Glycosaminoglycan synthase mutant as well as preparation method and application thereof

Abstract

The invention relates to a glycosaminoglycan synthase mutant and a preparation method and application thereof, and compared with glycosaminoglycan synthase shown in SEQ ID NO: 1, the glycosaminoglycan synthase mutant has amino acid mutation at the 108th site. Compared with wild type glycosaminoglycan synthetase, the mutant strain has the advantage that the yield of HA is obviously increased when the mutant strain is used for HA synthesis.

Description

Technical Field

[0001] The present application relates to the fields of protein engineering and genetic engineering, and specifically to glycosaminoglycan synthase mutants and preparation methods and applications thereof. Background Art

[0002] Glycosaminoglycans are a type of acidic mucopolysaccharides that play an important physiological role in the human body. Taking hyaluronic acid as an example, it (Hyaluronic acid, HA) is an acidic mucopolysaccharide composed of two monosaccharide units, D-glucuronic acid and N-acetylglucosamine, which are alternating. It has the characteristics of strong biocompatibility and significant moisturizing effect, and has been widely used in the pharmaceutical and cosmetic industries. The early production method of HA was mainly based on animal tissue extraction, but this method has the safety problems of limited tissue raw material sources, low yield, high purification cost and residual allergens. With the continuous development of synthetic biology, genetic engineering modification technology and fermentation technology, the synthesis of hyaluronic acid can be achieved by animal-free all-biological synthesis. HA prepared by microbial fermentation has become the main method for producing HA. The synthesis of HA using microbial cells is currently mainly concentrated in Streptococcus zooepidemicus, Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum and Lactococcus lactis, and the yield and molecular weight of HA produced by fermentation of different strains vary greatly. In order to further increase the production of HA, researchers have tried to regulate it at the cellular level, including strengthening key metabolic pathways, optimizing precursor concentrations, regulating the fermentation process, and adding additional degradation enzymes (Ma Yanqin et al., Biotechnology Bulletin, 2022, 38(2): 252-262.). Although there are many ways to regulate HA production, there are significant differences between different host cells, and a universal transformation strategy has not yet been formed.

[0003] Glycosaminoglycan synthase is responsible for polymerizing and transporting HA and is a key enzyme in the HA synthesis process. Researchers have modified the C-terminus of glycosaminoglycan synthase, which can significantly increase the molecular weight of the HA product, but the yield has decreased. In addition, glycosaminoglycan synthases from different sources also have different HA yields during the fermentation process. At present, the modification of glycosaminoglycan synthase at the enzyme molecular level to achieve the regulation of HA production still needs to be studied. Summary of the invention

[0004] Based on this, it is necessary to provide a glycosaminoglycan synthase mutant in one embodiment of the present application, which significantly improves the yield of synthesized HA.

[0005] The technical solutions include the following:

[0006] A glycosaminoglycan synthase mutant, wherein the glycosaminoglycan synthase mutant has an amino acid mutation occurring at position 108 compared with the glycosaminoglycan synthase shown in SEQ ID NO: 1.

[0007] In one embodiment, the amino acid at position 108 is mutated to glutamic acid or arginine.

[0008] In one embodiment, the glycosaminoglycan synthase mutant further has one or more amino acid mutations occurring at the following positions: position 113, position 147, position 199 and position 287 compared to the glycosaminoglycan synthase shown in SEQ ID NO: 1.

[0009] Optionally, the amino acids at positions 113, 147, 199 and 287 are each independently mutated to glutamic acid or arginine.

