Glycosaminoglycan synthetase mutants, methods of making and using the same

Abstract

The application relates to a glycosaminoglycan synthetase mutant and a preparation method and application thereof. The glycosaminoglycan synthetase mutant has an amino acid mutation occurring at the 108th site compared with the glycosaminoglycan synthetase shown in SEQ ID NO: 1. When the mutant strain is used for HA synthesis, the yield of HA is significantly improved compared with the wild-type glycosaminoglycan synthetase.

Description

Technical Field

[0001] This application relates to the fields of protein engineering and genetic engineering, specifically to glycosaminoglycan synthase mutants, their preparation methods, and applications. Background Technology

[0002] Glycosaminoglycans are a class of acidic mucopolysaccharides that play important physiological roles in the human body. Hyaluronic acid (HA) is an acidic mucopolysaccharide composed of alternating monosaccharide units of D-glucuronic acid and N-acetylglucosamine. It possesses strong biocompatibility and significant moisturizing effects, and has been widely used in the pharmaceutical and cosmetic industries. Early HA production methods primarily relied on animal tissue extraction, but this method suffers from limited raw material sources, low yields, high purification costs, and safety concerns regarding residual allergens. With the continuous development of synthetic biology, genetic engineering, and fermentation technologies, hyaluronic acid synthesis can now be achieved through a completely biological method without animal sources. Microbial fermentation has become the main method for HA production. Currently, the synthesis of HA using microbial cells mainly focuses on Streptococcus vesiculosus, Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Lactococcus lactis. However, the yield and molecular weight of HA produced by fermentation from different strains vary considerably. To further increase HA production, researchers have attempted to regulate it at the cellular level, including enhancing key metabolic pathways, optimizing precursor concentrations, regulating the fermentation process, and adding additional degrading enzymes (Ma Yanqin et al., Biotechnology Bulletin, 2022, 38(2): 252-262). Although there are various methods to regulate HA production, there are significant differences between different host cells, and a universally applicable modification strategy has not yet been formed.

[0003] Glycosaminoglycan synthases (GAs) are key enzymes in HA synthesis, responsible for the polymerization and transport of HA. Researchers have modified the C-terminus of GAs to significantly increase the molecular weight of the HA product, but this has resulted in a decrease in yield. Furthermore, HA yields vary among GAs from different sources during fermentation. Currently, further research is needed to modify GAs at the molecular level to regulate HA yield. Summary of the Invention

[0004] Based on this, one embodiment of this application needs to provide a glycosaminoglycan synthase mutant that significantly increases the yield of HA synthesis.

[0005] The technical solutions include the following:

[0006] A glycosaminoglycan synthase mutant having an amino acid mutation at position 108 compared to 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, compared to the glycosaminoglycan synthase shown in SEQ ID NO: 1, also has an amino acid mutation occurring at one or more of the following sites: position 113, position 147, position 199, and position 287.

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

[0010] In one embodiment, the glycosaminoglycan synthase mutant includes 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 that encodes the glycosaminoglycan synthase mutant or a fragment thereof.

[0012] Expression vectors, including the aforementioned nucleic acid molecules.

[0013] In one embodiment, the expression vector includes one or more of plasmid vectors, shuttle vectors, bacteriophages, and viral vectors.

[0014] In one embodiment, the plasmid vector includes one or more of the 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 the pEC-Ptac vector, pET-43a vector, pNV18.1 vector, and pXMJ19 vector.

[0016] The engineered bacteria express the glycosaminoglycan synthase mutant; or carry 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 may be Corynebacterium glutamicum, yeast, lactococcus, Bacillus subtilis, Escherichia coli, or streptococcus.

[0019] Optionally, the *Escherichia 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 includes Streptococcus vesicans.

[0023] Optionally, the lactococcus includes Lactococcus lactis.

[0024] Methods for preparing glycosaminoglycan synthase mutants include:

[0025] Cultivate the engineered bacteria; and,

[0026] The engineered bacteria were induced to express the glycosaminoglycan synthase mutant.

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

[0028] Methods for preparing glycosaminoglycans include:

[0029] The engineered bacteria were fermented to prepare glycosaminoglycans.

[0030] Optionally, the following steps are included:

[0031] The engineered bacteria were inoculated into a seed culture medium for seed culture to prepare bacterial solution;

[0032] The bacterial culture was inoculated into the fermentation medium at an inoculation rate of 1-10% v / v and fermented to prepare glycosaminoglycans.

[0033] In one embodiment, the glycosaminoglycan is hyaluronic acid or an analogue thereof.

