Cyclodextrin glycosyl transferase mutant and application thereof in synthesis of rebaudioside RI

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly relates to a cyclodextrin glycosyltransferase mutant and application thereof in synthesis of rebaudioside RI. Specifically, enzyme molecules are modified by utilizing a directed evolution theory, so that the cyclodextrin glycosyltransferase mutant is successfully obtained. Meanwhile, the cyclodextrin glycosyl transferase mutant is utilized, cheap and easily available cyclodextrin is used as a glycosyl donor to replace high-cost UDP-glucose, the catalytic efficiency is greatly improved, and the conversion rate reaches 98.8%. The reaction opens up a new way for preparing rebaudioside RI, the yield of the reaction product is high, the preparation process is simple, and the method is suitable for industrial production and has a wide application prospect.

Description

Technical Field

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

[0002] Steviol glycosides are diterpenoid compounds with a diterpenoid steviol as the basic skeleton. Steviol glycosides mainly exist in the leaves of Stevia rebaudiana, and its leaves contain various natural steviol glycosides such as stevioside, rebaudioside RA, rebaudioside RB, rebaudioside RD, rebaudioside RE, rebaudioside RI, rebaudioside RM, etc. Among them, stevioside is widely used. Although the sweetness of this kind of product reaches 300 - 600 times that of sucrose, there are still disadvantages such as bitter aftertaste, and the taste needs to be improved. Rebaudioside RI has good sweetness and taste, and low calories, and is currently recognized as an ideal steviol glycoside product. The content of rebaudioside RI in the original plant is very low, and the cost of plant extraction and purification is huge. The current production far from meets the market demand.

[0003] Cyclodextrin glycosyltransferase (abbreviated as CGTase, EC2.4.1.19), belonging to the α - cyclodextrinase family, has been proven to be a multifunctional enzyme that can catalyze four reactions, namely hydrolysis reaction, disproportionation reaction, cyclization reaction and coupling reaction. The transglycosylation reaction of CGTase is significant, and the hydrolysis reaction is weak, which is a typical transglycosidase. The disproportionation reaction and coupling reaction are intermolecular transglycosylation reactions that transfer maltooligosaccharides and cyclodextrins to various receptor molecules to produce glycosylated derivatives and modify the receptor molecules. Currently, the receptor molecules that can be used 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, L - ascorbic acid, etc. Rebaudioside RI is a polysaccharide glycoside formed by glycosylation modification of the aglycone steviol respectively. Therefore, cyclodextrin glycosyltransferase can be applied to the synthesis of rebaudioside RI. However, natural cyclodextrin glycosyltransferases generally have problems such as low enzyme activity, instability, and poor substrate promiscuity, so it is often difficult to meet the production requirements. Summary of the Invention

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

[0005] To achieve the above technical objectives, the present invention adopts the following technical solutions: In the first aspect of the present invention, a cyclodextrin glycosyltransferase mutant is provided, which can be cyclodextrin glycosyltransferase mutant M1 and / or cyclodextrin glycosyltransferase mutant M2; Among them, the cyclodextrin glycosyltransferase mutant M1 is obtained by mutating lysine at position 74 of the wild-type cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO.2 to histidine, proline at position 115 to asparagine, tyrosine at position 179 to serine, and glycine at position 397 to threonine; The cyclodextrin glycosyltransferase mutant M2 is obtained by mutating aspartic acid at position 128 of the wild-type cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO.4 to serine, serine at position 130 to asparagine, and aspartic acid at position 232 to glycine.

[0006] In the second aspect of the present invention, a nucleic acid molecule is provided, and the nucleic acid molecule encodes the cyclodextrin glycosyltransferase mutant described in the first aspect.

[0007] In the present invention, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0008] In the third aspect of the present invention, an expression cassette is provided, and the expression cassette contains the nucleic acid molecule described in the second aspect.

[0009] In the present invention, the expression cassette includes a promoter capable of initiating transcription of the nucleic acid molecule, and the nucleic acid molecule. Further, it may also include a termination sequence and an enhancer sequence, etc.

[0010] In the fourth aspect of the present invention, a recombinant vector is provided, and the recombinant vector contains the nucleic acid molecule described in the second aspect.