[0010] In one embodiment, the glycosaminoglycan synthase mutant comprises any one of the following amino acid mutations: K108R; K108E; K108R and M113E; K108E and M113E; K108R and S147R; K108E and S147R; K108R and I199E; K108E and I199E; K108R and N287E; K108E and N287E; K108E and M113R; K108E and S147E; K108E and I199R; K108E and N287R; K108R, M113R and S147R; K108R, I199R and N287E; K108R, S147R and I199R; K108R, N287R and M113E; K108R, M113E and S147R; K108E, I199E and N287R; K108E, S147E and I199E; K108R, N287E and M113E; K108R and M113R; K108R and S147E; K108R and I199R; K108R and N287R; K108E, M113R and S147E; K108E, S147E and I199R; K108E, I199R and N287R; K108E, N287R and I199E; K108R, M113R and S147E; K108R, S147E and I199R; K108R, I199R and N287R; K108R, N287R and I199E; K108E, M113R and I199R; K108E, M113R and N287R; K108E, M113R and N287E; K108R, M113R and I199R; K108R, M113R and N287R; K108R, M113R and N287E; K108E, M113R, I199R and S147E; K108E, S147E, N287R and I199R; K108E, I199R, M113R and N287R; K108R, M113R, I199R and S147E; K108R, S147E, N287R and I199R; K108R, I199R, M113R and N287R; K108R, M113R, I199R, S147E and N287R; K108E, M113R, I199R, S147E and N287R; K108E, S147E, N287R, I199R and M113E; K108E, I199R, M113R, N287R and S147R.

[0011] A nucleic acid molecule encoding the glycosaminoglycan synthase mutant or a fragment thereof.

[0012] An expression vector comprises the nucleic acid molecule.

[0013] In one embodiment, the expression vector comprises one or more of a plasmid vector, a shuttle vector, a phage and a viral vector.

[0014] In one embodiment, the plasmid vector includes one or more of pEC series vectors, pET series vectors, pNV series vectors, pXMJ19, pXZ10145, pZL071 and pXT01.

[0015] In one embodiment, the pEC series vectors include one or more of pEC-Ptac vector, pET-43a vector, pNV18.1 vector and pXMJ19 vector.

[0016] An engineered bacterium, wherein the engineered bacterium expresses the glycosaminoglycan synthase mutant; or carries the nucleic acid molecule or the expression vector.

[0017] Optionally, the host bacteria of the engineered bacteria are Escherichia coli, Rhodococcus, yeast, Nocardia, Bacillus subtilis, Lactococcus, Streptococcus or Corynebacterium glutamicum.

[0018] Optionally, the host bacteria is Corynebacterium glutamicum, yeast, lactococcus, Bacillus subtilis, Escherichia coli or Streptococcus.

[0019] Optionally, the E. coli includes E. coli BL21 (DE3) and / or E. coli K5.

[0020] Optionally, the Corynebacterium glutamicum includes Corynebacterium glutamicum ATCC13032.

[0021] Optionally, the Bacillus subtilis includes Bacillus subtilis 168.

[0022] Optionally, the Streptococcus comprises Streptococcus zooepidemicus.

[0023] Optionally, the lactococcus comprises Lactococcus lactis.

[0024] A method for preparing a glycosaminoglycan synthase mutant comprises:

[0025] Cultivating the engineered bacteria; and,

[0026] Induce the engineered bacteria to express the glycosaminoglycan synthase mutant.

[0027] The use of the glycosaminoglycan synthase mutant, the nucleic acid molecule, the expression vector or the engineering bacteria in the preparation of hyaluronic acid.

[0028] A method for preparing glycosaminoglycans, comprising:

[0029] The engineered bacteria are fermented and cultured to prepare glycosaminoglycan.

[0030] Optionally, the following steps are included:

[0031] Inoculating the engineered bacteria into a seed culture medium, performing seed culture, and preparing a bacterial solution;

[0032] The bacterial liquid is inoculated into a fermentation medium at an inoculation rate of 1-10% v / v to carry out fermentation culture to prepare glycosaminoglycan.

[0033] In one embodiment, the glycosaminoglycan is hyaluronic acid or its analogs.

[0034] Compared with the traditional technology, this application has the following beneficial effects:

[0035] The beneficial mutation sites designed for wild-type glycosaminoglycan synthase in the present application can significantly increase the yield of HA produced by glycosaminoglycan synthase. Compared with the wild-type glycosaminoglycan synthase, the yield of HA is significantly increased when the mutant strain is used for HA synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0037] Figure 1 This is the structural distribution of mutation sites of glycosaminoglycan synthase.

[0038] Figure 2 This is the process curve of HA production by fermentation of glycosaminoglycan synthase mutant strain.

