Pentamine-tolerant corynebacterium glutamicum mutant strain and mutation site application thereof

Abstract

The invention discloses a pentamethylene diamine tolerant corynebacterium glutamicum mutant strain and mutation site application thereof, and belongs to the field of microbial metabolic engineering. According to the invention, genes which have obvious influence on the pentanediamine tolerance of corynebacterium glutamicum are screened and identified, and a series of gene mutant strains are constructed. The pentanediamine tolerance concentration of the mutant strain constructed by the invention can reach 75 g / L under a shake flask fermentation condition, and is improved by 1.5 times compared with that of an original strain.

Description

Technical Field

[0001] This invention relates to a pentanediamine-resistant Corynebacterium glutamicum mutant strain and the application of its mutant site, belonging to the field of microbial metabolic engineering. Background Technology

[0002] Pentamethylene diamine, also known as cadaverine, is a crucial raw material for the production of nylon PA5X. Compared to energy-intensive traditional chemical synthesis processes, the biosynthesis of pentamethylene diamine offers advantages such as high reaction selectivity, environmentally friendly processes, and high resource utilization, making it a key direction for the development of bio-based nylon materials. Pentamethylene diamine prepared using bio-fermentation technology can replace the traditional petroleum-derived synthetic monomer hexamethylenediamine in nylon synthesis, helping to reduce my country's dependence on hexamethylenediamine products from the international market. With the increasing maturity of microbial fermentation technology, research on the biosynthesis of pentamethylene diamine is deepening. Constructing microbial cell factories and utilizing inexpensive sources such as glucose to produce various high-value-added products, including pentamethylene diamine, has broad prospects for production and application. In the main pathway for the microbial fermentation synthesis of pentanediamine, glucose is converted into oxaloacetic acid, a common precursor of various amino acids, via the EMP pathway and TCA cycle. After a multi-step enzymatic catalytic reaction, lysine is produced from aspartic acid. Then, L-lysine is used as a substrate for decarboxylation to generate pentanediamine. This pathway significantly reduces raw material costs and also helps to alleviate the current situation of lysine overcapacity in my country.

[0003] In the design and construction of efficient pentanediamine (PBD) cell factories, PBD toxicity is a significant limiting factor in PBD production. Identifying and screening PBD tolerance target proteins is crucial for overcoming this limitation. However, using mutagenesis breeding, modifying key synthases and regulating metabolic nodes in the pathway has proven insufficient to enhance PBD tolerance in *Corynebacterium glutamicum* strains, and random mutagenesis strategies suffer from individual genetic instability. Therefore, there is an urgent need to screen for a *Corynebacterium glutamicum* strain with high PBD tolerance while simultaneously revealing relevant potential key genes. Summary of the Invention

[0004] To address the problem of low tolerance of Corynebacterium glutamicum to pentanediamine in existing technologies, this invention provides a method for improving the pentanediamine tolerance of Corynebacterium glutamicum based on adaptive evolution, and further provides highly tolerant strains obtained by screening through this method and their key mutation sites.

[0005] This invention provides an engineered strain of *Corynebacterium glutamicum* with significantly enhanced tolerance to pentanediamine, possessing one or more of the following mutant genes: dnaA mutant gene dnaA D18Y dnaA mutant gene dnaA D365NfolP2 mutant gene folP2 M259K mqo mutant gene mqo W224C and the cgl1283 mutant gene cgl1283 G345D .

[0006] In one embodiment, the dnaA mutant gene dnaA D18Y The nucleotide sequence is shown in SEQ ID NO.6, dnaA mutant gene dnaA. D365N The nucleotide sequence is shown in SEQ ID NO.7, folP2 mutant gene folP2 M259K The nucleotide sequence is shown in SEQ ID NO.8, mqo mutant gene mqo W224C The nucleotide sequence is shown in SEQ ID NO.9, cgl1283 mutant gene cgl1283. G345D The nucleotide sequence is shown in SEQ ID NO.10.

[0007] In one embodiment, the engineered Corynebacterium glutamicum is based on ATCC13032 as the starting strain.

[0008] The present invention also provides a method for improving the tolerance of Corynebacterium glutamicum to pentanediamine, wherein the method involves introducing point mutations in the dnaA, folP2, mqo, or cgl1283 genes into the genome of Corynebacterium glutamicum.