[0011] The recombinant vector of the present invention is obtained by ligating a vector with the nucleic acid molecule. The vector can be constructed by any method known in the art or commercially purchased. For example, in one or more embodiments of the present invention, the vector is plasmid pET22b, which can be expressed in Escherichia coli.

[0012] In a fifth aspect of the present invention, there is provided a recombinant microorganism comprising the nucleic acid molecule described in the second aspect or the recombinant vector described in the fourth aspect.

[0013] The recombinant microorganism of the present invention can be a recombinant bacterium, specifically a recombinant Escherichia coli.

[0014] In a seventh aspect of the present invention, there is provided 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 the catalytic synthesis of rebaudioside RI.

[0015] In an eighth aspect of the present invention, there is provided a method for synthesizing rebaudioside RI, comprising: using the wet cells obtained by inducing and culturing the recombinant microorganism described in the fifth aspect above or the crude enzyme solution extracted by disrupting the wet cells as a catalyst, using stevioside ST as a substrate, using cyclodextrin as a co-substrate, and reacting in a liquid environment to synthesize rebaudioside RI; wherein, 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), so as to accelerate the reaction progress.

[0016] The dosage of the catalyst 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 addition amount of the co-substrate is 30-220 mM.

[0017] In the present invention, the wet cells can be prepared by the following method: inoculating the recombinant bacterium into an LB liquid medium containing benzylpenicillin resistance, culturing at 37 °C and 200 rpm for 12 h, then inoculating with an inoculum volume concentration of 1% into a fresh LB liquid medium containing 50 μg / mL ampicillin resistance, culturing at 37 °C and 200 rpm until the OD600 of the cells reaches 0.6-0.8, adding IPTG with a final concentration of 24 μg / mL, inducing and culturing at 25 °C for 16 h, then centrifuging at 4 °C and 8000 rpm for 20 min, discarding the supernatant, and collecting the precipitate to obtain the wet cells; Furthermore, the method for preparing the crude enzyme solution includes: resuspending the wet bacterial cells in a phosphate buffer solution with a pH of 7.5 and a concentration of 100 mM, and then subjecting them to ultrasonic disruption on an ice-water mixture for 5 min to obtain the crude enzyme solution; wherein, the conditions for ultrasonic disruption include: an ultrasonic power of 200 W, disrupting for 1 s and pausing for 2 s.

[0018] Advantageous technical effects of the above technical solution: (1) Through site-directed mutagenesis of wild-type cyclodextrin glycosyltransferase, the cyclodextrin glycosyltransferase mutants M1 and M2 were successfully obtained. Using stevioside ST as a substrate and in the presence of the auxiliary substrate cyclodextrin, the two cyclodextrin glycosyltransferase mutants were used to catalytically convert stevioside ST to rebaudioside RI in one pot. Finally, the co-expression strain completely catalyzed 70 mM of stevioside ST to produce rebaudioside RI in only 16 h. This method has a high raw material conversion rate, a high yield, and a short conversion time.

[0019] (2) Currently, the method for synthesizing rebaudioside RI by biocatalysis usually requires the addition of expensive UDP-glucose as a substrate. Through the action of UDP-glucosyltransferase, using stevioside ST as a substrate and UDP-glucose as a glycosyl donor to catalytically generate rebaudioside RI. Due to the extremely high price of UDP-glucose, it almost completely limits the feasibility of industrial preparation of rebaudioside RI, with poor economy and lack of market competitiveness. However, the above technical solution uses two co-expressed cyclodextrin glycosyltransferases to catalytically convert stevioside ST to rebaudioside RI in one pot, using inexpensive cyclodextrin as a glycosyl donor, which has good economic benefits. Therefore, it has great application value for the production of rebaudioside RI. Description of the Drawings

[0020] The attached drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0021] Figure 1 is E.coli The reaction route diagram of BL21(DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T(M1)-cgt3-D128S-S130N-D232G(M2) for catalytically synthesizing rebaudioside RI from stevioside ST.

[0022] Figure 2 is E.coli The reaction process diagram of BL21(DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T(M1)-cgt3-D128S-S130N-D232G(M2) for catalytically synthesizing rebaudioside RI. Detailed implementation manners

[0023] It should be noted that the following detailed description is exemplary and is 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 meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0024] It should be noted that the terms used herein are only for describing the specific implementation manners 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 forms are also intended to include the plural forms. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or their combinations.

