CN115975972A

CN115975972A - Glycosyltransferase mutant and coding gene thereof

Info

Publication number: CN115975972A
Application number: CN202211644724.0A
Authority: CN
Inventors: 郭保党; 吴炜坤; 武强强; 倪叶雯; 刘德川
Original assignee: Hangzhou Liwen Institute Biotechnology Co ltd
Current assignee: Hangzhou Liwen Institute Biotechnology Co ltd
Priority date: 2022-12-20
Filing date: 2022-12-20
Publication date: 2023-04-18
Anticipated expiration: 2042-12-20
Also published as: CN115975972B

Abstract

The invention discloses a glycosyltransferase mutant and a coding gene thereof, reb D has high sweetness, no bitter taste and licorice aftertaste, and is a very high-quality sugar substitute selection, and the high production cost of Reb D 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 microorganisms in a high amount which is about 3 times of that of wild type glycosyltransferases. Meanwhile, the heat stability of the mutant obtained by expression is obviously superior to that of the wild type. Therefore, the enzyme catalysis reaction system for generating Red D by the catalysis of Red A, which is participated by the mutants of three glycosyltransferases disclosed by the invention, can be carried out at a higher temperature, so that the catalysis efficiency of the reaction system can be obviously improved, and the production efficiency of the Red D is improved.

Description

Glycosyltransferase mutant and coding gene thereof

Technical Field

The invention relates to the field of bioengineering, in particular to a glycosyltransferase mutant and a coding gene thereof.

Background

Stevia sugar, also called stevioside, has the characteristics of high sweetness (300-450 times of cane sugar), low calorific value (1/300 of cane sugar) and the like, and natural cane sugar substitute is known as the third world's original sugar. Stevia sugar is extracted from stevia rebaudiana Bertoni, and most of the stevia sugar extracted from stevia rebaudiana Bertoni by manufacturers is Stevioside (STV) and Rebaudioside A (Rebaudianide A, reb A). However, STV and Reb a have a bitter taste and licorice aftertaste, which greatly limits the use of steviol glycosides as sweeteners in food products.

Researches find that Rebaudioside D (Rebaudioside D, reb D) maintains the advantages of high sweetness and low calorific value of stevioside, does not have bitter taste and licorice aftertaste, and is a very good sugar substitute. However, there is a large bottleneck for the scale production of Reb D: reb D accounts for less than 1% of stevia rebaudiana, and the production of Reb D by plant extraction is expensive. Glycosyltransferases are essentially of plant origin and are expressed in smaller amounts in microorganisms, which leads to the current high cost of Reb D production by microbial fermentation or enzymatic catalysis. 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 disadvantages of the prior art, it is an object of the present invention to provide an amino acid sequence of a plant-derived glycosyltransferase that can be expressed in a microorganism in a high amount.

In order to realize the purpose, the invention provides the following technical scheme: a glycosyltransferase, the wild type sequence of which is shown in SEQ ID NO. 1. The wild-type glycosyltransferase is derived from Solanum lycopersicum, NCBI Reference Sequence: XP _004250485.1. Carrying out the following site mutation on the wild glycosyltransferase to obtain a variant 1 with an amino acid sequence shown as 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。

carrying out the following site mutation on the wild type glycosyltransferase 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。

carrying out the following site mutation on the wild type glycosyltransferase 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 glycosyltransferases after mutation in escherichia coli is about 3 times of that of a wild type, the highest UGTSL2-2 is more 3.24 times, the expression quantity of the glycosyltransferase from a plant source in the escherichia coli is greatly improved, the production cost of Reb D is reduced, and the popularization of industrial production of the Reb D is facilitated.

In view of the disadvantages of the prior art, it is a second object of the present invention to provide a recombinant gene capable of expressing the glycosyltransferase in a microorganism.

In view of the disadvantages of the prior art, it is another object of the present invention to provide a recombinant plasmid comprising the above recombinant gene.