[0009] In one embodiment, the mutation is to mutate the dnaA gene (Gene ID: 1021144) to the mutant dnaA gene shown in SEQ ID NO. 6. D18Y .

[0010] In one embodiment, the mutation is to mutate the dnaA gene (Gene ID: 1021144) to the mutant gene dnaA shown in SEQ ID NO. 7. D365N .

[0011] In one embodiment, the mutation is to mutate the folP2 gene (Genbank accession number: QYO73341.1) to the mutated folP2 gene shown in SEQ ID NO.8. M259K .

[0012] In one embodiment, the mutation is to mutate the mqo gene (Gene ID: 1019958) to the mutant mqo gene shown in SEQ ID NO. 9. W224C .

[0013] In one embodiment, the mutation is to mutate the cgl1283 gene (Gene ID: 1019264) to the mutant gene cgl1283 shown in SEQ ID NO. 10. G345D .

[0014] In one embodiment, the engineered Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13032 as the starting strain.

[0015] The present invention also provides proteins with amino acid sequences as shown in any of SEQ ID NO. 1 to 5, or genes with nucleotide sequences as shown in any of SEQ ID NO. 6 to 10.

[0016] The present invention also provides the application of the engineered strain of Corynebacterium glutamicum as chassis cells.

[0017] In one embodiment, the application includes, but is not limited to, the preparation of pentanediamine-producing strains or their immobilized cells.

[0018] Beneficial effects: This invention obtained a mutant strain of *Corynebacterium glutamicum* through pentanediamine-mediated laboratory adaptive evolution screening. Under shake-flask fermentation conditions, this strain exhibited a pentanediamine tolerance concentration of up to 75 g / L, 1.5 times higher than the original strain, with a maximum OD562 of 10.9. Based on the genotypic changes of the mutant strain, this invention constructed a recombinant *Corynebacterium glutamicum* strain with enhanced pentanediamine tolerance. After 30 consecutive passages, its tolerance remained consistent with the initial strain, demonstrating good passage stability. Attached Figure Description

[0019] Figure 1 The growth of strain ATCC13032, the starting strain of Example 1 of this invention, during the in vitro passage pressure screening process is shown; wherein: a) 10 g / L pentamethylenediamine, b) 15 g / L pentamethylenediamine, c) 20 g / L pentamethylenediamine, d) 30 g / L pentamethylenediamine, e) 40 g / L pentamethylenediamine, f) 60 g / L pentamethylenediamine.

[0020] Figure 2 The growth of the evolved strain and the starting strain ATCC13032 in Example 2 of this invention under different concentrations of pentanediamine; wherein, a) 0 g / L pentanediamine, b) 15 g / L pentanediamine, c) 30 g / L pentanediamine, d) 45 g / L pentanediamine, e) 60 g / L pentanediamine, f) 75 g / L pentanediamine, g) 100 g / L pentanediamine, h) the maximum growth rate at different concentrations of pentanediamine.

[0021] Figure 3This describes the pentamethylenediamine tolerance of different single mutation sites and the cumulative five mutation sites in Example 3 of the present invention.

[0022] Figure 4 This describes the pentamidine tolerance of the five mutant site-accumulated strains and the evolved strains after 30 generations in Example 4 of the present invention. Detailed Implementation

[0023] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1: Pentylenediamine-mediated laboratory adaptive evolution Using *Corynebacterium glutamicum* ATCC 13032 as the starting strain, LBGB medium was used as the fermentation medium. The LBGB medium consisted of: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 5 g / L glucose, and 18.5 g / L brain and heart extract. Pentylene diamine was added at a concentration of 10–60 g / L (increasing with evolutionary stage) as needed during the culture process, with water as the solvent. The medium was prepared by thoroughly mixing all components and then sterilizing.

[0025] The specific evolutionary experimental procedure is as follows: First, a single colony was picked from a fresh LBGB solid plate and inoculated into 5 mL of LBGB liquid medium, and cultured overnight at 30°C with shaking. Then, a 5% (v / v) inoculum was transferred to 50 mL of LBGB medium containing 5 g / L pentamethylenediamine, and cultured for 12 h, during which OD600 was measured every 4 h. After the OD600 growth trend slowed down, the bacterial culture was diluted with physiological saline to OD600 = 0.1, and then a 5% (v / v) inoculum was transferred to 10 mL of fresh LBGB medium containing 10 g / L pentamethylenediamine for the next generation culture.