[0025] In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, to better understand the present invention, the definitions and explanations of related terms are provided below.

[0026] 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 contains a promoter, an MCS (multiple cloning site), and / or a terminator. The expression cassette may also include a gene of interest, a marker gene (such as the TK gene, the DHFR gene, the CAT gene, and the NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly(A) signal sequence, and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette can be directly connected or indirectly connected through a linker.

[0027] The term "vector" generally refers to a vector that can carry exogenous DNA or a target gene into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into the host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be amplified and / or expressed in the host cell. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the nature of the recipient cell. The vectors include, but are not limited to: plasmids, phages (such as phage λ or M13 phage), cosmids (i.e., cosmid plasmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC) or Ti plasmid artificial chromosomes (TAC)), viral vectors (such as baculovirus vectors, retroviruses, adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, polyomaviruses and herpesviruses). A vector can contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain an origin of replication.

[0028] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, etc. For example, the bacteria can be from the genera Corynebacterium, Brevibacterium, Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas and Bacillus, etc. The viruses can include rotaviruses, baculoviruses, retroviruses (such as lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, influenza viruses, polyomaviruses (such as SV40) and herpesviruses (such as herpes simplex virus), etc. The fungi can be from the genera Saccharomyces (such as Saccharomyces cerevisiae, Candida, Pichia methanolica, Pichia pastoris), Fusarium, Rhizoctonia, Verticillium, Penicillium, Aspergillus and Cephalosporium, etc. The actinomycetes can be from the genus Streptomyces. The algae can be from the phylum Cyanophyta, Fucus, Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas and Chrysosaccus, etc.

[0029] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by ligating an exogenous target gene and a vector in vitro, which can be constructed in any suitable way as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the ability for the exogenous target gene to replicate, integrate, amplify and / or express in the recipient cell.

[0030] The term "recombinant microorganism" generally refers to the operation and modification of the genes of a target microorganism to obtain a recombinant microorganism with changed functions. For example, introducing an exogenous target gene or a recombinant vector into the target microorganism, or directly performing gene editing on the endogenous genes of the target microorganism.

[0031] The term "mutation" generally refers to a change in the amino acid sequence or nucleotide sequence, which may include a change in the base pair composition or arrangement order in the structure of a gene, such as a point mutation caused by a single base change, or deletions, duplications, and insertions of multiple bases, etc., and may also include substitutions, deletions, and insertions (additions) of one or more amino acid residues of a protein, etc.

[0032] The term "site-directed mutation" generally refers to changing one or several bases in a certain gene through site-directed mutation methods, including addition, deletion, point mutation, etc., resulting in a change in the amino acid composition of the corresponding protein. Site-directed mutation methods include oligonucleotide primer-mediated site-directed mutation, PCR-mediated site-directed mutation, and cassette mutation, etc.

[0033] The term "homologous recombination" generally refers to a genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. Exemplarily, a site-directed mutation strategy based on homologous recombination can be achieved through the following steps: ligating homologous arms of the wild-type cyclodextrin glycosyltransferase gene to both ends of the nucleic acid molecule encoding the cyclodextrin glycosyltransferase mutant of the present invention and then introducing it into the recipient bacterium to replace the wild-type cyclodextrin glycosyltransferase gene, thereby achieving the purpose of site-directed mutation.

[0034] The term "fermentation" generally refers to a biological reaction process that produces and accumulates the required products through the growth, reproduction, and metabolic activities of organisms, including microbial fermentation.

[0035] 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 in conjunction with specific embodiments.

[0036] The culture medium formula and high-performance liquid chromatography detection method used in the following examples are as follows: LB medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 10 g / L, solvent is water, pH 7.4.

[0037] LB plate: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 10 g / L, 18 g / L agar, solvent is water, pH 7.4.

[0038] The concentration of the product rebaudioside RI was detected by high performance liquid chromatography (HPLC). The analytical method was as follows: Column model: QS-C18, 5 μm, 4.6×250 mm. The mobile phase was A (water):B (acetonitrile) = 68:32. The injection volume was 10 μL. The detection wavelength was 210 nm. The detection time was 18 min. The flow rate was 0.5 mL / min. The column temperature was 40°C.

[0039] Sample preparation: Take 20 μL of the sample after the reaction ended, dilute it 10 times, add 16 μL of 2M H2SO4 solution and 160 μL of 60% methanol solution to terminate the reaction, filter through a 0.22 μm filter membrane, and perform HPLC detection.

