Glucokinase mutant for enhancing glucose utilization rate of escherichia coli and application of glucokinase mutant

Abstract

The invention discloses a glucokinase mutant for enhancing the glucose utilization rate of escherichia coli and application of the glucokinase mutant. The invention belongs to the technical field of biology, and particularly relates to a glucokinase mutant for enhancing the glucose utilization rate of escherichia coli and application of the glucokinase mutant. The mutant protein disclosed by the invention is any one of the following proteins: 1) a protein obtained by mutating the 142 A of a sequence 1 into V or L and keeping other amino acid residues unchanged; 2) a protein obtained by mutating R in the 188th sequence of the sequence 1 into H and keeping other amino acid residues unchanged; and 3) a protein obtained by mutating the 142 of the sequence 1 into V or L, mutating R of the 188 of the sequence 1 into H and keeping other amino acid residues unchanged. The encoding gene of glucokinase in receptor escherichia coli is knocked out, and the encoding gene of mutant protein is introduced into the receptor escherichia coli, so that the enzyme activity of the obtained recombinant escherichia coli is 7.3 times that of wild escherichia coli, and the glucose utilization rate of escherichia coli is increased.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a glucokinase mutant that enhances glucose utilization in Escherichia coli and its applications. Background Technology

[0002] Lignocellulose is the world's most widely available renewable biomass resource. It is the richest biomass source for the renewable production of fuels and chemicals. Statistics show that lignocellulose accounts for approximately 50% of all biomass production annually, equivalent to about 10-50 billion tons. Utilizing lignocellulose hydrolysate for fermentation to produce bioenergy and bulk chemicals can partially reduce dependence on petroleum resources. Glucose and xylose are the main hydrolyzed monosaccharides in lignocellulose, with glucose accounting for 30%-50% and xylose for 5%-20%.

[0003] Escherichia coli has a clear genetic background and a full range of genetic modification tools, making it one of the commonly used model organisms in industry and a key research species in the utilization of lignocellulose. Solving the fundamental problem of xylose-glucose co-utilization to improve the economic benefits of lignocellulose biomass is essential. Establishing xylose-glucose co-consumption platform strains, understanding the key metabolic mechanisms, and identifying important modification targets will greatly promote and expand the development and utilization of lignocellulose biomass.

[0004] Studies have shown that knocking out the `glk`, `ptsG`, and `manZ` genes in *E. coli* yields strains capable of utilizing xylose, while knocking out `xylA` yields strains capable of utilizing glucose. Fermentation of these two strains together resulted in rapid consumption of both glucose and xylose. When the `ptsG` gene was deleted to avoid glucose inhibition, and `glk` was overexpressed to restore the glucose uptake rate, total glucose consumption increased in glucose-xylose medium, but xylose consumption and isobutanol production decreased. Therefore, designing the `glk` gene to improve glucose utilization in *E. coli*, and even to promote disaccharide utilization and increase product yield in glucose-xylose medium, is a crucial research topic worthy of investigation. Summary of the Invention

[0005] The main technical problem to be solved by this invention is how to enhance the glucose utilization rate of Escherichia coli.

[0006] To address the above problems, the present invention provides a mutant protein.

[0007] The mutant protein provided by this invention may be any of the following proteins:

[0008] 1) The protein obtained by mutating A at position 142 of sequence 1 to V or L while keeping other amino acid residues unchanged;

[0009] 2) The protein obtained by mutating R to H at position 188 of sequence 1 while keeping other amino acid residues unchanged;

[0010] 3) The protein obtained by mutating A at position 142 of sequence 1 to V or L, and mutating R at position 188 of sequence 1 to H, while keeping other amino acid residues unchanged.

[0011] The protein described in Sequence 1 is glucokinase.

[0012] The present invention also provides biological materials related to the proteins described above, comprising at least one of the following B1)-B4):

[0013] B1) Nucleic acid molecules that encode the proteins described above;

[0014] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0015] B3) A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2);

[0016] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0017] In the above-mentioned biological materials, the nucleic acid molecule described in B1) is the cDNA molecule or DNA molecule shown in sequence 2 of the sequence listing.

[0018] Furthermore, the recombinant vector described in B3) can be the recombinant vector pTrc99A-glk. WT pTrc99A-glk A142V pTrc99A-glk A142L pTrc99A-glk R188H pTrc99A-glk A142V,R188H or pTrc99A-glk A142L,R188H .