[0039] Figure 3 These are the results of the determination of HA production by glycosaminoglycan synthase mutants.

[0040] Figure 4 The results of molecular weight determination of HA synthesized by glycosaminoglycan synthase mutants. DETAILED DESCRIPTION

[0041] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0043] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0044] Herein, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0045] In this article, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally include", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0046] The terms "contain", "include" and "include" used herein are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0047] This application uses the wild-type glycosaminoglycan synthase from Streptococcus pyogenes serotype M18 as the object, and obtains multiple beneficial mutants through directional design and modification at the enzyme molecular level. And further discovered the distribution of these beneficial mutation sites in the spatial structure. The applicant used molecular docking to simulate the binding state of the substrate and the enzyme molecule on the basis of the structure, and studied the binding process of the substrate and the enzyme molecule through molecular dynamics simulation, analyzed the interaction force between the substrate and the enzyme molecule, determined the amino acid sites that may affect the catalytic performance of the enzyme, and combined with multiple sequence alignment analysis to lock the candidate amino acid residues, and conducted directional design based on the structural position and evolutionary conservation analysis, constructed a glycosaminoglycan synthase mutant library, and screened out multiple mutants with increased HA production and / or molecular weight through determination.

[0048] One embodiment of the present application provides a glycosaminoglycan synthase mutant, which, compared with the glycosaminoglycan synthase shown in SEQ ID NO: 1, has one or more amino acid mutations occurring at the following sites: position 108, position 113, position 147, position 199 and position 287. Compared with the wild-type glycosaminoglycan synthase, when the mutant strain is used for HA synthesis, the yield of HA is increased by 1-3 times, and the improvement effect is significant.

[0049] In a specific example, the glycosaminoglycan synthase mutant has an amino acid mutation occurring at position 108 compared to the glycosaminoglycan synthase shown in SEQ ID NO: 1.

[0050] In one specific example, the amino acid at position 108 is mutated to glutamic acid or arginine.

[0051] In a specific example, the glycosaminoglycan synthase mutant further has an amino acid mutation at one or more of the following positions: position 113, position 147, position 199 and position 287 compared to the glycosaminoglycan synthase shown in SEQ ID NO: 1.

[0052] In a specific example, the amino acids at positions 113, 147, 199 and 287 are each independently mutated to glutamic acid or arginine.

[0053] In a specific example, the glycosaminoglycan synthase mutant has at least one mutation in the following group:

[0054] (1) K108E or K108R;

[0055] (2) M113R or M113E;

[0056] (3) S147E or S147R;

[0057] (4) I199R or I199E;

[0058] (5) N287R or N287E.

[0059] In one specific example, the glycosaminoglycan synthase mutant has at least two mutations from the following groups:

[0060] (1) K108E or K108R;

[0061] (2) M113R or M113E;

[0062] (3) S147E or S147R;

[0063] (4) I199R or I199E;

[0064] (5) N287R or N287E.

[0065] In a specific example, the glycosaminoglycan synthase mutant has at least three mutations from the following groups:

[0066] (1) K108E or K108R;

[0067] (2) M113R or M113E;

[0068] (3) S147E or S147R;

[0069] (4) I199R or I199E;

[0070] (5) N287R or N287E.

[0071] In a specific example, the glycosaminoglycan synthase mutant has at least four mutations from the following groups:

[0072] (1) K108E or K108R;

[0073] (2) M113R or M113E;

[0074] (3) S147E or S147R;

[0075] (4) I199R or I199E;

[0076] (5) N287R or N287E.

[0077] In a specific example, the glycosaminoglycan synthase mutant has at least five groups of mutations as follows:

[0078] (1) K108E or K108R;

[0079] (2) M113R or M113E;

[0080] (3) S147E or S147R;

[0081] (4) I199R or I199E;

[0082] (5) N287R or N287E.