[0034] Compared with traditional technologies, this application has the following advantages:

[0035] The beneficial mutant sites designed in this application for wild-type glycosaminoglycan synthase significantly increased the HA production by the glycosaminoglycan synthase. Compared with wild-type glycosaminoglycan synthase, the HA production was significantly increased when using this mutant strain for HA synthesis. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 The structural distribution of mutation sites in glycosaminoglycan synthase.

[0038] Figure 2 The fermentation process curve for HA production by a glycosaminoglycan synthase mutant strain.

[0039] Figure 3 Results of HA production measurement for glycosaminoglycan synthase mutants.

[0040] Figure 4 The molecular weight of HA synthesized by the glycosaminoglycan synthase mutant was determined. Detailed Implementation

[0041] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0043] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0044] In this document, when referring to numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of selectable values ​​within a numerical interval is considered continuous, and includes the two endpoints (i.e., the minimum and maximum values) of the numerical interval, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every 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 subranges included therein. The "numerical value" in this numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0045] In this document, "optionally," "optionally," and "optional" mean that something is optional, that is, it is selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0046] The terms “containing,” “including,” and “comprise” as used herein are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0047] This application focuses on wild-type glycosaminoglycan synthase derived from *Streptococcus pyogenes* serotype M18. Through targeted design and modification at the enzyme molecule level, several beneficial mutants were obtained. Furthermore, the distribution of these beneficial mutation sites in the spatial structure was discovered. Based on the structure, the applicant used molecular docking to simulate the binding state of the substrate and enzyme molecules, and studied the binding process of the substrate and enzyme molecules through molecular dynamics simulations. The interaction forces between the substrate and enzyme molecules were analyzed, and amino acid sites that may affect the enzyme's catalytic performance were identified. Multiple sequence alignment analysis was used to pinpoint candidate amino acid residues, and targeted design was performed based on structural position and evolutionary conservation analysis to construct a glycosaminoglycan synthase mutant library. Through measurement, several mutants with increased HA yield and / or molecular weight were screened.

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

[0049] In one 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 either glutamic acid or arginine.

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

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

[0053] In one specific example, the glycosaminoglycan synthase mutant has at least one set of mutations from 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 one specific example, the glycosaminoglycan synthase mutant has at least three mutations from the following group:

[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 one specific example, the glycosaminoglycan synthase mutant has at least four of the following groups of mutations:

[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 one 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 groups 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. This mutant enables microbial fermentation to produce HA at a yield 1.0 to 1.2 times that of the wild-type enzyme.

[0085] In a specific example, the glycosaminoglycan synthase mutant has any of the following groups 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 and I199R; K108R, M113R and N287R; K108R, M113R and N287E; K108E, M113R and I199R and S147E; K10 The mutants are 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. The molecular weight of this mutant can reach 500,000-3,000,000 Da, enabling microbial fermentation to produce HA at a yield 1.3-2.8 times that of the wild-type enzyme.

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

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

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

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

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

[0092] One embodiment of this application also provides engineered bacteria that express the glycosaminoglycan synthase mutant; or carry the nucleic acid molecule or the expression vector.

[0093] This application focuses on the wild-type glycosaminoglycan synthase derived from Streptococcus pyogenes serotype M18. Starting with its structure, it analyzes the synthesis and transport mechanisms of glycosaminoglycans and obtains a new generation of production strains with significantly improved HA production through the design and construction of enzyme molecules.

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

[0095] In a specific example, the host bacteria are Corynebacterium glutamicum, yeast, lactococcus, Bacillus subtilis, Escherichia coli, or streptococcus.

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

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

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

[0099] In one specific example, streptococci include Streptococcus vesicans.

[0100] In a specific example, lactococci include lactococci with lactic acid bacteria.

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

[0102] An embodiment of this application also provides a method for preparing a glycosaminoglycan synthase mutant, including culturing engineered bacteria;

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

[0104] This application also provides the application 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 this application also provides a method for preparing glycosaminoglycans, comprising fermenting and culturing the engineered bacteria to prepare glycosaminoglycans.

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

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

[0108] The bacterial culture was inoculated into the fermentation medium at an inoculation rate of 1-10% v / v and fermented to prepare glycosaminoglycans.

[0109] In a specific example, the glycosaminoglycan is hyaluronic acid or its analogues (e.g., chondroitin, heparin).

[0110] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0111] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0112] Example 1: Design of Glycosaminoglycan synthase mutants

[0113] Based on the analysis of the glycosaminoglycan synthase protein structure of wild-type Streptococcus pyogenes (Serotype M18), molecular dynamics simulations were used to explore the substrate-enzyme binding process, and a strategy of mutation was proposed at the flexible structure of the protein surface region. Subsequently, structural analysis identified the amino acid sites on the glycosaminoglycan synthase surface region, including lysine at position 108, methionine at position 113, serine at position 147, isoleucine at position 199, and asparagine at position 287. Figure 1 Through structural analysis and multiple sequence alignment, the selected amino acids were mutated to either arginine (Arg, R) or glutamic acid (Glu, E). Based on this, combined mutations were performed on these sites to design combined mutants, as shown in Table 1.