[0019] The recombinant vector pTrc99A-glk WT The structure is described as follows: The recombinant expression vector is obtained by replacing the fragment between the 5'-aatttcacacaggaaacagacc-3' and 5'-aggtcgactctagaggatcc-3' recognition fragments of the vector pTrc99A with a DNA molecule whose nucleotide sequence is sequence 2, while keeping the other sequences of the pTrc99A vector unchanged. The recombinant vector pTrc99A-glk WT The nucleotide sequence is sequence 3.

[0020] pTrc99A-glk A142VpTrc99A-glk A142L pTrc99A-glk R188H With the recombinant vector pTrc99A-glk WT The only difference is that the GCG at positions 690-692 of sequence 3 is mutated to GTT, or the GCG at positions 690-692 of sequence 3 is mutated to TTA, or the G at position 829 of sequence 3 is mutated to A.

[0021] pTrc99A-glk A142V,R188H plasmid and recombinant vector pTrc99A-glk WT The only difference is that GCG is mutated to GTT at positions 690-692 of sequence 3 and G is mutated to A at position 829.

[0022] pTrc99A-glk A142L,R188H plasmid and recombinant vector pTrc99A-glk WT The only difference is that GCG at positions 690-692 of sequence 3 is mutated to TTA and G at position 829 is mutated to A.

[0023] Furthermore, the recombinant microorganism described in B4) is recombinant Escherichia coli.

[0024] The recombinant Escherichia coli was constructed according to the method described below.

[0025] The present invention provides a method for constructing the recombinant Escherichia coli described above. The method includes knocking out the glucokinase coding gene in the recipient Escherichia coli, introducing the coding gene of the mutant protein described above into the recipient Escherichia coli, and obtaining recombinant Escherichia coli, wherein the recipient Escherichia coli is Escherichia coli strain SL002.

[0026] The Escherichia coli SL002 strain was developed based on the ATCC 8739 strain by replacing the upstream promoter of the galP gene with the M1-93 promoter and the upstream promoter of the glk gene with the M1-37 promoter, and by knocking out the PTS system and the xylFGH gene cluster.

[0027] In the above construction method, the glucokinase is a protein with the amino acid sequence of sequence 1.

[0028] The nucleotide sequence of the gene encoding glucokinase is sequence 2 in the sequence listing.

[0029] The present invention also provides a method for improving glucokinase activity by mutating the 142nd position A of wild-type glucokinase with amino acid sequence 1 to V or L, or / and the 188th position R to H, to obtain a mutant protein with improved glucokinase activity.

[0030] The present invention also provides a method for preparing glucokinase, the method comprising fermenting the recombinant Escherichia coli described above to obtain the mutant protein described above, wherein the mutant protein is a glucokinase mutant.

[0031] The present invention also provides the aforementioned protein, the aforementioned biomaterial, and the aforementioned recombinant Escherichia coli for any of the following applications:

[0032] P1. Application in enhancing glucokinase activity;

[0033] P2, Application in the hydrolysis of glucose;

[0034] P3. Applications in hydrolyzed xylose;

[0035] P4. Applications in improving the utilization rate of lignocellulose.

[0036] The glucokinase mutation site disclosed in this invention can increase glucokinase activity, thereby improving glucose utilization and also improving the co-utilization of glucose and xylose. There are currently no reports of similar mutants. Attached Figure Description

[0037] Figure 1 pTrc99A-glk WT Plasmid map. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0041] The pRedCas9 plasmid used in the following examples is described in: Zhu et al. 2017, The CRISPR / Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway (Memb. Eng. 43, 37-45). This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0042] The pV4 plasmid used in the following examples is described in the specification 0131-0138 of the authorized patent: CN114058560A, Method for the Production of Glycine. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0043] The pTrc99A plasmid used in the following examples is described in the authorized patent: CN114350692B, "A method for whole-cell catalytic preparation of decarboxylated carnosine." This biological material is available to the public from the applicant and is intended solely for repeating the experiments of this invention; it may not be used for any other purpose.

[0044] The Escherichia coli ATCC 8739 described in the following examples can be obtained from the American Type Culture Collection Center (ATCC) (https: / / www.atcc.org).