[0040] Example 1: Construction of expression vector and engineering bacteria Two cyclodextrin glycosyltransferases derived from Thermoanaerobacterium saccharolyticum and Clostridium sp. USBA 49 were mined from the NCBI database. The NCBI accession numbers were WP_045409091.1 and WP_078681113.1. The nucleotide sequences were shown as SEQ ID NO.1 and SEQ ID NO.3 respectively, and the amino acid sequences were shown as SEQ ID NO.2 and SEQ ID NO.4 respectively.

[0041] Primers F1 and R1 were designed according to the nucleotide sequence shown in SEQ ID NO.1, and NdeI and NotI restriction enzyme cleavage sites were introduced into the primers respectively: F1: 5’-CATATGATGAAAAAAACGTTTAAATTGATATTG-3’; R1: 5’-GCGGCCGCCTACTGTTGCCAGTTTACAATTA-3’; Primers F2 and R2 were designed according to the nucleotide sequence shown in SEQ ID NO.3, and NdeI and NotI restriction enzyme cleavage sites were introduced into the primers respectively: F2: 5’-CATATGATGCGTAAATTTAATGCCTTAATAA-3’; R2: 5’-GCGGCCGCTTATTGTTGCCAATTAACAGTTAT-3’; Using the pET-22b plasmid as the expression vector, Escherichia coli E.coli BL21(DE3) / pET22b-cgt2 and E.coli BL21(DE3) / pET22b-cgt3 were constructed.

[0042] Construction of expression plasmid: Under the priming of the above primers, using the target gene as a template, the cyclodextrin glycosyltransferase gene sequence was amplified using high-fidelity Pfu DNA polymerase. After sequencing, the amplified fragment was treated with NdeI and NotI restriction endonucleases (TaKaRa), and the fragment was ligated to 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.

[0043] Preparation of competent cells: The glycerol stock of BL21(DE3) strain stored in a -80 °C refrigerator E. coli was streaked on an antibiotic-free LB plate and cultured at 37 °C for 10 h to obtain single colonies; a single colony from 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 the 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, taking care 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 containing 15% glycerol. 100 μL of the resuspended cells was aliquoted into sterilized 1.5 mL centrifuge tubes and stored in a -80 °C refrigerator, and taken out when needed.

[0044] Construction of recombinant Escherichia coli: First, the Escherichia coli BL21(DE3) (Invitrogen) competent cells stored at -80 °C were ice-bathed at 0 °C for 10 min, then 5 μL of the ligation product was added in a laminar flow hood, ice-bathed at 0 °C for 30 min, heat-shocked in a 42 °C water bath for 90 s, ice-bathed at 0 °C for 2 min, 600 μL of LB medium was added, and cultured in a shaker at 37 °C and 200 rpm for 1 h; spread on an LB plate containing 50 μg / ml ampicillin resistance and cultured at 37 °C for 8 - 12 h. Clones were randomly picked, plasmids were extracted and sequenced for identification, and recombinant Escherichia coli containing the expression recombinant plasmid was screened E.coli BL21(DE3) / pET22b-cgt2 and E.coli BL21(DE3) / pET22b-cgt3.

[0045] Example 2: Inductive expression of cyclodextrin glycosyltransferase Wet cells containing cyclodextrin glycosyltransferase gene: The recombinant Escherichia coli obtained in Example 1 was respectively E.coli BL21(DE3) / pET22b-cgt2 and E.coli BL21(DE3) / pET22b-cgt3 were respectively 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 an inoculation amount of 1% (v / v), cultured at 37 °C and 200 rpm until the OD600 of the cells reached 0.6 - 0.8, IPTG with a final concentration of 0.1 mM was added, and after inducing culture at 25 °C for 16 h, centrifuged at 4 °C and 8000 rpm for 20 min, the supernatant was discarded, the precipitate was collected, washed twice with phosphate buffer at pH 7.5 and 20 mM, and the recombinant bacteria E.coli BL21(DE3) / pET22b-cgt2 and E.coli BL21(DE3) / pET22b-cgt3 containing cyclodextrin glycosyltransferase were obtained. The wet cells were resuspended in phosphate buffer at pH 7.5 and 100 mM, and ultrasonically disrupted on an ice - water mixture for 5 min. The ultrasonic disruption conditions were: power of 200 W, disruption for 1 s, pause for 2 s, and a crude enzyme solution was obtained.

