High-yield B 12 Genetically engineered bacteria of halophilic red halophilic bacteria from Daqing and homologous recombination construction method and application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHIHEZI UNIVERSITY
- Filing Date
- 2026-07-03
- Publication Date
- 2026-08-07
AI Technical Summary
[0005]本发明是要解决现有大庆嗜盐碱红菌JX313的维生素B12产量较低，无法满足生产需求的问题，提供高产B12的大庆嗜盐碱红菌基因工程菌及其同源重组构建方法和应用
[0031]1、本发明成功实现nhaC1基因和nhaC2基因的双基因叠加敲除，敲除阳性率稳定，为该非模式嗜盐碱古菌的基因功能研究提供了成熟的技术方法。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial genetic engineering, and more particularly to high-yield B... 12 The genetically engineered Daqing halophilic red bacterium and its homologous recombination construction method and application. Background Technology
[0002] Extreme halophilic archaea are a characteristic group of microorganisms that inhabit highly saline-alkaline environments, capable of growing under conditions ranging from 10% NaCl to saturated NaCl concentrations and pH levels above 8.5. They maintain osmotic balance by accumulating high concentrations of K⁺ intracellularly through a "salt ingress" strategy, while simultaneously relying on Na⁺ / H⁺ antitransporters to expel excess Na⁺ from the cell, thus maintaining a low-sodium intracellular environment and pH homeostasis. This ion homeostasis regulation system is the core basis for their adaptation to extreme environments.
[0003] *Natronorubrum daqingense* JX313 is an extreme halophilic archaea isolated from saline-alkali soil in Daqing City. Taxonomically, it belongs to the genus *Natronorubrum*, and its public accession number is CGMCC 1.8909. Genome analysis confirmed that this strain naturally carries complete cobalamin (vitamin B1). 12 The biosynthetic pathway allows for the autonomous synthesis of vitamin B. 12 It is an ideal model strain for studying the cross-relationship between ion homeostasis and secondary metabolic regulation in halophilic archaea.
[0004] However, the vitamin B of Daqing halophilic erythrophyte JX313 12 Production volume is low and cannot meet production needs. Summary of the Invention
[0005] This invention aims to address the vitamin B deficiency of the existing Daqing halophilic erythrophorus bacterium JX313. 12 The problem of low output failing to meet production demand necessitates providing high-yield B. 12 The genetically engineered Daqing halophilic red bacterium and its homologous recombination construction method and application.
[0006] The Daqing halophilic erythromycete genetically engineered strain of the present invention is a double knockout strain of the nhaC1 and nhaC2 genes, that is, a genetically engineered strain in which the coding regions of the nhaC1 and nhaC2 genes are simultaneously deleted.
[0007] Furthermore, the method for preparing the double knockout strain of the nhaC1 and nhaC2 genes is as follows:
[0008] I. Using the genomic DNA of the nhaC1 gene knockout strain of Rhodopseudomonas halophilus JX313T from Daqing as a template, PCR amplification was performed using primers C2F-FP / C2F-RP to obtain the upstream homologous arm of the nhaC2 gene. PCR amplification was then performed using primers C2R-FP / C2R-RP to obtain the downstream homologous arm of the nhaC2 gene. Finally, using plasmid E-pUC57 as a template, PCR amplification was performed using primers C2E-FP / C2E-RP to obtain the erythromycin resistance fragment.
[0009] Fragment fusion was performed in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm" and ligated into the pUC18 cloning vector to obtain the double knockout plasmid pUC-nhaC2-dko.
[0010] Using the double knockout plasmid pUC-nhaC2-dko as a template, PCR amplification was performed with primers C2F-FP / C2R-RP to obtain the linear homologous recombination fragment of nhaC2.
[0011] 2. Using the nhaC1 gene knockout strain of Daqing halophilic erythrophorus JX313T as the starting strain, the bacterial suspension of the starting strain in the logarithmic growth phase was collected by centrifugation to prepare a protoplast suspension of strain ΔnhaC1; the nhaC2 knockout linear homologous recombination fragment was added to the protoplast suspension, and protoplast transformation was performed to obtain the double gene knockout strains ΔnhaC1&C2 of nhaC1 and nhaC2.
[0012] Furthermore, the method for preparing the nhaC1 gene knockout strain of *Rhodophyta daqingense* JX313T is as follows:
[0013] (1) Using wild-type genomic DNA as a template, PCR amplification was performed using primers C1F-FP / C1F-RP to obtain the upstream homologous arm, and PCR amplification was performed using primers C1R-FP / C1R-RP to obtain the downstream homologous arm. Using plasmid E-pUC57 as a template, PCR amplification was performed using primers C1E-FP / C1E-RP to obtain the erythromycin resistance fragment.
[0014] Fragment fusion was performed in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm" and ligated into the pUC18 cloning vector to obtain the knockout plasmid pUC-nhaC1-ko.
[0015] Using the knockout plasmid pUC-nhaC1-ko as a template, PCR amplification was performed with primers C1F-FP / C1R-RP to obtain the linear homologous recombination fragment of nhaC1.
[0016] (2) Add the nhaC1 linear homologous recombination fragment to the protoplast suspension of wild-type strains, perform protoplast transformation, and obtain the nhaC1 gene knockout strain ΔnhaC1.
[0017] The Daqing halophilic erythromycete genetically engineered strain of the present invention is an nhaC1 gene knockout strain, that is, a genetically engineered strain with the coding region of the nhaC1 gene missing.
