A method for improving biomass and cell protein content of bacillus amyloliquefaciens by knocking out global transcription factor codY

Abstract

The application belongs to the technical field of genetic engineering, and discloses a method for improving biomass and cell protein content of Bacillus amyloliquefaciens by knocking out global transcription factor codY. The engineering strain is obtained by taking Bacillus amyloliquefaciens HZ-12 as a starting bacterium and knocking out the global transcription factor coding gene codY, and the biomass and cell protein content of the Bacillus amyloliquefaciens engineering bacterium HZ-12ΔcodY are increased by 37.44% and 6.88% respectively relative to those of the control strain HZ-12.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a method for increasing the biomass and cell protein content of engineered Bacillus amyloliquefaciens by knocking out the global transcription factor codY. Background Technology

[0002] Dietary protein is an essential nutrient for humans, playing a vital role in maintaining normal physiological metabolism, immune function, and tissue repair. In recent years, with the continuous growth of the global population and the improvement of living standards, the demand for high-quality protein has increased dramatically, making it difficult for traditional planting and animal husbandry production models to meet the demand. Therefore, exploring and developing new protein resources as a powerful supplement to traditional protein supplies is of significant strategic importance for alleviating the current global protein shortage crisis and promoting sustainable agricultural development. Currently, microbial protein is widely recognized as a highly promising new alternative protein source due to its significant advantages such as high production efficiency, low energy consumption, and comprehensive nutrition.

[0003] Bacillus amyloliquefaciens, a food-grade safe strain, possesses rapid growth, strong tolerance, and efficient secretory expression capabilities, making it an ideal candidate strain for enhancing microbial cell protein content. The global transcription factor CodY participates in regulating multiple key physiological processes in Gram-positive bacteria, including carbon and nitrogen metabolism, amino acid synthesis, and cell division. Its deletion may relieve the inhibition of related metabolic pathways, thereby promoting cell proliferation and protein accumulation. Based on this, this invention significantly increases the biomass and cell protein content of Bacillus amyloliquefaciens by knocking out the global transcription factor encoding gene codY in Bacillus amyloliquefaciens HZ-12, providing an effective strategy for increasing Bacillus amyloliquefaciens protein production and laying a genetic resource foundation for the discovery and application of novel alternative proteins. Summary of the Invention

[0004] The purpose of this invention is to provide a method for increasing the biomass and cell protein content of Bacillus amyloliquefaciens by knocking out the global transcription factor codY. By using genetic engineering technology, the global transcription factor encoding gene codY is knocked out in Bacillus amyloliquefaciens HZ-12, which significantly increases the cell biomass and cell protein content of Bacillus amyloliquefaciens.

[0005] To achieve the above objectives, the present invention employs the following technical measures: A strain of Bacillus amyloliquefaciens engineered to increase cell biomass and cell protein content was obtained by knocking out the global transcription factor coding gene codY in Bacillus amyloliquefaciens HZ-12. The specific construction method is as follows: 1) Using the genomic DNA of Bacillus amyloliquefaciens HZ-12 as a template, the upstream and downstream homologous arms of the codY gene were amplified by PCR. 2) The upstream homologous arm (SEQ ID NO.2) and the downstream homologous arm (SEQ ID NO.3) of the codY gene were ligated together by overlap extension PCR to form a homologous arm fusion fragment; 3) The homologous arm fusion fragment and plasmid T2(2)-ori were double-digested with XbaI and SmaI restriction endonucleases to obtain the digested gene fragment and linear plasmid fragment. The fragments were then ligated with DNA ligase to obtain the knockout plasmid T2(2)-ΔcodY. 4) The knockout plasmid T2(2)-ΔcodY was transformed into Bacillus amyloliquefaciens HZ-12, and positive transformants were obtained by screening with kanamycin as a selection marker; 5) After transferring the positive transformants to a culture medium containing kanamycin at 45°C for several generations, colony PCR detection was performed (the sequences of the detection primers are shown in SEQ ID NO.4 and 7, or in SEQ ID NO.5 and 6). Positive single-exchange binding strains in which the upstream or downstream homologous arm of the codY gene completely exchanges with the genomic DNA of Bacillus amyloliquefaciens HZ-12 were screened. 6) Select positive single-exchange conjugate strains and inoculate them into a 37°C medium without kanamycin. After several transfer cultures, use PCR (with the sequences of the detection primers shown in SEQ ID NO. 8 and 9) to screen for Bacillus amyloliquefaciens with the codY gene knocked out to obtain HZ-12ΔcodY.

