Construction method and application of bacillus amyloliquefaciens chassis strain

Abstract

The invention provides a construction method and application of a bacillus amyloliquefaciens chassis strain, bacillus amyloliquefaciens HZ12 is taken as a starting strain, two key genes of a non-PTS system are screened and identified from a glucose transport pathway, and the bacillus amyloliquefaciens chassis strain is obtained. According to the present invention, the L-tyrosine genes are respectively a glucose osmose gene Bao-glcP1 derived from bacillus amyloliquefaciens and a glucokinase gene Cgb-glK derived from corynebacterium glutamicum, and after the genes are subjected to overexpression, the L-tyrosine yields of the engineering bacteria respectively achieve 395.82 mg / L and 385.37 mg / L, and are increased by 36% and 33% compared with the original strain; then, by strengthening a non-PTS system, weakening the PTS system and reconstructing glucose uptake and phosphorylation pathways of the strain, the phosphorylation process of glucose and PEP are decoupled, a chassis strain with high yield of L-tyrosine is obtained, and the yield of L-tyrosine is increased to 407.16 mg / L and is increased by 43% compared with that of an original strain. Key rate-limiting enzyme genes aroA and tyrA for synthesizing L-tyrosine are further overexpressed in the chassis strain, so that the yield of the L-tyrosine is increased to 1400.53 mg / L and is increased by 3.82 times compared with that of an original strain.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, specifically relating to a method for constructing a Bacillus amyloliquefaciens chassis strain and its application. Background Technology

[0002] L-Tyrosine, an important aromatic amino acid, is not only a key component of protein synthesis but also a crucial precursor for many high-value bioactive compounds (such as levodopa, resveratrol, and various alkaloids), possessing broad market prospects in the pharmaceutical, nutritional supplement, and fine chemical industries. With the rapid development of synthetic biology and metabolic engineering, the green bio-manufacturing of L-tyrosine using microbial cell factories has become a mainstream alternative to traditional chemical extraction methods. Among numerous microbial hosts, *Bacillus amyloliquefaciens*, with its superior protein secretion capacity, clear genetic background, and recognized safe strains, is considered an excellent platform for producing amino acids and their derivatives. However, the efficient synthesis of L-tyrosine using the strain's natural metabolic network, particularly its carbon source utilization efficiency, especially glucose uptake and primary metabolic pathways, often becomes a key bottleneck limiting the accumulation of the target product.

[0003] In *Bacillus amyloliquefaciens*, glucose uptake and phosphorylation primarily rely on the phosphotransferase system (PTS). The PTS is a highly efficient but metabolically costly system: simultaneously with glucose transport into the cell, phosphoenolpyruvate (PEP) is directly used as a phosphate donor, phosphorylating it to glucose-6-phosphate. While this process achieves simultaneous glucose transport and activation, it also leads to irreversible PEP consumption. Notably, PEP is a key metabolic precursor in the initiation stage of aromatic amino acid synthesis pathways. In the shikimic acid pathway, PEP condenses with erythrose 4-phosphate under the catalysis of DAHP synthase, which is the rate-limiting step of the entire pathway. In *E. coli*, 50% of intracellular PEP flows to the PTS system for glucose transport. Therefore, there is direct competition between PTS-mediated glucose uptake and L-tyrosine synthesis for PEP; this inherent metabolic conflict is considered a core factor limiting further increases in L-tyrosine production, as illustrated in the diagram below. Figure 1 As shown. Based on this, replacing PTS with an uptake mechanism and phosphorylation function independent of phosphoenolpyruvate may help enhance L-tyrosine synthesis in Bacillus amyloliquefaciens. Summary of the Invention

[0004] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to provide a method for constructing a Bacillus amyloliquefaciens chassis strain and its application, reconstructing the glucose transport system of Bacillus amyloliquefaciens to increase L-tyrosine production. By identifying and targeting key genes involved in glucose uptake and phosphorylation in Bacillus amyloliquefaciens, and by screening and introducing high-performance heterologous glucose transporters and glucokinases, a novel, highly efficient glucose utilization pathway adapted to L-tyrosine synthesis is constructed in Bacillus amyloliquefaciens. Further optimization of the UTR enhances the expression levels of key genes. Finally, by weakening the endogenous PTS system and strengthening non-PTS pathways, the glucose phosphorylation process is decoupled from PEP, relying instead on ATP for energy, providing a new strategy and material basis for the efficient microbial production of this amino acid and its derivatives.

[0005] Technical solution: A Bacillus amyloliquefaciens chassis strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH Using Bacillus amyloliquefaciens as the starting strain, the glucose permease gene was overexpressed. glcP 1 and glucokinase gene glK It also lacks the gene encoding the phosphoryl carrier protein HPr. ptsH The Bacillus amyloliquefaciens chassis strain HZ12:: was constructed. Bao - glcP 1:: Cgb - glK Δ ptsH .

[0006] Preferably, the glucose permease gene glcP 1 is derived from Bacillus amyloliquefaciens, and its nucleotide sequence is shown in SEQ ID NO.2.

[0007] Preferably, the glucokinase gene glK It is derived from Corynebacterium glutamicum, and its nucleotide sequence is shown in SEQ ID NO.7.