[0083] In a specific example, the glycosaminoglycan synthase mutant has any of the following sets of mutations:

[0084] K108R; K108E; K108R and M113E; K108E and M113E; K108R and S147R; K108E and S147R; K108R and I199E; K108E and I199E; K108R and N287E; K108E and N287E; K108E, S147E and I199E; K108R, N287E and M113E; K108R and M113R; K108R and S147E; K108R and I199R; K108R and N287R; K108E, M113R and S147E; K108E, S147E and I199R; K108E, I199R and N287R; K108E, N287R and I199E; K108E and M113R; K108E and S147E; K108E and I199R; K108E and N287R; K108R, M113R and S147R; K108R, I199R and N287E; K108R, S147R and I199R; K108R, N287R and M113E; K108R, M113E and S147R; K108E, I199E and N287R. The mutant can make the microbial fermentation produce HA with an output of 1.0 to 1.2 times that of the wild enzyme.

[0085] In a specific example, the glycosaminoglycan synthase mutant has any of the following sets of mutations:

[0086] K108R, M113R and S147E; K108R, S147E and I199R; K108R, I199R and N287R; K108R, N287R and I199E; K108E, M113R and I199R; K108E, M113R and N287R; K108E, M113R and N287E; K108R, M113R and I199R; K108R, M113R and N287R; K108R, M113R and N287E; K108E, M113R, I199R and S147E; K10 8E, S147E, N287R and I199R; K108E, I199R, M113R and N287R; K108R, M113R, I199R and S147E; K108R, S147E, N287R and I199R; K108R, I199R, M113R and N287R; K108R, M113R, I199R, S147E and N287R; K108E, M113R, I199R, S147E and N287R; K108E, S147E, N287R, I199R and M113E. The molecular weight of the mutant can reach 500,000-300,000 Da, and the yield of HA produced by microbial fermentation is 1.3-2.8 times that of the wild enzyme.

[0087] One embodiment of the present application also provides a nucleic acid molecule encoding the glycosaminoglycan synthase mutant or a fragment thereof. The nucleic acid sequence of the nucleic acid molecule is obtained by base mutation based on the sequence of the wild-type glycosaminoglycan synthase.

[0088] One embodiment of the present application also provides an expression vector, comprising the nucleic acid molecule. The expression vector selected in the present application can stably exist and autonomously replicate in various hosts of prokaryotic or eukaryotic cells, and optionally, the expression vector includes one or more of a plasmid vector, a shuttle vector, a phage and a viral vector.

[0089] In a specific example, the plasmid vector includes one or more of pEC series vectors, pET series vectors, pNV series vectors, pXMJ19, pXZ10145, pZL071 and pXT01.

[0090] In a specific example, the pEC series vectors include one or more of a pEC-Ptac vector, a pET-43a vector, a pNV18.1 vector, and a pXMJ19 vector.

[0091] Through molecular biological operations such as enzyme cutting and ligation, the nucleotide sequence of the wild-type glycosaminoglycan synthase was inserted into pEC-Ptac to construct a recombinant expression plasmid, which was named pEC-Ptac-HAS; the coding genes of the glycosaminoglycan synthase mutants described in the present application were constructed into the recombinant expression plasmids, which were named HA-1, 2, 3, and so on.

[0092] One embodiment of the present application also provides an engineered bacterium, which expresses the glycosaminoglycan synthase mutant; or carries the nucleic acid molecule or the expression vector.

[0093] This application takes the wild-type glycosaminoglycan synthase from Streptococcus pyogenes serotype M18 as the object, analyzes its synthesis and transport mechanism from the perspective of structure, and obtains a new generation of production strains with significantly improved HA production through design and construction at the enzyme molecular level.

[0094] In a specific example, the host bacteria of the engineered bacteria are selected from Escherichia coli, Rhodococcus, Saccharomyces, Nocardia, Bacillus subtilis, Lactococcus, Streptococcus or Corynebacterium glutamicum.

[0095] In a specific example, the host bacteria is Corynebacterium glutamicum, Saccharomyces cerevisiae, Lactococcus, Bacillus subtilis, Escherichia coli or Streptococcus.

[0096] In a specific example, the E. coli includes E. coli BL21 (DE3) and / or E. coli K5.

[0097] In a specific example, the Corynebacterium glutamicum includes Corynebacterium glutamicum ATCC13032.