[0018] Furthermore, the method for preparing the nhaC1 gene knockout strain is as follows:
[0019] (1) Using wild-type genomic DNA as a template, PCR amplification was performed using primers C1F-FP / C1F-RP to obtain the upstream homologous arm, and PCR amplification was performed using primers C1R-FP / C1R-RP to obtain the downstream homologous arm. Using plasmid E-pUC57 as a template, PCR amplification was performed using primers C1E-FP / C1E-RP to obtain the erythromycin resistance fragment.
[0020] Fragment fusion was performed in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm" and ligated into the pUC18 cloning vector to obtain the knockout plasmid pUC-nhaC1-ko.
[0021] Using the knockout plasmid pUC-nhaC1-ko as a template, PCR amplification was performed with primers C1F-FP / C1R-RP to obtain the linear homologous recombination fragment of nhaC1.
[0022] (2) Add the nhaC1 linear homologous recombination fragment to the protoplast suspension of wild-type strains, perform protoplast transformation, and obtain the nhaC1 gene knockout strain ΔnhaC1.
[0023] The Daqing halophilic erythromycete genetically engineered strain of the present invention is an nhaC2 gene knockout strain, that is, a genetically engineered strain with the coding region of the nhaC2 gene missing.
[0024] Furthermore, the method for preparing the nhaC2 gene knockout strain is as follows:
[0025] (1) Using wild-type genomic DNA as a template, the upstream homologous arm was obtained by PCR amplification using primers C2F-FP / C2F-RP, the downstream homologous arm was obtained by PCR amplification using primers C2R-FP / C2R-RP, and the erythromycin resistance fragment was obtained by PCR amplification using plasmid E-pUC57 as a template and primers C2E-FP / C2E-RP.
[0026] Fragment fusion was performed in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm" and ligated into the pUC18 cloning vector to obtain the knockout plasmid pUC-nhaC2-ko.
[0027] Using the knockout plasmid pUC-nhaC2-ko as a template, PCR amplification was performed with primers C2F-FP / C2R-RP to obtain the linear homologous recombination fragment of nhaC2.
[0028] (2) Add the nhaC2 linear homologous recombination fragment to the protoplast suspension of wild-type strains, perform protoplast transformation, and obtain the nhaC2 gene knockout strain ΔnhaC2.
[0029] The above-mentioned Daqing halophilic erythromycin genetically engineered bacteria is used to improve B... 12 Production.
[0030] The beneficial effects of this invention are:
[0031] 1. This invention successfully achieved dual-gene knockout of the nhaC1 and nhaC2 genes with a stable knockout positive rate, providing a mature technical method for gene function research of this non-model halophilic archaea.
[0032] 2. This invention, through comparative studies of single-gene knockout and double-gene knockout, clarifies the individual contributions and synergistic effects of nhaC1 and nhaC2 in the salt and alkali tolerance of bacterial strains: nhaC2 contributes more significantly to the alkali tolerance of the strains, and both genes work together to maintain the high salt tolerance of the strains. The inhibitory effect of double-gene knockout on the growth of the strains is additive. This finding fills a gap in the in vivo functional research of NhaC family proteins in halophilic archaea.
[0033] 3. Both knockout of the nhaC1 gene and knockout of the nhaC2 gene can significantly increase vitamin B1 levels. 12 The transcriptional level of key synthetic genes was increased, thereby enhancing the vitamin B content of the strain. 12 Yield; the yield-enhancing effect of double gene knockout is more significant. This discovery provides two novel metabolic regulatory targets for the construction of high-yield cobalamin engineered bacteria, and the two targets can be used in combination, which has important application value. Attached Figure Description
[0034] Figure 1 The construction and identification results of the Daqing halophilic red bacterium knockout strain ΔnhaC1;
[0035] Figure 2 The results of the construction and identification of the +nhaC1 complement strain;
[0036] Figure 3 The construction and identification results of the Daqing halophilic red bacterium knockout strain ΔnhaC2;
[0037] Figure 4 The results of the construction and identification of the +nhaC2 complement strain;
[0038] Figure 5 Electrophoresis diagrams for the construction and validation of the double knockout strain ΔnhaC1&C2;
[0039] Figure 6 Physiological growth characteristics of various strains related to the nhaC1 gene;
[0040] Figure 7 Physiological growth characteristics of various strains related to the nhaC2 gene;
[0041] Figure 8 Physiological growth characteristics of ΔnhaC1&C2 double knockout strain
[0042] Figure 9 For each strain and vitamin B 12 The results of the composite association analysis. Detailed Implementation
[0043] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.
[0044] Example 1: Construction of ΔnhaC1 single knockout strain
[0045] 1. Experimental materials
[0046] Experimental strain: wild strain of Rhodophyta daqingense JX313 (this strain has been published, with accession number CGMCC 1.8909).
[0047] Vectors and reagents: pUC18 cloning vector, CloneExpress Ultra One Step Cloning Kit (Vazyme C115), erythromycin, protoplast preparation buffer and reagent kit, DNA gel extraction kit.
[0048] The primer sequences are shown in the table below:
[0049] Table 1
[0050]
[0051] 2. Operating Procedures
[0052] (1) Using the genomic DNA of wild-type JX313 as a template, PCR amplification was performed using primers C1F-FP / C1F-RP to obtain the upstream homologous arm (500 bp), and PCR amplification was performed using primers C1R-FP / C1R-RP to obtain the downstream homologous arm (500 bp). Using plasmid E-pUC57 as a template, PCR amplification was performed using primers C1E-FP / C1E-RP to obtain the 735 bp erythromycin resistance fragment (ermE). The fragments were purified by gel extraction after agarose gel electrophoresis.
