CN115975972B - Glycosyltransferase mutant and encoding gene thereof - Google Patents
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Abstract
The invention discloses a glycosyltransferase mutant and a coding gene thereof, and Reb D has high sweetness, no bitter taste and no liquorice aftertaste, is a very high-quality sugar substitute choice, and has high production cost, which is a main reason for limiting the popularization of Reb D as a sweetener. The three plant-derived mutant glycosyltransferases designed by the invention can be expressed in high quantity in microorganisms in about 3 times of wild type. Meanwhile, the thermal stability of the mutant obtained by expression is obviously better than that of a wild type. Therefore, the enzyme catalytic reaction system for catalyzing Red A to generate Red D by using the mutant of the three glycosyltransferases disclosed by the invention can be carried out at a higher temperature, so that the catalytic efficiency of the reaction system can be obviously improved, and the production efficiency of Red D can be improved.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a glycosyltransferase mutant and a coding gene thereof.
Background
Stevioside, also known as stevioside, has the characteristics of high sweetness (300-450 times of sucrose), low calorific value (1/300 of sucrose) and the like, and a natural sucrose substitute is known as 'world third glycogen'. Stevioside is extracted from stevia rebaudiana, and most of steviosides extracted from stevia rebaudiana by manufacturers are Stevioside (STV) and Rebaudioside a (Rebaudioside a, reb a). STV and Reb A however have bitter and licorice aftertastes, which greatly limits the use of steviol glycosides as sweeteners in foods.
Research shows that Rebaudioside D (Reb D) maintains the advantages of high sweetness and low calorific value of stevioside, has no bitter taste and no licorice aftertaste, and is a very high-quality sugar substitute choice. However, the mass production of Reb D presents a major bottleneck: reb D is less than 1% in stevia rebaudiana, and the cost of producing Reb D by plant extraction is high. Glycosyltransferases are essentially of plant origin and are expressed in relatively small amounts in microorganisms, which results in the current costs of producing Reb D by microbial fermentation or enzymatic catalysis also being at a relatively high level. Therefore, how to increase the expression level of plant-derived glycosyltransferases in microorganisms is a technical problem to be solved.
Disclosure of Invention
In view of the shortcomings of the prior art, one of the purposes of the present invention is to provide an amino acid sequence of a plant-derived glycosyltransferase that can be expressed in microorganisms in high amounts.
In order to achieve the above purpose, the present invention provides the following technical solutions: a glycosyltransferase has a wild-type sequence shown in SEQ ID NO. 1. The wild-type glycosyltransferase is derived from Solanum lycopersicum, NCBI Reference Sequence:XP_ 004250485.1. The following site mutations were performed on the above wild-type glycosyltransferase to obtain variant 1 having an amino acid sequence shown in SEQ ID NO. 2:
L5I-L34H-R41P-I48K-A56S-I62V-N87M-P88N-H91K-P99S-R103K-N127S-E128S-Q129H-C140G-S149H-E167D-V168Y-V171K-T202E-C211L-T212R-G244D-T245K-D266E-V270I-F272H-S277A-A285V-K301E-R312I-T329I-G331A-A341L-I356F-P361F-G387E-T392V-L393I-S395K-K414E-I419K-A426E。
the wild type glycosyltransferase is subjected to the following site mutation to obtain a variant 2 with an amino acid sequence shown as SEQ ID NO. 3:
L5I-L34H-I48K-I62V-N87M-P88N-H91K-P99S-N127S-E128S-Q129H-C140G-S149H-V168Y-T202E-C211L-T212R-G244D-D266E-V270I-F272H-S277A-A285V-R312I-T329I-G331A-A341L-I356F-P361F-G387E-T392V-L393I-S395K-I419K-A426E。
the wild type glycosyltransferase is subjected to the following site mutation to obtain a variant 3 with an amino acid sequence shown as SEQ ID NO. 4:
L5I-I48K-N87L-P99S-S149H-T202E-C211L-G244D-V270I-S277T-A285V-I356F-P361F-G387E-L393I-I419K。
the expression quantity of the three mutated glycosyltransferases in the escherichia coli is about 3 times that of the wild type, the highest UGTSL2-2 is 3.24 times more, the expression quantity of the plant-derived glycosyltransferases in the escherichia coli is greatly improved, the production cost of the Reb D is reduced, and the popularization of the industrial production of the Reb D is facilitated.
