Bacillus velezensis engineering strain for improving iturin a production and construction method thereof

Abstract

The application provides a bacillus velezensis engineering bacterium for improving iturin A production and a construction method thereof. ituA The gene is inserted in front of a promoter P bacA And the genome is knocked out of a proline degradation related gene fadM The bacillus velezensis engineering bacterium ZD-P2Δ fadM Compared with the bacillus velezensis ZD122, the engineering strain ZD-P2Δ fadM fadM The production of iturin A is improved by 2 times.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an engineered Bacillus velezensis bacterium capable of improving iturin A production and a construction method thereof. Background Art

[0002] Iturin A is a biosurfactant composed of a hydrophobic fatty acid and a hydrophilic peptide chain. Its unique amphiphilic structure and biodegradability have attracted widespread attention. Iturin A is widely used in oil recovery, agricultural biocontrol, cosmetics, and food. Iturin A has great commercial value, but its low yield is the main factor limiting its industrial production (see Xu Yuxiang. Metabolic Engineering of Bacillus amyloliquefaciens HZ-12 for High Iturin A Production [D]. Hubei University, 2020.).

[0003] Currently, there are no reports of chemically synthesizing iturin A, so industrial production of this valuable compound must rely on biosynthesis. Bacillus is the most efficient known bacterial species for producing iturin A, but the production capacity of wild strains is limited. Therefore, developing strategies to increase the yield of iturin A in these strains is crucial. Summary of the Invention

[0004] The present invention aims to provide an engineered Bacillus velezine strain for increasing iturin A production and a method for constructing the same. By genetic engineering technology, the promoter PbacA is inserted before the ituA gene of Bacillus velezine ZD122, and the fadM gene, a gene related to proline degradation in the genome, is knocked out, thereby significantly increasing iturin A production.

[0005] In order to achieve the above object, the present invention adopts the following technical measures:

[0006] An engineered Bacillus velezensis strain with increased iturin A production is constructed by inserting the promoter PbacA before the ituA gene of Bacillus velezensis ZD122 and knocking out the fadM gene, a gene involved in proline degradation in the genome. The specific construction method is as follows:

[0007] 1) Using the genomic DNA of Bacillus velezensis ZD122 as a template, the upstream homology arm and downstream homology arm of the ituA gene were amplified by PCR. The upstream homology arm of the ituA gene, the promoter PbacA and the downstream homology arm of the ituA gene were connected together by overlap extension PCR. The resulting homology arm fusion fragment was ligated with the plasmid T2(2)-ori to obtain the promoter-inserted vector T2(2)-PbacA-ituA;

[0008] 2) The vector T2(2)-PbacA-ituA was transformed into Bacillus velezensis ZD122, and kanamycin was used as a selection marker to obtain positive transformants. The positive transformants were screened by single and double crossover to obtain a strain in which the promoter PbacA was inserted in front of the ituA gene. The strain was named Bacillus velezensis ZD-P2;

[0009] 3) Using the genomic DNA of Bacillus velezensis ZD122 as a template, the upstream homology arm and the downstream homology arm of the fadM gene were amplified by PCR, and the upstream homology arm and the downstream homology arm of the fadM gene were connected together by overlap extension PCR. The resulting homology arm fusion fragment was ligated with the plasmid T2(2)-ori to obtain the knockout plasmid T2(2)-ΔfadM;

[0010] 4) The knockout plasmid T2(2)-ΔfadM was transformed into Bacillus velezensis ZD-P2, and kanamycin was used as a screening marker to obtain positive transformants. The positive transformants were screened by single and double crossover to obtain Bacillus velezensis with the fadM gene successfully knocked out. The strain was named ZD-P2ΔfadM;

[0011] Furthermore, the primer sequences of the upstream homologous arms of the ituA gene in step 1) are shown as SEQ ID NOs. 7 and 8, the primer sequences of the downstream homologous arms of the ituA gene are shown as SEQ ID NOs. 9 and 10, the primer sequences of the upstream homologous arms of the fadM gene in step 3) are shown as SEQ ID NOs. 11 and 12, and the primer sequences of the downstream homologous arms of the fadM gene are shown as SEQ ID NOs. 13 and 14.

