Gamma-glutamyl kinase mutants, nucleic acid molecules, recombinant plasmids, host cells, methods for producing l-proline and uses

Abstract

The application discloses a kind of gamma-glutamyl kinase mutants, nucleic acid molecules, recombinant plasmids, host cells, the method and application of producing L-proline, belong to genetic engineering and enzyme engineering technical field.The gamma-glutamyl kinase mutant, the gamma-glutamyl kinase mutant is based on the mutation of amino acid sequence SEQ ID NO.2, the mutation is one or two of E74K and V150M;Or, the mutation is E74K, G149A and V150R.In addition, the application also proposes the application of the above-mentioned gamma-glutamyl kinase mutant in preparing L-proline or proline derivative.The gamma-glutamyl kinase mutant proposed in the application enhances the catalytic performance of enzyme, significantly improves the yield of L-proline.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to a γ-glutamyl kinase mutant, nucleic acid molecule, recombinant plasmid, host cell, method for producing L-proline, and its application. Background Technology

[0002] L-proline is an important functional amino acid widely used in food, medicine, feed additives, biomaterials, and cosmetics. Currently, industrial L-proline is mainly produced through microbial fermentation. Corynebacterium glutamicum, with its clear genetic background, stable metabolism, and strong amino acid synthesis capabilities, has become an important host for the modification of L-proline industrial production strains. In Corynebacterium glutamicum, L-proline is mainly synthesized from glutamate through a multi-step enzymatic reaction. Gamma-glutamyl kinase (ProB) catalyzes the conversion of glutamate to gamma-glutamyl phosphate, and is the key rate-limiting enzyme in the L-proline synthesis pathway, significantly influencing the conversion efficiency of metabolic flux to L-proline. However, the natural gamma-glutamyl kinase ProB is subject to feedback regulation by the end product L-proline. When the intracellular L-proline concentration increases, it inhibits ProB enzyme activity, leading to a decrease in the metabolic flux of the proline synthesis pathway and limiting further accumulation of L-proline. Therefore, relieving ProB feedback inhibition and enhancing its catalytic activity has become an important strategy for increasing L-proline production. Currently, functional modification of ProB mainly focuses on the feedback regulation region. Studies have found that amino acid residues at positions 149 and 150 of the ProB protein loop region are involved in the regulation of L-proline feedback inhibition. By replacing amino acids in this region, feedback-resistant ProB mutants can be obtained, such as ProB-G149K, ProB-V150N, and ProB-G149A / V150R mutants, which can effectively reduce the inhibitory effect of L-proline on ProB activity. However, existing technologies mainly focus on modifying the feedback binding site. Research on whether there are novel functional sites in other regions of the ProB protein that can enhance catalytic performance is still lacking. Furthermore, simply relieving feedback inhibition cannot fully meet the requirements for constructing high-yield L-proline engineered bacteria.

[0003] The existing technology has the following main shortcomings:

[0004] (1) ProB modification strategy is singular

[0005] Currently, relieving L-proline feedback inhibition mainly relies on mutations in the loop regions at positions 149 and 150. However, the range of mutation sites is limited, making it difficult to further improve the catalytic performance of ProB.

[0006] (2) It is difficult to balance feedback resistance and catalytic efficiency.

[0007] While existing feedback resistance mutations can reduce L-proline inhibition, some mutations may lead to: decreased enzyme catalytic efficiency; reduced protein stability; and limited metabolic enhancement effects.

[0008] (3) Lack of ProB distal functional enhancement site mining

[0009] No novel regulatory sites other than the feedback regulatory region have yet been discovered that can promote enhanced ProB enzyme activity.

[0010] (4) Lack of multi-site collaborative optimization strategy

[0011] Existing studies mainly employ mutations at single sites or in local regions, lacking a strategy of synergistic modification of feedback release sites and catalytic enhancement sites.

[0012] Therefore, developing new ProB functional enhancement sites and simultaneously improving ProB feedback resistance and catalytic ability through multi-site synergistic modification strategies are of great significance for further improving the fermentation level of L-proline. Summary of the Invention

[0013] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a γ-glutamyl kinase mutant, nucleic acid molecule, recombinant plasmid, host cell, method for producing L-proline and its application, thereby solving the technical problem of how to increase L-proline production through new mutants in the prior art.