Compared with the prior art, the invention has the advantages that: the three plant-derived mutated glycosyltransferases designed by the invention can be expressed in microorganisms in high quantity. Specifically, the expression level of wild glycosyltransferase in colibacillus is 3.4g/L, and the expression levels of the three mutant after mutation in colibacillus reach 10.2g/L,11.0g/L and 8.5g/L respectively. The expression quantity of the three glycosyltransferases after mutation in escherichia coli is about 3 times of that of the wild type glycosyltransferase, the highest mutant reaches 3.24 times, the expression quantity of the glycosyltransferase from plant sources in the escherichia coli is greatly improved, and the production cost of Reb D is reduced. Meanwhile, wild glycosyltransferase is incubated for 30min at 40 ℃, the enzyme activity is reduced to about 55 percent, three mutants obtained after mutation are incubated for 30min at 40 ℃, the enzyme activity is reduced to about 79 percent and about 92 percent respectively, and the enzyme activity is not reduced. Therefore, through mutation modification, 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 catalysis reaction system which participates in the enzyme catalysis reaction of Red A to generate Red D can react at a higher temperature, so that the catalysis efficiency of the reaction system can be obviously improved, and the production efficiency of the Red D is improved.

Drawings

FIG. 1 is a protein gel diagram 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 the residual enzyme activity of UGTSL2 incubated at 30 deg.C, 40 deg.C, 50 deg.C for various periods of time;

FIG. 4 is a graph of residual enzyme activity of UGTSL2, UGTSL2-1, UGTSL2-2, and UGTSL2-3 incubated at 40 deg.C for 30min, respectively.

Detailed Description

A DNA construct is a sequence capable of expressing the glycosyltransferase and glycosyltransferase variants of the present 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 the 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 a heterologous polynucleotide fragment in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. It is within the knowledge of the skilled person to select a suitable expression vector.

The term "host cell" refers to a suitable host vector for expression comprising the DNA of the invention. The host may comprise any organism capable of containing and expressing the nucleic acids or genes disclosed herein, but is not limited thereto. The host may be a prokaryote or eukaryote, a single cell or multicellular, including mammalian cells, plant cells, fungi, and the like. Examples of unicellular 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, MM294, NM522, NM554, TGI, χ 1776, 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 waterman, advanced applied math, 2 482, 1981, needleman and Wunsch, j.mol. Biol., 48, 443, 1970; pearson and lippman, proceedings of the american academy of sciences 85. The latest version, such as 2.2.16, 2.2.17, 2.2.18, 2.2.19 or the latest version, includes blastp for protein-protein comparisons, blastn for nucleotide-nucleotide comparisons, tblastn for protein-nucleotide comparisons, or blastx for nucleotide-protein comparisons, with the parameters as follows: the maximum sequence number returned is 10,000 or 100,000, the value of E (expected value) is 1e ⁻² Or 1e ⁻⁵ The number of words is 3, the scoring matrix BLOSUM62, the gap cost is 11, and the gap cost is extended by 1, which may be appropriate. For example, the value of E is 1E ⁻⁵ Indicating that the probability of a random homology match is about 1/10,000, indicating a high confidence of true homology.

The term "identity" means that the residues in 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 be 50% identical if, when properly aligned, their corresponding fragments have identical residues at 5 of 10 positions. Most bioinformatic programs report the percent identity of regions of aligned sequences, which are usually not the entire molecule. If the alignment is long enough and contains enough identical residues, then an expectation can be calculated, indicating that the same order in the alignment is unlikely to occur randomly.

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Example 1

The protein sequence contains abundant evolution information and has a crucial effect on improving the thermal stability of the protein. Many 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 homologous sequences based on homologous sequence alignment files. However, noise from the phylogenetic tree is present in the homologous sequences, which severely affects the accuracy of the model. Therefore, the inventor proposes a post-processing method for mean product correction, but the method is only effective for predicting protein contact maps and cannot eliminate the influence of noise on a coevolution protein design algorithm. The inventors propose a spectral norm penalty term based on the classical protein coevolution analysis method Gremlin. In the optimization process of the Gremlin, the spectral norm penalty term can inhibit the maximum characteristic value of the coevolution matrix, and further, the system generated noise is reduced. Based on the evolution matrix obtained by calculation, the inventor designs a greedy algorithm of generation-by-generation optimal single-point mutation, continuously enhances the evolution signal in wild-type glycosyltransferase (the amino acid sequence is shown as SEQ ID NO. 1), and finally obtains the optimal enzyme variants (the amino acid sequences are shown as SEQ ID NO.2, SEQ ID NO.3 and SEQID NO. 4) on the calculation level.