[0026] like Figure 1As shown, the strain was continuously passaged four times at the same pentanediamine concentration until the average growth rate stabilized within 36 h. Then, it was transferred to LBGB medium with a one-level increase in pentanediamine concentration for further cultivation. This passage process was repeated sequentially in LBGB medium with pentanediamine concentrations of 10, 15, 20, 30, 40, and 60 g / L until the final tolerant concentration reached 60 g / L. Finally, the bacterial culture after cultivation in 60 g / L pentanediamine LBGB medium was spread onto LBGB solid plates containing the same concentration of pentanediamine. After 36 h of cultivation, single colonies were isolated to obtain the adaptively evolved strain.

[0027] Example 2: Determination of pentamethylenediamine inhibitory concentration in evolved strains The starting strain *Corynebacterium glutamicum* ATCC13032 and the strain obtained through adaptive evolution in Example 1 were streaked onto fresh LBGB solid medium and incubated statically at 30°C for 36 h. Single colonies were then picked and inoculated into 5 mL of LBGB liquid medium and incubated overnight at 30°C and 200 rpm. Subsequently, 5% (v / v) inoculum was transferred to 30 mL of LBGB liquid medium containing different concentrations of pentanediamine, with pentanediamine concentration gradients of 0, 15, 30, 45, 60, 75, and 100 g / L. All shake flasks were incubated at 30°C and 200 rpm for 48 h. After incubation, the maximum OD600 and growth rate of the bacteria under each condition were determined according to standard methods.

[0028] The results are as follows Figure 2 As shown, compared with the starting strain, the evolved strain exhibited increased maximum growth rate and maximum OD600 at different pentanediamine concentrations, while no significant difference in growth behavior was observed between the two strains under pentanediamine-free conditions. The evolved strain achieved a maximum tolerance concentration of 75 g / L for pentanediamine in LBGB medium during shake-flask fermentation, 1.5 times higher than the starting strain's maximum tolerance concentration of 30 g / L. Under these conditions, the evolved strain had a maximum OD600 of 7.21 and a maximum specific growth rate of 0.281; while the starting strain had a maximum OD600 of 0.32 and a maximum specific growth rate of 0.037.

[0029] Example 3: Determination of Mutation Sites in Evolved Strains To elucidate the molecular mechanism by which evolved strains enhance tolerance to pentanediamine, we performed whole-genome resequencing on the original strain *Corynebacterium glutamicum* ATCC13032 and its evolved mutant strains. Alignment analysis identified 25 single nucleotide mutations, including 5 missense mutations that may affect protein function. To verify the role of these mutations in pentanediamine tolerance, we constructed 5 single-point mutant recombinant strains using reverse metabolic engineering, namely: dnaA... D18Y Mutant strains AC1 and dnaAD365N Mutant strains AC2 and folP2 M259K Mutant strains AC3 and cgl1283 G345D Mutant strains AC4, mqo W224C The mutant strain AC5 was used to verify the function of these mutations using a reverse metabolic engineering strategy.

[0030] (1) Construction of single-point mutant strains AC1~AC5 Shuttle plasmid construction: Recombinant plasmids containing specific point mutations (nucleotide sequences SEQ ID NO. 6~SEQ ID NO. 10) were constructed using seamless cloning technology. Using the commercially available plasmid pK18mobSacB as a vector, recombinant plasmids AC1~AC5 containing the five mutation sites mentioned above were constructed: dnaA D18Y plasmid AC1, dnaA D365N plasmids AC2 and folP2 M259K plasmids AC3 and cgl1283 G345D plasmids AC4 and mqo W224C Plasmid AC5. As shown in Tables 1-5, primers covering upstream and downstream homologous arms and vector linearization primers were designed for each mutation site. After PCR amplification of the corresponding fragments, each fragment was inserted into the linearized vector using seamless cloning technology to construct the corresponding mutant plasmid.

[0031] Table 1 dnaA D18Y Primers used for constructing shuttle plasmid AC1

[0032] Table 2 dnaA D365N Primers used for constructing shuttle plasmid AC2

[0033] Table 3 folP2 M259K Primers used for constructing shuttle plasmid AC3

[0034] Table 4 cgl1283 G345D Primers used for constructing shuttle plasmid AC4

[0035] Table 5 mqo W224C Primers used for constructing shuttle plasmid AC5

[0036] Prepare the PCR amplification system according to Table 6 and perform PCR amplification.