[0046] Example 3: Establishment of a cyclodextrin glycosyltransferase gene mutation library 1. Site - directed mutagenesis Using E.coli BL21(DE3) / pET22b-cgt2 and E.coli BL21(DE3) / pET22b-cgt3 constructed in Example 1 as the starting strains.

[0047] It was transformed by the method of directed evolution theory. The crystal structure of cyclodextrin glycosyltransferase obtained by homology modeling was docked to obtain a complex model. And the amino acid residues around the substrate stevioside ST in the cgt2 structure were analyzed, and sites 74, 114, 115, 116, 197, 397 were selected for site - directed mutagenesis, and the primer design is shown in Table 1.

[0048] It was transformed by the method of directed evolution theory. The crystal structure of cyclodextrin glycosyltransferase obtained by homology modeling was docked to obtain a complex model. And the amino acid residues around the substrate rebaudioside RA in the cgt3 structure were analyzed, and sites 126, 128, 130, 231, 232 were selected for site - directed mutagenesis, and the primer design is shown in Table 2.

[0049] The mutant PCR system (100 μL) is as follows: 25 μL of 2*Phanta Max buffer, 1 μL of dNTPs, 1 μL each of the upstream and downstream mutant primers, 1 μL of the template (starting strain), 0.5 μL of Pfu DNA polymerase, and ddH2O is added to make up to 50 μL. The PCR conditions are: pre-denaturation at 95°C for 3 min, followed by 30 cycles: 95°C for 15 s, 60°C for 15 s, 72°C for 7 min 20 s, and finally a final extension at 72°C for 10 min. The PCR products are respectively verified by positive DNA agarose gel electrophoresis. The PCR products are digested with DpnI enzyme for the template at 37°C for 1 hour at 200 rpm, inactivated at 65°C for 1 minute, and the PCR products are heat-shocked and transformed, and Escherichia coli E. coli BL21(DE3) is activated, placed at 37°C and 200 rpm, cultured for 1 hour, and spread on an LB plate containing 50 μg / mL ampicillin resistance, and cultured overnight at 37°C in an inverted position.

[0050] Table 1 Design of site-directed mutation primers for cyclodextrin glycosyltransferase cgt2

[0051] Table 2 Design of site-directed mutation primers for cyclodextrin glycosyltransferase cgt3

[0052] Example 4: Screening of the cyclodextrin glycosyltransferase gene mutation library Single colonies are picked from the plate obtained in Example 3, inoculated into an LB liquid medium containing 50 μg / mL ampicillin resistance, cultured at 37°C and 200 rpm for 12 h, the bacterial strains are preserved and sent to a sequencing company for sequencing verification. After correct sequencing verification, the preserved bacterial strains are inoculated into an LB liquid medium containing 50 μg / mL ampicillin resistance at an inoculation amount of 0.2%, cultured at 37°C and 200 rpm for 12 h, and then inoculated into a fresh LB liquid medium containing 50 μg / mL ampicillin resistance at an inoculation amount of 1% (v / v), cultured at 37°C and 200 rpm until the OD600 of the bacteria reaches 0.6 - 0.8, IPTG with a final concentration of 0.1 mM is added, induced and cultured at 25°C for 16 h, then centrifuged at 4°C and 8000 rpm for 20 min, the supernatant is discarded, and the precipitate is collected to obtain the wet bacteria containing the cyclodextrin glycosyltransferase cgt2 and cgt3 gene mutation libraries. The wet bacteria are resuspended in a phosphate buffer at pH 7.5 and 100 mM, and ultrasonically disrupted on an ice-water mixture for 5 min. The ultrasonic disruption conditions are: power of 200 W, disrupted for 1 s, paused for 2 s, to obtain a crude enzyme solution 1. Primary screening of cgt2: Prepare the reaction solution (200 μL): The final concentration of the substrate stevioside ST is 20 mM, the final concentration of cyclodextrin is 30 mM, the catalyst dosage is 5 g / L based on the total weight of the wet bacterial cells before disruption, and the reaction solution is composed with a phosphate buffer at pH 7.5 as the reaction medium. Reaction conditions: React on a reactor at 40 °C and 500 rpm for 15 hours. After the reaction ends, take 20 μL of the sample at the end of the reaction, dilute it 10 times, add 16 μL of 2 M H2SO4 solution and 160 μL of 60% methanol solution to terminate the reaction, filter through a 0.22 μm filter membrane, and perform HPLC detection. The detection results are shown in Table 3.