[0114] Table 1 List of mutants

[0115]

[0116] The amino acid sequence of the glycosaminoglycan synthase of wild-type Streptococcus pyogenes (Serotype M18):

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

[0118] Example 2 Construction of a glycosaminoglycan synthase mutant library

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

[0120] First, a recombinant plasmid pEC-XK99E-Ptac-SY containing wild-type enzymes was constructed. Then, using pEC-XK99E-Ptac-SY as a template, forward and reverse primers containing mutation sites were designed, and the whole plasmid was amplified using a high-fidelity enzyme.

[0121] The 10 μL PCR system includes:

[0122] 1 μL pEC-XK99E-Ptac-SY plasmid template (approximately 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 ddH2O.

[0127] Combinatorial mutants are constructed by performing one or more rounds of mutations on top of single-point mutants, and specific primers are used to introduce mutation sites.

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

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

[0130] (2) Denaturation at 98 ℃ for 30 s;

[0131] (3) Anneal at ℃ for 10 s (Tm-5 of primers);

[0132] (4) Extend at 72 ℃ for 7 min;

[0133] The above steps (2)-(4) are performed for a total of 30 cycles, and finally extended at 72 ℃ for 10 min.

[0134] The recombinant plasmid obtained by PCR amplification was digested with Dpn I enzyme, transformed into competent cells, and plated on solid plates containing antibiotics (50 μg / mL). The plates were then incubated upside down at 37°C for approximately 12 hours. Single clones were picked for sequencing verification, and the recombinant plasmid pEC-XK99E-Ptac-SY1 of the mutant was extracted and stored at -20°C.

[0135] Table 2 Primer sequences used for constructing mutant libraries

[0136]

[0137] Combinatorial mutants are obtained through multiple rounds of PCR reactions based on single-point mutants, following the construction method described above. Taking the combinatorial mutant K108R / S147E / N287R / I199R as an example, firstly, using the plasmid sequence of the wild-type enzyme as a template, primers K108R-U and K108R-D are used to amplify the entire plasmid to obtain the K108R single-point mutant. Subsequently, following the same method, using the plasmid sequence of the K108R mutant as a template, primers S147E-U and S147E-D are used to amplify the entire plasmid to obtain the K108R / S147E single-point mutant. Finally, multiple rounds of mutagenesis can yield multi-point mutants.

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

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

[0140] Example 4: Production of HA by shake-flask fermentation of genetically engineered bacteria with glycosaminoglycan synthase mutants

[0141] Genetically engineered bacterial colonies containing glycosaminoglycan synthase mutants were picked from LB agar plates and transferred to LBG medium containing the corresponding antibiotics. The colonies were incubated at 30°C. o Incubate overnight at 200 rpm at C. Add 5% inoculum to a 300 mL Erlenmeyer flask containing 50 mL of fermentation medium, and add the appropriate antibiotic. Incubate at 28°C. o C. After culturing for 3 h, IPTG was added to a final concentration of 1 mM to induce expression. The mixture was then cultured at 30 ℃ and 200 rpm for 48 h, with the pH of the fermentation broth maintained between 6.0 and 8.0. OD was measured at specified time points. 600 And HA concentration, and HA molecular weight.

[0142] Figure 2 The fermentation process curve for HA production by a glycosaminoglycan synthase mutant strain.

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

[0144] Take an appropriate amount of fermentation broth, add 3 times its volume of ethanol, mix well, and let stand for 2 hours. Centrifuge, discard the supernatant, retain the precipitate, and dry at room temperature for 1 hour. Add an equal volume of deionized water and accelerate the hydrolysis using a vortex mixer. Centrifuge to discard the bacterial cells and insoluble impurities, and obtain the supernatant, which is the HA aqueous solution.

[0145] The HA concentration was then determined using the CTAB (trimethylhexylammonium bromide) turbidimetric method. The reaction system consisted of the sample, acetate buffer, and CTAB solution. After a reaction of 5 min, the OD was measured. 400 Absorbance value. According to the formula: HA(g / L) = OD 400 Use +0.009 to calculate the HA concentration.

[0146] A total of 50 glycosaminoglycan synthase mutants were obtained through site-directed mutagenesis and combinatorial mutagenesis. HA was then produced by fermentation using the constructed glycosaminoglycan synthase mutant genetically engineered bacteria. The concentration of HA was measured, and the relative yields of HA produced by the mutants compared to wild-type glycosaminoglycan synthase are shown in Table 3. Figure 3 As shown.