[0012] The application of the engineered Bacillus velezinii ZD-P2 prepared by the above method in the production of iturin A: In a specific embodiment of the present invention, the colony ZD-P2 was picked and inoculated into LB medium for seed culture, and then inoculated into the fermentation medium (corn starch 30 g / L, soybean meal 70 g / L, K2HPO4·3H2O 1 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 1 g / L, MnSO4·H2O 0.01 g / L) according to a certain inoculum size, and cultured at 37°C and 180 rpm for 72 hours. The results showed that the yield of iturin A produced by the engineered Bacillus velezinii ZD-P2ΔfadM was 2 times higher than that of the original bacterium Bacillus velezinii ZD122.

[0013] Compared with the prior art, the present invention has the following advantages and effects:

[0014] 1. The present invention discovered for the first time that inserting the promoter PbacA before the ituA gene and knocking out the proline degradation-related gene fadM in the genome can significantly increase the yield of iturin A produced by Bacillus velezini. Compared with the original strain Bacillus velezini ZD122, the yield of iturin A produced by the engineered Bacillus velezini ZD-P2ΔfadM constructed by the present invention was increased by 2 times.

[0015] 2. The product iturin A has high potential application value in agricultural production, biomedicine and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the construction process of the promoter insertion vector T2(2)-PbacA-ituA.

[0017] Figure 2 Schematic diagram of the construction process of the knockout vector T2(2)-ΔfadM.

[0018] Figure 3 This is the verification band of the engineered Bacillus velez ZD-P2 in Example 2, in which the promoter PbacA was inserted before the ituA gene of Bacillus velez ZD122. Lane M is a DNA marker, and lanes 1 and 2 are PCR products using PbacA-F and ituA-KYR as primers, and Bacillus velez ZD-P2 and ZD122 as templates, respectively.

[0019] Figure 4 This is the verification band of the Bacillus velez engineered strain ZD-P2ΔfadM in which the fadM gene was knocked out in Bacillus velez ZD-P2 in Example 4. Lane M is a DNA marker, and lanes 1 and 2 are PCR products using ΔfadM-KYF and ΔfadM-KYR as primers, and Bacillus velez ZD-P2 and ZD-P2ΔfadM as templates, respectively.

[0020] Figure 5 The yield of ituA produced by the engineered Bacillus velezensis strain ZD-P2ΔfadM. DETAILED DESCRIPTION

[0021] In the following examples, molecular biology experimental methods without specific conditions specified were performed under conventional conditions according to the Molecular Cloning Laboratory Manual (New York: Cold Spring Harbor).

[0022] Biomaterial Description:

[0023] Bacillus velezensis ZD122 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20241480 and the deposit date July 4, 2024; Bacillus licheniformis DW2 is a publicly available biological material provided by the Microbial Engineering Laboratory of Huazhong Agricultural University.

[0024] Example 1: Construction of promoter-inserted vector

[0025] Using the genomic DNA of Bacillus licheniformis DW2 as a template, primers PbacA-F and PbacA-R were used to PCR amplify the 335bp promoter PbacA. Based on the ituA gene sequence in the genomic DNA sequence of Bacillus velez ZD122, upstream homology arm primers (itu-F1, ituR1) and downstream homology arm primers (ituA-F2, ituA-R2) were designed; and using the genomic DNA of Bacillus velez ZD122 as a template, PCR amplification was performed using the upstream homology arm primers itu-F1 / ituA-R1 and the downstream homology arm primers ituA-F2 / ituA-R2 to obtain the upstream homology arm fragment and the downstream homology arm fragment of the ituA gene. The primer sequences are as follows: PbacA-F: 5'-ATTTTAAAATAAAGCGCCCAGCCTGCGATTTCGGCG-3'

[0026] PbacA-R: 5'-GGTTTGGAATTGACTGGTATACATATAAAAATTCTCCTTTTTGA TAAAAT-3'

[0027] ituA-F1: 5'-GGACTAGTTTCATTTCAAATGTCACCGC-3' (contains SpeI restriction site) ituA-R1: 5'-CGCCGAAATCGCAGGCTGGGCGCTTTATTTTAAAAT-3'

[0028] ituA-F2: 5'-ATTTTATCAAAAAGGAGAATTTTTATATGTATACCAGTCAATTCC AAACC-3'

[0029] ituA-R2: 5'-GCTCTAGAACTCCTTTTGGATCTCCTGTC-3' (containing XbaI restriction enzyme cleavage site).