[0045] The *Escherichia coli* strain SL002 described in the following examples is described in: Zhu X, Zhao D, Qiu H, Fan F, Man S, Bi C, Zhang X. The CRISPR / Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the *Escherichia coli* xylose utilization pathway. Metab Eng. 2017 Sep; 43(Pt A):37-45. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0046] The strains used in this invention are shown in Table 1, the primers used are shown in Table 2, and the plasmids involved are shown in Table 3.

[0047] Table 1. Strains used in this invention

[0048] strain genotype SL002 ATCC 8739,PTS-,FRT::M1-93-galP,FRT::M1-37-glk,ΔxylFGH CW005 ATCC 8739,PTS-,FRT-M1-93-galP,ΔglkΔxylFGH CW006 <![CDATA[CW005 pTrc99A-glk WT ]]> CW007 <![CDATA[CW005 pTrc99A-glk A142V ]]> CW008 <![CDATA[CW005 pTrc99A-glk A142L ]]> CW009 <![CDATA[CW005 pTrc99A-glk R188H ]]> CW010 <![CDATA[CW005 pTrc99A-glk A142V,R188H <!-- 3 -->]]> CW011 <![CDATA[CW005 pTrc99A-glk A142L,R188H ]]>

[0049] Table 2. Primer information used in this invention

[0050]

[0051]

[0052] Table 3. Plasmids used and constructed in this invention

[0053]

[0054] Example 1: Effect of Glk mutant on glucose utilization in Escherichia coli

[0055] 1. pTrc99A-glk WT plasmid construction

[0056] pTrc99A-glk WT The specific steps of the construction process are as follows:

[0057] The first step was to obtain the pTrc99A plasmid backbone fragment. Using the pTrc99A plasmid as a template, PCR amplification was performed using primers pTrc99A-F and pTrc99A-R (specific sequences are shown in Table 2), yielding a PCR product of approximately 4.1 kb, which is the pTrc99A plasmid backbone fragment A. This fragment contains amp, pBR322, Plac-lacI, and Ptrc.

[0058] The amplification system consisted of: 10 μL New England Biolabs Phusion 5X buffer, 1 μL dNTPs (2.5 mM each), 20 ng DNA template, 2 μL primers (10 μM each), 0.5 μL Phusion High-Fidelity DNA polymerase (2.5 U / μL), and 33.5 μL distilled water, for a total volume of 50 μL.

[0059] The amplification conditions were as follows: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, 56℃ annealing for 10 seconds, 72℃ extension for 2.5 minutes (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0060] The second step is to obtain the wild-type glk sequence fragment B. Using the ATCC8739 genome as a template, PCR amplification was performed using primers Glk-F and Glk-R (specific sequences are shown in Table 2) to obtain a PCR product of about 1 kb, which is the wild-type glk sequence fragment B.

[0061] The third step is to use the Gibson technique (Hieff) to process the above fragments A and B. The Universal II OneStep Cloning Kit (assembled by Yisheng Biotechnology (Shanghai) Co., Ltd.) was used to chemically transform the clones into competent TransT1 cells (Beijing TransGen Biotech Co., Ltd.). The resulting clones were verified by PCR using primers lacI-600F and Glk-600R (specific sequences are shown in Table 2). The band size was approximately 1.2 kb. Plasmid DNA was extracted from positive clones and sent for sequencing analysis, yielding the correct pTrc99A-glk clone. WT Plasmid.

[0062] pTrc99A-glk WT The plasmid structure is described as follows: The recombinant expression vector pTrc99A-glk is obtained by replacing the sequence between the 5'-AATTTCACACAGGAAACAGACC3' and 5'-AGGTCGACTCTAGAGGATCC3' recognition fragments in the vector pTrc99A with a DNA molecule whose nucleotide sequence is sequence 2, while keeping the other sequences of the pTrc99A vector unchanged. WT The plasmid's nucleotide sequence is sequence 3 in the sequence listing, and the vector map is shown below. Figure 1 .

[0063] 2. Construction of overexpression mutant glk plasmid

[0064] First, using pTrc99A-glk obtained in (1) WT Using the plasmid as a template, PCR amplification was performed using primer pairs Glk-A142V-F / Glk-A142V-R, Glk-A142L-F / Glk-A142L-R, and Glk-R188H-F / Glk-R188H-R, respectively, yielding a PCR product of approximately 5.1 kb, namely pTrc99A-glk. A142V pTrc99A-glk A142L With pTrc99A-glk R188H The corresponding segment.