[0014] To achieve the above-mentioned technical objectives, the present invention provides a γ-glutamyl kinase mutant, wherein the γ-glutamyl kinase mutant is mutated based on the amino acid sequence SEQ ID NO. 2, and the mutation is one or both of E74K and V150M; or, the mutation is E74K, G149A and V150R.

[0015] In any embodiment, the mutant is ProB-E74K, whose gene sequence is shown in SEQ ID NO. 5 and amino acid sequence is shown in SEQ ID NO. 9.

[0016] In any embodiment, the mutant is ProB-V150M, whose gene sequence is shown in SEQ ID NO. 6 and amino acid sequence is shown in SEQ ID NO. 10.

[0017] In any embodiment, the mutant is ProB-E74K-V150M, whose gene sequence is shown in SEQ ID NO. 3 and amino acid sequence is shown in SEQ ID NO. 11.

[0018] In any embodiment, the mutant is ProB-E74K-G149A-V150R, whose gene sequence is shown in SEQ ID NO. 8 and amino acid sequence is shown in SEQ ID NO. 13.

[0019] Furthermore, the present invention also proposes a nucleic acid molecule that encodes the aforementioned γ-glutamyl kinase mutant.

[0020] In addition, the present invention also proposes a recombinant plasmid comprising the above-mentioned nucleic acid molecules.

[0021] Furthermore, the present invention also proposes a host cell containing the aforementioned nucleic acid molecule or the aforementioned recombinant plasmid.

[0022] Furthermore, the present invention also proposes a method for producing L-proline, which is obtained through the above-mentioned host cell fermentation.

[0023] Furthermore, the present invention also proposes the application of the above-mentioned γ-glutamyl kinase mutant, or the above-mentioned nucleic acid molecule, or recombinant plasmid, or the above-mentioned host cell in the preparation of L-proline or proline derivatives.

[0024] Compared with the prior art, the beneficial effects of the present invention include: the γ-glutamyl kinase mutant proposed in the present invention is a mutation based on the gene sequence SEQ ID NO. 1, wherein the mutation is one or both of E74K and V150M; or, the mutation is E74K, G149A and V150R. This mutant can relieve L-proline feedback inhibition, enhance the catalytic performance of the enzyme, and significantly increase the yield of L-proline.

[0025] In addition, this invention combines the E74K mutation with the existing feedback resistance mutation G149A / V150R to obtain a ProB enhanced mutant with higher L-proline synthesis capacity; and constructs a high-yield L-proline engineered strain to improve the level of industrial fermentation production. Attached Figure Description

[0026] Figure 1 This is a map of gRNA expression and homologous repair template plasmid carrying in Example 11 of the present invention.

[0027] Figure 2This is the proline fermentation result of strains carrying different proB mutants in Example 12 of the present invention; wherein, WT: strains 9-12 carry unmutated proB; E74K: strains 9-12 carry mutated proB-E74K; V150M: strains 9-12 carry mutated proB-V150M; E74K-V150M: strains 9-12 carry mutated proB-E74K-V150M; G149A-V150R: strains 9-12 carry mutated proB-G149A-V150R; E74K-G149A-V150R: strains 9-12 carry mutated proB-E74K-G149A-V150R. Detailed Implementation

[0028] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0029] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0030] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0031] This specific embodiment provides a γ-glutamyl kinase mutant, which is a mutation based on the amino acid sequence SEQ ID NO. 2, wherein the mutation is one or both of E74K and V150M; or, the mutation is E74K, G149A and V150R.

[0032] In some embodiments, the mutant is ProB-E74K, whose gene sequence is shown in SEQ ID NO. 5 and amino acid sequence is shown in SEQ ID NO. 9; in some embodiments, the mutant is ProB-V150M, whose gene sequence is shown in SEQ ID NO. 6 and amino acid sequence is shown in SEQ ID NO. 10; in some embodiments, the mutant is ProB-E74K-V150M, whose gene sequence is shown in SEQ ID NO. 3 and amino acid sequence is shown in SEQ ID NO. 11; in some embodiments, the mutant is ProB-E74K-G149A-V150R, whose gene sequence is shown in SEQ ID NO. 8 and amino acid sequence is shown in SEQ ID NO. 13.