[0008] The above-mentioned Bacillus amyloliquefaciens chassis strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH The construction method includes the following steps:

[0009] S1. Using the genome of Bacillus amyloliquefaciens as a template, amplify homologous arm A, homologous arm B, and gene fragment P43+UTR1+. Bao - glcP1+T amyL Homologous arms A and B were coupled with P43+UTR1+ via SOE-PCR. Bao - glcP 1+T amyL Fragments are joined together to obtain a fusion fragment, which is then processed using a restriction endonuclease. Xba I and BamH The fusion fragment and the temperature-sensitive knockout plasmid T2(2)-ori were digested with enzyme I. The digested fusion fragment and T2(2)-ori were ligated with T4 ligase. The ligation product was transformed into competent E. coli cells to obtain the gene. Bao - glcP The integrated expression vector T2(2)-1 XKDG :: Bao - glcP 1;

[0010] S2. Integrate expression vector T2(2)- XKDG :: Bao - glcP 1. Electroporation was performed into competent Bacillus amyloliquefaciens cells, and positive transformants were identified. Single-crossover and double-crossover strains were screened and identified from the positive transformants to obtain the integrative strain HZ12:: Bao - glcP 1;

[0011] S3. Using the genome of Bacillus amyloliquefaciens as a template, amplify homologous arm A, homologous arm B, and gene fragment P43+. Cgb - glK +T amyL Homologous arms A and B were linked to gene fragment P43+ by SOE-PCR. Cgb - glK +T amyL Fragments are joined together to obtain a fusion fragment, which is then processed using a restriction endonuclease. Xba I and BamH The fusion fragment and the temperature-sensitive knockout plasmid T2(2)-ori were digested with enzyme I. The digested fusion fragment and T2(2)-ori were ligated with T4 ligase. The ligation product was transformed into competent E. coli cells to obtain the gene. Cgb - glK The integrated expression vector T2(2)- XKDE :: Cgb - glK ;

[0012] S4. Integrate expression vector T2(2)- XKDE :: Cgb - glK The integrated strain HZ12:: was constructed by electroporation to S2 Bao - glcPIn step 1, the integrated strain HZ12:: was obtained. Bao - glcP 1:: Cgb - glK ;

[0013] S5. Using the genome of Bacillus amyloliquefaciens as a template, amplify homologous arm A and homologous arm B, ligate homologous arm A and homologous arm B together by SOE-PCR to obtain the fusion fragment, and then use restriction endonucleases... Xba I and BamH The fusion fragment and the temperature-sensitive knockout plasmid T2(2)-ori were digested with enzyme I. The digested fusion fragment and T2(2)-ori were ligated with T4 ligase. The ligation product was transformed into competent E. coli cells to obtain the gene. ptsH Knockout vector T2(2)Δ ptsH ;

[0014] S6. Knockout vector T2(2)Δ ptsH The integrated strain HZ12:: was constructed by electroporation to S4 Bao - glcP 1:: Cgb - glK In the process, chassis strain HZ12:: was obtained. Bao - glcP 1:: Cgb - glK Δ ptsH .

[0015] A genetically engineered Bacillus amyloliquefaciens strain, HZ13 / tyrA - aroA In the above-mentioned Bacillus amyloliquefaciens chassis strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH Based on this, overexpression of the DAHP synthase gene aroA and prephenyl acid dehydrogenase gene tyrA The genetically engineered Bacillus amyloliquefaciens strain HZ13 / was constructed. tyrA - aroA .

[0016] The above-mentioned Bacillus amyloliquefaciens chassis strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH Or genetically engineered Bacillus amyloliquefaciens HZ13 / tyrA - aroA Applications in the production of L-tyrosine.

[0017] The specific method for producing L-tyrosine described above includes the following steps:

[0018] S1. Bacillus amyloliquefaciens chassis strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH Or genetically engineered Bacillus amyloliquefaciens HZ13 / tyrA - aroA After activation on LB plates, single colonies are picked and inoculated into LB liquid medium and cultured at 35-38℃ and 180-220 rpm for 8-12 h to obtain seed culture;

[0019] S2. The seed culture is inoculated into the fermentation medium at an inoculum of 3-5%, and fermented at 35-38℃ and 143-170 rpm for 36-40 h to produce L-tyrosine.

[0020] Preferably, the fermentation medium is composed of: glucose 20-25 g / L, (NH4)2SO4 2.5-4.5 g / L, K2HPO4 6-7 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 1-2 g / L, sodium citrate 4-6 g / L, peptone 4-6 g / L, yeast extract 5-7 g / L, and trace elements 1 mL / L.

[0021] Preferably, the composition of the trace elements is as follows: sodium citrate 9~12 g / L, FeSO4·7H2O 3~5 g / L, CaCl2 3~5 g / L, MnSO4·5H2O 0.5~2 g / L, CoCl2·6H2O 0.2~0.6 g / L, ZnSO4·7H2O 0.1~0.4 g / L, AlCl3·6H2O 0.05~0.2 g / L, CuCl2·H2O 0.05~0.2 g / L, and H3BO4 0.001~0.006 g / L.