[0065] Using Gibson technology (Hieff) After self-assembly and ligation using the Universal IIOne Step Cloning Kit (Yisheng Biotechnology (Shanghai) Co., Ltd.), the clones were chemically transformed into competent TransT1 cells (Beijing TransGen Biotech Co., Ltd.). The resulting clones were subjected to PCR using primers lacI-600F and Glk-600R (specific sequences are shown in Table 2) to obtain a fragment of approximately 1.2 kb. After sequencing analysis, positive clones were selected, and plasmid DNA was extracted to obtain the correct pTrc99A-glk clones. A142V pTrc99A-glk A142L With pTrc99A-glk R188H Plasmid.

[0066] pTrc99A-glk A142V pTrc99A-glk A142L pTrc99A-glk R188H With the recombinant vector pTrc99A-glk WT The only difference is that the GCG at positions 690-692 of sequence 3 is mutated to GTT, or the GCG at positions 690-692 of sequence 3 is mutated to TTA, or the G at position 829 of sequence 3 is mutated to A.

[0067] Subsequently, pTrc99A-glk was used respectively. A142V With pTrc99A-glk A142L Using the plasmid as a template, PCR amplification was performed using the Glk-R188H-F / Glk-R188H-R primer pair, yielding PCR products of approximately 5.1 kb, namely pTrc99A-glk. A142V,R188H With pTrc99A-glk A142L,R188H Plasmid fragments. Using Gibson technology (Hieff) After self-assembly and ligation using the UniversalIIOne Step Cloning Kit (Yisheng Biotechnology (Shanghai) Co., Ltd.), the clones were chemically transformed into competent TransT1 cells (Beijing TransGen Biotech Co., Ltd.). The obtained clones were subjected to PCR using primers lacI-600F and Glk-600R (specific sequences are shown in Table 2) to obtain fragments of approximately 1.2 kb. After sequencing analysis, positive clones were selected for plasmid DNA extraction, yielding the correct pTrc99A-glk clones. A142V,R188H With pTrc99A-glk A142L,R188H Plasmid.

[0068] pTrc99A-glk A142V,R188H With pTrc99A-glk A142L,R188H plasmid and recombinant vector pTrc99A-glkWT The only difference is that: the GCG at positions 690-692 of sequence 3 is mutated to GTT and the G at position 829 is mutated to A, or the GCG at positions 690-692 of sequence 3 is mutated to TTA and the G at position 829 is mutated to A.

[0069] 3. Construction of the Glk mutant based on SL002 and the construction of the starting strain CW005.

[0070] The first step is to construct the pV4-del-glk plasmid.

[0071] I. Obtain pV4 plasmid backbone fragment A.

[0072] Using pV4 plasmid as a template, reverse PCR amplification was performed using primers HA-F and N20-glkR (specific sequences are shown in Table 2) to obtain a PCR product of approximately 4.1 kb, which is the pV4 plasmid backbone fragment A. This fragment contains cat, P15A, and the self-cleaving element lacI-Ptrc-cat-N20-gRNA.

[0073] The amplification system consisted of: 10 μL New England Biolabs Phusion 5X buffer, 1 μL dNTPs (2.5 mM each), 20 ng DNA template, 2 μL primers (10 μM each), 0.5 μL Phusion High-Fidelity DNA polymerase (2.5 U / μL), and 33.5 μL distilled water, for a total volume of 50 μL.

[0074] The amplification conditions were as follows: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, 56℃ annealing for 10 seconds, 72℃ extension for 2.5 minutes (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0075] II. Obtain plasmid fragment B between the glk-N20 sequence and the upstream homologous arm of glk.

[0076] Using pV4 plasmid as a template, PCR amplification was performed using primers N20-glkF and HA-R (specific sequences are shown in Table 2) to obtain a PCR product of approximately 400 bp, which is DNA fragment B. The amplification system and amplification conditions are as described in the first step of Example 1(1).

[0077] III. Obtain upstream and downstream homologous arm fragments C and D of the glk gene. Using genomic DNA from *E. coli* ATCC 8739 as a template, PCR amplification was performed using primers Glkdel-UF and Glkdel-UR (specific sequences are shown in Table 2) to obtain the upstream homologous arm fragment C, approximately 200 bp. Similarly, PCR amplification was performed using primers Glkdel-DF and Glkdel-DR (specific sequences are shown in Table 2) to obtain the downstream homologous arm fragment D, approximately 200 bp. The amplification system and conditions are the same as in step (1), step one.