[0033] This specific embodiment also proposes a nucleic acid molecule that encodes the above-mentioned γ-glutamyl kinase mutant.

[0034] This specific embodiment also proposes a recombinant plasmid comprising the aforementioned nucleic acid molecules.

[0035] This specific embodiment also proposes a host cell containing the above-described nucleic acid molecule or recombinant plasmid or the above-described recombinant plasmid.

[0036] This specific embodiment also proposes a method for producing L-proline, obtained through the above-mentioned host cell fermentation.

[0037] This specific embodiment also proposes the application of the above-mentioned γ-glutamyl kinase mutant, the above-mentioned nucleic acid molecule, or the above-mentioned host cell in the preparation of L-proline or proline derivatives.

[0038] Obtaining the ProB-E74K / V150M mutant of this invention:

[0039] A random mutant library was constructed using the proB gene from Corynebacterium glutamicum as a template via error-prone PCR.

[0040] Specific steps:

[0041] ① Amplify the proB encoding gene;

[0042] ② Introduce random mutations using error-prone PCR;

[0043] ③ Construct a proB mutant library;

[0044] ④ Introduce the mutant library into Corynebacterium glutamicum;

[0045] ⑤ High-throughput screening of L-proline-producing bacteria by paper chromatography;

[0046] ⑥ The L-proline-producing bacteria that were initially screened were subjected to shake-flask re-screening, and the strain with the highest yield was sequenced for analysis of the proB carried by it.

[0047] Obtained: ProB-E74K / V150M mutant

[0048] in:

[0049] E74K: The 74th glutamic acid (E) is mutated to lysine (K);

[0050] V150M: Valine (V) at position 150 is mutated to methionine (M).

[0051] E74K Function Enhancement Verification

[0052] By comparing genomes carrying the mutant ProB E74K ProB V150M and ProB E74K / V150M 9-12proB E74K 、 9-12proB V150M 、 9-12proB E74KV150M Proline fermentation performance of strains and starting strains 9-12; such as Figure 2 As shown, approximately 0.4 g / L of proline accumulates in cells 9-12, and proB in cells 9-12... E74K The strain accumulated approximately 1.73 g / L proline and 9-12 proB. V150M Accumulated approximately 3.47 g / L proline, however 9-12 proB E74KV150M It can accumulate approximately 6.7 g / L of L-proline. These results indicate that E74K not only participates in feedback resistance regulation but also enhances the catalytic activity of ProB.

[0053] ProB E74K / G149A / V150R Construction of combined mutant engineered bacteria

[0054] Further, the E74K mutation was introduced into the existing ProB... G149A / V150R Feedback-resistant mutants yielded the ProB mutant. E74K / G149A / V150R This was introduced into 9-12 to obtain the mutant strain 9-12proB. E74K / G149A / V150R .like Figure 2 As shown, the fermentation results indicate that 9-12proBE74K / G149A / V150R Mutant strains compared to 9-12proB G149A / V150R Mutant bacteria are able to accumulate more proline.

[0055] This invention discovers a novel ProB functional enhancement site, E74K. Unlike previous studies that focused on the feedback regulatory regions at positions 149 and 150, this invention is the first to demonstrate that the E→K mutation at position 74 of ProB can enhance enzyme catalytic performance. The resulting ProB-E74K-G149A-V150R more effectively increases L-proline accumulation. Additionally, the discovered site V150M also enhances enzyme catalytic performance; the resulting mutants ProB-E74K, ProB-V150M, and ProB-E74K-V150M all effectively increase L-proline accumulation.

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0057] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0058] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0059] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0060] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0061] Table 1. Plasmids and sequences used in the examples