[0030] PCR system: ddH2O 25.0μL, 5×TransStart TMFastPfu Buffer 5.0μL, dNTPs 2.5μL, FastPfu DNA Polymerase 1.0μL, forward primer (10μmol / L) 1.0μL, reverse primer (10μmol / L) 1.0μL, template DNA (200ng / μL) 0.5μL.

[0031] PCR reaction conditions: 95°C for 5 min; 95°C for 30 s, 50-60°C for 30 s, 72°C for 30-60 s, 30-35 cycles; 72°C for 5 min.

[0032] According to the above PCR reaction system and conditions, PCR amplification was performed using primers PbacA-F and PbacA-R to obtain the promoter PbacA (335 bp), the sequence of which is shown in SEQ ID NO. 1. PCR amplification was performed using primers ituA-F1 / ituA-R1 to obtain the upstream homology arm fragment (530 bp), the sequence of which is shown in SEQ ID NO. 3. PCR amplification was performed using primers ituA-F2 / ituA-R2 to obtain the downstream homology arm fragment (545 bp), the sequence of which is shown in SEQ ID NO. 4. The upstream homology arm, promoter PbacA, and downstream homology arm were ligated by overlap extension PCR to form a homology arm fusion fragment (1408 bp).

[0033] The homology arm fusion fragment and plasmid T2(2)-ori were double-digested with SpeI and XbaI restriction endonucleases to obtain the digested gene fragment and the linearized plasmid fragment. The digested gene fragment and the linearized plasmid fragment were ligated with T4 DNA ligase to obtain the ligation product ( Figure 1 ); The ligation product was transformed into Escherichia coli DH5α by the calcium chloride transformation method, and screened at 37°C in LB medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH 7.2) containing kanamycin (20 μg / mL) resistance to obtain transformants, which were verified by colony PCR and plasmid PCR (primers used were: T2-F and T2-R) to obtain the promoter-inserted vector T2(2)-PbacA-ituA.

[0034] Among them, the sequences of T2-F and T2-R are:

[0035] T2-F: 5'-ATGTGATAACTCGGCGTA-3'

[0036] T2-R: 5'-GCAAGCAGCAGATTACGC-3'.

[0037] Example 2: Construction of promoter-inserted engineered bacteria ZD-P2

[0038] The vector T2(2)-PbacA-ituA was transferred into Bacillus velezensis ZD122 and screened at 37°C in LB medium containing kanamycin (20 μg / mL) to obtain transformants. The transformants were verified by colony PCR (primers used were: T2-F and T2-R) to obtain positive transformants, namely Bacillus velezensis ZD122 transferred with the vector T2(2)-PbacA-ituA.

[0039] The positive transformants were transferred and cultured three times on LB medium containing kanamycin resistance at 45°C, each culture for 12 h. Single-crossover strains were detected by colony PCR using T2-F and ituA-KYR as primers or T2-R and ituA-KYF as primers. A single-crossover strain was obtained by amplifying a band of 1683 bp or 1792 bp in length.

[0040] Among them, the sequences of ituA-KYF and ituA-KYR are:

[0041] ituA-KYF:5'-AGTTCGCTGATACGATCAGC-3'

[0042] ituA-KYR: 5'-AAGAAAGAGTCTTTCGAATGAAC-3'.

[0043] The single exchange strain was inoculated and cultured in LB medium without kanamycin at 37°C for several times. The transformants were selected for colony PCR verification (primers PbacA-F and ituA-KYR). The strain that amplified a 959 bp band was selected ( Figure 3 ). Subsequently, DNA sequencing was performed on the positive transformants to further verify the results, and a strain with a successful double crossover and the promoter PbacA inserted before the ituA gene was obtained, namely Bacillus velezensis ZD-P2.