[0098] In one specific example, the Bacillus subtilis includes Bacillus subtilis 168.

[0099] In one specific example, the Streptococcus includes Streptococcus Zooepidemicus.

[0100] In one specific example, the lactococcus includes Lactococcus lactis.

[0101] In a specific example, the nucleic acid molecule described in the present application is directly inserted into the chromosome of the host bacteria, or the expression vector is introduced into the host bacteria using the calcium chloride method or electroporation transformation method.

[0102] One embodiment of the present application also provides a method for preparing a glycosaminoglycan synthase mutant, comprising culturing an engineered bacterium;

[0103] Induce engineered bacteria to express glycosaminoglycan synthase mutants.

[0104] One embodiment of the present application also provides the use of the glycosaminoglycan synthase mutant, the nucleic acid molecule, the expression vector or the engineered bacteria in the preparation of hyaluronic acid. The glycosaminoglycan synthase mutant can be used to synthesize HA, and the yield is increased by 1-3 times compared with the wild-type enzyme.

[0105] One embodiment of the present application also provides a method for preparing glycosaminoglycan, comprising fermenting and culturing the engineered bacteria to prepare glycosaminoglycan.

[0106] In a specific example, the molecular weight of hyaluronic acid is 10 kDa to 3000 kDa.

[0107] In a specific example, the method includes: inoculating the engineered bacteria into a seed culture medium, performing seed culture, and preparing a bacterial solution;

[0108] The bacterial liquid is inoculated into a fermentation medium at an inoculation rate of 1-10% v / v to carry out fermentation culture to prepare glycosaminoglycan.

[0109] In one specific example, the glycosaminoglycan is hyaluronic acid or an analog thereof (eg, chondroitin, heparin).

[0110] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0111] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0112] Example 1 Design of glycosaminoglycan synthase mutants

[0113] Based on the analysis of the protein structure of wild-type Streptococcus pyogenes serotype M18 glycosaminoglycan synthase, molecular dynamics simulation was used to explore the substrate binding process between substrate molecules and enzyme molecules, and a strategy for mutation of the flexible structure on the surface of the protein was proposed. Subsequently, through structural analysis, the amino acid sites on the surface of the glycosaminoglycan synthase were identified, including the 108th lysine site, the 113th methionine site, the 147th serine site, the 199th isoleucine site, and the 287th asparagine site ( Figure 1 ). The selected amino acids were mutated to arginine (Arg, R) or glutamic acid (Glu, E) by structural analysis and multiple sequence alignment. On this basis, these sites were combined with mutations to design combined mutants as shown in Table 1.

[0114] Table 1 List of mutants

[0115]

[0116] Glycosaminoglycan synthase from wild-type Streptococcus pyogenes serotype M18, with the amino acid sequence:

[0117] MEKLKNLITFTTFIILWVLMIVLNTYVFGSKGSLTPYGILLLIYLTLKMSLSFFYRPYKGKVGNYKVAAIIPSYNEDGESLLETLKSVQSQTYPIAEIFVVDDGSA DKTGIKMIEDYIANNPDATNVIAHRLPENVGKRHAQAWAFSRSDADVFLTVDSDSYIYPDALEELLKSFNDKDVYAATGHLNARNRDVNLLTILTDIRYDNAFGVE RAAQSVTGNILVCSGPLSVYRREVVVPNIERYTSQTFLGIPVSIGDDRCLTNYATDLGKTVYQSTARCDTDVPFNFKTYLKQQNRWNKSFFRESIISVKKMMQTPL VAVWTVLEVSMFIMLLYSVLDLFVGEAQEFNGLKILAFLTLIFLVALCRNIHYMVKHPLAFLLSPIYGILHLFVLQPLRLYSLFTIRNADWGTRKAEEEEPL (SEQ ID NO: 1).

[0118] Example 2 Construction of a Glycosaminoglycan Synthase Mutant Library

[0119] The glycosaminoglycan synthase mutant in Example 1 was selected to construct a recombinant expression vector.

[0120] First, a recombinant plasmid pEC-XK99E-Ptac-SY containing the wild enzyme was constructed, and then the pEC-XK99E-Ptac-SY recombinant plasmid was used as a template to design forward primers and reverse primers containing the mutation site, and a high-fidelity enzyme was used to amplify the entire plasmid.