[0022] Beneficial effects:

[0023] This invention uses food-grade Bacillus amyloliquefaciens HZ12 as the starting strain. To reduce the consumption of L-tyrosine PEP, a novel, highly efficient glucose utilization pathway adapted to L-tyrosine synthesis was constructed in Bacillus amyloliquefaciens. First, a novel glucose utilization pathway derived from Bacillus amyloliquefaciens was screened and identified. Bao - glcP 1 with Corynebacterium glutamicum Cgb - glKTwo key genes were identified. Overexpression of these genes in *Bacillus amyloliquefaciens* HZ12 resulted in L-tyrosine production of 395.82 mg / L and 385.37 mg / L, respectively, representing increases of 36% and 33% compared to the starting strain. Systematic optimization of the 5'-UTR further increased L-tyrosine synthesis to 459.64 mg / L. By reconstructing the glucose uptake and phosphorylation pathways of the strain, a high-yielding L-tyrosine chassis strain, HZ12::, was obtained. Bao - glcP 1:: Cgb - glK Δ [[ID=I06]]ptsH Its L-tyrosine production increased to 407.16 mg / L, a 43% increase compared to the starting strain. In the chassis strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH Based on this, the key rate-limiting enzyme gene for L-tyrosine synthesis was further overexpressed. aroA and tyrA The L-tyrosine production was further increased to 1400.53 g / L, which is 3.82 times higher than that of the original strain. Attached Figure Description

[0024] Figure 1 A schematic diagram illustrating glucose transport in Bacillus amyloliquefaciens. Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0026] The *Bacillus amyloliquefaciens* HZ12 involved in the following examples and comparative examples was preserved in the Microbial Engineering Laboratory of Huazhong Agricultural University; the overexpression vector pHY- [[ID=1I5]]tyrA - aroA It is kept in the inventor's laboratory.

[0027] Example 1.

[0028] This embodiment is based on Bacillus amyloliquefaciens (Bacillus). Bacillus amyloliquefaciens The overexpression vector pHY- of glucose permease from ) Bao - glcP 1 and engineered strain HZ / Bao - glcP The construction of 1 includes the following steps:

[0029] S1. Using the primers shown in Table 1, Bacillus subtilisUsing the 168 genome as a template and P43-F and P43-R as primers, the P43 promoter (SEQ ID NO.1) was amplified; B. amyloliquefaciens Using the genome as a template, ​ - ​ 1-F and ​ - ​ 1-R is a primer for amplification. ​ - ​ 1 gene fragment (SEQ ID NO.2); ​ ​ Using the WX-02 genome as a template, with T ​ -F and T ​ -R is a primer, amplifying T ​ The termination fragment (SEQ ID NO.3) was amplified in the following system as shown in Table 2. The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 34 cycles, 72℃ final extension for 5 min, and incubation at 4℃.

[0030] S2. Recover the PCR products as P43-F and T ​ -R is a primer used for SOE-PCR to deliver the P43 promoter fragment, ​ - ​ 1 gene fragment, T ​ The terminator fragments were connected together, and the SOE-PCR system is shown in Table 3. The SOE-PCR conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ annealing for 2 min, 7 cycles; 72℃ extension for 10 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ annealing for 1 min, 30 cycles; 72℃ final extension for 5 min, and incubation at 4℃ (the annealing temperature varied according to the Tm value of the primers, and the extension time varied according to the size of the target fragment).

[0031] S3. Purify and recover the fusion fragment using restriction endonucleases. ​ I and ​ The fusion fragment and pHY300PLK vector were double-digested with enzyme I. After product recovery, the digested fusion fragment and pHY300PLK vector were ligated using T4 ligase at 25°C for 2 h. The ligation system is shown in Table 4. The ligation product was then transformed. ​ DH5α competent cells, and transformants were verified by colony PCR using single colonies with pHY300-F / R primers;

[0032] S4. After preliminary identification as a positivist, plasmid was extracted for further processing. ​ I and ​Double enzyme digestion and DNA sequencing verification confirmed the presence of glucose permease from Bacillus amyloliquefaciens. After verification, the overexpression vector pHY- from Bacillus amyloliquefaciens was obtained. ​ - ​ 1;

[0033] S5. The successfully constructed overexpression vector pHY- ​ - ​ 1. Electroporation was performed into Bacillus amyloliquefaciens HZ12 competent cells, and the cells were plated on tetracycline-resistant plates and cultured at 37℃ for 16–24 h to obtain transformants. Colony PCR was performed using pHY300-F / R primers to verify the transformations, and the engineered strain HZ / was finally constructed. ​ - ​ 1.

[0034] Table 1. PCR primers.

[0035]

[0036] Note: The underlined areas represent the overlapping regions of the SOE-PCR splicing.

[0037] Table 2 PCR amplification system

[0038]

[0039] Table 3 SOE-PCR reaction system

[0040]

[0041] Table 4 Connection System

[0042]

[0043] Example 2

[0044] This embodiment uses the engineered strain HZ / constructed in Example 1. ​ - ​ 1. A method for producing L-tyrosine by fermentation, comprising the following steps:

[0045] S1. The engineered strain HZ / ​ - ​ 1. Activate the culture on LB agar plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar), then pick a single colony and inoculate it into 50 mL of liquid LB medium. Incubate at 37°C and 143 rpm until OD500. 600 A seed solution should reach approximately 4.0.