[0044] Example 3: Construction of temperature-sensitive knockout vector

[0045] Based on the fadM gene sequence in the genome of Bacillus velez ZD122, upstream homology arm primers (fadM-F1, fadM-R1) and downstream homology arm primers (fadM-F2, fadM-R2) of the fadM gene were designed; and using the genomic DNA of Bacillus velez ZD122 as a template, PCR amplification was performed using the upstream homology arm primers and downstream homology arm primers of the fadM gene to obtain the upstream homology arm fragment of the fadM gene and the downstream homology arm fragment of the fadM gene. The primer sequences are as follows:

[0046] fadM-F1: 5'-CGGGATCCAGCAGCTTTTGCATGGC-3' (containing BamHI restriction site)

[0047] fadM-R1: 5'-AAAGGAGGGAGTTCATCCTCGGCTGCTCCAAAAAG-3'

[0048] fadM-F2: 5'-CTTTTTGGAGCAGCCGAGGATGAACTCCCTCCTTT-3'

[0049] fadM-R2: 5'-GCTCTAGAATGGATAACTCAGGCAGGTC-3' (containing XbaI restriction enzyme cleavage site).

[0050] PCR system: ddH2O 25.0μL, 5×TransStart TM FastPfu Buffer 5.0 μL, dNTPs 2.5 μL, FastPfu DNA Polymerase 1.0 μL, Primer (10 μmol / L) 1 1.0 μL, Primer (10 μmol / L) 2 1.0 μL, Template DNA (200 ng / μL) 0.5 μL.

[0051] PCR reaction conditions: 95°C for 5 min; 95°C for 30 s, 50-60°C for 30 s, 72°C for 30-60 s, 30-35 cycles; 72°C for 5 min.

[0052] According to the above PCR reaction system and conditions, primer pair fadM-F1 / fadM-R1 was used to perform PCR amplification to obtain the upstream homology arm fragment of fadM (555bp), the sequence of which is shown in SEQ ID NO.5, and primer pair fadM-F2 / fadM-R2 was used to perform PCR amplification to obtain the downstream homology arm fragment of fadM (534bp), the sequence of which is shown in SEQ ID NO.6. The upstream homology arm and the downstream homology arm were connected together by overlapping extension PCR to form a homology arm fusion fragment (1089bp); the homology arm fusion fragment and plasmid T2(2)-ori were double-digested by XbaI and BamHI restriction endonucleases to obtain a digested gene fragment and a linear plasmid fragment. The digested gene fragment and the linear plasmid fragment were ligated by T4 DNA ligase to obtain a ligation product ( Figure 2); The ligation product was transformed into Escherichia coli DH5α by the calcium chloride transformation method, and screened at 37°C in LB medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH 7.2) containing kanamycin (20 μg / mL) resistance to obtain transformants, which were verified by colony PCR and plasmid PCR (primers used were: T2-F and T2-R) to obtain the gene knockout vector T2(2)-ΔfadM.

[0053] Example 4: Construction of gene knockout engineered bacteria ZD-P2ΔfadM

[0054] The knockout vector T2(2)-ΔfadM was transferred into Bacillus velezensis ZD-P2 and screened at 37°C using LB medium containing kanamycin (20 μg / mL) to obtain transformants. The transformants were verified by colony PCR (primers used were: T2-F and T2-R) to obtain positive transformants, namely Bacillus velezensis ZD-P2 that had been transferred with the knockout vector T2(2)-ΔfadM.

[0055] Positive transformants were transferred and cultured three times on LB medium containing kanamycin resistance at 45°C, each culture for 12 h. Single-crossover strains were detected by colony PCR using T2-F and ΔfadM-KYR as primers or T2-R and ΔfadM-KYF as primers. Single-crossover strains were obtained by amplifying a band of 1333 bp or 1442 bp in length.

[0056] The sequences of ΔfadM-KYF and ΔfadM-KYR are:

[0057] ΔfadM-KYF:5'-CAGCCGAAGTCAGCGG-3'

[0058] ΔfadM-KYR: 5'-TTTAGATTTTCCTGTTGAATCCA-3'.

[0059] The single-exchange strain was inoculated and cultured several times in LB medium without kanamycin at 37°C, and the transformants were selected for colony PCR verification (primers ΔfadM-KYF and ΔfadM-KYR) ( Figure 4 The positive transformants were then further verified by DNA sequencing, and a fadM knockout strain with successful double crossover was obtained, namely Bacillus velezensis ZD-P2ΔfadM.