Specifically, the wild-type glycosyltransferase (UGTSL 2, the amino acid sequence of which is shown in SEQ ID NO. 1) is used as a parent, and the following point mutations are carried out: 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 285V-K301E-R312I-T329I-G331A 341L-I356F-P361F-G387E-T392V-L393I-S395K-K414E-I419K-A426E to give glycosyltransferase variant-1 (UGTSL 2-1, with the amino acid sequence shown in SEQ ID NO. 2).

Or wild glycosyltransferase (the amino acid sequence is shown as SEQ ID NO. 1) is used as a parent to carry out 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-285V-R312I-T329I-G331A-341L-I356F-P361F-G387E-T392V-L393I-S395K-I419K-A426E to obtain glycosyltransferase variant-2 (UGL 2-2, amino acid sequence shown in SEQ ID NO. 3).

Or wild glycosyltransferase (the amino acid sequence is shown as SEQ ID NO. 1) is used as a parent to carry out the following point mutation: L5I-I48K-N87L-P99S-S149H-T202E-C211L-G244D-V270I-S277T-A285V-I356F-P361F-G387E-L393I-I419K to obtain 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 target genes (UGTSL 2, UGTSL2-1, UGTSL2-2 and UGTSL 2-3) are synthesized by Beijing Ongchou Biotechnology Co., ltd, and the nucleotide sequences are inserted into an expression vector, specifically, inserted into NdeI and XhoI enzyme cutting sites of pET21b (+), so as to obtain corresponding plasmids. Subsequently, the synthesized plasmids pET21b-UGTSL2, pET21b-UGTSL2-1, pET21b-UGTSL2-2 and pET21b-UGTSL2-3 are transferred into host cells (E. coliBL21 (DE 3)), and thus E.coli strains containing different plasmids were constructed.

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), expressing strain shake, OD g/L sodium chloride ₆₀₀ When the concentration reaches 0.6-0.8, IPTG (final concentration of 0.1 mM) is added at 16 deg.C, and induction is carried out at 180rpmThe culture was conducted overnight. 8000 g, centrifuging for 10 minutes to collect cells, and weighing and quantifying the thalli. The cells were resuspended in lysis buffer 20mM Tris,300mM NaCl (pH = 8) at 1/10 of the medium. Sonication (1 min/mL lysate) followed by centrifugation at 15000 g for 30min at 4 ℃ separates the supernatant and the pellet. The hetero-protein was eluted with NTA-0 (30 mL), NTA-20 (30 mL), the eluate was checked with Coomassie brilliant blue until no blue color was observed, and 20. Mu.L of the supernatant of the target protein was collected with NTA-40 (5 mL), NTA-60 (10 mL), and NTA-150 (20 mL), followed by 4. Mu.L of the electrophoretic loading buffer, water bath at 100 ℃ for 5 min, 1. Mu.L of the loading sample, and electrophoresis with 130V for 70 min, followed by SDS-PAGE analysis, and the results of the determination are shown in FIG. 1.

Protein expression amount determination: overnight induced UGTSL2, UGTSL2-1, UGTSL2-2 and UGTSL2-3 were harvested and weighed, resuspended in lysis buffer 20mM Tris,300mM NaCl (pH = 8.0) at 1g of phage/10 mL, sonicated (lysate 1 min/mL) for each mutant, centrifuged at 4 ℃ for 30min at 15000 ℃ for g, 20. Mu.L of supernatant sample was applied to 5. Mu.L of loading buffer (electrophoretic loading buffer), washed in 100 ℃ for 5 min in water, 1. Mu.L of supernatant was applied, electrophoresed at 130V for about 70 min (time was appropriately extended), and then subjected to Mass blue staining, and the expression level of each mutant target protein in the protein gel (gel graph is shown in FIG. 1) was analyzed by software Bandscan 5.0, the results are shown in Table 1.

TABLE 1

Protein	UGTSL2	UGTSL2-1	UGTSL2-2	UGTSL2-3
					Expression level (g/L)	3.4	10.2	11.0	8.5

As can be seen from Table 1, the expression quantity of the three glycosyltransferases after mutation in Escherichia coli is about 3 times of that of a wild type glycosyltransferase, and the highest UGTSL2-2 reaches 3.24 times, so that the expression quantity of the plant-derived glycosyltransferase in Escherichia coli is greatly improved, the production cost of Reb D is reduced, and the popularization of the industrial production of Reb D is facilitated.

