Bacillus velezensis engineering bacterium for improving yield of iturin A and construction method of bacillus velezensis engineering bacterium

Abstract

The invention provides a bacillus velezensis engineering bacterium for improving yield of iturin A. A promoter PacA is inserted in front of an ituA gene of bacillus velezensis ZD122 through a genetic engineering method, and a proline degradation related gene fadM in a genome is knocked out, so that the bacillus velezensis engineering bacterium ZD-P2 [delta] fadM is successfully obtained, and the bacillus velezensis engineering bacterium ZD-P2 [delta] fadM can be used for improving the yield of iturin A. The invention further provides a construction method of the bacillus velezensis engineering bacterium ZD-P2 [delta] fadM. Compared with the bacillus velezensis ZD122, the yield of iturin A produced by the engineering strain ZD-P2 delta fadM constructed by the invention is increased by two times.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an engineering bacillus velezine for improving iturin A production and a construction method thereof. Background Art

[0002] Iturin A is a biosurfactant composed of hydrophobic fatty acids and hydrophilic peptide chains. Its special amphiphilic structure and biodegradability have attracted widespread attention. Iturin A is widely used in oil recovery, agricultural biological control, cosmetics and food. Iturin A has great commercial value, but its low yield is the main factor limiting its industrial production (see reference: Xu Yuxiang. Metabolic engineering of Bacillus amyloliquefaciens HZ-12 to produce high-yield iturin A [D]. Hubei University, 2020.).

[0003] There is no report on the chemical synthesis of iturin A, so the industrial production of high-value iturin A must rely on biosynthesis. Bacillus is the most efficient known strain for producing iturin A, but the production capacity of wild strains is limited. Therefore, it is of great significance to adopt strategies to increase the yield of iturin A produced by strains. Summary of the invention

[0004] The present invention aims to provide an engineered bacillus velezine for improving the yield of iturin A and a construction method thereof. The promoter PbacA is inserted before the ituA gene of the bacillus velezine ZD122 through genetic engineering technology, and the fadM gene related to proline degradation in the genome is knocked out, thereby significantly improving the yield of iturin A.

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

[0006] A strain of Bacillus velezensis engineered bacteria for improving iturin A production is prepared by inserting a promoter PbacA before the ituA gene of Bacillus velezensis ZD122, and knocking out the fadM gene, a gene related to 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, PCR was used to amplify the upstream homology arm and downstream homology arm of the ituA gene. 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 connected to 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 velez 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 a strain in which the promoter PbacA was inserted in front of the ituA gene. The strain was named Bacillus velez ZD-P2;

[0009] 3) Using the genomic DNA of Bacillus velezensis ZD122 as a template, PCR was used to amplify the upstream homology arm and the downstream homology arm of the fadM gene. 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 connected to 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 sequence of the upstream homologous arm of the ituA gene in the step 1) is shown as SEQ ID NOs. 7 and 8, the primer sequence of the downstream homologous arm of the ituA gene is shown as SEQ ID NOs. 9 and 10, the primer sequence of the upstream homologous arm of the fadM gene in the step 3) is shown as SEQ ID NOs. 11 and 12, and the primer sequence of the downstream homologous arm of the fadM gene is shown as SEQ ID NOs. 13 and 14.

[0012] Application of the engineered Bacillus belezii 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 inoculated into the fermentation medium (corn starch 30g / L, soybean meal 70g / L, K2HPO4·3H2O 1g / L, MgSO4·7H2O 1g / L, FeSO4·7H2O 1g / L, MnSO4·H2O 0.01g / L) according to a certain inoculum amount, and cultured at 37°C and 180rpm for 72h. The results showed that the yield of iturin A produced by the engineered Bacillus belezii ZD-P2ΔfadM was 2 times higher than that of the original bacterium Bacillus belezii ZD122.

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

[0014] 1. The present invention first discovered that inserting the promoter PbacA before the ituA gene and knocking out the fadM gene related to proline degradation in the genome can significantly increase the yield of iturin A produced by Bacillus velez. Compared with the original bacterium Bacillus velez ZD122, the yield of iturin A produced by the Bacillus velez engineered bacterium ZD-P2ΔfadM constructed by the present invention is 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 Bacillus velez engineered bacteria ZD-P2 in Example 2 in which the promoter PbacA was inserted in front of 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, respectively, and Bacillus velez ZD-P2 and ZD122 as templates.