[0037] Table 6 PCR amplification system

[0038] PCR amplification conditions: 1) Pre-denaturation: 98 ℃ for 30 s; 2) Denaturation: 98 ℃ for 10 s; Annealing: 55 ℃ for 5 s; Extension: 72 ℃ for 30 s; 34 cycles in total; 3) Post-extension: 72 ℃ for 1 min; 4) Storage at 4 ℃.

[0039] Prepare a seamless cloning system according to Table 7 and perform seamless cloning.

[0040] Table 7 Seamless Cloning System

[0041] Seamless cloning conditions: react at 50℃ for 30-60 min, and store the reaction product at 4℃.

[0042] Transformation and Validation: Five groups of seamless clones were transformed into [a specific technology / system] using the thermal shock method. E. coli BL21(DE3) competent cells. Competent cells were removed from -80 °C and thawed on ice for 2 min. 10 μL of the ligation product was then mixed with 100 μL of... E. coli BL21(DE3) competent cells were mixed and incubated on ice for 15 min. Centrifuge tubes were then heat-shocked in a 42 ℃ water bath for 90 s, immediately removed, and incubated on ice for 2 min. In a sterile environment, 800 μL of LB medium was added to the centrifuge tubes, mixed, and incubated at 37 ℃ and 200 rpm for 45 min to revive the cells. After revival, the cells were centrifuged at 5000 rpm at room temperature for 2 min. Part of the supernatant was discarded in a clean bench, and the remaining liquid (approximately 100 μL) was mixed with the cells. The mixture was then evenly spread onto LB solid medium containing Kans antibiotic using a disposable spreader. After complete absorption, the plates were inverted and incubated at 37 ℃ for approximately 16 h. Single colonies were picked from each plate for sequencing verification.

[0043] (2) Preparation of competent cells transformed from Corynebacterium glutamicum: 1) Inoculate 400 μL of Corynebacterium glutamicum into seed culture medium and incubate overnight (approximately 12 h) at 200 rpm and 30 ℃. 2) Transfer 5 mL of bacterial culture to 50 mL of Epo medium and incubate at 200 rpm and 30 ℃ for 5 h; 3) Incubate the bacterial culture in an ice-water bath for 30 minutes, then centrifuge at 6000 rpm and 4 ℃ for 6 minutes; 4) Take about 30 mL of pre-cooled 10% glycerol, fully suspend the bacterial cells, centrifuge at 5000 rpm and 4 ℃ for 6 min; then take pre-cooled 10% glycerol and wash twice more.

[0044] 5) Resuspend the cells in 400 μL of pre-cooled 10% glycerol, aliquot into 1.5 mL centrifuge tubes, 100 μL per tube, approximately 5 tubes, freeze at -80 ℃ for 2 h, and then electroporate.

[0045] 6) Take 10 μL of pre-chilled plasmid on ice and mix it with competent cells, then incubate on ice for 10 min; 7) Add to a pre-cooled 2 mm shock cup and shock twice at 2.5 KV; add 1 mL of preheated LBHIS medium, mix well, transfer to a 1.5 mL centrifuge tube, and heat shock at 46 ℃ for 6 min. 8) Incubate at 30 ℃ and 100 rpm for 2 hours; 9) Centrifuge at 5000 rpm for 2 min and collect the bacterial cells; 10) Aspirate the supernatant until approximately 100 μL of bacterial culture remains, mix well, and spread onto LBHIS plates containing kanamycin; incubate at 30°C for approximately 36 h. Pick a single colony from each plate for sequencing verification.

[0046] (3) Verification of the effect of single-point mutation The starting strain, AC1–AC5, and the evolutionary mutant strain were inoculated into LBGB medium containing different concentrations of pentanediamine according to the method described in Example 2, and cultured at 30°C and 200 rpm for 36 hours. Their maximum OD values ​​were measured, and the results are as follows: Figure 3 As shown, dnaA D18Y dnaA D365N folP2 M259K cgl1283 G345D and mqo W224C The maximum OD values ​​at 75 g / L pentanediamine concentrations were 2.01, 1.87, 1.96, 2.26, and 2.03, respectively, all higher than the original strain's maximum OD of 0.32 at this concentration. The maximum OD of the evolved mutant strain reached 7.21. These results indicate that all five mutation sites positively contribute to pentanediamine tolerance. Therefore, dnaA D18Y dnaA D365N folP2 M259K cgl1283 G345D and mqo W224C It has been identified as a novel target for pentamethylenediamine tolerance modification in this invention.