[0053] (2) Prepare a seamless cloning reaction system according to the molar ratio of vector to insert fragment 1:3, perform fragment fusion in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm", ligate to pUC18 cloning vector, and ligate at 50℃ for 15~30 min; transform the ligation product into Escherichia coli DH5α competent cells, screen with 100 μg / mL ampicillin, pick transformants to extract plasmid, and perform PCR identification, double enzyme digestion and sequencing verification with primers M13-47F / M13R to obtain the knockout plasmid pUC-nhaC1-ko;
[0054] (3) Using the knockout plasmid pUC-nhaC1-ko as a template, PCR amplification was performed with primers C1F-FP / C1R-RP to obtain the linear homologous recombination fragment of nhaC1 (1735 bp), which was then purified by gel extraction for later use.
[0055] (4) Preparation of wild-type protoplasts: take protoplasts during the logarithmic growth phase (OD). 600 Collect 5 mL of wild-type bacterial culture (≈0.8%) by centrifugation at 5000×g; wash 3 times with protoplast formation buffer (Solution I); resuspend the bacterial cells in 150 μL of protoplast formation solution (Solution II), add 15 μL of 0.5M EDTA (pH 8.0), and let stand at room temperature for 10 min to prepare a protoplast suspension.
[0056] (5) Add 2 μg of linear homologous recombination fragment to the protoplast suspension and let stand at room temperature for 15 min; add 175 μL of 60% PEG600 solution preheated at 37℃, mix gently, and let stand at room temperature for 30 min to mediate homologous recombination; add 1 mL of protoplast dilution buffer (Solution Ⅲ) to wash the cells, centrifuge at 3500×g for 2 min, and repeat the washing once.
[0057] (6) Resuspend the bacterial cells in 1 mL of protoplast regeneration solution (Solution IV), let stand at 37℃ for 1.5 h, and then revive at 37℃ and 40 rpm for 12 h; wash the bacterial cells twice with protoplast transformation dilution solution (Solution V), resuspend them, spread them on HLB solid medium containing 200 μg / mL erythromycin (LB medium containing 17.5% NaCl, pH 10.0), and incubate upside down at 37℃ for 10 days.
[0058] (7) Select a single colony to extract genomic DNA, and perform PCR amplification using primers C1Y-FP / C1Y-RP (expected product 2136 bp). Combine the sequencing results to verify that the nhaC1 coding region has been completely replaced by the resistance gene.
[0059] 3. Results
[0060] A stable, heritable nhaC1 knockout strain, ΔnhaC1, was successfully obtained. The knocked-out gene is the CDS sequence of nhaC1, as shown in SEQ ID NO: 1 in the sequence listing.
[0061] The construction and identification results of the Daqing halophilic erythrocyte knockout strain ΔnhaC1 are as follows: Figure 1 As shown in the figure, A represents the amplification results of each precursor fragment of the linear DNA fragment required for nhaC1 knockout; lane 1: upstream homologous arm of nhaC1, lane 2: erythromycin resistance fragment, lane 3: downstream homologous arm of nhaC1; B represents the PCR identification of the recombinant knockout plasmid pUC-nhaC1-ko, lanes 1 and 2 are the amplification products of the M13 universal primers; C represents the electrophoresis results of the plasmid pUC-nhaC1-ko after double digestion with EcoRI and HindIII, lanes 1 and 2 are the digestion products; D represents the electrophoresis of the purified nhaC1 knockout linear fragment, lanes 1-3 are the purified products; E represents the genomic PCR verification of the positive knockout transformant, lanes 1-3 are the amplification products of the C1Y-FP / C1Y-RP primers.
[0062] Example 2: Construction of nhaC1 gene complementation strain
[0063] 1. Experimental materials
[0064] Experimental strain: ΔnhaC1 knockout strain
[0065] Vectors and reagents: pUC18 cloning vector, hygromycin B, seamless cloning kit
[0066] 2. Operating Procedures
[0067] (1) Using wild-type genomic DNA as a template, the upstream homologous arm of nhaC1 (500 bp) was amplified by PCR using primers C1F-FP / C1BF-RP; the hygromycin resistance gene (1026 bp) was amplified by PCR using plasmid pSilentI as a template using primers C1H-FP / C1H-RP; and the full length of nhaC1 and the downstream homologous arm (2201 bp) were amplified by PCR using primers C1BR-FP / C1R-RP.
[0068] (2) The above three fragments were ligated into the pUC18 vector by seamless cloning, transformed into Escherichia coli DH5α, and after screening with ampicillin, the complement plasmid pUC-nhaC1-c was obtained by PCR identification, double enzyme digestion identification and sequencing verification.
[0069] (3) Using the complement plasmid pUC-nhaC1-c as a template, the linear complement fragment (4128 bp) was amplified by PCR using primers C1F-FP / C1R-RP, and purified by gel extraction for later use.
[0070] (4) Following the protoplast transformation method in Example 1, the linear complemented fragment was transformed into the protoplasts of strain ΔnhaC1, spread on HLB solid medium containing 50 μg / mL hygromycin B, and cultured upside down at 37°C for 10 days.
[0071] (5) Select a single colony to extract genomic DNA, perform PCR verification, and combine with sequencing to confirm that the supplemented fragment is correctly integrated.