In view of the shortcomings of the prior art, a second object of the present invention is to provide a recombinant gene capable of expressing the above glycosyltransferase in a microorganism.
In view of the shortcomings of the prior art, it is a third object of the present invention to provide a recombinant plasmid comprising the recombinant gene.
Compared with the prior art, the invention has the advantages that: the three plant source mutant glycosyltransferases designed by the invention can be expressed in high quantity in microorganisms. Specifically, the expression level of the wild glycosyltransferase in the escherichia coli is 3.4g/L, and the expression levels of the three mutants after mutation in the escherichia coli reach 10.2g/L,11.0g/L and 8.5g/L respectively. The expression quantity of the three mutated glycosyltransferases in the escherichia coli is about 3 times that of the wild type, the highest mutant is 3.24 times that of the mutant, the expression quantity of the plant-derived glycosyltransferases in the escherichia coli is greatly improved, and the production cost of the Reb D is reduced. Meanwhile, the wild glycosyltransferase is incubated for 30min at 40 ℃, the enzyme activity is reduced to about 55%, three mutants obtained after mutation are incubated for 30min at 40 ℃, the enzyme activity is respectively reduced to about 79%, 92% and the enzyme activity is not reduced. Therefore, through mutation transformation, the three glycosyltransferase variants provided by the invention not only improve the expression quantity of the glycosyltransferase variants in escherichia coli, but also obviously improve the thermal stability, and an enzyme catalytic reaction system for catalyzing and generating Red D by the participating Red A can react at a higher temperature, so that the catalytic efficiency of the reaction system can be obviously improved, and the production efficiency of Red D is improved.
Drawings
FIG. 1 is a diagram of a protein glue of UGTSL2, UGTSL2-1, UGTSL2-2 and UGTSL 2-3;
FIG. 2 is a reaction scheme for rebaudioside D;
FIG. 3 is a graph of residual enzyme activity of UGTSL2 incubated at 30℃at 40℃for different times at 50 ℃;
FIG. 4 is a graph of residual enzyme activities of UGTSL2, UGTSL2-1, UGTSL2-2 and UGTSL2-3 incubated at 40℃for 30min, respectively.
Detailed Description
DNA constructs refer to sequences capable of expressing the glycosyltransferases and glycosyltransferase variants of the invention. Typically, the DNA construct is synthesized in vitro by PCR or other suitable techniques known in the art. In certain embodiments, the DNA construct further comprises other accessory elements, such as control elements (e.g., promoters, etc.). The DNA construct may also include a labeling substance (e.g., fluorescence, etc.). The DNA construct may also include other sequences that do not affect expression of the gene of interest.
The term "expression" refers to the process by which DNA is transcribed into messenger RNA (mRNA) and then translated into protein.
An "expression vector" has the ability to incorporate and express heterologous polynucleic acid fragments in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The choice of an appropriate expression vector is within the knowledge of the skilled person.
The term "host cell" refers to a suitable host vector for expressing a DNA comprising the invention. The host may comprise any organism capable of comprising and expressing the nucleic acids or genes disclosed herein, but is not limited thereto. The host may be a prokaryote or eukaryote, single or multiple cells, including mammalian cells, plant cells, fungi, and the like. Examples of single cell hosts include cells of Escherichia, salmonella, bacillus, clostridium, streptomyces, staphylococcus, neisseria, lactobacillus, shigella and Mycoplasma. Suitable E.coli strains (including many others) include BL21 (DE 3), C600, DH 5. Alpha. F',1113101, JM83, JM101, JM103, JM105, JM107, JM109, JM110, MC1061, MC4100, M294, NM522, NM554, TGI, χ1776, XL1-Blue and Y1089+, all of which are commercially available.