[0053] Table 3 Primary screening reaction results

[0054] 2. Primary screening of cgt3: Prepare the reaction solution (200 μL): The final concentration of the substrate rebaudioside RA is 10 mM, the final concentration of cyclodextrin is 30 mM, the catalyst dosage is 5 g / L based on the total weight of the wet bacterial cells before disruption, and the reaction solution is composed with a phosphate buffer at pH 7.5 as the reaction medium. Reaction conditions: React on a reactor at 40 °C and 500 rpm for 16 hours. After the reaction ends, take 20 μL of the sample at the end of the reaction, dilute it 10 times, add 16 μL of 2 M H2SO4 solution and 160 μL of 60% methanol solution to terminate the reaction, filter through a 0.22 μm filter membrane, and perform HPLC detection. The detection results are shown in Table 4.

[0055] Table 4 Primary screening reaction results

[0056] 3. Re-screening of cgt2: Perform re-screening on the strains obtained from the primary screening. Combine and mutate the re-screened mutants and send them to a sequencing company for sequencing verification. After the sequencing verification is correct, then perform activity verification. Prepare the re-screening reaction solution (10 mL): The final concentration of the substrate stevioside ST is 80 mM, the final concentration of cyclodextrin is 120 mM, the catalyst dosage is 20 g / L based on the total weight of the wet bacterial cells before disruption, and the reaction solution is composed with a phosphate buffer at pH 7.5 as the reaction medium. Reaction conditions: React on a reactor at 40 °C and 500 rpm for 15 hours. After the reaction ends, take 20 μL of the sample at the end of the reaction, dilute it 10 times, add 16 μL of 2 M H2SO4 solution and 160 μL of 60% methanol solution to terminate the reaction, filter through a 0.22 μm filter membrane, and perform HPLC detection. The detection results are shown in Table 5, and the strain with the highest activity is obtained E.coli BL21(DE3) / pET22b-cgt2-K74H-P115N-Y179S-G397T(M1).

[0057] Table 5 Results of the second-round screening reaction

[0058] 4. Second-round screening of cgt3: The strains obtained from the first-round screening were subjected to second-round screening. The combined mutant was sent to a sequencing company for sequencing verification. After correct sequencing verification, viability verification was carried out. The reaction solution for the second-round screening (10 mL) was prepared as follows: the final concentration of substrate rebaudioside RA was 70 mM, the final concentration of cyclodextrin was 200 mM, the dosage of the catalyst was 20 g / L based on the total weight of the wet bacterial cells before disruption, and the reaction medium was a phosphate buffer at pH 7.5 to form the reaction solution. Reaction conditions: React on a reactor at 40 °C and 500 rpm for 16 hours. After the reaction ended, 20 μL of the sample at the end of the reaction was taken, diluted 10 times, 16 μL of 2M H2SO4 solution and 160 μL of 60% methanol solution were added to terminate the reaction, and the mixture was filtered through a 0.22 μm filter membrane for HPLC detection. The detection results are shown in Table 6, and the strain with the highest viability was obtained E.coli BL21(DE3) / pET22b-cgt3-D128S-S130N-D232G(M2).

[0059] Table 6 Results of the second-round screening reaction

[0060] Example 5: Construction of a co-expression strain of cyclodextrin glycosyltransferase cgt2 and cyclodextrin glycosyltransferase cgt3 A co-expression strain was constructed by combining the recombinant cyclodextrin glycosyltransferase cgt2 with the highest viability obtained in Example 4 and 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