[0046] S2. The seed culture was transferred at an inoculum of 3% to the fermentation medium (glucose 22 g / L, (NH4)2SO4 3 g / L, K2HPO4 6.75 g / L, KH2PO4 1.25 g / L, MgSO4·7H2O 1.5 g / L, sodium citrate 5 g / L, peptone 3 g / L, yeast extract 6 g / L; trace elements: sodium citrate 10 g / L, FeSO4·7H2O 4 g / L, CaCl2 4 g / L, MnSO4·5H2O 1 g / L, CoCl2·6H2O 0.4 g / L, ZnSO4·7H2O 0.2 g / L, AlCl3·6H2O 0.1 g / L, CuCl2·H2O 0.1 g / L, H3BO4 0.05 g / L), with a trace element addition of 1... mL / L, fermented at 37℃ and 143 rpm for 36 h, with 3 replicates per group;

[0047] S3. Detection method of L-tyrosine: 1 mL of fermentation broth was added to 0.6 mL of 1 mol / L HCl and vortexed for 1 h. After centrifugation at 12000 rpm for 3 min, the supernatant was collected and filtered through a 0.22 μm microporous membrane. An Agilent 1100 HPLC system was used with a ZORBAX Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm) column. The mobile phase was 10% methanol-90% sodium acetate (100 mmol / L, pH 4.0), the flow rate was 0.6 mL / min, the injection volume was 10 μL, the column temperature was 30°C, and the detection wavelength was 280 nm using a UV detector.

[0048] Example 3

[0049] The difference between this embodiment and embodiment 2 is that the inoculation amount of seed liquid in this embodiment is 5%, while the remaining steps are the same as in embodiment 2.

[0050] Example 4

[0051] The difference between this embodiment and embodiment 2 is that the fermentation temperature in this embodiment is 35°C and the fermentation time is 40 hours, while the remaining steps are the same as in embodiment 2.

[0052] Example 5

[0053] The difference between this embodiment and Embodiment 2 is that the inoculation amount of seed liquid in this embodiment is 4%, the fermentation temperature is 36°C, and the fermentation time is 38 h. The remaining steps are the same as in Embodiment 2.

[0054] Comparative Example 1

[0055] The difference between this embodiment and Embodiment 2 is that the strain used in this comparative example is Bacillus amyloliquefaciens HZ / pHY300 transformed into an empty pHY300 vector, and the remaining steps are the same as in Embodiment 2.

[0056] Comparative Example 2

[0057] The difference between this comparative example and Example 2 is that the strain used in this comparative example is Bacillus subtilis overexpressing (…). ​ Engineered strain HZ / of glucose permease from [source missing] ​ - ​ The remaining steps are the same as in Example 2, using Bacillus subtilis ( ​ The overexpression vector pHY- of glucose permease from ) ​ - ​ and engineered strain HZ / ​ - ​ The construction method includes the following steps:

[0058] S1. Using the primers shown in Table 1, ​ Using the 168 genome as a template and P43-F and P43-R as primers, the P43 promoter (SEQ ID NO.1) was amplified; ​ Using the 168 genome as a template, ​ - ​ -F and ​ - ​ -R is a primer, used for amplification. ​ - ​ Gene fragment (SEQ ID NO.4); with ​ Using the WX-02 genome as a template, with T ​ -F and T ​ -R is a primer, amplifying T ​ The termination fragment (SEQ ID NO.3) was amplified using the system shown in Table 2, and the PCR amplification conditions were the same as in Example 1.

[0059] S2~S5. Same as S2~S5 in Example 1, to construct Bacillus subtilis ( ​ The overexpression vector pHY- of glucose permease from ) ​ - ​ and engineered strain HZ / ​ - ​ .

[0060] Comparative Example 3

[0061] The difference between this comparative example and Example 2 is that the strain used in this comparative example is Bacillus licheniformis overexpressing (… ​ Engineered strain HZ / of glucose permease from [source missing] ​ - ​The remaining steps are the same as in Example 2, Bacillus licheniformis ( ​ The overexpression vector pHY- of glucose permease from ) ​ - ​ and engineered strain HZ / ​ - ​ The construction method includes the following steps:

[0062] S1. Using the primers shown in Table 1, ​ Using the 168 genome as a template and P43-F and P43-R as primers, the P43 promoter (SEQ ID NO.1) was amplified; ​ Using the WX-02 genome as a template, ​ - ​ -F and ​ - ​ -R is a primer, used for amplification. ​ - ​ Gene fragment (SEQ ID NO.5); with ​ Using the WX-02 genome as a template, with T ​ -F and T ​ -R is a primer, amplifying T ​ The termination fragment (SEQ ID NO.3) was amplified using the system shown in Table 2, and the PCR amplification conditions were the same as in Example 1.

[0063] S2~S5. Same as S2~S5 in Example 1, to construct Bacillus licheniformis ( ​ The overexpression vector pHY- of glucose permease from ) ​ - ​ and engineered strain HZ / ​ - ​ .

[0064] Comparative Example 4

[0065] The difference between this comparative example and Example 2 is that the strain used in this comparative example is Corynebacterium glutamicum overexpressing glutamate (… ​ Engineered strain HZ / of glucose permease from [source missing] ​ - ​ 1. The remaining steps are the same as in Example 2, Corynebacterium glutamicum ( ​ The overexpression vector pHY- of glucose permease from ) ​ - ​ 1 and engineered strain HZ / ​ - ​ The construction method of 1 includes the following steps:

[0066] S1. Using the primers shown in Table 1, B. subtilis Using the 168 genome as a template and P43-F and P43-R as primers, the P43 promoter (SEQ ID NO.1) was amplified; C. glutamicum Using the genome as a template, Cgb - iolT 1-F and Cgb - iolT 1-R is a primer for amplification. Cgb - iolT 1 gene fragment (SEQ ID NO.6); B. lichenformis Using the WX-02 genome as a template, with T amyL -F and T amyL -R is a primer, amplifying T amyL The termination fragment (SEQ ID NO.3) was amplified using the system shown in Table 2, and the PCR amplification conditions were the same as in Example 1.