[0078] IV. The above fragments A, B, C, and D were assembled using the Golden Gate technique (sequentially and seamlessly linked in the order of A, B, C, and D), and chemically transformed into competent TransT1 cells (Beijing TransGen Biotech Co., Ltd.). The resulting clones were verified by PCR using primers P15A-UP and Glkdel-DR (specific sequences are shown in Table 2). The band size was approximately 820 bp. Plasmid DNA was extracted from positive clones and sent for sequencing analysis to obtain the correct pV4-del-glk plasmid.

[0079] The structure of the pV4-del-glk plasmid is described as follows: A DNA molecule with the nucleotide sequence of sequence 4 is inserted between the 5'-CTTCCGTATTTAGCCAGCGGTTG-3' and 5'GTGCGCCATGAGAACGAACCAT-3' recognition fragments of the vector pV4; a DNA molecule with the nucleotide sequence of sequence 5 (the N20 sequence targeting glk) is inserted between the 5'-CTTCCGTATTTAGCCAGCGGT-3' and 5'GTGCGCCATGAGAACGAACCAT-3' recognition fragments, while keeping the other sequences of the pV4 vector unchanged, to obtain the recombinant expression vector. The nucleotide sequence of the pV4-del-glk plasmid is sequence 6 in the sequence listing.

[0080] The second step, the specific steps for glk gene knockout, are as follows:

[0081] Starting with the engineered *E. coli* strain SL002, plasmids pRedCas9 and pV4-del-glk were simultaneously transformed into SL002 electrotransformed competent cells. The cells were plated on kanamycin and chloramphenicol plates and incubated overnight at 30°C. Single colonies were picked and incubated in 2 mL LB agar (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose), induced homologous recombination, and cleaved non-recombinant DNA at 250 rpm overnight at 30°C. The incubated DNA was diluted and plated on LB agar (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose) plates and incubated overnight at 30°C. Single colonies were picked for colony PCR verification using primers glk-YZ-up and glk-YZ-down (specific sequences are shown in Table 2). Positive clones yielded PCR products of approximately 400 bp in size, and these positive clones were named recombinant *E. coli* CW005.

[0082] 4. Construction of Glk mutant strain

[0083] The plasmid pTrc99A-glk constructed in steps 1 and 2 WT pTrc99A-glk A142V pTrc99A-glk A142L pTrc99A-glk R188H pTrc99A-glk A142V,R188H and pTrc99A-glk A142L,R188H The cells were chemically transformed into the chemically competent cells of the recombinant Escherichia coli CW005 constructed in step 3, respectively, to obtain CW006, CW007, CW008, CW009, CW010 and CW011 (see Table 3 for details).

[0084] Example 2: The regulatory effect of the Glk mutant on glucose metabolism

[0085] The seed culture medium consists of the following components (solvent is water): NaCl: 10 g / L, Tryptone: 10 g / L, yeast extract: 5 g / L.

[0086] The fermentation medium consists of the following components:

[0087] Macroelements: Glucose 50g / L, NH4H2PO4 17.56g / L, (NH4)2HPO4 52.6g / L, MgSO4·7H2O 37g / L, Betaine-HCl 15.361g / L, KCl 18.75g / L.

[0088] Trace elements: FeCl3·6H2O 2.4g / L, CoCl2·6H2O 0.3g / L, CuCl2·2H2O 0.15g / L, ZnCl2 0.3g / L, Na2MoO4·2H2O 0.3g / L, MnCl2·4H2O 0.5g / L, H3BO3 0.072g / L.

[0089] Seed culture: 30 mL of seed culture medium was prepared in a 250 mL Erlenmeyer flask and sterilized at 121 °C for 20 min. After cooling, single colonies of the recombinant Escherichia coli CW006, CW007, CW008, CW009, CW010, and CW011 constructed in Example 1 were picked from the plates and inoculated into seed culture medium supplemented with 100 μg / mL ampicillin. The cultures were incubated at 37 °C and 250 rpm for 12 hours to obtain seed culture, which was then used for inoculation of fermentation medium.