[0060] Example 5: Effect of inserting the promoter PbacA before the ituA gene and simultaneously blocking proline degradation on the production of iturin A by ZD122

[0061] Velezella ZD122 and ZD-P2ΔfadM were selected and inoculated into LB medium, cultured at 37°C and 180 rpm for 12 h, and the OD 600 When the pH value was between 3.0 and 4.0, 25 mL of fermentation medium (corn starch 30 g / L, soybean meal 70 g / L, K2HPO4·3H2O 1 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 1 g / L, MnSO4·H2O 0.01 g / L) was inoculated at 3% inoculum and cultured at 37°C and 180 rpm for 72 h. 0.3 mL of the fermentation supernatant was mixed with 1.2 mL of methanol, shaken for 1 h, and centrifuged at 10,000 g for 10 min for iturin A determination. The concentration of iturin A was determined by high performance liquid chromatography using a LiChrospher C18 reverse phase column (4.6 mm × 250 mm, 5 μm), a mobile phase of 10 mmol / L ammonium acetate / acetonitrile (65:35, V / V), a flow rate of 1.0 mL / min, an injection volume of 10 μL, and a detection wavelength of 210 nm. Figure 5 It showed that the iturin A production of the engineered Bacillus velezensis ZD-P2ΔfadM was as high as 1.10 g / L, which was 2 times higher than that of the original Bacillus velezensis ZD122.

Claims

1. An engineered strain of Bacillus velezensis for increasing iturin A production, characterized in that: The engineering bacteria is in the form of Bacillus velez ( Bacillus velezensis )ZD122 ituA Insert promoter P before gene bacA , and knocked out genes related to proline degradation in the genome fadM The deposit number of the Bacillus velezensis ZD122 is CCTCC NO: M20241480, and the promoter P bacA The nucleotide sequence is shown in SEQ ID NO.1, fadM The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

2. The method for constructing the Bacillus velezensis engineered bacteria according to claim 1, characterized in that: The following steps are involved: 1) Using genomic DNA of Bacillus velezensis ZD122 as template, PCR amplification ituA The upstream and downstream homology arms of the gene were overlapped by PCR. ituA Gene upstream homology arm, promoter P bacA and ituA The downstream homology arms of the gene were connected together, and the resulting homology arm fusion fragment was connected to the plasmid T2(2)-ori to obtain the promoter insertion vector T2(2)-P bacA - ituA ; 2) The vector T2(2)-PbacA-ituA was transformed into Bacillus velezensis ZD122, and kanamycin was used as a screening marker to obtain positive transformants. The positive transformants were screened by single and double crossover to obtain ituA Insert promoter P before gene bacA strain, the strain being named Bacillus velez ZD-P2; 3) PCR amplification using genomic DNA of Bacillus velezensis ZD122 as template fadM The upstream and downstream homology arms of the gene were separated by overlap extension PCR. fadM The upstream homology arm and the downstream homology arm of the gene were connected together, and the resulting homology arm fusion fragment was connected to the plasmid T2(2)-ori to obtain the knockout plasmid T2(2)-Δ fadM ; 4) The knockout plasmid T2(2)-Δ fadM The strain was transformed into Bacillus velezensis ZD-P2 and kanamycin was used as a screening marker to obtain positive transformants. The positive transformants were successfully knocked out after single and double crossover screening. fadM Velez-Bacillus sp., the strain was named ZD-P2Δ fadM .

3. The construction method according to claim 2, characterized in that In step 1) ituA The primer sequences of the upstream homology arms of the gene are shown in SEQ ID NO. 7 and 8. ituA The primer sequences of the downstream homology arms of the gene are shown in SEQ ID NO.9 and 10. In step 3) fadM The primer sequences of the upstream homology arms of the gene are shown in SEQ ID NO. 11 and 12. fadM The primer sequences of the downstream homology arms of the gene are shown in SEQ ID NOs. 13 and 14.

4. Use of the engineered Bacillus velezensis bacteria according to claim 1 in producing iturin A.

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