[0062] Example 1: Culture medium and culture conditions

[0063] E. coli JM109 for plasmid cloning was cultured aerobically in LB (Luria–Bertani) medium at 37°C. If appropriate, 50 mg·L⁻¹ was added to the medium. -1 Kanamycin (Kan) or 20 mg / L -1 Chloramphenicol (Cm). Corynebacterium glutamicum and its derivatives in LBG medium at 30°C (LB medium supplemented with 5 g·L⁻¹). -1 Aerobic culture was carried out in Epo medium (LBG medium supplemented with 30 g·L⁻¹ glucose). -1 Glycine, 1 g·L -1 Tween 80 and 4 g·L -1 Isoniazid was used to culture competent Corynebacterium glutamicum cells. LBH medium (2.5 g·L⁻¹) -1 Yeast extract, 5 g·L -1 Yeast extract, 5 g·L -1 Sodium chloride, 18.5 g·L -1 Brain and heart extract, and 91 g·L -1 Sorbitol was used to obtain Corynebacterium glutamicum transformants. CM medium (10 g·L⁻¹) -1 Yeast extract, 10 g·L -1 Beef extract, 10 g·L -1 Sodium chloride and 5 g·L -1 Glucose was used for plasmid elimination. When transforming Corynebacterium glutamicum using a plasmid carrying the kanamycin resistance gene, 25 mg / L glucose was added to LBH medium. -1 Kan.

[0064] Example 2 Plasmid Construction

[0065] The plasmids were constructed using recombination or T4 DNA ligase. The construction process is shown in Table 1.

[0066] Example 3 Preparation and transformation of competent Corynebacterium glutamicum

[0067] Preparation of *Corynebacterium glutamicum* competent cells: The preparation of electrotransformation competent cells for *Corynebacterium glutamicum* was performed according to known methods with appropriate modifications. First, the preserved *Corynebacterium glutamicum* glycerol tubes were activated on CM plates. The activated strain was inoculated into 50 mL Erlenmeyer flasks containing 10 mL LBG medium and incubated at 200 rpm. -1 Incubate at 30°C for 10–13 h at 200 r·min -1 (For seed culture. Then, transfer 3 mL of seed culture to a 500 mL Erlenmeyer flask containing 100 mL of fresh Epo medium.) Incubate at 30°C for 5 h. When the ΔOD of the culture... 600 When the pH reaches 0.4-0.5, place the Erlenmeyer flask in an ice bath for 20 minutes, followed by incubation at 4°C and 4000 r·min. -1 Centrifuge for 5 min to harvest cells. After washing three times with 10% glycerol pre-cooled at 4°C, suspend the cells in 0.3 mL of 10% glycerol pre-cooled for use as competent cells.

[0068] Transformation method for Corynebacterium glutamicum: Plasmids were added to 30–40 μL of competent cells, mixed thoroughly, and transferred to an electroporation cuvette pre-chilled on ice for 5 min at 1 mm (Bio-Rad Laboratories, Shanghai, China). GenePulserXcell was used. TM (Bio-Rad Laboratories, Shanghai, China) Electroporation was performed with parameters set at 1800 V, 5 ms, and 1 mm. Immediately afterwards, 1 mL of LBH medium was added, and the washed cells were rapidly incubated at 46°C for 6 min. Cells were then incubated at 30°C for 2 h, followed by plating with antibiotics and IPTG as needed on LBH plates, and incubated at 30°C until single colonies appeared.

[0069] Example 4 Gene editing of Corynebacterium glutamicum

[0070] One μg of pZF2 series plasmids carrying fgRNA and two ~1 kb homologous arms (HA) were transformed into Corynebacterium glutamicum by electroporation; subsequently, the plasmids were prepared in a solution containing 25 mg·L⁻¹ of glutamic acid. -1 Transformants were screened on LBH agar plates containing kanamycin; finally, after incubation at 30°C for 48 h, single colonies grew on the plates. The correct edited strain was confirmed by colony PCR and sequencing. The pZF2 plasmid possesses a temperature-sensitive replicon pBL. tsThe transformant replicates at 30°C but not at 37°C. Therefore, this characteristic can be used for plasmid elimination. Transformants were inoculated into antibiotic-free LBG medium and incubated overnight at 37°C. The next day, the culture was diluted and plated onto antibiotic-free CM medium plates and incubated at 30°C until single colonies appeared. Plasmid elimination was determined by the colony's sensitivity to antibiotics. Each grown single colony was transferred to a container containing 50 mg·L⁻¹ antibiotics. -1 On kanamycin CM plates and corresponding CM plates. A single colony that cannot grow on the corresponding resistance plate indicates successful plasmid elimination.