"homology" refers to sequence similarity or sequence identity. Homology is determined using standard techniques known in the art (see, e.g., smith and Watermann, advanced applied mathematics, 2:482, 1981;Needleman and Wunsch, J.Mol. biol., 48:443, 1970; proc. Natl. Acad. Sci. USA 85:2444, 1988; programs such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin genetics software package (the Madison genetics computer group, wis.), and Del. Fuler et al, nucleic acids research, 12:387-395, 1984.) one non-limiting example includes the use of BLAST programs (Altschul et al, gapped BLAST and PSI-BLAST: new generation protein database search programs, nucleic Acid Res. 25:3389-3402, 1997) to identify sequences that can be said to be "homologous". The latest versions, such as 2.2.16, 2.2.17, 2.2.18, 2.2.19 or the latest versions, include blastp for protein-protein comparison, blastn for nucleotide-nucleotide comparison, tblastn for protein-nucleotide comparison or blastx for nucleotide-protein comparison, the parameters are as follows: the maximum number of sequences returned is 10,000 or 100,000, and the E value (expected value) is 1e −2 Or 1e −5 A word number of 3, a scoring matrix BLOSUM62, a gap cost of 11, a gap cost extension of 1, may be suitable. For example, E has a value of 1E −5 Indicating a probability of random homology matches of about 1/10,000, thus marking a high confidence of true homology.
The term "identity" means that the residues in the two sequences are identical when aligned for maximum correspondence, as measured using sequence comparison or analysis algorithms such as those described herein. For example, two sequences are said to have 50% identity if, when properly aligned, the corresponding fragments of the two sequences have identical residues at 5 of the 10 positions. Most bioinformatic programs report percent identity of aligned sequence regions, which are typically not the entire molecule. If the alignment is long enough and contains enough identical residues, then the expected value can be calculated, indicating that the same level in alignment is unlikely to occur randomly.
The invention will now be described in further detail with reference to the drawings and examples.
Example 1
The protein sequence contains abundant evolutionary information, and plays a vital role in improving the thermal stability of the protein. A number of classical co-evolution analysis methods have been developed in the art that use a Potts model based on physical energy to obtain co-evolution signals in protein sequences by continuously reducing the energy of the homologous sequences based on homologous sequence alignment files. However, the presence of noise in the homologous sequence, which originates from the phylogenetic tree, severely affects the accuracy of the model. For this reason, the inventors propose a post-processing method for mean product correction, but this method is effective only for predicting protein contact patterns, and cannot eliminate the influence of noise on the co-evolutionary protein design algorithm. The inventor proposes a spectral norm penalty term based on the classical protein co-evolution analysis method Gremlin. In the Gremlin optimizing process, the spectrum norm penalty term can restrain the maximum eigenvalue of the co-evolution matrix, and further, the noise generated by the system is reduced. Based on the evolution matrix obtained by calculation, the inventor designs a greedy algorithm of optimal single point mutation generation by generation, continuously enhances the evolution signals in wild glycosyltransferase (the amino acid sequence is shown as SEQ ID NO. 1), and finally obtains the optimal enzyme variants (the base acid sequences are shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4) on the calculation level.
Specifically, the invention takes wild glycosyltransferase (UGTSL 2, the amino acid sequence of which is shown as SEQ ID NO. 1) as a parent, and carries out the following point mutation: L5I-L34H-R41P-I48K-A56S-I62V-N87M-P88N-H91K-P99S-R103K-N127S-E128S-Q129H-C140G-S149H-E167D-V168Y-V171K-T202E-C211L-T212R-G244D-T245K-D266E-V270I-F272H-S277A-A285V-K301E-R312I-T329I-G331A-A341L-I356F-P361F-G387E-T392V-L393I-S395K-K414E-I419K-A426E to give glycosyltransferase variant-1 (UGTSL 2-1, amino acid sequence shown in SEQ ID NO. 2).