[0121] The 10 μL PCR system includes:

[0122] 1 μL pEC-XK99E-Ptac-SY plasmid template (about 50 ng / μL);

[0123] 5 μL high-fidelity DNA polymerase;

[0124] 1.0 μL Primer 1 (10 μM);

[0125] 1.0 μL Primer 2 (10 μM);

[0126] 2 μL ddHO 2 O.

[0127] The construction of combined mutants is obtained by performing multiple rounds of mutations on the basis of single-point mutants, and specific primers are also used to introduce mutation sites.

[0128] The conditions for the PCR reaction were as follows:

[0129] (1) Pre-denaturation at 98 °C for 1 min;

[0130] (2) Denaturation at 98°C for 30 seconds;

[0131] (3) Anneal at (primer Tm - 5) °C for 10 s;

[0132] (4) Extension at 72 °C for 7 min;

[0133] The above steps (2)-(4) were repeated for 30 cycles, with a final extension at 72°C for 10 min.

[0134] The recombinant plasmid obtained by PCR amplification was digested with Dpn I enzyme, transformed into competent cells, and spread on a solid plate containing antibiotics (50 μg / mL) and placed in an incubator at 37°C for about 12 hours. Single clones were selected for sequencing verification, and the recombinant plasmid pEC-XK99E-Ptac-SY1 of the mutant was extracted and stored in a -20°C refrigerator.

[0135] Table 2 Primer sequences used to construct mutant library

[0136]

[0137] The combined mutants were obtained by multiple rounds of PCR reactions based on the single point mutants according to the above construction method. Taking the combined mutant K108R / S147E / N287R / I199R as an example, the K108R single point mutant was first constructed by whole plasmid amplification using primers K108R-U and K108R-D with the plasmid sequence of the wild-type enzyme as a template; then, according to the same method, the K108R mutant plasmid sequence was used as a template, and primers S147E-U and S147E-D were used to construct the K108R / S147E single point mutant by whole plasmid amplification. Finally, multiple point mutants can be obtained through multiple rounds of mutations.

[0138] Example 3 Construction of genetically engineered bacteria of glycosaminoglycan synthase mutants

[0139] The recombinant expression vector pEC-XK99E-Ptac-SY1 prepared in Example 2 was transformed into competent cells (Corynebacterium glutamicum) by heat shock method, coated on a solid plate containing antibiotics (50 μg / mL), cultured at 37°C overnight, and then a single colony was picked and transferred to LB liquid culture containing 50 μg / mL kanamycin, cultured at 37°C for 12 h, and sent for sequencing. The correct clones were stored in a -70°C refrigerator to obtain genetically engineered bacteria for glycosaminoglycan synthase mutants.

[0140] Example 4 Production of HA by shake flask fermentation of genetically engineered bacteria expressing mutant glycosaminoglycan synthase

[0141] The genetically engineered bacterial lawn of glycosaminoglycan synthase mutants was picked from the LB plate medium and transferred into LBG medium containing the corresponding antibiotics. o Cultivate overnight at 200 rpm. Add the seed solution to a 300 mL conical flask containing 50 mL of fermentation medium at a 5% inoculation rate and add the corresponding antibiotics. o After 3 h of culture, IPTG was added at a final concentration of 1 mM to induce expression. The culture was carried out at 30 °C and a shaking speed of 200 rpm for 48 h. During this period, the pH of the fermentation liquid was controlled between 6.0 and 8.0. Samples were taken at the specified time to measure the OD 600 and HA concentration, and the molecular weight of HA.

[0142] Figure 2 This is the process curve of HA production by fermentation of glycosaminoglycan synthase mutant strain.

[0143] 1. Isolation, extraction and concentration determination of HA:

[0144] Take an appropriate amount of fermentation stock solution, add 3 times the volume of ethanol, mix well and let stand for 2 hours. Centrifuge and discard the supernatant, retain the precipitate, and dry it at room temperature for 1 hour. Add an equal volume of deionized water and accelerate the dissolution with a vortex oscillator. Centrifuge and discard the bacteria and insoluble impurities, and the supernatant obtained is the HA aqueous solution.