[0071] Example 5: Fermentation production of L-proline in a 96-well plate

[0072] Seed culture medium (SM medium) (g·L) -1 Fermentation medium (FM medium): Glucose 30-35, corn steep liquor powder 12-15, soybean meal hydrolysate 6-10, (NH4)2SO4 5-7, K2HPO4·3H2O 1-2, MgSO4·7H2O 0.5-1, FeSO4·7H2O 0.01-0.03, ZnSO4·7H2O 0.01-0.03, Biotin 0.001-0.004, Vitamin B1 0.001-0.004, MOPS 20-25, pH 7-7.2. (g·L) -1 The formula consisted of: glucose 120-150 g, corn steep liquor powder 10-15 g, soybean meal hydrolysate 5-10 g, (NH4)2SO4 35-40 g, K2HPO4·3H2O 1-1.5 g, MgSO4·7H2O 0.5-1.0 g, FeSO4·7H2O 0.03-0.05 g, ZnSO4·7H2O 0.01-0.03 g, MnSO4·4H2O 0.01-0.03 g, MOPS 20-25 g, biotin 0.001-0.004 g, vitamin B1 0.001-0.004 g, and pH 7.0-7.2. The mutant library transformants were inoculated into 96-well plates containing 600 μL of SM medium. The plates were incubated at 30°C and 300 r / min in a constant temperature shaking incubator. -1 Seed cultures were obtained after culturing for 12 h. The seed cultures were then inoculated into 96-well plates, with each well containing 300 μL of FM medium. Initial OD... 600 The value was 0.1. A 96-well plate was tested at 30 °C and 300 r·min. -1 After 48 h of incubation, the culture was stopped and L-proline and glucose were measured.

[0073] Example 6: Production of L-proline by fermentation in shake flasks

[0074] Seed culture was performed using SM medium. FM medium is a fermentation medium. The strain was cultured in 250 mL shake flasks containing 40 mL of SM medium at 30 °C and a rotation speed of 200 r·min. -1 The culture time was 15 h. Then, the seed culture was inoculated into a 500 mL shake flask containing 25 mL of FM medium, with an initial OD of [missing value]. 600 The value is 0.1. At 30℃ and 200 r·min -1 After 48 hours of fermentation, the culture was stopped and glucose and L-proline levels were measured.

[0075] Example 7 Analysis Method

[0076] Amino acids were determined using reversed-phase high-performance liquid chromatography (HPLC) on an Agilent 1290 system. Paper chromatography was used to identify L-proline-producing bacteria. The developing solvent for paper chromatography was a mixture of n-butanol, glacial acetic acid, and water in a volume ratio of 4:1:2. The colorimetric reagent was 5 g·L⁻¹. -1 The ninhydrin-acetone solution was used. After paper chromatography, the filter paper was dried in an oven at 80°C, then sprayed with a colorimetric reagent, and dried again in an oven at 80°C for color development. Finally, the concentration of L-proline was determined based on the size and intensity of the yellow spots.

[0077] Example 8: Construction of proB mutant plasmid library

[0078] The mutant was constructed using a ready-to-use error-prone PCR kit (Beisheng Jingze (Qidong) Biotechnology Co., Ltd.). The Corynebacterium glutamicum genome was used as a template, and Ep-proB-F and Ep-proB-R were used as primers. The error-prone PCR amplification system is shown in Table 2 below.

[0079] Table 2

[0080]

[0081] The error-prone PCR procedures are shown in Table 3 below.

[0082] Table 3

[0083]

[0084] Forty-five cycles were performed. After the reaction, 1 μL of the reaction product was used as a template for another 45 cycles of error-prone PCR. Agarose gel electrophoresis was performed, and the PCR product P1, approximately 1200 fragments in size, obtained from the error-prone PCR amplification, was recovered from the gel. Fragment P2 was amplified using the pH36-VF / R primers with Novizumab P510 high-fidelity enzyme. Subsequently, fragments P1 and P2 were recombined using the Novizumab one-step cloning kit (C112). The recombinant product was transferred into E. coli JM109 competent cells. After heat shock at 42°C for 90 s, the competent cells were directly transferred into 50 mL Erlenmeyer flasks containing 10 mL LB. After recovery at 37°C for 1 h, 50 mg / L kanamycin was added to the flasks, and the cells were cultured for another 10–16 h. The cells were collected by centrifugation, and the plasmid library was extracted.