[0072] 3. Results
[0073] A stable nhaC1 complement strain was obtained, and its growth phenotype was not significantly different from that of the wild strain, confirming that the phenotypic changes after nhaC1 knockout were caused by the specificity of this gene deletion.
[0074] The construction and identification results of the Daqing halophilic erythrophorus + nhaC1 supplemented strain are as follows: Figure 2 As shown in the diagram. A represents the amplification of each precursor fragment of the nhaC1 complemented linear fragment; lane 1: upstream homologous arm; lane 2: hygromycin resistance fragment; lane 3: full-length nhaC1 + downstream homologous arm; B represents PCR identification of the complemented plasmid pUC-nhaC1-c, lane 3 is the amplification product of primer M13, expected size 3727 bp; C represents the results of double digestion of pUC-nhaC1-c with BamHI and HindIII, lanes 1-3 are the digestion products; D represents the purified nhaC1 complemented linear fragment, lane 1 is the purified product; E represents multiplex PCR verification of the complemented strain: lanes 1-3 are the full-length verification primer C1Y-FP / C1Y-RP products; lanes 4, 7, and 10 are the upstream homologous arm primer products; lanes 5, 8, and 11 are the hygromycin resistance primer products; lanes 6, 9, and 12 are the downstream homologous arm primer products.
[0075] Example 3: Construction of ΔnhaC2 single knockout strain
[0076] 1. Experimental materials
[0077] Experimental strain: wild strain of Rhodophyta daqingense JX313 (preservation number CGMCC1.8909).
[0078] Vectors and reagents: pUC18 cloning vector, CloneExpress Ultra One Step Cloning Kit (Vazyme C115), erythromycin, protoplast preparation buffer and reagent kit, DNA gel extraction kit.
[0079] The primer sequences are shown in the table below:
[0080] Table 2
[0081]
[0082] 2. Operating Procedures
[0083] (1) Using wild-type genomic DNA as a template, upstream homologous arm (500 bp) was obtained by PCR amplification using primers C2F-FP / C2F-RP, and downstream homologous arm (500 bp) was obtained by PCR amplification using primers C2R-FP / C2R-RP. Using plasmid E-pUC57 as a template, 735 bp erythromycin resistance fragment (ermE) was obtained by PCR amplification using primers C2E-FP / C2E-RP. The target fragments were purified by gel extraction after agarose gel electrophoresis.
[0084] (2) Prepare a seamless cloning reaction system according to the molar ratio of vector to insert fragment 1:3, perform fragment fusion in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm", ligate to pUC18 cloning vector, and ligate at 50℃ for 15~30 min; transform the ligation product into Escherichia coli DH5α competent cells, screen with 100 μg / mL ampicillin, pick transformants to extract plasmid, identify by PCR, double enzyme digestion and sequencing to obtain knockout plasmid pUC-nhaC2-ko;
[0085] (3) Using the knockout plasmid pUC-nhaC2-ko as a template, PCR amplification was performed with primers C2F-FP / C2R-RP to obtain the linear homologous recombination fragment of nhaC2, which was then purified by gel extraction for later use.
[0086] (4) Preparation of wild-type protoplasts: take protoplasts during the logarithmic growth phase (OD). 600 Collect 5 mL of wild-type bacterial culture (≈0.8%) by centrifugation at 5000×g; wash 3 times with protoplast formation buffer (Solution I); resuspend the bacterial cells in 150 μL of protoplast formation solution (Solution II), add 15 μL of 0.5M EDTA (pH 8.0), and let stand at room temperature for 10 min to prepare a protoplast suspension.
[0087] (5) Add 2 μg of linear homologous recombination fragment to the protoplast suspension and let stand at room temperature for 15 min; add 175 μL of 60% PEG600 solution preheated at 37℃, mix gently, and let stand at room temperature for 30 min to mediate homologous recombination; add 1 mL of protoplast dilution buffer (Solution Ⅲ) to wash the cells, centrifuge at 3500×g for 2 min, and repeat the washing once.
[0088] (6) Resuspend the bacterial cells in 1 mL of protoplast regeneration solution (Solution IV), let stand at 37℃ for 1.5 h, and then revive at 37℃ and 40 rpm for 12 h; wash the bacterial cells twice with protoplast transformation dilution solution (Solution V), resuspend them, spread them on HLB solid medium containing 200 μg / mL erythromycin (LB medium containing 17.5% NaCl, pH 10.0), and incubate upside down at 37℃ for 10 days.
[0089] (7) Select a single colony to extract genomic DNA, and perform PCR amplification using primers C2Y-FP / C2Y-RP (expected product 2135 bp). Combine the sequencing results to verify that the nhaC2 coding region has been completely replaced by the resistance gene.
[0090] 3. Results
[0091] A stably inherited nhaC2 knockout strain, ΔnhaC2, was successfully obtained. The knockout gene is the CDS sequence of nhaC2, as shown in SEQ ID NO: 2 in the sequence listing. The strain can grow stably on HLB medium containing 200 μg / mL erythromycin.
[0092] The construction and identification results of the Daqing halophilic erythrocyte knockout strain ΔnhaC2 are as follows: Figure 3 As shown in the figure. A represents the amplification results of each precursor fragment of the linear DNA fragment required for nhaC2 knockout; lane 1: upstream homologous arm of nhaC2, lane 2: erythromycin resistance fragment, lane 3: downstream homologous arm of nhaC2; B represents the PCR identification of the recombinant knockout plasmid pUC-nhaC2-ko, lanes 2 and 3 are amplification products using the M13 universal primers; C represents the single enzyme digestion verification results of plasmid pUC-nhaC2-ko, lanes 1 and 2 are enzyme digestion products; D represents the electrophoresis of the purified nhaC2 knockout linear fragment, lanes 1-3 are the purified products; E represents the genomic PCR verification of the positive knockout transformant, lanes 1-3 are amplification products using the C2Y-FP / C2Y-RP primers.