[0047] (4) Verification of the effect of five-point mutation Referring to the method described in Example (2), the mutation sites of AC1–AC5 were sequentially transformed into the starting strain to construct the five-point mutant strain AC6. Following the method described in Example 2, the strain was inoculated into LBGB medium containing different concentrations of pentanediamine and cultured at 30°C and 200 rpm for 36 hours. The maximum OD value was then measured, and the results are as follows: Figure 3 As shown, the maximum OD of strain AC6 is consistent with that of the evolutionary mutant strain. This result indicates that the combination of the five mutation sites exhibits a synergistic effect, significantly enhancing the strain's tolerance to pentanediamine.

[0048] Example 4: Determination of the stability of the strain after passage The strain constructed in Example 3 was cultured according to the method in Example 2 and passaged 30 times. The passaged strain AC6 was inoculated into LBGB medium containing different concentrations of pentanediamine and cultured at 30°C and 200 rpm for 36 hours. Its maximum OD value was measured and compared with the tolerance test of the primary strain under the same conditions. The results are as follows: Figure 4 As shown, the maximum OD of strain AC6 is consistent with that of the primary strain. This result indicates that the evolved mutant strain and the mutation site exhibit good passage stability.

[0049] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Corynebacterium glutamicum engineered bacteria with enhanced tolerance to pentamethylenediamine, characterized in that, having one or more of the following mutant genes: a dnaA mutant gene, a folP2 mutant gene folP2 M259K , a mqo mutant gene mqo W224C , a cgl1283 mutant gene cgl1283 G345D ; the dnaA mutant gene comprises a D18Y and / or D365N mutation.

2. Corynebacterium glutamicum engineered bacterium according to claim 1, characterized in that, The nucleotide sequence of the dnaA mutant gene is shown as SEQ ID NO. 6 or SEQ ID NO. 7, the nucleotide sequence of the folP2 mutant gene folP2 M259K is shown as SEQ ID NO. 8, the nucleotide sequence of the mqo mutant gene mqo W224C is shown as SEQ ID NO. 9, and the nucleotide sequence of the cgl1283 mutant gene cgl1283 G345D is shown as SEQ ID NO.

10.

3. The Corynebacterium glutamicum engineered bacterium of claim 1 or 2, characterized in that, ATCC13032 as the starting strain.

4. A method for increasing glutamate resistance in Corynebacterium glutamicum, characterized by, introducing a point mutation of dnaA, folP2, mqo or cgl1283 gene in the genome of Corynebacterium glutamicum, including but not limited to Corynebacterium glutamicum ATCC13032; the point mutation is selected from one or more of the following: D18Y and / or D365N mutation of the dnaA gene, M259K mutation of the folP2 gene, W224C mutation of the mqo gene, and G345D mutation of the cgl1283 gene.

5. The method of claim 4, wherein, the mutation is that the dnaA gene shown in Gene ID: 1021144 is mutated into the sequence shown in SEQ ID NO. 6 or SEQ ID NO.

7.

6. The method of claim 4, wherein, the mutation is that the coding gene of folP2 shown in Genbank Accession No: QYO73341.1 is mutated into the sequence shown in SEQ ID NO.

8.

7. The method of claim 4, wherein, the mutation is that the mqo gene shown in Gene ID: 1019958 is mutated into the sequence shown in SEQ ID NO.

9.

8. The method of claim 4, wherein, the mutation is that the cgl1283 gene shown in Gene ID: 1019264 is mutated into the sequence shown in SEQ ID NO.

10.

9. A biomaterial, characterized in that, (a) or (b): (a) a protein with an amino acid sequence shown in any one of SEQ ID NO. 1-5; (b) a gene with a nucleotide sequence shown in any one of SEQ ID NO. 6-10.

10. Corynebacterium glutamicum engineering bacteria as claimed in any one of claims 1-3 for use as a chassis cell.

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