[0067] S2~S5. Same as S2~S5 in Example 1, to construct Corynebacterium glutamicum ( C. glutamicum The overexpression vector pHY- of glucose permease from ) Cgb - iolT 1 and engineered strain HZ / Cgb - iolT 1.

[0068] Table 5. Effects of overexpression of glucose permease from different sources on L-tyrosine production.

[0069]

[0070] As shown in Table 5, Examples 2-5 overexpressed the glucose permease gene derived from Bacillus amyloliquefaciens (BAM). Bao - glcP 1) After that, the L-tyrosine production increased by 30-39% compared with Comparative Example 1 (starting strain). However, after overexpressing glucose permease genes from other strains (Comparative Examples 2-3), the L-tyrosine production was lower than that of Examples 2-5 and Comparative Example 1 (starting strain).

[0071] Example 6

[0072] This example uses Corynebacterium glutamicum (… C. glutamicum overexpression vector pHY- of glucokinase derived from ) Cgb - glK and engineered strain HZ / Cgb - glK The construction of [the system] includes the following steps:

[0073] S1. Using the primers shown in Table 6, B. subtilis Using the 168 genome as a template and P43-F and P43-R as primers, the P43 promoter (SEQ ID NO.1) was amplified; C. glutamicum Using the genome as a template, Cgb - glK -F and Cgb - glK -R is a primer, used for amplification. Cgb - glK Gene fragment (SEQ ID NO.7); with B. lichenformis Using the WX-02 genome as a template, with T amyL -F and T amyL -R is a primer, amplifying T amyL The termination fragment (SEQ ID NO.3) was amplified using the system shown in Table 2, and the PCR amplification conditions were the same as in Example 1.

[0074] S2~S5. Same as S2~S5 in Example 1, to construct Corynebacterium glutamicum ( C. glutamicum overexpression vector pHY- of glucokinase derived from ) Cgb - glK and engineered strain HZ / Cgb - glK .

[0075] Table 6 PCR Primers

[0076]

[0077] Note: The underlined areas represent the overlapping regions of the SOE-PCR splicing.

[0078] Example 7

[0079] This embodiment uses the engineered strain HZ / constructed in Example 6. Cgb - glK The fermentation process for producing tyrosine follows the same method as in Example 2.

[0080] Example 8

[0081] The difference between this embodiment and embodiment 7 is that the inoculation amount of seed liquid in this embodiment is 2%, while the remaining steps are the same as in embodiment 7.

[0082] Example 9

[0083] The difference between this embodiment and embodiment 7 is that the fermentation temperature in this embodiment is 36°C and the fermentation time is 48 hours, while the remaining steps are the same as in embodiment 7.

[0084] Example 10

[0085] The difference between this embodiment and embodiment 7 is that the inoculation amount of seed liquid in this embodiment is 3%, the fermentation temperature is 37°C, and the fermentation time is 42 h. The remaining steps are the same as in embodiment 7.

[0086] Comparative Example 5

[0087] The difference between this comparative example and Example 7 is that the strain used in this comparative example is Bacillus amyloliquefaciens overexpressing (Bacillus). B. amyloliquefaciens Engineered strain HZ / of glucokinase from [source missing] Bao - glcK The remaining steps are the same as in Example 7, using Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). B. amyloliquefaciens The overexpression vector pHY- of glucose permease from ) Bao - glcK and engineered strain HZ / Bao - glcK The construction method includes the following steps:

[0088] S1. Using the primers shown in Table 6, B. subtilis Using the 168 genome as a template and P43-F and P43-R as primers, the P43 promoter (SEQ ID NO.1) was amplified; B. amyloliquefaciens Using the genome as a template, Bao - glcK -F and Bao - glcK -R is a primer, used for amplification. Bao - glcK Gene fragment (SEQ ID NO.8); with B. lichenformis Using the WX-02 genome as a template, with T amyL -F and T amyL -R is a primer, amplifying T amyL The termination fragment (SEQ ID NO.3) was amplified using the system shown in Table 2, and the PCR amplification conditions were the same as in Example 1.

[0089] S2~S5. Same as S2~S5 in Example 1, to construct Bacillus amyloliquefaciens ( B. amyloliquefaciens The overexpression vector pHY- of glucose permease from ) Bao - glcK and engineered strain HZ / Bao - glcK .

[0090] Comparative Example 6

[0091] The difference between this comparative example and Example 7 is that the strain used in this comparative example is Bacillus licheniformis overexpressing (… B. licheniformis Engineered strain HZ / - of glucokinase Bli - glcK The remaining steps are the same as in Example 7, Bacillus licheniformis ( B. licheniformis overexpression vector pHY- of glucokinase derived from ) Bli - glcK and engineered strain HZ / - Bli - glcK The construction method includes the following steps:

[0092] S1. Using the primers shown in Table 6, B. subtilis Using the 168 genome as a template and P43-F and P43-R as primers, the P43 promoter (SEQ ID NO.1) was amplified; B. licheniformis Using the genome as a template, Bli - glcK -F and Bli - glcK -R is a primer, used for amplification. Bli - glcK Gene fragment (SEQ ID NO.9); with B. lichenformis Using the WX-02 genome as a template, with T amyL -F and T amyL -R is a primer, amplifying T amyL The termination fragment (SEQ ID NO.3) was amplified using the system shown in Table 2, and the PCR amplification conditions were the same as in Example 1.

[0093] S2~S5. Same as S2~S5 in Example 1, to construct Bacillus licheniformis ( B. licheniformis overexpression vector pHY- of glucokinase derived from ) Bli - glcK and engineered strain HZ / - Bli - glcK .