Thermal stability assay of UGTSL2, UGTSL2-1, UGTSL2-2, and UGTSL 2-3:

reaction System 3 mM MgCl ₂ 0.5 mMReda,0.1g/L glycosyltransferase, 2 mMUDP-glucose, 100 mM sulfate buffer (pH 8);

standard curve: reb D was formulated into different concentration solutions: 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6mM, 1mM, HPLC detection statistical area, standard curve is drawn.

HPLC conditions: LC-2030C HT system (SHIMADZU, japan), water (0.1% formic acid)/acetonitrile (0.1% formic acid) gradient elution (0 min:25% acetonitrile, 75% water; 12 min.

Rebaudioside A and UDP-glucose can be catalyzed by glycosyltransferase to obtain rebaudioside D and UDP, and the specific reaction equation is shown in figure 2.

UGTSL2 is incubated at 30 ℃,40 ℃ and 50 ℃ for different times (0 h,0.5 h, 1h, 2h, 4h, 6h and 24 h), reaction liquid is prepared according to the reaction system, the reaction liquid is placed in a 30 ℃ water bath kettle for reaction for 1h, the reaction system is placed in a 95 ℃ water bath for 10 min to terminate the reaction, the reaction is filtered by a 0.22 mu M filter membrane, HPLC detection and analysis are carried out, and 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%, and at 40 ℃ for 30 minutes, the enzyme activity was reduced to about 55%, and the enzyme was rapidly inactivated at 50 ℃. As described above, wild-type glycosyltransferases have poor thermostability.

UGTSL2, UGTSL2-1, UGTSL2-2 and UGTSL2-3 are respectively incubated at 40 ℃ for 30min, reaction liquid is prepared according to the reaction system, the reaction liquid is placed in a water bath kettle at 30 ℃ for reaction for 1h, the reaction system is placed in a water bath at 95 ℃ for 10 min to terminate the reaction, the reaction is filtered by a filter membrane at 0.22 mu M, and HPLC detection analysis is carried out, wherein the detection result is shown in figure 4.

As can be seen from FIG. 4, after mutation, the glycosyltransferase variant-1 (UGTSL 2-1) obtained is incubated at 40 ℃ for 30min, the enzyme activity is reduced to about 79%, the enzyme activity of the glycosyltransferase variant-2 (UGTSL 2-2) is reduced to about 92%, and the enzyme activity of the glycosyltransferase variant-3 (UGTSL 2-3) is not reduced (experimental data shows that the data is about 102%, and the deviation of the data is within the experimental error range). In conclusion, through mutation modification, the thermal stability of the three glycosyltransferase variants provided by the invention is obviously improved, and the enzyme catalysis reaction system which participates in the Red A catalysis to generate the Red D can be carried out at a higher temperature, so that the catalysis efficiency of the reaction system can be obviously improved, and the production efficiency of the 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 embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to those skilled in the art without departing from the principles of the present invention may be made without departing from the scope of the present invention.

Claims

1. A glycosyltransferase mutant, wherein the wild-type glycosyltransferase of SEQ ID No.1 is mutated as follows: 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 285V-K301E-R312I-T329I-G331A 341L-I356F-P361F-G387E-T392V-L393I-S395K-K414E-I419K-A426E;

or L5I-L34H-I48K-I62V-N87M-P88N-H91K-P99S-N127S-E128S-Q129H-C140G-S149H-V168Y-T202E-C211L-T212R-G244D-D266E-V270I-F272H-S277A-285V-R312I-T329I-G331A 341L-I356F-P361F-G387E-T392V-L393I-S395K-I419K-A426E;

or L5I-I48K-N87L-P99S-S149H-T202E-C211L-G244D-V270I-S277T-A285V-I356F-P361F-G387E-L393I-I419K to obtain variant 1 with the amino acid sequence shown in SEQ ID NO.2 or variant 2 with the amino acid sequence shown in SEQ ID NO.3 or variant 3 with the amino acid sequence shown in SEQ ID NO. 3.

2. A recombinant gene capable of expressing the DNA or RNA of the glycosyltransferase mutant of claim 1.

3. A recombinant plasmid comprising the recombinant gene according to claim 2.