[0006] The application of *Bacillus amyloliquefaciens* HZ-12ΔcodY prepared by the above method in improving bacterial biomass and bacterial protein content: Colonies were picked and inoculated into LB medium for seed culture, and then inoculated into fermentation medium at a 5% inoculum (xylose 50 g / L, sodium citrate 15 g / L, ammonium sulfate 10 g / L, KH2PO4 1 g / L, CaCl2 1 g / L, MgSO4·7H2O 1 g / L, MnSO4·H2O 0.15 g / L, ZnSO4·7H2O 1 g / L), and cultured at 37℃ and 230 rpm for 36 h. The results showed that the biomass and bacterial protein content of the engineered strain HZ-12ΔcodY were significantly increased compared with the control strain HZ-12, by 37.44% and 6.88%, respectively.

[0007] This invention is the first attempt to knock out the global transcription factor encoding gene codY in Bacillus amyloliquefaciens HZ-12. Compared with the control strain HZ-12, it significantly increased the biomass and cell protein content, providing new gene resources for the discovery of novel alternative proteins.

[0008] Compared with the prior art, the present invention has the following advantages and effects: 1. This invention is the first to discover that knocking out the global transcription factor encoding gene codY in Bacillus amyloliquefaciens HZ-12 can significantly increase the biomass and cell protein content of Bacillus amyloliquefaciens.

[0009] 2. Compared with the control strain, the engineered Bacillus amyloliquefaciens strain HZ-12ΔcodY constructed in this invention has a 37.44% higher biomass and a 6.88% higher cell protein content.

[0010] 3. The gene knockout technology used in this invention leaves no antibiotic selection markers or other exogenous DNA fragments. The engineered strain HZ-12ΔcodY still meets the relevant food safety and biosafety standards in terms of genetic background and can be directly applied to the production of alternative proteins in the food, feed and fermentation industries. Attached Figure Description

[0011] Figure 1 To investigate the effect of knocking out the global transcription factor encoding gene codY on bacterial biomass in Bacillus amyloliquefaciens HZ-12.

[0012] Figure 2 To investigate the effect of knocking out the global transcription factor encoding gene codY on the protein content of Bacillus amyloliquefaciens HZ-12. Detailed Implementation

[0013] Unless otherwise specified, the molecular biology experimental methods used in the following examples were performed under standard conditions, referring to "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor).

[0014] Description of biological materials: Bacillus amyloliquefaciens HZ-12 and plasmid T2(2)-ori are publicly available biological materials, which have been reported in the article “Metabolic engineering of Bacillus amyloliquefaciens for enhanced production of S-adenosylmethionine by coupling of an engineered S-adenosylmethionine pathway and the tricarboxylic acid cycle” (Biotechnol.Biofuels 12, 211) and are currently deposited in the Microbial Engineering Laboratory of Huazhong Agricultural University.

[0015] Example 1: Construction of a temperature-sensitive knockout vector Using the upstream and downstream gene sequences of codY from the genomic DNA sequence of Bacillus amyloliquefaciens HZ-12, primers for the upstream homologous arm of the codY gene (codY-AF, codY-AR) and the downstream homologous arm (codY-BF, codY-BR) were designed. Using the genomic DNA of Bacillus amyloliquefaciens HZ-12 as a template, PCR amplification was performed using the upstream and downstream homologous arm primers of the codY gene, respectively, to obtain the upstream and downstream homologous arm fragments of the codY gene. The primer sequences are as follows: codY-AF:GCCCCGGGTTGACATGCTGCAAGGCT (containing SmaII restriction site); codY-AR:CAGGGATCCTGCTTTTGTCGATAAATAATCCTCCTAGAATTCCTC; codY-BF: GAGGAATTCTAGGAGGATTATTTATCGACAAAAGCAGGATCCCTG; codY-BR: GCTCTAGACCAGGTTGGACGATACGAC (containing XbaI restriction site).