[0093] Example 4: Construction of the +nhaC2 gene complementation strain
[0094] 1. Experimental materials
[0095] Experimental strain: ΔnhaC2 knockout strain
[0096] Vectors and reagents: pUC18 cloning vector, hygromycin B, seamless cloning kit
[0097] 2. Operating Procedures
[0098] (1) Using wild-type genomic DNA as a template, the upstream homologous arm (500 bp) of nhaC2 was amplified by PCR using primers C2F-FP / C2BF-RP; using plasmid pSilentI as a template, the hygromycin resistance gene (1026 bp) was amplified by PCR using primers C2H-FP / C2H-RP; and the full length of nhaC2 and the downstream homologous arm were amplified by PCR using primers C2BR-FP / C2R-RP.
[0099] (2) The above three fragments were ligated into the pUC18 vector by seamless cloning, transformed into Escherichia coli DH5α, and after screening with ampicillin, the complement plasmid pUC-nhaC2-c was obtained by PCR identification, double enzyme digestion identification and sequencing verification.
[0100] (3) Using the complement plasmid pUC-nhaC2-c as a template, the linear complement fragment (4001 bp) was amplified by PCR using primers C2F-FP / C2R-RP, and purified by gel extraction for later use.
[0101] (4) Following the protoplast transformation method in Example 1, the linear complemented fragment was transformed into the protoplasts of strain ΔnhaC2, which were then spread on HLB solid medium containing 50 μg / mL hygromycin B and cultured upside down at 37°C for 10 days.
[0102] (5) Select a single colony to extract genomic DNA, perform PCR verification, and combine with sequencing to confirm that the supplemented fragment is correctly integrated.
[0103] 3. Results
[0104] A stable nhaC2 complement strain was obtained, and its growth phenotype was not significantly different from that of the wild-type strain, confirming that the phenotypic changes after nhaC2 knockout were caused by the specific deletion of this gene.
[0105] The construction and identification results of the Daqing halophilic erythrophorus + nhaC2 supplemented strain are as follows: Figure 4 As shown in the diagram. A represents the amplification of each precursor fragment of the nhaC2 complemented linear fragment; lane 1: upstream homologous arm; lane 2: hygromycin resistance fragment; lane 3: full-length nhaC1 + downstream homologous arm; B represents PCR identification of the complemented plasmid pUC-nhaC2-c; lanes 3 and 4 are amplification products using M13 primers; C represents single enzyme digestion verification of the complemented plasmid; lanes 1-3: BamHI single enzyme digestion products; D represents the purified nhaC2 complemented linear fragment; lane 1 is the purified product; E represents multiplex PCR verification of the complemented strain: lanes 1-3 are full-length verification primer C2Y-FP / C2Y-RP products (4001 bp); lanes 4, 6, and 8: C2Y-FP / C2BH-RP products (1726 bp); lanes 5, 7, and 9: C2BH-FP / C2Y-RP products (3301 bp).
[0106] Example 5: Construction of ΔnhaC1&C2 double knockout strain
[0107] (1) Construction of double knockout vector
[0108] Using genomic DNA from the knockout strain ΔnhaC1 as a template, PCR amplification was performed using primers C2F-FP / C2F-RP to obtain the upstream homologous arm (500 bp) of the nhaC2 gene. PCR amplification was then performed using primers C2R-FP / C2R-RP to obtain the downstream homologous arm (500 bp) of the nhaC2 gene. Using plasmid E-pUC57 as a template, PCR amplification was performed using primers C2E-FP / C2E-RP to obtain a 1026 bp erythromycin resistance fragment. The fragments were purified by agarose gel electrophoresis and recovery.
[0109] A seamless cloning reaction system was prepared according to a 1:3 molar ratio of vector to insert fragment. Fragment fusion was performed in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm" and ligated into the pUC18 cloning vector. Ligation was carried out at 50℃ for 15-30 min. The ligation product was transformed into E. coli DH5α competent cells, screened with 100 μg / mL ampicillin, and plasmids were extracted from transformants. The plasmids were identified by PCR, double enzyme digestion and sequencing to obtain the double knockout plasmid pUC-nhaC2-dko.
[0110] Using the double knockout plasmid pUC-nhaC2-dko as a template, PCR amplification was performed with primers C2F-FP / C2R-RP to obtain the linear homologous recombination fragment of nhaC2, which was then purified by gel extraction for later use.
[0111] (2) Protoplast transformation
[0112] Starting with the ΔnhaC1 single knockout strain, the logarithmic growth phase (OD) was used. 600 Collect 5 mL of ΔnhaC1 knockout strain culture (≈0.8%) by centrifugation at 5000×g; wash 3 times with protoplast formation buffer (Solution I); resuspend the bacterial cells in 150 μL of protoplast formation solution (Solution II), add 15 μL of 0.5M EDTA (pH 8.0), and let stand at room temperature for 10 min to prepare ΔnhaC1 strain protoplast suspension.