[0094] Table 7. Effects of overexpression of glucokinase from different strains on L-tyrosine production.

[0095]

[0096] As shown in Table 7, Examples 7-10 overexpressed the glucokinase gene derived from Corynebacterium glutamicum (GMO). Cgb - glK After that, the L-tyrosine production was 27-33% higher than that of Comparative Example 1 (starting strain), while the L-tyrosine production of overexpression of glucokinase genes from other strains (Comparative Examples 5-6) was lower than that of Examples 7-10 and Comparative Example 1 (starting strain).

[0097] Example 11

[0098] This embodiment uses glucose permease derived from Bacillus amyloliquefaciens (BAM). Bao - glcP 1 ） The integrated expression vector T2(2)- XKDG :: Bao - glcP 1 and integrated strain HZ12:: Bao - glcP The construction of 1 includes the following steps:

[0099] S1. Using the primers shown in Table 8, use *Bacillus amyloliquefaciens* (… B. amyloliquefaciens Using the genome of ) as a template, homologous arm A, homologous arm B, and plasmid pHY- were amplified. Bao - glcP 1 is the template for amplifying gene fragment P43+UTR1+ Bao - glcP 1+T amy L, the amplification system is shown in Table 2, and the PCR amplification conditions are the same as in Example 1;

[0100] S2. Recover the PCR product and use SOE-PCR to connect the upstream and downstream homologous arms to P43+UTR1+. Bao - glcP 1+T amyL The fragments were linked together, and the SOE-PCR system is shown in Table 3. The SOE-PCR conditions were the same as in Example 1.

[0101] S3. Purify and recover the fusion fragment using restriction endonucleases. Xba I and BamH The fusion fragment and the temperature-sensitive knockout plasmid T2(2)-ori were digested with enzyme I. The digested fusion fragment and T2(2)-ori were ligated using T4 ligase at 4°C for 6-8 h. The ligation system is shown in Table 4. The ligation product was transformed. E. coli DH5α competent cells were used to transform the cells. Colony PCR was performed on single colonies using the T2-F / R primer pair. After preliminary identification as positive transformants, plasmids were extracted for DNA sequencing verification. Once verified, the gene was obtained. Bao - glcP The integrated expression vector T2(2)- XKDG :: Bao - glcP 1;

[0102] S4. Express vector T2(2)- XKDG :: Bao - glcP 1. Electroporation was performed on Bacillus amyloliquefaciens HZ12 competent cells, which were then plated on Kan resistance plates and cultured at 37°C for 16-20 h. Transformants were verified using primers T2-F / T2-R, and the products were detected by agarose gel electrophoresis.

[0103] S5. Select the verified positive transformants containing the knockout plasmid and inoculate them into liquid LB medium containing 20 μg / mL Kan. Incubate at 45°C for 6 h as one generation. Transfer to new LB medium containing Kan and culture for a total of 3 generations. Then, streak the bacterial culture on Kan-resistant plates and incubate at 45°C. After the single colonies grow, select a small number of bacterial cells for colony PCR verification to confirm the complete single-exchange strain.

[0104] S6. Inoculate the single-exchange strain into 5 mL of antibiotic-free liquid LB medium and subculture at 37°C for 6 hours per generation; the inoculation volume for each subculture is 50-100 μL, and the culture from each generation is diluted (10 μL). -6 ~10 -7 Spread the culture onto LB agar plates and incubate at 37°C until single colonies appear. Pick single colonies and inoculate them onto LB agar plates containing Kan and those without Kan, respectively. Incubate at 37°C for 10–12 h. Select single colonies that grow on non-resistant LB agar plates but not on Kan-containing LB agar plates for colony PCR identification to preliminarily determine the gene-integrated strain HZ12:: Bao - glcP 1. Liquid culture, extraction of genomic DNA for PCR identification.

[0105] Example 12

[0106] This example uses glucokinase derived from Corynebacterium glutamicum (GGlutamate). Cgb - glK The integrated expression vector T2(2)- XKDE :: Cgb - glK and integrated strain HZ12:: Bao - glcP 1:: Cgb - glK The construction of [the system] includes the following steps:

[0107] S1. Using the primers shown in Table 8, use *Bacillus amyloliquefaciens* (… B. amyloliquefaciens Using the genome of ) as a template, homologous arm A and homologous arm B were amplified using plasmid pHY- Cgb - glK P43+ gene fragment amplification template Cgb - glK +T amyL The amplification system is shown in Table 2, and the PCR amplification conditions are the same as in Example 1.

[0108] S2~S3. Same as Example 11 S2~S3, construct expression vector T2(2)- XKDE :: Cgb - glK ;

[0109] S4. Express vector T2(2)- XKDE :: Cgb - glK Electroporation to strain HZ12 constructed in Example 11:: Bao - glcP 1. Spread the transformants on Kan resistance plates and incubate at 37°C for 16-20 h. Verify the transformants using primers T2-F / T2-R, and detect the products by agarose gel electrophoresis.

[0110] S5~S6. Same as S5~S6 in Example 11, to construct the integrated strain HZ12:: Bao - glcP 1:: Cgb - glK .

[0111] Table 8 PCR Primers

[0112]

[0113] Note: Bold areas represent restriction enzyme sites, and underlined areas represent overlapping regions in SOE-PCR splicing.