[0061] Primers were designed based on the cgt2-K74H-P115N-Y179S-G397T and cgt3-D128S-S130N-D232G nucleotide sequences and the pET-Duet vector sequence. Using homologous recombination, cgt2-K74H-P115N-Y179S-G397T was constructed between the NcoI-NotI restriction enzyme sites of the first cloning site of the pET-Duet vector, and cgt3-D128S-S130N-D232G was constructed between the NdeI-XhoI restriction enzyme sites of the second cloning site of the pET-Duet vector. Construction of the expression plasmid: Under the priming of primers F3 / R3, using the target gene as a template, high-fidelity Pfu DNA polymerase was used for amplification to obtain the cgt2-K74H-P115N-Y179S-G397T gene sequence with homologous arms. Under the priming of primers F4 / R4, using the pETduet plasmid as a template, high-fidelity Pfu DNA polymerase was used for amplification to obtain the linearized vector sequence, and the target gene was homologously recombined with the linearized vector using a homologous recombinase. The transformation process of the recombinant product referred to Example 1. After sequencing verification, plasmid pETduet-cgt2-K74H-P115N-Y179S-G397T was obtained. Then, under the priming of primers F5 / R5, using the target gene as a template, high-fidelity Pfu DNA polymerase was used for amplification to obtain the cgt3-D128S-S130N-D232G gene sequence with homologous arms. Under the priming of primers F6 / R6, using the pETduet-cgt2-K74H-P115N-Y179S-G397T plasmid as a template, high-fidelity Pfu DNA polymerase was used for amplification to obtain the linearized vector sequence, and the target gene was homologously recombined with the linearized vector using a homologous recombinase. The transformation process of the recombinant product referred to Example 1. After sequencing verification, the co-expression strain E.coli BL21(DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T-cgt3-D128S-S130N-D232G was obtained.

[0062] Table 7 Primer Design

[0063] Example 6: Application of the co-expression strain in the catalytic synthesis of rebaudioside RI 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. It was inoculated into LB liquid medium containing 50 μg / mL ampicillin at a final concentration and cultured at 37 °C for 9 hours to obtain a seed solution, which was then inoculated into a 5-L fermenter containing 3 L of fermentation medium at an inoculation amount of 3.5% (v / v). It was cultured at 37 °C and 500 rpm for about 3 - 4 h until the cell density OD reached 6 - 8. After the temperature of the fermenter was reduced to 25 °C, lactose at a final concentration of 5 g / L was added as an inducer, and then it was cultured at 25 °C and 500 rpm for 12 h. The cultured fermentation broth was centrifuged at 8000 rpm for 10 min to obtain a co-expression strain containing two cyclodextrin glycosyltransferase mutants. E.coli The wet cells of BL21(DE3) / pETduet-cgt2-K74H-P115N-Y179S-G397T-cgt3-D128S-S130N-D232G obtained were disrupted using a high-pressure homogenizer.

[0064] Among them, the composition of the fermenter medium: 45 g of tryptone, 36 g of yeast extract, 30 g of sodium chloride, 4.08 g of potassium dihydrogen phosphate, 45 g of glycerol, 6.84 g of dipotassium hydrogen phosphate trihydrate, 15 g of ammonium sulfate, 1.125 g of magnesium sulfate, 4 g of antifoaming agent, and distilled water was added to make up to 3 L for dissolution.

[0065] The dosage of the catalyst was 20 g / L based on the total weight of the wet cells before high-pressure homogenization, the final concentration of the substrate stevioside ST was 70 mM, the final concentration of cyclodextrin was 200 mM, and a phosphate buffer solution with pH 7.5 was used as the reaction medium to make the total volume of the reaction solution 1 L. Reaction conditions: 40 °C, 500 rpm for 16 h. After the reaction ended, 20 μL of the sample at the end of the reaction was taken, diluted 10 times, 16 μL of 2M H2SO4 solution and 160 μL of 60% methanol solution were added to terminate the reaction, and it was filtered through a 0.22 μm filter membrane for HPLC detection. The reaction progress curve is as Figure 2 shown. After the reaction ended, the concentration of rebaudioside RI reached 69.2 mM, and the conversion rate reached 98.8%.