[0113] Add 2 μg of nhaC2 knockout linear homologous recombination fragment to the protoplast suspension and let stand at room temperature for 15 min; add 175 μL of 60% PEG600 solution preheated at 37℃, mix gently, and let stand at room temperature for 30 min to mediate homologous recombination; add 1 mL of protoplast dilution buffer (Solution III) to wash the cells, centrifuge at 3500×g for 2 min, and repeat the washing once.
[0114] The bacterial cells were resuspended in 1 mL of protoplast regeneration solution (Solution IV), incubated at 37°C for 1.5 h, and then revived at 37°C and 40 rpm for 12 h. The bacterial cells were washed twice with protoplast transformation dilution solution (Solution V), resuspended, and spread onto HLB solid medium containing 200 μg / mL erythromycin (LB medium containing 17.5% NaCl, pH 10.0), and incubated upside down at 37°C for 10 days.
[0115] (3) Validation of positive strains
[0116] Genomic DNA was extracted from single colonies picked from antibiotic-free plates and amplified by PCR using primers C2Y-FP / C2Y-RP to verify that the nhaC2 coding region had been completely replaced by the hygromycin resistance gene. Simultaneously, combined with the verification results at the nhaC1 site, it was confirmed that both genes were successfully knocked out, resulting in the successful acquisition of the nhaC1 and nhaC2 double-gene knockout strain ΔnhaC1&C2. This strain can stably grow on HLB medium containing 200 μg / mL erythromycin and 50 μg / mL hygromycin B.
[0117] Electrophoresis diagram of the construction and validation of the double gene knockout strain ΔnhaC1&C2 is shown below. Figure 5 As shown in the figures. Figure A shows the amplification of the nhaC2 knockout linear fragment precursor (used for double knockout), lane 1: upstream homologous arm, lane 2: hygromycin resistance fragment (1026 bp), lane 3: downstream homologous arm; Figure B shows the PCR verification of the double knockout plasmid pUC-nhaC2-dko, lanes 1 and 2: amplification products using the M13 universal primers; Figure C shows the enzyme digestion verification of the double knockout plasmid, lanes 1 and 2: enzyme digestion products; Figure D shows the purified double knockout linear fragment, lanes 1-3: purified products; Figure E shows the genomic PCR verification of the positive double knockout strain, lanes 1-3: C2Y-FP / C2Y-RP amplification products (hygromycin replacement type).
[0118] Example 6: Functional Validation of Strains and Optimization of Fermentation Process
[0119] 1. Verification of the physiological function of the strain
[0120] Experimental setup: A NaCl concentration gradient of 10%–35% and a pH gradient of 7.0–11.0 were established. Wild-type strains WT, ΔnhaC1, ΔnhaC2, ΔnhaC1 & ΔnhaC2, +nhaC1, and +nhaC2 were cultured separately and incubated at 35℃ for 7 days. OD was then measured. 600 .
[0121] Experimental results:
[0122] Physiological growth characteristics of various strains related to the nhaC1 gene, such as Figure 6 As shown in the figure. Figure A compares the growth of three strains (WT, ΔnhaC1, and +nhaC1) under different NaCl concentration gradients; Figure B compares the growth of the three strains under different initial pH gradients; Figure C shows the growth curve of strain ΔnhaC1. The physiological growth characteristics of each strain related to the nhaC2 gene are as follows: Figure 7 As shown in the figure. Figure A compares the growth of the three strains WT, ΔnhaC2, and +nhaC2 under different NaCl concentration gradients; Figure B compares the growth of the three strains under different initial pH gradients; Figure C shows the growth curve of strain ΔnhaC2. The physiological growth characteristics of the ΔnhaC1 & C2 double knockout strains are as follows: Figure 8 As shown in the figure. Figure A compares the growth of WT and ΔnhaC1&C2 under different NaCl concentration gradients; Figure B compares the growth of WT and ΔnhaC1&C2 under different initial pH gradients; Figure C shows the growth curve of the double knockout strain ΔnhaC1&C2.
[0123] The results showed that the optimal salinity for ΔnhaC1 growth was still 17.5% NaCl, and the biomass decreased by approximately (26.11±3.77)% compared to WT; the pH tolerance range was 8.0~10.5, which was 0.5 pH units lower than WT.
[0124] The optimal salinity for ΔnhaC2 growth remains 17.5% NaCl, with biomass decreasing by approximately (32.71±2.13)% compared to WT; the upper limit of salt tolerance is 30%; the optimal pH is 9.5, and the pH tolerance range is 8.0~10.5.
[0125] The optimal salinity for growth of ΔnhaC1 & ΔnhaC2 remains at 15% NaCl, with a biomass decrease of approximately (39.09±2.50)%; the upper limit of salt tolerance is 27.5%; the optimal pH is 9.5, and the pH tolerance range is 7.0~10.5.
[0126] The growth phenotypes of +nhaC1 and +nhaC2 were basically consistent with those of WT, confirming that the phenotypic changes were caused by the deletion of the corresponding genes.
[0127] 2. Gene expression and vitamin B 12 Content detection
[0128] Experimental setup: Two groups were set up: 17.5% NaCl (optimal growth) and 22.5% NaCl (salt stress), and WT, ΔnhaC1, ΔnhaC2, and ΔnhaC1&C2 were cultured respectively. Total RNA was extracted and reverse transcribed into DNA, which was then detected by RT-qPCR. The primers used are shown in Table 3. Intracellular vitamin B was determined by HPLC. 12 content.
[0129] Detection method: HPLC was performed using a Waters Symmetry C18 column (4.6 mm × 250 mm, 5 μm), with a mobile phase of methanol: 0.028 M Na2HPO4 = 26:74 (pH 3.5), a flow rate of 1.0 mL / min, a column temperature of 30℃, and a detection wavelength of 361 nm.