[0114] Example 13

[0115] This embodiment is based on carrier T2(2)Δ ptsH and engineered strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH The construction of [the system] includes the following steps:

[0116] S1. Using the primers shown in Table 9, use *Bacillus amyloliquefaciens* (… B. amyloliquefaciens Using the genome of ) as a template, homologous arm A and homologous arm B were amplified. The amplification system is shown in Table 2. The PCR amplification conditions were the same as in Example 1.

[0117] S2. The PCR product was recovered, and the upstream and downstream homologous arms were linked together by SOE-PCR. The SOE-PCR system is shown in Table 3. The SOE-PCR conditions are the same as in Example 1.

[0118] S3. Purify and recover the fusion fragment using restriction endonucleases. Xba I and BamH The fusion fragment and the temperature-sensitive knockout plasmid T2(2)-ori were digested with enzyme I. The digested fusion fragment and T2(2)-ori were ligated using T4 ligase at 4°C for 6-8 h. The ligation system is shown in Table 4. The ligation product was transformed. E.coli DH5α competent cells were used to transform the cells. Colony PCR was performed on single colonies using the T2-F / R primer pair. After preliminary identification as positive transformants, plasmids were extracted for DNA sequencing verification. Once verified, the gene was obtained. ptsH Knockout vector T2(2)Δ ptsH ;

[0119] S4. Knockout vector T2(2)Δ ptsH Electroporation to the integrated strain HZ12 constructed in Example 12:: Bao - glcP 1:: Cgb - glK The goal is to decouple glucose phosphorylation from PEP by weakening the endogenous PTS system and strengthening non-PTS pathways.

[0120] Table 9 PCR Primers

[0121]

[0122] Note: The underlined areas represent the overlapping regions of the SOE-PCR splicing.

[0123] Example 14

[0124] This example uses the engineered strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH / tyrA - aroA (Renamed to: HZ13 / ) tyrA - aroA The construction of ) is specifically as follows: the laboratory-preserved overexpression vector pHY- tyrA - aroA Electroporation was performed into Bacillus amyloliquefaciens HZ12 competent cells, and the cells were plated on tetracycline-resistant plates and cultured at 37°C for 16–24 h to obtain transformants. Colony PCR was performed using pHY300-F / R primers for verification, and the engineered strain HZ13 / was finally constructed. tyrA - aroA .

[0125] Example 15

[0126] This embodiment utilizes the integrated strain HZ12:: constructed in Example 11. Bao - glcP The fermentation process for producing tyrosine follows the same method as in Example 2.

[0127] Example 16

[0128] This embodiment utilizes the integrated strain HZ12 constructed in Example 12:: Bao - glcP :: Cgb - glcK The fermentation process for producing tyrosine follows the same method as in Example 2.

[0129] Example 17

[0130] This embodiment uses the engineered strain HZ12:: constructed in Example 13. Bao - glcP :: Cgb - glcK Δ ptsH The fermentation process for producing tyrosine follows the same method as in Example 2.

[0131] Example 18

[0132] This embodiment uses the engineered strain HZ13 / constructed in Example 14. tyrA - aroA The fermentation process for producing L-tyrosine follows the same method as in Example 2.

[0133] Table 10 L-tyrosine production of the integrative strains

[0134]

[0135] As shown in Table 10, strain HZ12:: Bao - glcP 1. HZ12:: Bao - glcP 1:: Cgb - glK The L-tyrosine production reached 328.32 mg / L and 357.01 mg / L, respectively, which were 15% and 51% higher than those of the control strain HZ / pHY300, indicating that the glucose permease gene from Bacillus amyloliquefaciens was effective. Bao - glcP 1 and the glucokinase gene derived from Corynebacterium glutamicum Cgb - glK The synergistic expression of [a specific ingredient] enhances the synthesis of L-tyrosine. (Strain HZ12::) Bao - glcP 1:: Cgb - glK Δ ptsH The L-tyrosine yield was 407.16 mg / L, indicating that knockout... ptsH Attenuating the endogenous PTS system and enhancing non-PTS pathways can significantly increase L-tyrosine production. In the chassis strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsHOverexpression of DAHP synthase gene aroA and prephenyl acid dehydrogenase gene tyrA (Example 18) can increase the yield of tyrosine to 1400.53 mg / L, which is 3.82 times higher than that of the starting strain HZ / pHY300.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A Bacillus amyloliquefaciens chassis strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH Its features are: The Bacillus amyloliquefaciens chassis strain HZ12:: Bao - glcP 1:: Cgb - glK Δ ptsH Using Bacillus amyloliquefaciens as the starting strain, glucose permease gene was overexpressed. glcP 1 and glucokinase gene glK It also lacks the gene encoding the phosphoryl carrier protein HPr. ptsH The Bacillus amyloliquefaciens chassis strain HZ12:: was constructed. Bao - glcP 1:: Cgb - glK Δ ptsH .

2. The Bacillus amyloliquefaciens chassis strain HZ12:: according to claim 1 Bao - glcP 1:: Cgb - glK Δ ptsH Its features are: The glucose permease gene glcP 1 is derived from Bacillus amyloliquefaciens, and its nucleotide sequence is shown in SEQ ID NO.

2.

3. The Bacillus amyloliquefaciens chassis strain HZ12:: according to claim 1 Bao - glcP 1:: Cgb - glK Δ ptsH Its features are: The glucokinase gene glK It is derived from Corynebacterium glutamicum, and its nucleotide sequence is shown in SEQ ID NO.

7.

