVAINRNLTTSYNITGLYTALPAGTYTDVLGGLLNGNSISVASNGSVTSFTLGAGEVAVWQYVSSSNSPLIGHVGPTMTKAGQTITIDGRGFGTTAGQVLFGSTAGTIVSWDDTEVKVKVPSVTPGKYNVSLKTSSGATSNTYNNINILTGNQVCVRFVVNNANTVYGENVYLTGNVAELGNWDTSKAIGPMFNQVVYQYPTWYYDVSVPAGTTIQFKFIKKNGSTITWEGGSNHTYTVPSSGTGTVIVNWQQ (SEQ ID NO.2)

[0102] Clostridium sp.

[0103] Clostridium sp. Amino acid sequence of cyclodextrin glycosyltransferase from USBA 49 (SEQ ID NO. 4)

[0104] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A cyclodextrin glycosyltransferase mutant, characterized in that: The cyclodextrin glycosyltransferase mutant is cyclodextrin glycosyltransferase mutant M1 and / or cyclodextrin glycosyltransferase mutant M2; The cyclodextrin glycosyltransferase mutant M1 is obtained by mutating the lysine at position 74 to histidine, the proline at position 115 to asparagine, the tyrosine at position 179 to serine, and the glycine at position 397 to threonine of the wild-type cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO. 2; The cyclodextrin glycosyltransferase mutant M2 is obtained by mutating the 128th aspartic acid to serine, the 130th serine to asparagine, and the 232nd aspartic acid to glycine of the wild-type cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO.

4.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the cyclodextrin glycosyltransferase mutant according to claim 1.

3. An expression cassette, characterized in that The expression cassette comprises the nucleic acid molecule of claim 2.

4. A recombinant vector, characterized in that The recombinant vector comprises the nucleic acid molecule according to claim 2.

5. A recombinant microorganism, characterized in that The recombinant microorganism comprises the nucleic acid molecule according to claim 2 or the recombinant vector according to claim 4.

6. The recombinant microorganism according to claim 5, characterized in that The recombinant microorganism is recombinant Escherichia coli.

7. Use of the cyclodextrin glycosyltransferase mutant according to claim 1 and / or the recombinant microorganism according to any one of claims 5 to 6 in catalytic synthesis of rebaudioside RI.

8. A method for synthesizing rebaudioside RI, characterized in that: include: Rebaudioside RI is synthesized in a liquid environment using wet cells obtained by induced culture of the recombinant microorganism according to any one of claims 5 to 6 or a crude enzyme solution extracted by crushing the wet cells as a catalyst, stevioside ST as a substrate, and cyclodextrin as a co-substrate.

9. The method according to claim 8, wherein The liquid environment is a phosphate buffer solution with a pH of 7-8; the reaction conditions include: reacting at 35-45° C. and 200-800 rpm.

10. The method according to claim 8, wherein The amount of the catalyst used is 5-25 g / L based on the total weight of the wet cells, the initial concentration of the substrate is 10-80 mM, and the amount of the auxiliary substrate added is 30-220 mM.

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