Method for knocking out regulatory factor gene yeiE in escherichia coli to improve production of corac acid

Abstract

The invention discloses a method for knocking out a regulatory factor gene yeiE in escherichia coli to improve production of corac acid, and belongs to the field of gene engineering. According to the invention, a LysR type transcriptional regulatory factor yeiE gene is knocked out from an escherichia coli genome Escherichia coli K-12 MG1655, so that the yield of the coralic acid of the constructed mutant strain MG1655 [delta] yeiE is obviously increased, the yield of the coralic acid of the mutant strain is 1.5 times that of the coralic acid of a wild bacterium Escherichia coli K-12 MG1655, and the gene expression level of a gene cluster in a coralic acid synthesis route is obviously increased at the transcriptional level.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and molecular biology, and in particular to a method for knocking out a regulatory factor gene of Escherichia coli. yei Method for increasing colanic acid production. Background Art

[0002] Colanic acid (CA) is a mucoid extracellular polysaccharide that exists in polymeric form. High molecular weight CA polymers can be β -1,4 Fucoside hydrolase hydrolyzes it into the minimum repeating unit of CA. The hydrolyzed CA monomer is mainly composed of one molecule of glucose, two molecules of fucose, two molecules of galactose and one molecule of glucuronic acid. CA is a defense mechanism established by bacteria when facing environmental stress. It can effectively protect bacteria from the stress of harsh environments such as high temperature, hypertonicity, and acid. Its products have broad application prospects in the fields of food, medicine, and cosmetics. The synthesis of Escherichia coli CA is regulated by the Rcs phosphorylation system. Transcription factors bind to target gene sites in the CA gene cluster, initiate the transcription of CA genes and control their transcription efficiency, thereby regulating the biosynthesis of bacterial CA. Among the methods to promote CA biosynthesis, the yield is relatively high by knocking out negative regulatory factors or overexpressing positive regulatory factors.

[0003] YeiE is a LysR-type transcriptional regulator that consists of a DNA binding domain and a regulatory domain that recognizes an unknown ligand. The target genes regulated by YeiE are involved in a variety of biological processes, including cell wall / membrane biogenesis, transport and metabolism, signal transduction, and transcriptional regulation. yei After the gene is expressed, it affects the biofilm formation, adhesion and flagella assembly of the strain.

[0004] The present invention knocks out the LysR type transcriptional regulatory factor in the Escherichia coli genome yei The gene enables the colanic acid production of the engineered bacteria to reach 0.971 g / L, which is 1.5 times that of the original bacteria, significantly improving the colanic acid production. Summary of the invention

[0005] The present invention aims to knock out yei The invention discloses a method and application for constructing a high-yield colanic acid engineering strain. The colanic acid engineering strain constructed by the invention can significantly improve the colanic acid yield, and the construction method has simple steps.

[0006] To achieve the above-mentioned object of the invention, the present invention provides the following solutions: The present invention provides a method for knocking out a yei Gene, a method for constructing a high-yield colanic acid producing strain, comprising the following steps: (1) Using the pKD3 plasmid as a template, PCR amplification was performed to obtain the FRT-chloramphenicol resistance gene-FRT fragment; yei 500 bp of upstream and downstream homology arms were obtained by PCR amplification; the above-mentioned fragments were purified by a gel recovery kit, and the three purified DNA fragments were used as templates to obtain upstream-chloramphenicol-downstream genome site-specific integration fusion fragments by PCR amplification using the upstream primer of the upstream fragment and the downstream primer of the downstream fragment, and the fusion fragments were purified and recovered; (2) The temperature-sensitive plasmid pTKRed was transferred into Escherichia coli and cultured in a 30°C constant temperature incubator until a single colony grew. During the culture process, 25 μg / mL spectinomycin was added to maintain the stability of the pTKRed plasmid, induce the expression of the recombinase, and prepare electroporated competent cells; (3) The genomic integration DNA fragment obtained in step (1) is electroporated into the electroporation competent cells prepared in step (2), and the cells are cultured in SOC medium containing 0.5 mM IPTG at 37°C for 12 h, and then plated on a solid LB plate containing chloramphenicol and cultured at 37°C until a single colony grows. Positive clones are screened and the resistance gene is removed after sequencing is correct to obtain an engineered strain; (4) The engineered strain was cultured and activated in LBG liquid medium, and then transferred to 50 mL LBG liquid medium for culture to produce colanic acid by fermentation.

[0007] Furthermore, the knockout gene yei From Escherichia coli K-12 MG1655.

[0008] Furthermore, the knockout gene yei The nucleotide sequence is shown in SEQ ID No.1.

[0009] Furthermore, the knockout yei The key gene has one of the following nucleotide sequences: (1) yei The nucleotide sequence of Up is shown in SEQ ID NO.2; (2) The nucleotide sequence of CM is shown in SEQ ID NO.3; (3) yei The Down nucleotide sequence is shown in SEQ ID NO.4.

[0010] Furthermore, in step (4), the engineered bacteria were cultured and activated in LBG liquid culture medium at 25°C for 12 h, and 2% of the inoculum was transferred to 50 mL LBG liquid culture medium and cultured at 25°C for 48 h for the fermentation production of colanic acid.

[0011] The present invention also provides a method for preparing a high-yield colanic acid producing strain using the Red homologous recombination system.

[0012] The present invention also provides an application of the high-yield colanic acid producing strain obtained by the Red homologous recombination system preparation method in colanic acid fermentation production.

[0013] The invention also provides the application of colanic acid produced by the high-yield colanic acid engineering strain in food, cosmetics and medicine.