[0090] 1. The role of the Glk mutant in glucose metabolism

[0091] Fermentation culture: The fermentation medium volume in the 500mL anaerobic tank is 250mL. The seed culture is added according to the final OD concentration. 550nm An inoculum of 0.1 μg / mL was inoculated into fermentation medium supplemented with 100 μg / mL ampicillin. Fermentation was carried out at 37°C and 150 rpm for 4 days to obtain the fermentation broth. 6M KOH was used as the neutralizing agent to maintain the pH of the fermenter at 7.0. The fermentation broth consisted of all substances within the fermenter. No gas was introduced during the cultivation process.

[0092] Analytical methods: Components in the fermentation broth after 24 hours were determined using an Agilent-1200 high-performance liquid chromatograph. Glucose concentration in the fermentation broth was determined using an Aminex HPX-87H organic acid column from Biorad. Lactic acid optical purity was analyzed using a SUMICHIRALOA-6000 chiral column from Sumika Chemical Analysis Service, Japan.

[0093] Glucose standards were purchased from Merck Life Sciences, catalog number 50-99-7. The specific detection procedures were performed according to the materials and methods described in the following literature: Zhu, XN; Tan, ZG; Xu, HT; Chen, J.; Tang, JL; Zhang, XL, Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli. Metab Eng 2014, 24, 87-96. The results are shown in Table 4.

[0094] Table 4. Evaluation of glucose fermentation by recombinant Escherichia coli

[0095] strain 24-hour glucose consumption (g / L) Glucose utilization rate (%) CW006 15.95±1.05 31.9±2.1 CW007 35.26±2.08 70.52±4.16 CW008 31.50±2.05 63±4.1 CW009 34.76±1.77 69.52±3.54 CW010 42.80±2.90 85.6±5.8 CW011 39.80±1.90 79.6±3.8

[0096] Table 4 shows that all three point mutants can enhance glucose utilization, with overexpression of glk being the most effective. A142V The mutant (CW007) showed the strongest enhancement of glucose utilization. When the mutation was superimposed, glucose utilization was further improved compared to the single-point mutant. A142V，R188H The mutant (CW011) has a higher glucose utilization rate than glk. A142L，R188H The mutant (CW010) has a high mutation rate, reaching 85.6±5.8%, which is 1.21 times that of the wild-type glk (CW006).

[0097] 2. The effect of the Glk mutant on glucose-xylose co-utilization in Escherichia coli

[0098] In this experiment, the seed culture medium for the Glk mutant was the same as above. The fermentation medium was prepared by adding 50 g / L xylose to the medium used in step 1. The fermentation method was the same as above.

[0099] Analytical methods: Components in the fermentation broth after 24 hours were determined using an Agilent-1200 high-performance liquid chromatograph. Glucose and xylose concentrations in the fermentation broth were determined using a Biorad Aminex HPX–87H organic acid column. Lactic acid optical purity was analyzed using a Sumika Chemical Analysis Service (SMI) SUMICHIRALOA-6000 chiral column.

[0100] Glucose and xylose standards were purchased from Merck Life Sciences, catalog numbers 50-99-7 and 58-86-6, respectively. The specific detection procedures were performed according to the materials and methods described in the literature: Zhu, XN; Tan, ZG; Xu, HT; Chen, J.; Tang, JL; Zhang, XL, Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli. Metab Eng 2014, 24, 87-96. The glucose and xylose consumption after 24 hours of fermentation is shown in Table 5.

[0101] Table 5. Evaluation of glucose-xylose fermentation in recombinant Escherichia coli

[0102] strain 24-hour glucose consumption (g / L) 24-hour xylose consumption (g / L) CW006 10.62±1.40 3.85±0.85 CW007 27.97±2.43 10.12±1.41 CW008 26.51±2.68 8.45±0.62 CW009 22.48±2.02 6.56±0.87 CW010 36.67±3.32 21.70±2.90 CW011 29.80±1.79 16.60±3.95

[0103] Table 5 shows that the three point mutants also exhibited positive effects in the co-utilization of glucose and xylose, among which overexpression of glk showed the best results. A142V The mutant (CW007) showed the most significant enhancement in glucose and xylose utilization. When the mutation was superimposed, both glucose and xylose utilization were further improved compared to the single-point mutant. A142V，R188H The mutant (CW011) had the highest glucose and xylose utilization rates, which were 3.45 and 5.63 times that of the wild-type control, respectively, showing a significant improvement.