[0016] PCR system: 25.0 mL ddH2O, 5×TransStart TM FastPfu Buffer 5.0 mL, dNTPs 2.5mL, FastPfu DNA Polymerase 1.0 mL, forward primer (10 mM) 1.0 mL, reverse primer (10 mM) 1.0 mL, template DNA (289 ng / mL) 0.5 mL.

[0017] PCR reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 50~60℃ for 10 s, 72℃ for 30~60 s, 30~35 cycles; 72℃ for 5 min, 25℃ for 5 min.

[0018] Based on the PCR reaction system and conditions described above, PCR amplification using primer pair (codY-AF / codY-AR) yielded the upstream homologous arm fragment of codY (822 bp), sequence shown in SEQ ID NO.2. PCR amplification using primer pair (codY-BF / codY-BR) yielded the downstream homologous arm fragment of codY (827 bp), sequence shown in SEQ ID NO.3. Overlap extension PCR was then used to ligate the upstream and downstream homologous arms together, forming a homologous arm fusion fragment (1649 bp).

[0019] The homologous arm fusion fragment and plasmid T2(2)-ori were double-digested with SmaII and BamHI restriction endonucleases to obtain the digested gene fragment and linear plasmid fragment. The digested gene fragment and linear plasmid fragment were ligated with T4 DNA ligase to obtain the ligation product. The ligation product was transformed into E. coli competent cells DH5α by CaCl2 transformation and screened at 37°C on LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.2) containing kanamycin (20 μg / mL) resistance. The transformed individuals were verified by colony PCR and plasmid PCR (using primers T2-F and T2-R) to obtain the gene knockout vector T2(2)-ΔcodY.

[0020] The sequences for T2-F and T2-R are as follows: T2-F: ATGTGATAACTCGGCGTA; T2-R: GCAGAGCAGCAGATTACGC.

[0021] Example 2: Construction of Bacillus amyloliquefaciens gene knockout engineered strain HZ-12ΔcodY The knockout vector T2(2)-ΔcodY was transformed into Bacillus amyloliquefaciens HZ-12 and screened at 37°C on LB medium containing kanamycin (20 μg / mL). The transformed strains were verified by colony PCR (using primers T2-F and T2-R) and positive transformants (i.e. Bacillus amyloliquefaciens HZ-12 transformed with the knockout vector T2(2)-ΔcodY) were obtained.

[0022] Positive transformants were cultured several times at 45℃ on LB medium containing kanamycin resistance, each time for 12 h. Colony PCR was performed using T2-F and codY-BR primers (or T2-R and codY-AF primers) to detect single-exchange strains. The PCR-verified single-exchange strains were then cultured several times at 37℃ on LB medium without kanamycin. Transformants were selected for colony PCR verification (primers codY-VF / codY-VR). Single colonies with a band size of 2011 bp were selected; compared to the control strain, the codY knockout strain lacked 780 bp. DNA sequencing of the positive transformants further verified the double-exchange success, yielding a codY knockout strain (i.e., Bacillus amyloliquefaciens HZ-12ΔcodY). The sequences of primers codY-VF and codY-VR are as follows: codY-VF: GGTGTGAAAAATCCGTTTGA; codY-VR:GACGGGTCTTCCGTAACG.

[0023] Example 3: Effects of gene knockout (codY) on biomass and cell protein content in Bacillus amyloliquefaciens HZ-12 Single colonies of *Bacillus amyloliquefaciens* HZ-12 and HZ-12ΔcodY were picked and inoculated into 5 mL of LB medium and cultured at 37℃ and 180 rpm for 12 h. Then, 5% of the inoculum was transferred to 25 mL of fermentation medium (xylose 50 g / L, sodium citrate 15 g / L, ammonium sulfate 10 g / L, KH₂PO₄ 1 g / L, CaCl₂ 1 g / L, MgSO₄·7H₂O 1 g / L, MnSO₄·H₂O 0.15 g / L, ZnSO₄·7H₂O 1 g / L) and cultured at 37℃ and 230 rpm for 36 h. Subsequently, the bacterial cells were collected by centrifugation at 10,000 rpm for 10 min, resuspended in 10 mL of sterile water, and washed three times to remove medium components. The cells were then dried in a thermostatic drying oven at 103℃ for 4 h until their mass remained constant.