[0145] Then the HA concentration was determined by CTAB (trimethylhexadecyl ammonium bromide) turbidimetric method. The reaction system included the sample to be tested, acetic acid buffer and CTAB solution. After 5 min of reaction, the OD 400 Absorbance value. According to the formula: HA (g / L) = OD 400 +0.009 to calculate HA concentration.

[0146] A total of 50 glycosaminoglycan synthase mutants were obtained through site-directed mutagenesis and combined mutagenesis. The glycosaminoglycan synthase mutant genetically engineered bacteria were used for HA fermentation production by fermentation induction culture, and the concentration of HA was measured. Compared with the wild-type glycosaminoglycan synthase, the relative yield of HA produced by the mutant fermentation is shown in Table 3 and Figure 3 shown.

[0147] Table 3 Relative yields of HA produced by mutant fermentation

[0148]

[0149] Note: The mutant "K108R / M113E" in the table means that the mutant has two mutation modes, K108R and M113E. The same applies to other mutants. Standard substitution notation is used, i.e. K108R means that K at position 15 from the starting N-terminus is replaced by R.

[0150] In Table 3, each mutant shown represents a mutant obtained after a specific mutation occurred in the corresponding amino acid site in the amino acid sequence shown in SEQ ID NO: 1. Through the determination of HA production, it was found that the production of HA produced by the constructed mutants was improved compared with the wild-type strain, and the molecular weight improvement effect was more obvious after the combined mutation.

[0151] 2. HA molecular weight determination

[0152] The molecular weight of hyaluronic acid was determined by SEC-MALS. HA was separated by molecular size exclusion chromatography, and then the molecular weight of the HA sample was determined by coupling the optical analysis technology method multi-angle laser scattering. The chromatographic column selected was the TSK-GEL GMPWXL column, the mobile phase was 23.38 g / L NaCl solution, and the results were analyzed using the Wyatt MALS system ( Figure 4Single point mutants and some combination mutants can maintain the same molecular weight as the wild type while increasing the yield, ranging from 0.2-1 million Da. Among them, the combined mutants K108R / M113R / S147E, K108R / S147E / I199R, K108R / I199R / N287R, K108R / N287R / I199E, K108E / M113R / I199R, K108E / M113R / N287R, K108E / M113R / N287E, K108R / M113R / I199R, K108R / M113R / N287R, K108R / M113R / N287E, K108R / M113R / I199R, K108R / M113R / N287R, K108R / M113R / N287E The HA molecular weight of / I199R / M113R / N287R, K108R / M113R / I199R / S147E, K108R / S147E / N287R / I199R, K108R / I199R / M113R / N287R, K108R / M113R / I199R / S147E / N287R, K108E / M113R / I199R / S147E / N287R, K108E / S147E / N287R / I199R / M113E and K108E / I199R / M113R / N287R / S147R can reach 500,000-3 million Da, with a significant increase in molecular weight.

[0153] In summary, the results show that the designed amino acid site mutations in Table 1 can significantly increase the yield of HA. In addition, the combined mutation of some sites not only has a more significant effect on increasing HA yield, but also can significantly increase HA molecular weight.

[0154] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0155] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A glycosaminoglycan synthase mutant, characterized in that: Compared with the glycosaminoglycan synthase shown in SEQ ID NO: 1, the glycosaminoglycan synthase mutant has an amino acid mutation occurring at position 108.

2. The glycosaminoglycan synthase mutant according to claim 1, characterized in that The amino acid at position 108 is mutated to glutamic acid or arginine.

3. The glycosaminoglycan synthase mutant according to claim 1 or 2, characterized in that Compared with the glycosaminoglycan synthase shown in SEQ ID NO: 1, the glycosaminoglycan synthase mutant further has one or more amino acid mutations occurring at the following positions: position 113, position 147, position 199 and position 287; Optionally, the amino acids at positions 113, 147, 199 and 287 are each independently mutated to glutamic acid or arginine.