[0085] The proB gene sequence SEQ ID NO. 1 is shown below:

[0086]

[0087] The amino acid sequence of ProB, SEQ ID NO. 2, is as follows:

[0088] MRERISNAKRVVVKIGSSSSLTNDEDGHTVDPNRINTIVNALQARMEAGSDLIVVSSGAVAAGMAPLGLSTRPTELAVKQAAAAVGQVHLMHQWGRSFARYGRPIGQVLLTAADAGKRDRARNAQRTIDKLRILGAVPIVNENDTVATTGVNFGDNDRLAAIVAHLVSADALVLLSDVDGLFDKN PTDPTAKFISEVRDGNDLKGVIAGDGGKVGTGGMASKVSAARLASRSGVPVLLTSAANIGPALEDAQVGTVFHPKDNRLSAWKFWALYAADTAGKIRLDDGAVEAVTSGGKSLLAVGITEIIGDFQQGEIVEILGPAGQIIGRGEVSYDSDTLQSMVGMQTQDLPDGMQRPVVHADYLSNYASRA

[0089] Example 9: Introduction of mutant plasmid library into Corynebacterium glutamicum 9-12

[0090] Corynebacterium glutamicum 9-12 is formed by knocking out the lactate dehydrogenase encoding gene ldh in the genome of Corynebacterium glutamicum ATCC13032 and replacing it with P. lacM The -cas12a-rrnBT1T2 gene cassette, where the proline dehydrogenase encoding gene putA is knocked out and replaced with P... tac The gene cassette -cas9-rrnBT1T2 was modified. 0.5 μg of the plasmid library was electroporated into Corynebacterium glutamicum strain 9-12. After heat shock at 46℃ for 6 min, the culture was recovered for 1.5 h and plated onto LBHIS plates containing 25 mg / L. The plates were then incubated at 30℃ for 36-48 h to obtain single clones containing the library plasmid.

[0091] Example 10 Screening of proline-producing bacteria

[0092] Wild-type ProB is subject to strict feedback inhibition by proline, and proline does not accumulate during fermentation. (Proline paper chromatography semi-quantitative screening) Proline is an imino acid that reacts with cinnamone to form a yellow compound. The monoclonal strain obtained in Example 9 was fermented according to the method in Example 5, and the proline-producing bacteria were initially screened using paper chromatography as described in Example 7. Compared to wild-type proB-overexpressing bacteria, 19 strains with significantly larger and brighter yellow spots on paper chromatography were screened. These strains were then subjected to shake-flask rescreening, and proline was determined using liquid chromatography as described in Example 7. The strain with the highest yield was sequenced, and the sequencing results showed that ProB carries E74K and V150M mutations. The sequencing alignment results are as follows: The mutation at position 30, A, is changed to T, corresponding to a synonymous mutation; the mutation at position 220, G, is changed to A, corresponding to a mutation at position 74, where glutamic acid residue E is changed to lysine residue K; the mutation at position 448, G, is changed to A, corresponding to a mutation at position 150, where valine residue V is changed to methionine residue M; the mutation at position 636, A, is changed to G, corresponding to a synonymous mutation.

[0093] Example 11 Construction of proB point mutant bacteria

[0094] Strains 9-12 were derived from Corynebacterium glutamicum ATCC13032 carrying the Cas9 and Cas12a encoding genes. Competent cells of strain 9-12 were prepared according to Example 3, and mutant strains were obtained according to Example 4, i.e., the plasmid pZF2-fgRNA-proB was applied to each strain. E74K pZF2-fgRNA-proB V150M pZF2-fgRNA-proB E74KV150M pZF2-fgRNA-proB G149AV150R and pZF2-fgRNA-proB E74KG149AV150R The repair template plasmid was transferred into strains 9-12, and plasmid elimination was verified by strain P, resulting in strains 9-12proB. E74K 9-12proB V150M 9-12proB E74KV150M 9-12proB G149AV150R and 9-12proB E74KG149AV150R Plasmid map as follows Figure 1 As shown.