[0104] Example 3: Enzyme activity assay of Glk mutant

[0105] Crude extracts were prepared using recombinant E. coli CW006, CW007, CW008, CW009, CW010, and CW011 cells harvested after 24 hours of fermentation in Example 2. The collected cells were first washed twice with 50 mM Tris buffer (pH 7.0), then suspended in the buffer with a protease inhibitor (Roche, Switzerland) to adjust the cell concentration to OD. 550 The sample was sonicated at a concentration of 10 nm (using 50 mM Tris buffer (pH 7.0) as a blank control). After centrifugation at 12,000 × g and 4 °C for 20 minutes, the supernatant was transferred to a new tube. The protein concentration of the crude extract was determined using a Bio-Rad protein assay kit (Bio-Rad, USA).

[0106] To determine glucokinase (EC 2.7.1.1), the crude extract was added to 1 ml of a buffer containing 100 mM Tris buffer (pH 7.5), 60 mM MgCl2, 1.0 mM MTT, 2 mM ATP, and 0.5 mM NADP, according to a previously reported method (Fraenkel and Horecker, 1964). + The reaction mixture contained 15 mM glucose and 2 U glucose-6-β dehydrogenase. Enzyme-specific activity was calculated from the linear fraction of the reaction, determined by at least three separate measurements at room temperature. These reactions were initiated by the addition of the crude extract. NADP + The wavelength and millimolecular extinction coefficient are 340 nm and 6.23 cm⁻¹, respectively. -1 mM -1 One unit (U) of enzyme activity represents the amount of enzyme that catalyzes the conversion of 1 μmol of substrate into a specific product per minute (1 U / mg = μmol / min / mg protein).

[0107] The results of glucokinase activity assays for CW006-CW011 are shown in Table 6. After modification, the enzyme activity of the recombinant strain CW010 is approximately 7.3 times that of CW006.

[0108] Table 6. Enzyme activity of glucokinase in recombinant Escherichia coli

[0109] strain Enzyme activity (U / mg protein) CW006 90.85±9.05 CW007 345.16±16.81 CW008 281.45±26.32 CW009 324.56±18.7 CW010 663.70±22.90 CW011 546.60±13.95

[0110] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A mutant protein, said mutant protein being any of the following proteins: 1) The protein obtained by mutating the A at position 142 of sequence 1 to V or L while keeping other amino acid residues unchanged; 2) The protein obtained by mutating R to H at position 188 of sequence 1 while keeping other amino acid residues unchanged; 3) The protein obtained by mutating the 142nd position of sequence 1 to V or L and the 188th position of sequence 1 to H while keeping other amino acid residues unchanged.

2. The biological material associated with the protein of claim 1 is at least one of B1)-B4) below: B1) A nucleic acid molecule encoding the protein of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule is the cDNA molecule or DNA molecule shown in sequence 2 of the sequence listing.

4. The biomaterial according to claim 2 or 3, characterized in that: The recombinant microorganism is recombinant Escherichia coli.

5. A method for constructing recombinant Escherichia coli in the biomaterial of claim 4, characterized in that, The method includes knocking out the glucokinase coding gene in the recipient Escherichia coli, introducing the coding gene of the mutant protein described in claim 1 into the recipient Escherichia coli to obtain recombinant Escherichia coli, wherein the recipient Escherichia coli is Escherichia coli strain SL002.

6. The method according to claim 5, characterized in that: The glucokinase is a protein whose amino acid sequence is sequence 1.

7. A method for improving glucokinase activity, wherein the amino acid sequence of wild-type glucokinase with position 142A is mutated to V or L, or position 188R is mutated to H, to obtain a mutant protein with improved glucokinase activity.

8. A method for preparing glucokinase, the method comprising fermenting the recombinant Escherichia coli of claim 4 to obtain the mutant protein of claim 1, wherein the mutant protein is glucokinase.

9. The protein of claim 1, the biological material of claim 2 or 3, and the recombinant Escherichia coli of claim 4, in any of the following applications: P1. Application in enhancing glucokinase activity; P2, Application in the hydrolysis of glucose; P3. Applications in hydrolyzed xylose; P4. Applications in improving the utilization rate of lignocellulose.

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