4. The glycosaminoglycan synthase mutant according to claim 1 or 2, characterized in that The glycosaminoglycan synthase mutant has any of the following amino acid mutations: K108R; K108E; K108R and M113E; K108E and M113E; K108R and S147R; K108E and S147R; K108R and I199E; K108E and I199E; K108R and N287E; K108E and N287E; K108E and M113R; K108E and S147E; K108E and I199R; K108E and N287R; K108R, M113R and S147R; K108R, I199R and N287E; K108 R, S147R and I199R; K108R, N287R and M113E; K108R, M113E and S147R; K108E, I199E and N287R; K108E, S147E and I199E; K108R, N287E and M113E; K108R and M113R; K108R and S147E; K108R and I199R; K108R and N287R; K108E, M113R and S147E; K108E, S147E and I199R; K108E, I199R and N287R; K108E, N287R and I199E ; K108R, M113R and S147E; K108R, S147E and I199R; K108R, I199R and N287R; K108R, N287R and I199E; K108E, M113R and I199R; K108E, M113R and N287R; K108E, M113R and N287E; K108R, M113R and I199R; K108R, M113R and N287R; K108R, M113R and N287E; K108E, M113R, I199R and S147E; K108E, S147E, N287R and I199R; K108E, I199R, M113R, and N287R; K108R, M113R, I199R, and S147E; K108R, S147E, N287R, and I199R; K108R, I199R, M113R, and N287R; K108R, M113R, I199R, S147E, and N287R; K108E, M113R, I199R, S147E, and N287R; K108E, S147E, N287R, I199R, and M113E; K108E, I199R, M113R, N287R, and S147R.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the glycosaminoglycan synthase mutant or a fragment thereof according to any one of claims 1 to 4.

6. An expression vector, characterized in that comprising the nucleic acid molecule of claim 5; The expression vector includes one or more of a plasmid vector, a shuttle vector, a phage and a virus vector; Optionally, the plasmid vector includes one or more of pEC series vectors, pET series vectors, pNV series vectors, pXMJ19, pXZ10145, pZL071 and pXT01; Optionally, the pEC series vectors include one or more of a pEC-Ptac vector, a pET-43a vector, a pNV18.1 vector and a pXMJ19 vector.

7. An engineered bacterium, characterized in that: The engineered bacteria expresses the glycosaminoglycan synthase mutant according to any one of claims 1 to 4; or carries the nucleic acid molecule according to claim 5 or the expression vector according to claim 6; Optionally, the host bacteria of the engineered bacteria are Escherichia coli, Rhodococcus, yeast, Nocardia, Bacillus subtilis, Lactococcus, Streptococcus or Corynebacterium glutamicum; Optionally, the host bacteria is Corynebacterium glutamicum, yeast, lactococcus, Bacillus subtilis, Escherichia coli or Streptococcus; Optionally, the E. coli includes E. coli BL21 (DE3) and / or E. coli K5; Optionally, the Corynebacterium glutamicum includes Corynebacterium glutamicum ATCC13032; Optionally, the Bacillus subtilis includes Bacillus subtilis 168; Optionally, the streptococcus comprises Streptococcus zooepidemicus; Optionally, the lactococcus comprises Lactococcus lactis.

8. A method for preparing a glycosaminoglycan synthase mutant, characterized in that: include: Cultivating the engineered bacteria according to claim 7; and, Induce the engineered bacteria to express the glycosaminoglycan synthase mutant.

9. Use of the glycosaminoglycan synthase mutant according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 or 6, or the engineered bacteria according to claim 7 or 8 in the preparation of hyaluronic acid.

10. A method for preparing glycosaminoglycan, characterized in that: include: Fermenting and culturing the engineered bacteria according to claim 7 to prepare glycosaminoglycans; Optionally, the following steps are included: Inoculating the engineered bacteria into a seed culture medium, performing seed culture, and preparing a bacterial solution; Inoculating the bacterial solution into a fermentation medium at an inoculation rate of 1-10% v / v to perform fermentation culture to prepare glycosaminoglycans; Optionally, the glycosaminoglycan is hyaluronic acid or an analog thereof.

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