Or using wild glycosyltransferase (amino acid sequence shown in SEQ ID NO. 1) as parent, performing the following point mutation: L5I-L34H-I48K-I62V-N87M-P88N-H91K-P99S-N127S-E128S-Q129H-C140G-S149H-V168Y-T202E-C211L-T212R-G244D-D266E-V270I-F272H-S277A-A285V-R312I-T329I-G331A-A341L-I356F-P361F-G387E-T392V-L393I-S395K-I419K-A426E to give glycosyltransferase variant-2 (UGTSL 2-2, amino acid sequence shown in SEQ ID NO. 3).
Or using wild glycosyltransferase (amino acid sequence shown in SEQ ID NO. 1) as parent, performing the following point mutation: L5I-I48K-N87L-P99S-S149H-T202E-C211L-G244D-V270I-S277T-A285V-I356F-P361F-G387E-L393I-I419K, resulting in glycosyltransferase variant-3 (UGTSL 2-3, amino acid sequence shown in SEQ ID NO. 4).
Construction of the DNA construct:
the nucleotide sequences of the genes of interest (UGTSL 2, UGTSL2-1, UGTSL2-2 and UGTSL 2-3) were all synthesized by Beijing-department Biotechnology, inc., and these nucleotide sequences were inserted into expression vectors, specifically, ndeI and XhoI cleavage sites of pET21b (+) to obtain corresponding plasmids. Then transferring the synthesized plasmids pET21b-UGTSL2, pET21b-UGTSL2-1, pET21b-UGTSL2-2 and pET21b-UGTSL2-3 into host cellsE. coliBL21 (DE 3)) and thus E.coli strains containing different plasmids.
Expression and purification of UGTSL2, UGTSL2-1, UGTSL2-2 and UGTSL 2-3:
inoculating 1% of the strain into 2 YT culture medium (16 g/L peptone, 10 g/L yeast extract, 5g/L sodium chloride), shaking the expressed strain, and OD 600 IPTG (final concentration 0.1. 0.1 mM) was added at 16℃to 0.6-0.8 and induced at 180rpm for overnight incubation. 8000 Cells were collected by centrifugation for 10 minutes, and the cells were weighed and quantified. The cells were resuspended in lysis buffer 20mM Tris,300mM NaCl (ph=8) at 1/10 of the medium. Sonication (lysate 1 min/mL) followed by centrifugation at 15000 g at 4℃for 30min, the supernatant and pellet were separated. The hetero protein was eluted with NTA-0 (30 mL), NTA-20 (30 mL), and Coomassie Brilliant blue was detected to be non-blue, NTA-40 (5 mL), NTA-60 (10 mL)) NTA-150 (20 mL) was used for collecting 20. Mu.L of the target protein supernatant, adding 4. Mu.L of electrophoresis loading buffer, carrying out water bath at 100 ℃ for 5 min, taking 1. Mu.L of loading, carrying out electrophoresis for 70 min at 130V, and carrying out SDS-PAGE analysis, wherein the measurement result is shown in FIG. 1.
Protein expression amount measurement: the overnight induced UGTSL2, UGTSL2-1, UGTSL2-2 and UGTSL2-3 were collected and weighed, resuspended in 1g of cell/10 mL lysis buffer 20mM Tris,300mM NaCl (pH=8.0), and after sonication of each mutant (lysate 1 min/mL), centrifuged at 15000 g for 30min at 4℃to take 20. Mu.L of supernatant, 5. Mu.L of loading buffer (electrophoresis loading buffer), water-bath at 100℃for 5 min, 1. Mu.L of loading, 130V electrophoresis for about 70 min (time can be prolonged appropriately), followed by Coomassie staining with coomassie-blue, and analysis of the protein expression level of each mutant in protein gel (protein gel pattern as shown in FIG. 1) by software BandScan 5.0, the analysis results are shown in Table 1.