[0024] Collect 1 mL of bacterial culture by centrifugation at 10,000 rpm for 5 min, discard the supernatant, resuspend the bacterial cells in 1 mL of sterile water, repeat the washing once, discard the supernatant, and then serially dilute to OD0.05. 600 The value is approximately 0.2-0.8, and the OD is measured using a UV spectrophotometer. 600 The result is as follows Figure 1 As shown, the biomass OD of the engineered Bacillus amyloliquefaciens strain HZ-12ΔcodY 600 The value was 7.455, compared to the control strain HZ-12 (OD). 600(It was 5.424), an increase of 37.44%.

[0025] Weigh 2-4 mg of Bacillus amyloliquefaciens cell powder, wrap it tightly in a tin boat, and use a German Elementar Vario PYRO cube and Isoprime 100 elemental analyzer with acetanilide as a standard. The combustion tube temperature was set to 950℃, the reduction tube temperature to 600℃, the helium flow rate to 228 L / min, and the oxygen flow rate to 20 L / min. The nitrogen content (%) in the sample was determined, and 6.25 times the nitrogen content was taken as the bacterial protein content. Results are as follows: Figure 2 As shown, the bacterial protein content of the engineered Bacillus amyloliquefaciens strain HZ-12ΔcodY was 64.721%, which was 6.88% higher than that of the control strain HZ-12 (crude protein content 60.554%).

Claims

1. An engineered strain of Bacillus amyloliquefaciens, characterized in that, The engineered bacteria were obtained by knocking out the global transcription factor encoding gene codY in Bacillus amyloliquefaciens HZ-12.

2. The engineered Bacillus amyloliquefaciens strain according to claim 1, characterized in that, The nucleotide sequence of the gene codY, which is the global transcription factor that was knocked out, is shown in SEQ ID NO.

1.

3. The method for constructing the engineered Bacillus amyloliquefaciens according to claim 1, comprising the following steps: 1) Using the genomic DNA of Bacillus amyloliquefaciens HZ-12 as a template, the upstream and downstream homologous arms of the codY gene were amplified by PCR. 2) The upstream and downstream homologous arms of the codY gene were joined together by overlap extension PCR to form a homologous arm fusion fragment. 3) The homologous arm fusion fragment and plasmid T2(2)-ori were double-digested with XbaI and SmaI restriction endonucleases to obtain the digested gene fragment and linear plasmid fragment. The fragments were then ligated with DNA ligase to obtain the knockout plasmid T2(2)-ΔcodY. The knockout plasmid T2(2)-ΔcodY was transformed into Bacillus amyloliquefaciens HZ-12, and positive transformants were obtained by screening with kanamycin as a selection marker. 4) After transferring the positive transformants to a medium containing kanamycin at 45°C for several generations, positive single-exchange conjugate strains that completely exchange the upstream or downstream homologous arm of the codY gene with the genomic DNA of Bacillus amyloliquefaciens HZ-12 were screened. 5) Select positive single-exchange conjugate strains and inoculate them into a 37°C medium without kanamycin. After several transfer cultures, Bacillus amyloliquefaciens HZ-12ΔcodY with the codY gene knocked out was obtained.

4. The construction method according to claim 3, characterized in that, The sequences of the upstream and downstream homologous arms of the codY gene in step 1) are shown in SEQ ID NO.2 and SEQ ID NO.

3.

5. The construction method according to claim 3, characterized in that, The sequences of the PCR detection primers for the positive single-exchange binding strains in step 4) are shown in SEQ ID NO.4 and 7, or in SEQ ID NO.5 and 6.

6. The construction method according to claim 3, characterized in that, The sequences of the PCR detection primers for Bacillus amyloliquefaciens HZ-12ΔcodY in step 5) are shown in SEQ ID NO. 8 and 9.

7. The application of the engineered Bacillus amyloliquefaciens strain according to claim 1 in improving bacterial biomass and bacterial protein content.

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