[0147] Table 3. Relative yield of HA produced by fermentation of mutants

[0148]

[0149] Note: In the table, the mutant “K108R / M113E” indicates that the mutant has two mutation modes, K108R and M113E. Other mutants can be understood similarly. Using standard substitution notation, K108R means that the K at position 15 from the start N end is replaced by R.

[0150] Table 3 shows that each mutant represents a mutant obtained by making a specific mutation at the corresponding amino acid site in the amino acid sequence shown in SEQ ID NO: 1. The determination of HA yield revealed that the HA produced by the constructed mutants was increased compared to the wild-type strain, with the molecular weight increase being more significant after combined mutations.

[0151] 2. Determination of HA molecular weight

[0152] The molecular weight of hyaluronic acid (HA) was determined using SEC-MALS coupled with molecular size exclusion chromatography. HA was separated by size exclusion chromatography, followed by multi-angle laser scattering (LSS) optical analysis to determine the molecular weight of the HA sample. A TSK-GEL GMPWXL column was used, with a mobile phase of 23.38 g / L NaCl solution. The results were analyzed using a Wyatt MALS system. Figure 4(An example result of the mutant). Single-point mutants and some combined mutants increased yield while maintaining the same molecular weight as the wild type, ranging from 200,000 to 1,000,000 Da. The combined mutants include 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, K108E / M113R / I199R / S147E, K108E / S147E / N287R / I199R, and K108E. The HA molecular weights 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 the level of 500,000 to 3,000,000 Da, showing a significant increase in molecular weight.

[0153] The results in summary indicate that the amino acid site mutations designed in Table 1 can significantly increase HA production. Furthermore, combined mutations at certain sites not only have a more significant effect on increasing HA production but also significantly increase the molecular weight of HA.

[0154] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A glycosaminoglycan synthase mutant, characterized in that, The glycosaminoglycan synthase mutant described herein is an amino acid mutation occurring at position 108, compared to the glycosaminoglycan synthase shown in SEQ ID NO:

1. The amino acid at position 108 is glutamic acid or arginine.

2. The glycosaminoglycan synthase mutant according to claim 1, characterized in that, Compared with the glycosaminoglycan synthase shown in SEQ ID NO: 1, the glycosaminoglycan synthase mutant also has an amino acid mutation occurring at one or more of the following sites: positions 113, 147, 199 and 287, where each amino acid at positions 113, 147, 199 and 287 is independently mutated to glutamic acid or arginine.

3. The glycosaminoglycan synthase mutant according to any one of claims 1 to 2, characterized in that, The glycosaminoglycan synthase mutant has any of the following sets of 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, and 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.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a glycosaminoglycan synthase mutant as described in any one of claims 1 to 3.

5. An expression vector, characterized in that, Includes the nucleic acid molecule as described in claim 4; The expression vector includes one or more of plasmid vectors, shuttle vectors, bacteriophages, and viral vectors.

6. The expression vector according to claim 5, characterized in that, The plasmid vectors include one or more of the following: pEC series vectors, pET series vectors, pNV series vectors, pXMJ19, pXZ10145, pZL071, and pXT01.

7. The expression vector according to claim 6, characterized in that, The pEC series vectors include one or more of the following: pEC-Ptac vector, pET-43a vector, pNV18.1 vector, and pXMJ19 vector.

8. Engineered bacteria, characterized in that, The engineered bacteria express the glycosaminoglycan synthase mutant of any one of claims 1 to 3; or carry the nucleic acid molecule of claim 4 or the expression vector of any one of claims 5 to 7.

9. The engineered bacteria according to claim 8, characterized in that, The host bacterium of the engineered bacteria is Corynebacterium glutamicum.

10. A method for preparing a glycosaminoglycan synthase mutant, characterized in that, include: Cultivate the engineered bacteria as described in claim 8 or 9; and, The engineered bacteria were induced to express the glycosaminoglycan synthase mutant.

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

12. A method for preparing glycosaminoglycans, characterized in that, include: The engineered bacteria of claim 8 or 9 are fermented to prepare glycosaminoglycans.

13. The method for preparing glycosaminoglycans according to claim 12, characterized in that, Includes the following steps: The engineered bacteria were inoculated into a seed culture medium for seed culture to prepare bacterial solution; The bacterial culture was inoculated into the fermentation medium at an inoculation rate of 1-10% v / v and fermented to prepare glycosaminoglycans.

14. The method for preparing glycosaminoglycans according to claim 13, characterized in that, The glycosaminoglycan is hyaluronic acid or an analogue thereof.

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