[0130] Table 3
[0131]
[0132] Experimental results:
[0133] Various strains and vitamin B 12 Association analysis of synthesis, such as Figure 9 As shown in the figure. Figure A shows the effect of salt stress on the expression level of the JX313 gene in *Rhodophyton daqingense*. The significance analysis of gene expression levels under optimal and additional 5% salt stress conditions is presented. This indicates that the result is highly significant, i.e., p < 0.001; A value of p < 0.05 indicates significance; no label indicates non-significance. Figure B shows intracellular vitamin B1. 12 Content detection; lowercase letters a, b, c, etc., are used in the figures to represent different statistical groups, where the same letter indicates no significant difference between groups, and different letters indicate significant differences (p<0.05). Figure C shows the effect of gene knockout on gene expression levels of *Rhodophyton daqingense* under optimal conditions. Figure D shows the effect of an additional 5% salt stress environment; The figure shows the significance analysis of gene expression levels between the treatment group and the control group. The treatment group consisted of gene knockout strains, while the control group consisted of wild-type strain JX313T. This indicates that the result is highly significant, i.e., p < 0.001; This indicates that the result is significant, i.e., p < 0.01.
[0134] 1. Salt stress can simultaneously induce a significant upregulation of the expression of nhaC1, nhaC2, and cobC genes. Figure 9 A)
[0135] Compared to optimal growth conditions, the expression of wild-type strain was significantly upregulated (p<0.05) and nhaC2 was significantly upregulated (p<0.05) after 5% salt stress treatment, along with vitamin B1. 12 The key gene cobC was significantly upregulated (p<0.001). This indicates that under high-salt conditions, the strain activates the nhaC1 / nhaC2-mediated Na+ efflux system to expel excess intracellular sodium ions, and simultaneously initiates cobC-mediated vitamin B1 synthesis. 12 The two pathways work together to respond to salt stress, balancing intracellular and extracellular osmotic pressure through the accumulation of compatible solutes.
[0136] 2. Knockout of nhaC1 and nhaC2 can promote intracellular vitamin B1. 12 Accumulation, the synthesis of ΔnhaC2 was most significantly increased under salt stress ( Figure 9 B)
[0137] Regardless of the optimal environment or 5% salt stress, intracellular vitamin B1 and ΔnhaC2... 12 The content of vitamin B in all strains was significantly higher than that in the wild type; salt stress further increased the intracellular vitamin B content in all strains. 12 Levels, including intracellular vitamin B 12 The content reached its peak under stress, significantly higher than that of the wild type and ΔnhaC1. This demonstrates that when the nhaC gene deletion causes impaired Na+ efflux, the strain compensates by enhancing vitamin B. 12 Synthesis to compensate for salt tolerance deficiency; knocking out nhaC2 increases the body's vitamin B content. 12 Effective gene modification strategies for intracellular enrichment.
[0138] 3. Knocking out nhaC1, nhaC2, or cobC will reversely regulate the transcriptional levels of the other two genes, indicating a close regulatory interaction between the two salt tolerance pathways. Figure 9 C represents the optimal condition; Figure 9 D represents 5% salt stress)
[0139] Optimal conditions ( Figure 9 C): Knocking out cobC, nhaC1, or nhaC2 alone significantly upregulated their expression; knocking out nhaC1 or nhaC2 alone, or knocking out both ΔnhaC1 and C2, all resulted in a significant increase in cobC expression; there is also mutual regulation between nhaC1 and nhaC2, and knocking out one of them can induce the upregulation of the other gene.
[0140] 5% salt stress ( Figure 9D): This regulatory pattern was further strengthened. Compared with the wild type, the transcriptional levels of nhaC1, nhaC2, and cobC were all significantly upregulated to varying degrees in various knockout strains. Double knockout of ΔnhaC1 & C2 had the strongest activation effect on cobC, indicating that the greater the degree of obstruction of the sodium ion efflux pathway, the more vitamin B1 was activated. 12 The more pronounced the compensatory transcriptional activation effect of synthetic genes.
[0141] 4. Summary
[0142] The halophilic alkali-loving red bacterium *Rhodotorula daqingensis* exhibits two complementary salt tolerance pathways in response to high salt stress: ① an active Na+ efflux pathway dominated by nhaC1 / nhaC2; ② a vitamin B1+ pathway dominated by cobC. 12 Compatible solute osmotic balance pathway. Both pathways exhibit negative feedback compensatory regulation: when nhaC1 / nhaC2-mediated sodium efflux is lost, cobC transcription is strongly activated, driving vitamin B1 uptake. 12 Large amounts of vitamin B are synthesized and accumulated to resist osmotic stress; conversely, vitamin B is inhibited. 12 Synthesis also induces the upregulation of sodium transport genes. From an application perspective, the ΔnhaC2 strain, combined with a salt stress induction strategy, possesses the capability for high-yield industrial fermentation of vitamin B1. 12 This discovery also provides a theoretical basis for the modification of the chassis cells of this halophilic erythrophyll bacterium to synthesize other metabolites.
Claims
1. High-yield B 12 The genetically engineered *Rhodophyton daqingense* strain is characterized by... The genetically engineered bacteria are double knockout strains of the nhaC1 and nhaC2 genes, that is, genetically engineered strains in which the coding regions of the nhaC1 and nhaC2 genes are simultaneously deleted.