[0095] Error-prone PCR results in proB E74K / V150M The gene sequence (i.e., the mutant ProB-E74K-V150M) SEQ ID NO. 3 is as follows:

[0096]

[0097] genome integration proB E74K / V150M The gene sequence of the mutant ProB-E74K-V150M (SEQ ID NO. 4) is as follows:

[0098]

[0099] genome integration proB E74K The gene sequence (SEQ ID NO. 5) of the mutant ProB-E74K is as follows:

[0100]

[0101] genome integration proB V150M The gene sequence (SEQ ID NO. 6) of the mutant ProB-V150M is as follows:

[0102]

[0103] genome integration proB G149A / V150R The gene sequence of the mutant ProB-G149A-V150R (SEQ ID NO. 7) is as follows:

[0104]

[0105] genome integration proB E74K / G149A / V150R The gene sequence of the mutant ProB-E74K-G149A-V150R (SEQ ID NO. 8) is as follows:

[0106]

[0107] ProB E74K (i.e., the amino acid sequence of mutant ProB-E74K) SEQ ID NO. 9 is as follows:

[0108] MRERISNAKRVVVKIGSSSLTNDEDGHTVDPNRINTIVNALQARMEAGSDLIVVSSGAVAAGMAPLGLSTRPTKLAVKQAAAAVGQVHLMHQWGRSFARYGRPIGQVLLTAADAGKRDRARNAQRTIDKLRILGAVPIVNENDTVATTGVNFGDNDRLAAIVAHLVSADALVLLSDVDGLFDKNPTDPTAKFISEVRDGNDLKGVIAGDGGKVGTGGMASKVSAARLASRSGVPVLLTSAANIGPALEDAQVGTVFHPKDNRLSAWKFWALYAADTAGKIRLDDGAVEAVTSGGKSLLAVGITEIIGDFQQGEIVEILGPAGQIIGRGEVSYDSDTLQSMVGMQTQDLPDGMQRPVVHADYLSNYASRA

[0109] ProB V150M (i.e., the amino acid sequence of mutant ProB-V150M) SEQ ID NO. 10 is as follows:

[0110] MRERISNAKRVVVKIGSSSLTNDEDGHTVDPNRINTIVNALQARMEAGSDLIVVSSGAVAAGMAPLGLSTRPTELAVKQAAAAVGQVHLMHQWGRSFARYGRPIGQVLLTAADAGKRDRARNAQRTIDKLRILGAVPIVNENDTVATTGMNFGDNDRLAAIVAHLVSADALVLLSDVDGLFDKNPTDPTAKFISEVRDGNDLKGVIAGDGGKVGTGGMASKVSAARLASRSGVPVLLTSAANIGPALEDAQVGTVFHPKDNRLSAWKFWALYAADTAGKIRLDDGAVEAVTSGGKSLLAVGITEIIGDFQQGEIVEILGPAGQIIGRGEVSYDSDTLQSMVGMQTQDLPDGMQRPVVHADYLSNYASRA

[0111] ProB E74K / V150M(i.e., mutant ProB-E74K-V150M) the amino acid sequence of SEQ ID NO. 11 is as follows:

[0112] MRERISNAKRVVVKIGSSSLTNDEDGHTVDPNRINTIVNALQARMEAGSDLIVVSSGAVAAGMAPLGLSTRPTKLAVKQAAAAVGQVHLMHQWGRSFARYGRPIGQVLLTAADAGKRDRARNAQRTIDKLRILGAVPIVNENDTVATTGMNFGDNDRLAAIVAHLVSADALVLLSDVDGLFDKNPTDPTAKFISEVRDGNDLKGVIAGDGGKVGTGGMASKVSAARLASRSGVPVLLTSAANIGPALEDAQVGTVFHPKDNRLSAWKFWALYAADTAGKIRLDDGAVEAVTSGGKSLLAVGITEIIGDFQQGEIVEILGPAGQIIGRGEVSYDSDTLQSMVGMQTQDLPDGMQRPVVHADYLSNYASRA

[0113] ProB G149A / V150R (i.e., mutant ProB-G149A-V150R) the amino acid sequence of SEQ ID NO. 12 is as follows:

[0114] MRERISNAKRVVVKIGSSSLTNDEDGHTVDPNRINTIVNALQARMEAGSDLIVVSSGAVAAGMAPLGLSTRPTELAVKQAAAAVGQVHLMHQWGRSFARYGRPIGQVLLTAADAGKRDRARNAQRTIDKLRILGAVPIVNENDTVATTARNFGDNDRLAAIVAHLVSADALVLLSDVDGLFDKNPTDPTAKFISEVRDGNDLKGVIAGDGGKVGTGGMASKVSAARLASRSGVPVLLTSAANIGPALEDAQVGTVFHPKDNRLSAWKFWALYAADTAGKIRLDDGAVEAVTSGGKSLLAVGITEIIGDFQQGEIVEILGPAGQIIGRGEVSYDSDTLQSMVGMQTQDLPDGMQRPVVHADYLSNYASRA

[0115] ProB E74K / G149A / V150RThe amino acid sequence of the mutant ProB-E74K-G149A-V150R (SEQ ID NO. 13) is as follows:

[0116] MRERISNAKRVVVKIGSSSSLTNDEDGHTVDPNRINTIVNALQARMEAGSDLIVVSSGAVAAGMAPLGLSTRPTKLAVKQAAAAVGQVHLMHQWGRSFARYGRPIGQVLLTAADAGKRDRARNAQRTIDKLRILGAVPIVNENDTVATTARNFGDNDRLAAIVAHLVSADALVLLSDVDGLFDKN PTDPTAKFISEVRDGNDLKGVIAGDGGKVGTGGMASKVSAARLASRSGVPVLLTSAANIGPALEDAQVGTVFHPKDNRLSAWKFWALYAADTAGKIRLDDGAVEAVTSGGKSLLAVGITEIIGDFQQGEIVEILGPAGQIIGRGEVSYDSDTLQSMVGMQTQDLPDGMQRPVVHADYLSNYASRA

[0117] Example 12: Validation of Fermentation Performance of Mutant Strains

[0118] 9-12proB E74K 9-12proB V150M 9-12proB E74KV150M 9-12proB G149AV150R and 9-12proB E74KG149AV150R Strains 9-12 were fermented for proline according to the method in Example 6, and proline was detected by liquid chromatography according to the method in Example 7. The results are as follows. Figure 2 As shown in the figure. Fermentation results indicated that, compared to the wild-type proB strain 9-12 (WT), it accumulated 0.40 ± 0.10 g·L⁻¹. -1 L-proline, carrying proB E74K Strain 9-12proB E74K (E74K) Accumulation 1.73 ± 0.15 g·L -1 L-proline levels increased by 332.5%, indicating that E74K significantly promotes L-proline accumulation. The V150 site is a modified site proposed in existing technologies to relieve proline feedback inhibition; V150M exhibits a certain effect in relieving proline feedback inhibition, accumulating 3.47 ± 0.14 g·L⁻¹. -1 L-proline was not tolerated; however, the E74K-V150M mutant significantly increased proline accumulation, reaching 6.71 ± 0.19 g·L⁻¹. -1L-proline, these results indicate that the E74K mutation may contribute to the performance of feedback inhibition mutants. Therefore, the E74K mutation was introduced into the mutant proB-G149A-V150R to obtain proB-E74K-G149A-V150R. The results showed that the mutant strain 9-12 carrying proB-E74K-G149A-V150R (E74K-G149A-V150R) achieved a yield of 8.57 ± 0.31 g·L⁻¹. -1 L-proline was significantly higher than that of the mutant strain 9-12proB-G149A-V150R (G149A-V150R), which carried proB-G149A-V150R (7.87 ± 0.12 g·L⁻¹). -1 L-proline increased by 8.77%.

[0119] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A γ-glutamyl kinase mutant, characterized in that, The γ-glutamyl kinase mutant is a mutation based on the amino acid sequence SEQ ID NO. 2; the mutant is ProB-E74K-G149A-V150R, and its gene sequence is shown in SEQ ID NO.

8.

2. The γ-glutamyl kinase mutant according to claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

13.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the γ-glutamyl kinase mutant of claim 1.

4. A recombinant plasmid, characterized in that, Includes the nucleic acid molecule as described in claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the recombinant plasmid of claim 4.

6. A method for producing L-proline, characterized in that, Obtained by host cell fermentation as described in claim 5.

7. The use of the γ-glutamyl kinase mutant of claim 1, the nucleic acid molecule of claim 3, the recombinant plasmid of claim 4, or the host cell of claim 5 in the preparation of L-proline or proline derivatives.

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