TABLE 1
| Proteins | UGTSL2 | UGTSL2-1 | UGTSL2-2 | UGTSL2-3 |
| Expression level (g/L) | 3.4 | 10.2 | 11.0 | 8.5 |
As shown in Table 1, the expression level of the three mutated glycosyltransferases in the invention in the escherichia coli is about 3 times that of the wild type, and the highest UGTSL2-2 is 3.24 times that of the mutant glycosyltransferases, so that the expression level of the plant-derived glycosyltransferases in the escherichia coli is greatly improved, the production cost of the Reb D is reduced, and the popularization of the industrial production of the Reb D is facilitated.
Thermal stability determination of UGTSL2, UGTSL2-1, UGTSL2-2 and UGTSL 2-3:
reaction System 3 mM MgCl 2 0.5 mM R edA,0.1g/L glycosyltransferase, 2 mM RDP-glucose, 100 mM sulfate buffer (pH 8);
standard curve: reb D was formulated as a solution of varying concentration: 0.01 Standard curves were drawn for the statistical areas of hplc detection at mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6mM, 1 mM.
HPLC conditions: LC-2030C HT system (SHIMADZU, japan), gradient elution with water (0.1% formic acid)/acetonitrile (0.1% formic acid) (0 min:25% acetonitrile, 75% water; 12 min:47% acetonitrile, 53% water; 15 min:100% acetonitrile, 0% water; 17 min:100% ACN,0% water; 17.1min:25% acetonitrile, 75% water; 20 min:25% acetonitrile, 75% water), flow rates 0.8mL/min,210 nm, TC-C18 chromatographic column (250 mm ×4.6 mm), column temperature 40 ℃.
Rebaudioside A and UDP-glucose are catalyzed by glycosyltransferase to yield rebaudioside D and UDP, the specific reaction equations are shown in FIG. 2.
After incubating UGTSL2 at 30 ℃,40 ℃ and 50 ℃ for different time (0 h,0.5 h,1 h,2 h,4 h,6 h,24 h), preparing a reaction solution according to the reaction system, placing the reaction solution in a water bath kettle at 30 ℃ for reaction for 1h, placing the reaction system in a water bath at 95 ℃ for 10 min for stopping the reaction, filtering by a filter membrane of 0.22 mu M, and detecting and analyzing by HPLC, wherein the detection result is shown in figure 3.
As can be seen from FIG. 3, the wild-type glycosyltransferase was incubated at 30℃for 6 hours, the enzyme activity was reduced to about 70%, the enzyme activity was incubated at 40℃for 30 minutes, the enzyme activity was reduced to about 55%, and the enzyme activity was rapidly inactivated at 50 ℃. In summary, the wild-type glycosyltransferases have poor thermostability.
Incubating UGTSL2, UGTSL2-1, UGTSL2-2 and UGTSL2-3 at 40deg.C for 30min respectively, preparing reaction solution according to the reaction system, placing the reaction solution in a water bath kettle at 30deg.C for reacting for 1h, placing the reaction system in a water bath at 95deg.C for 10 min to terminate the reaction, filtering with a 0.22 μm filter membrane, and detecting and analyzing by HPLC, wherein the detection result is shown in figure 4.
As can be seen from FIG. 4, the glycosyltransferase variant-1 (UGTSL 2-1) obtained after mutation was incubated at 40℃for 30min, the enzyme activity was reduced to about 79%, the glycosyltransferase variant-2 (UGTSL 2-2) was reduced to about 92%, and the glycosyltransferase variant-3 (UGTSL 2-3) was not reduced (experimental data showed about 102%, and the deviation of the data was within experimental error). In conclusion, through mutation transformation, the thermal stability of the three glycosyltransferase variants provided by the invention is obviously improved, and an enzyme catalytic reaction system for catalyzing Red A to generate Red D with the three glycosyltransferase variants can be carried out at a higher temperature, so that the catalytic efficiency of the reaction system can be obviously improved, and the production efficiency of Red D is improved.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention can be made by one of ordinary skill in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (3)
1. A glycosyltransferase mutant, which is characterized in that the glycosyltransferase mutant is a variant 1 with an amino acid sequence shown as SEQ ID NO.2 or a variant 2 with an amino acid sequence shown as SEQ ID NO.3 or a variant 3 with an amino acid sequence shown as SEQ ID NO. 4.