2. The high-yield B as described in claim 1 12 The method for preparing the genetically engineered Daqing halophilic erythromycete is characterized by, The method is as follows: I. Using the genomic DNA of the nhaC1 gene knockout strain of Rhodopseudomonas halophilus JX313T from Daqing as a template, PCR amplification was performed using primers C2F-FP / C2F-RP to obtain the upstream homologous arm of the nhaC2 gene. PCR amplification was then performed using primers C2R-FP / C2R-RP to obtain the downstream homologous arm of the nhaC2 gene. Finally, using plasmid E-pUC57 as a template, PCR amplification was performed using primers C2E-FP / C2E-RP to obtain the erythromycin resistance fragment. Fragment fusion was performed in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm" and ligated into the pUC18 cloning vector to obtain the double knockout plasmid pUC-nhaC2-dko. Using the double knockout plasmid pUC-nhaC2-dko as a template, PCR amplification was performed with primers C2F-FP / C2R-RP to obtain the linear homologous recombination fragment of nhaC2.
2. Using the nhaC1 gene knockout strain of Daqing halophilic erythrophorus JX313T as the starting strain, the bacterial suspension of the starting strain in the logarithmic growth phase was collected by centrifugation to prepare a protoplast suspension of strain ΔnhaC1; the nhaC2 linear homologous recombination fragment was added to the protoplast suspension, and protoplast transformation was performed to obtain the double gene knockout strains ΔnhaC1&C2 of nhaC1 and nhaC2.
3. The high-yield B according to claim 2 12 The method for preparing the genetically engineered Daqing halophilic erythromycete is characterized by, The method for preparing the nhaC1 gene knockout strain of Daqing halophilic erythrophorus JX313T is as follows: (1) Using wild-type genomic DNA as a template, PCR amplification was performed using primers C1F-FP / C1F-RP to obtain the upstream homologous arm, and PCR amplification was performed using primers C1R-FP / C1R-RP to obtain the downstream homologous arm. Using plasmid E-pUC57 as a template, PCR amplification was performed using primers C1E-FP / C1E-RP to obtain the erythromycin resistance fragment. Fragment fusion was performed in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm" and ligated into the pUC18 cloning vector to obtain the knockout plasmid pUC-nhaC1-ko. Using the knockout plasmid pUC-nhaC1-ko as a template, PCR amplification was performed with primers C1F-FP / C1R-RP to obtain the linear homologous recombination fragment of nhaC1. (2) Add the nhaC1 linear homologous recombination fragment to the protoplast suspension of wild-type strains, perform protoplast transformation, and obtain the nhaC1 gene knockout strain ΔnhaC1.
4. High-yield B 12 The genetically engineered *Rhodophyton daqingense* strain is characterized by... The genetically engineered bacteria are nhaC1 gene knockout strains, that is, genetically engineered strains with the coding region of the nhaC1 gene missing.
5. The high-yield B according to claim 4 12 The method for preparing the genetically engineered Daqing halophilic erythromycete is characterized by, The method for preparing the nhaC1 gene knockout strain is as follows: (1) Using wild-type genomic DNA as a template, PCR amplification was performed using primers C1F-FP / C1F-RP to obtain the upstream homologous arm, and PCR amplification was performed using primers C1R-FP / C1R-RP to obtain the downstream homologous arm. Using plasmid E-pUC57 as a template, PCR amplification was performed using primers C1E-FP / C1E-RP to obtain the erythromycin resistance fragment. Fragment fusion was performed in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm" and ligated into the pUC18 cloning vector to obtain the knockout plasmid pUC-nhaC1-ko. Using the knockout plasmid pUC-nhaC1-ko as a template, PCR amplification was performed with primers C1F-FP / C1R-RP to obtain the linear homologous recombination fragment of nhaC1. (2) Add the nhaC1 linear homologous recombination fragment to the protoplast suspension of wild-type strains, perform protoplast transformation, and obtain the nhaC1 gene knockout strain ΔnhaC1.
6. High-yield B 12 The genetically engineered *Rhodophyton daqingense* strain is characterized by... The genetically engineered bacteria are nhaC2 gene knockout strains, that is, genetically engineered strains with the coding region of the nhaC2 gene missing.
7. The high-yield B according to claim 6 12 The method for preparing the genetically engineered Daqing halophilic erythromycete is characterized by, The method for preparing the nhaC2 gene knockout strain is as follows: (1) Using wild-type genomic DNA as a template, the upstream homologous arm was obtained by PCR amplification using primers C2F-FP / C2F-RP, the downstream homologous arm was obtained by PCR amplification using primers C2R-FP / C2R-RP, and the erythromycin resistance fragment was obtained by PCR amplification using plasmid E-pUC57 as a template and primers C2E-FP / C2E-RP. Fragment fusion was performed in the order of "upstream homologous arm - erythromycin resistance fragment - downstream homologous arm" and ligated into the pUC18 cloning vector to obtain the knockout plasmid pUC-nhaC2-ko. Using the knockout plasmid pUC-nhaC2-ko as a template, PCR amplification was performed with primers C2F-FP / C2R-RP to obtain the linear homologous recombination fragment of nhaC2. (2) Add the nhaC2 linear homologous recombination fragment to the protoplast suspension of wild-type strains, perform protoplast transformation, and obtain the nhaC2 gene knockout strain ΔnhaC2.
8. The Daqing halophilic erythromycete genetically engineered strain as described in claim 1, 4, or 6, in improving B... 12 Applications in production.