[0019] Figure 4 This is the verification band of the Bacillus velez engineered bacteria 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 strain of Bacillus velez ZD-P2ΔfadM. DETAILED DESCRIPTION

[0021] The molecular biology experimental methods in the following examples without specifying specific conditions are all based on conventional conditions and refer 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 a deposit number of CCTCC NO: M 20241480 and a deposit date of 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 insertion vector

[0025] Using the genomic DNA of Bacillus licheniformis DW2 as a template, PCR amplification with primers PbacA-F and PbacA-R was performed to obtain the 335bp promoter PbacA. According to 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 with upstream homology arm primers itu-F1 / ituA-R1 and downstream homology arm primers ituA-F2 / ituA-R2 to obtain the upstream homology arm fragment and 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 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, the promoter PbacA (335 bp) was obtained by PCR amplification using primers PbacA-F and PbacA-R, and the sequence is shown in SEQ ID NO.1. The upstream homology arm fragment (530 bp) was obtained by PCR amplification using primers ituA-F1 / ituA-R1, and the sequence is shown in SEQ ID NO.3. The downstream homology arm fragment (545 bp) was obtained by PCR amplification using primers ituA-F2 / ituA-R2, and the sequence is shown in SEQ ID NO.4. The upstream homology arm, the promoter PbacA and the downstream homology arm were connected together by overlapping 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 a restriction-digested gene fragment and a linear plasmid fragment. The restriction-digested gene fragment and the linear plasmid fragment were ligated with T4 DNA ligase to obtain a ligation product ( Figure 1 ); the ligation product was transformed into Escherichia coli DH5α by the calcium chloride transformation method, and the cells were 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 the cells were screened at 37°C in an LB medium containing kanamycin (20 μg / mL) resistance to obtain transformants. The transformants were verified by colony PCR (primers used were: T2-F and T2-R) to obtain positive transformants, i.e., 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 time for 12 hours, and colony PCR was performed to detect single-exchange strains using T2-F and ituA-KYR as primers or T2-R and ituA-KYF as primers. Amplified bands of 1683 bp or 1792 bp in length indicated that single-exchange strains were obtained.

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

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

[0045] According to the fadM gene sequence in the genome of Bacillus velez ZD122, the upstream homology arm primers (fadM-F1, fadM-R1) and downstream homology arm primers (fadM-F2, fadM-R2) of the fadM gene were designed; and the genomic DNA of Bacillus velez ZD122 was used as a template, and the upstream homology arm primers and downstream homology arm primers of the fadM gene were used for PCR amplification 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 site).

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

[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, the 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 the 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 connected by T4 DNA ligase to obtain a connection product ( Figure 2); the ligation product was transformed into Escherichia coli DH5α by the calcium chloride transformation method, and the cells were 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) resistance to obtain transformants. The transformants were verified by colony PCR (primers used were: T2-F and T2-R) to obtain positive transformants, i.e., Bacillus velezensis ZD-P2 into which the knockout vector T2(2)-ΔfadM had been transferred.

[0055] The positive transformants were transferred and cultured three times on LB medium containing kanamycin resistance at 45°C, each time for 12 h, and colony PCR was performed to detect single-exchange strains using T2-F and ΔfadM-KYR as primers or T2-R and ΔfadM-KYF as primers. Amplification of a band of 1333 bp or 1442 bp in length indicated that a single-exchange strain was obtained.

[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 in LB medium without kanamycin at 37°C for several times, and the transformants were selected for colony PCR verification (primers were ΔfadM-KYF and ΔfadM-KYR) ( Figure 4 ). Subsequently, DNA sequencing was performed on the positive transformants for further verification, and a fadM knockout strain with successful double exchange was obtained, namely Bacillus velezensis ZD-P2ΔfadM.

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

[0061] Velezella ZD122 and ZD-P2ΔfadM were selected and inoculated into LB medium, and cultured at 37°C and 180 rpm for 12 h. 600 When the pH value was 3.0-4.0, 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% inoculation and cultured at 37°C and 180 rpm for 72 h. 0.3 mL of 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 was shown that the iturin A production of the engineered Bacillus velezinii ZD-P2ΔfadM was as high as 1.10 g / L, which was 2 times higher than that of the original Bacillus velezinii ZD122.

Claims

1. An engineered bacillus velezine for increasing the yield of iturin A, characterized in that: The engineered bacteria is in Bacillus velez ( Bacillus velezensis )ZD122 iuda 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.

2. The Bacillus Velezii engineered bacteria according to claim 1, characterized in that 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.

3. The method for constructing the Bacillus Velezii engineered bacteria according to claim 1, characterized in that: The following steps are involved: 1) PCR amplification using genomic DNA of Bacillus velez ZD122 as template iuda The upstream and downstream homology arms of the gene were overlapped by PCR. iuda Gene upstream homology arm, promoter P bacA and iuda 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 - iuda ; 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 iuda Insert promoter P before gene bacA strain, the strain being named Bacillus velez ZD-P2; 3) PCR amplification using genomic DNA of Bacillus velez ZD122 as template fadM The upstream and downstream homology arms of the gene were overlapped by 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 exchange screening. fadM Velezella sp., the strain was named ZD-P2Δ fadM .

4. The use according to claim 3, characterized in that In step 1) iuda The primer sequences of the upstream homology arms of the gene are shown in SEQ ID NOs. 7 and 8. iuda The primer sequences of the downstream homology arms of the gene are shown in SEQ ID NOs. 9 and 10. In step 3) fadM The primer sequences of the upstream homology arms of the gene are shown in SEQ ID NOs. 11 and 12. fadM The primer sequences of the downstream homology arms of the gene are shown in SEQ ID NOs. 13 and 14.

5. Use of the Bacillus Velezii engineered bacteria according to claim 1 in producing iturin A.

Images