[0066] The nucleotide and amino acid sequence information used in the present invention Thermoanaerobacterium saccharolyticum Thermoanaerobacterium saccharolyticum Amino acid sequence of cyclodextrin glycosyltransferase from the source: MKKTFKLILVLMLSLTLVFGLTAPIQAASDTAVSNVVNYSTDVIYQIVTDRFVDGNTSNNPTGDLYDPTHTSLKKYFGGDWQGIINKINDGYLTGMGVTAIWISQPVENIYAVLPDSTFGGSTSYHGYWARDFKRTNPYFGSFTDFQNLINTAHAHNIKVIIDFAPNHTSPASETDPTYAENGRLYDNGTLLGGYTNDTNGYFHHYGGTDFSSYEDGIYRNLFDLADLNQQNSTIDSYLKSAIKVWLDMGIDGIRLDAVKHMPFGWQKNFMDSILSYRPVFTFGEWFLGTNEIDANNTYFANESGMSLLDFRFSQKVRQVFRDNTDTMYGLDSMIQSTASDYNFINDMVTFIDNHDMDRFYNGGSTRPVEQALAFTLTSRGVPAIYYGTEQYMTGNGDPYNRAMMTSFNTATTAYNVIKKLAPLRKSNPAIAYGTTQQRWINNDVYIYERKFENNVALVAINRNLTTSYNITGLYTALPAGTYTDVLGGLLNGNSISVASNGSVTSFTLGAGEVAVWQYVSSSNSPLIGHVGPTMTKAGQTITIDGRGFGTTAGQVLFGSTAGTIVSWDDTEVKVKVPSVTPGKYNVSLKTSSGATSNTYNNINILTGNQVCVRFVVNNANTVYGENVYLTGNVAELGNWDTSKAIGPMFNQVVYQYPTWYYDVSVPAGTTIQFKFIKKNGSTITWEGGSNHTYTVPSSGTGTVIVNWQQ (SEQ ID NO.2) Clostridium sp. Clostridium sp. Amino acid sequence of cyclodextrin glycosyltransferase from USBA 49: MRKFNALIKNKLFPFILSFILLFSLYINNPPKALTASTYSGDSSVSNKVNFSTDVIYQIVTDRFYDGNTSNNPTGTLYDSSKTQLKKYFGGDWQGIINKINDGYLSDMGITAIWISQPIENIYSVLSDGSTSYHGYWARDFKKTNPFFGSFTDFQNLINAAHAKNIKVIIDFAPNHTSPADEDNVNYAENGRLYDNGSLIAGYSNDTNGIFHHNGGTNFSTLEDGIYKNLFDLADLNHQNNIVDSYIKESIKKWLDMGIDGIRMDAVKHMPMGWQKNFMDSIYSYKPVFTFGEWFLGTNEVDLNNHYFANNSGMSLLDFRFAQKVRQVFRDNTDTMYGLNSMITDTYNSYEDINDQVTFIDNHDMDRFTITNGNTRKTDLALTLLLTSRGVPAIYYGTEQYMTGNGDPNNRAMMASFNKTTTAYKIIQKLAPLRKTNPALSYGTTTEKWINNDVYTYERKFGNSVVLVAINKNLNTSVSVNGIYTSLPSGTYSDFLSGIVSGNSITVNSNGSVNSFTLPAGSAAVWQYTSNTNNPTIGHIGHPMAKPGQTIVIDGRGFGSTKGSVYFGSTAATVISWSDTEIKVTVPSIQPGKIGVYLKTASGLTSNTFNNFDVLTSSQVLVRFVVNNANTNLGENLYLVGNTAELGNWDTNKAIGPIFNQVVYKYPTWYYDVNVPSNTNIQFKFIKKNGSSVVWEGGSNHTYTTPTNSTGIITVNWQQ (SEQ ID NO.4) The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope 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; Among them, the cyclodextrin glycosyltransferase mutant M1 is obtained by mutating lysine at position 74, proline at position 115, tyrosine at position 179, and glycine at position 397 of the wild-type cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO.2 into histidine, asparagine, serine, and threonine, respectively; The cyclodextrin glycosyltransferase mutant M2 is obtained by mutating aspartic acid at position 128, serine at position 130, and aspartic acid at position 232 of the wild-type cyclodextrin glycosyltransferase with the amino acid sequence shown in SEQ ID NO.4 into serine, asparagine, and glycine, respectively.

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 contains the nucleic acid molecule according to claim 2.

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

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

6. The recombinant microorganism according to claim 5, wherein The recombinant microorganism is recombinant Escherichia coli.

7. Use of the cyclodextrin glycosyltransferase mutant according to claim 1, the nucleic acid molecule according to claim 2, and / or the recombinant microorganism according to any one of claims 5-6 in the catalytic synthesis of rebaudioside RI.

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

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 dosage of the catalyst 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 addition amount of the co-substrate is 30-220 mM.

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