2. A recombinant gene, characterized in that it is a DNA capable of expressing the glycosyltransferase mutant of claim 1.
3. A recombinant plasmid comprising the recombinant gene of claim 2.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107105734A (en) * | 2014-09-19 | 2017-08-29 | 谱赛科有限责任公司 | High-purity steviol glycoside |
| WO2019090336A1 (en) * | 2017-11-06 | 2019-05-09 | Purecircle Usa Inc. | High-purity steviol-glycosides |
| CN109750072A (en) * | 2019-01-31 | 2019-05-14 | 南京工业大学 | Method for preparing rebaudioside E by enzyme method |
| CN113462670A (en) * | 2021-08-23 | 2021-10-01 | 南京工业大学 | Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside M by using same |
| CN114164191A (en) * | 2021-11-17 | 2022-03-11 | 江南大学 | Method for efficiently biosynthesizing rebaudioside D by utilizing glycosyltransferase |
| CN114574460A (en) * | 2022-03-15 | 2022-06-03 | 江南大学 | Method for efficiently biosynthesizing rebaudioside M by utilizing glycosyltransferase UGT76G1 mutant |
| WO2022257983A1 (en) * | 2021-06-08 | 2022-12-15 | 弈柯莱生物科技(上海)股份有限公司 | β-1,2-GLYCOSYLTRANSFERASE, AND APPLICATION THEREOF |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112080480B (en) * | 2019-06-14 | 2023-01-03 | 中国科学院分子植物科学卓越创新中心 | Glycosyltransferase mutants and uses thereof |
| US11396646B2 (en) * | 2020-05-29 | 2022-07-26 | QTG Development, Inc. | Steviol glycosyltransferases and genes encoding the same |
-
2022
- 2022-12-20 CN CN202211644724.0A patent/CN115975972B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107105734A (en) * | 2014-09-19 | 2017-08-29 | 谱赛科有限责任公司 | High-purity steviol glycoside |
| CN113549664A (en) * | 2014-09-19 | 2021-10-26 | 谱赛科有限责任公司 | High purity steviol glycosides |
| WO2019090336A1 (en) * | 2017-11-06 | 2019-05-09 | Purecircle Usa Inc. | High-purity steviol-glycosides |
| CN109750072A (en) * | 2019-01-31 | 2019-05-14 | 南京工业大学 | Method for preparing rebaudioside E by enzyme method |
| WO2022257983A1 (en) * | 2021-06-08 | 2022-12-15 | 弈柯莱生物科技(上海)股份有限公司 | β-1,2-GLYCOSYLTRANSFERASE, AND APPLICATION THEREOF |
| CN113462670A (en) * | 2021-08-23 | 2021-10-01 | 南京工业大学 | Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside M by using same |
| CN114164191A (en) * | 2021-11-17 | 2022-03-11 | 江南大学 | Method for efficiently biosynthesizing rebaudioside D by utilizing glycosyltransferase |
| CN114574460A (en) * | 2022-03-15 | 2022-06-03 | 江南大学 | Method for efficiently biosynthesizing rebaudioside M by utilizing glycosyltransferase UGT76G1 mutant |
Non-Patent Citations (2)
| Title |
|---|
| Production of rebaudioside D from stevioside using a UGTSL2 Asn358Phe mutant in a multi-enzyme system;Liangliang Chen et al;《Microb Biotechnol》;第13卷(第4期);第973-984页 * |
| 植物源UDP-糖基转移酶及其分子改造;郭芳 等;《中国生物工程杂志》;第41卷(第9期);第78-91页 * |
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