4. The Bacillus amyloliquefaciens chassis strain HZ12 according to any one of claims 1 to 3. Bao - glcP 1:: Cgb - glK Δ ptsH The construction method is characterized by, Includes the following steps: S1. Using the genome of Bacillus amyloliquefaciens as a template, amplify homologous arm A, homologous arm B, and gene fragment P43+UTR1+. Bao - glcP 1+T amyL Homologous arms A and B were coupled with P43+UTR1+ via SOE-PCR. Bao - glcP 1+T amyL Fragments are joined together to obtain a fusion fragment, which is then processed using a restriction endonuclease. Xba I and BamH The fusion fragment and the temperature-sensitive knockout plasmid T2(2)-ori were digested with enzyme I. The digested fusion fragment and T2(2)-ori were ligated with T4 ligase. The ligation product was transformed into competent E. coli cells to obtain the gene. Bao - glcP The integrated expression vector T2(2)-1 XKDG :: Bao - glcP 1; S2. Integrate expression vector T2(2)- XKDG :: Bao - glcP 1. Electroporation was performed into competent Bacillus amyloliquefaciens cells, and positive transformants were identified. Single-crossover and double-crossover strains were screened and identified from the positive transformants to obtain the integrative strain HZ12:: Bao - glcP 1; S3. Using the genome of Bacillus amyloliquefaciens as a template, amplify homologous arm A, homologous arm B, and gene fragment P43+. Cgb - glK +T amyL Homologous arms A and B were linked to gene fragment P43+ by SOE-PCR. Cgb - glK +T amyL Fragments are joined together to obtain a fusion fragment, which is then processed using a restriction endonuclease. Xba I and BamH The fusion fragment and the temperature-sensitive knockout plasmid T2(2)-ori were digested with enzyme I. The digested fusion fragment and T2(2)-ori were ligated with T4 ligase. The ligation product was transformed into competent E. coli cells to obtain the gene. Cgb - glK The integrated expression vector T2(2)- XKDE :: Cgb - glK ; S4. Integrate expression vector T2(2)- XKDE :: Cgb - glK The integrated strain HZ12:: was constructed by electroporation to S2 Bao - glcP In step 1, the integrated strain HZ12:: was obtained. Bao - glcP 1:: Cgb - glK ; S5. Using the genome of Bacillus amyloliquefaciens as a template, amplify homologous arm A and homologous arm B, ligate homologous arm A and homologous arm B together by SOE-PCR to obtain the fusion fragment, and then use restriction endonucleases... Xba I and BamH The fusion fragment and the temperature-sensitive knockout plasmid T2(2)-ori were digested with enzyme I. The digested fusion fragment and T2(2)-ori were ligated with T4 ligase. The ligation product was transformed into competent E. coli cells to obtain the gene. ptsH Knockout vector T2(2)Δ ptsH ; S6. Knockout vector T2(2)Δ ptsH The integrated strain HZ12:: was constructed by electroporation to S4 Bao - glcP 1:: Cgb - glK In the process, chassis strain HZ12:: was obtained. Bao-glcP 1:: Cgb-glK Δ ptsH .

5. A genetically engineered Bacillus amyloliquefaciens strain, HZ13 / tyrA - aroA Its features are: The Bacillus amyloliquefaciens chassis strain HZ12:: as described in any one of claims 1 to 3 Bao - glcP 1:: Cgb - glK Δ ptsH Based on this, overexpression of the DAHP synthase gene aroA and prephenyl acid dehydrogenase gene tyrA The genetically engineered Bacillus amyloliquefaciens strain HZ13 / was constructed. tyrA - aroA .

6. The Bacillus amyloliquefaciens chassis strain HZ12 according to any one of claims 1 to 3: Bao - glcP 1:: Cgb - glK Δ ptsH Or the Bacillus amyloliquefaciens genetically engineered strain HZ13 / as described in claim 5 tyrA - aroA Applications in the production of L-tyrosine.

7. The application according to claim 6, characterized in that, The specific method for producing L-tyrosine includes the following steps: S1. The *Bacillus amyloliquefaciens* chassis strain HZ12 as described in any one of claims 1 to 3: Bao - glcP 1:: Cgb - glK Δ ptsH Or the Bacillus amyloliquefaciens genetically engineered strain HZ13 / as described in claim 5 tyrA - aroA After activation on LB plates, single colonies are picked and inoculated into LB liquid medium and cultured at 35-38℃ and 180-220 rpm for 8-12 h to obtain seed culture; S2. The seed culture is inoculated into the fermentation medium at an inoculum of 3-5%, and fermented at 35-38℃ and 143-170 rpm for 36-40 h to produce L-tyrosine.

8. The application according to claim 7, characterized in that, The fermentation medium consists of: glucose 20-25 g / L, (NH4)2SO4 2.5-4.5 g / L, K2HPO4 6-7 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 1-2 g / L, sodium citrate 4-6 g / L, peptone 4-6 g / L, yeast extract 5-7 g / L, and trace elements 1 mL / L.

9. The application according to claim 8, characterized in that, The trace element composition is as follows: sodium citrate 9~12 g / L, FeSO4·7H2O 3~5 g / L, CaCl2 3~5 g / L, MnSO4·5H2O 0.5~2 g / L, CoCl2·6H2O 0.2~0.6 g / L, ZnSO4·7H2O 0.1~0.4 g / L, AlCl3·6H2O 0.05~0.2 g / L, CuCl2·H2O 0.05~0.2 g / L, H3BO4 0.001~0.006 g / L.

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