[0014] The present invention discloses the following technical effects: The present invention utilizes the Red homologous recombination system to knock out the yei The gene was used to initially obtain a high-yield colanic acid strain. The colanic acid production of the constructed colanic acid engineered bacteria was 1.5 times that of the original bacteria, significantly improving the colanic acid production.

[0015] The high-yield colanic acid engineering bacteria constructed by the present invention enhances the colanic acid synthesis-related gene cluster at the transcription level manA , manB , gD , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below. The drawings described below are only some embodiments of the present invention.

[0017] Figure 1 To amplify the genome site-specific integration fusion fragment map.

[0018] Figure 2 This is the PCR map of MG1655ΔyeiE colony.

[0019] Figure 3 OD of colanic acid fermentation for high colanic acid production engineered strain 600 result.

[0020] Figure 4 These are the results of colanic acid fermentation production of the high-yield colanic acid engineering strain.

[0021] Figure 5These are the transcriptional results of the engineered bacteria with high colanic acid production. DETAILED DESCRIPTION

[0022] Those skilled in the art can make appropriate modifications to the methods and applications herein without departing from the content of the present invention to implement and apply the technology of the present invention.

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The embodiments described herein are only part of the embodiments of the present invention, rather than all the embodiments.

[0024] The present invention knocks out the LysR type transcriptional regulatory factor in the Escherichia coli genome yei The gene was used to construct a high-yield colanic acid engineered bacterium with a colanic acid yield of 0.971 g / L, which is 1.5 times that of the original bacterium, significantly improving the colanic acid yield.

[0025] Example 1 Obtaining genes and constructing high-colanic acid-producing engineered strains 1. Acquisition of genes From Escherichia coli yei The gene, whose nucleotide sequence is shown in SEQ ID No.1.

[0026] The knockout yei The key gene has one of the following nucleotide sequences: (1) yei The nucleotide sequence of Up is shown in SEQ ID NO.2; (2) The nucleotide sequence of CM is shown in SEQ ID NO.3; (3) yei The Down nucleotide sequence is shown in SEQ ID NO.4.

[0027] 2. Construction of MG1655ΔyeiE high-yield colanic acid engineering strain (1) ΔYeiE-up gene amplification Using MG1655 as a template, primers ΔYeiE-up-F and primers ΔYeiE-up-R, polymerase chain reaction (PCR) was performed to amplify the ΔYeiE-up fragment. The PCR amplification system is shown in the following table: template 100 ng Primer ΔYeiE-up-F 2.5 μL Primer ΔYeiE-up-R 2.5 μL 2×SuperNova PCR Mix (GenStar, Cat. No. ZA065-101) 25 μL Ultrapure water Make up to 50 μL The PCR program was: 98ºC for 10 min; 30 cycles × (98ºC for 15 s, 55ºC for 15 s, 72ºC for 38 s); 72ºC for 5 min; 16ºC for 10 min.

[0028] The ΔYeiE-up-F primer sequence is shown in SEQ ID No.5, and the ΔYeiE-up-R primer sequence is shown in SEQ ID No.6.

[0029] The PCR product was purified by gel recovery using a gel recovery kit (Vazyme, catalog number DC301-01).

[0030] (2) ΔYeiE-down gene amplification Using MG1655 as a template, primers ΔYeiE-down-F and primers ΔYeiE-down-R, polymerase chain reaction (PCR) was performed to amplify the ΔYeiE-down fragment. The PCR amplification system is as follows: template 100 ng Primer ΔYeiE-down-F 2.5 μL Primer ΔYeiE-down-R 2.5 μL 2×SuperNova PCR Mix (GenStar, Cat. No. ZA065-101) 25 μL Ultrapure water Make up to 50 μL The PCR program was: 98ºC for 10 min; 30 cycles × (98ºC for 15 s, 55ºC for 15 s, 72ºC for 38 s); 72ºC for 5 min; 16ºC for 10 min.

[0031] The ΔYeiE-down-F primer sequence is shown in SEQ ID No.7, and the ΔYeiE-down-R primer sequence is shown in SEQ ID No.8.

[0032] The PCR product was purified by gel recovery using a gel recovery kit (Vazyme, catalog number DC301-01).

[0033] (3) FRT-CM-FRT gene amplification Using pKD3 as a template, primers CM-F and CM-R, polymerase chain reaction (PCR) was performed to amplify the FRT-CM-FRT fragment. The PCR amplification system is as follows: template 100 ng Primer CM-F 2.5 μL Primer CM-R 2.5 μL 2×SuperNova PCR Mix (GenStar, Cat. No. ZA065-101) 25 μL Ultrapure water Make up to 50 μL The PCR program was: 98ºC for 10 min; 30 cycles × (98ºC for 15 s, 55ºC for 15 s, 72ºC for 38 s); 72ºC for 5 min; 16ºC for 10 min.

[0034] The CM-F primer sequence is shown in SEQ ID No.9, and the CM-R primer sequence is shown in SEQ ID No.10.

[0035] The PCR product was purified by gel recovery using a gel recovery kit (Vazyme, catalog number DC301-01).

[0036] (4) Overlap extension Through polymerase chain reaction (PCR), overlap amplification of target gene fragments, primers ΔYeiE-up-F and ΔYeiE-down-R, the PCR reaction system is as follows: template 100 ng Primer ΔYeiE-up-F 2.5 μL Primer ΔYeiE-down-R 2.5 μL 2×SuperNova PCR Mix (GenStar, Cat. No. ZA065-101) 25 μL Ultrapure water Make up to 50 μL The PCR program was: 98 ºC for 10 min; 10 cycles × (98 ºC for 15 s, 55 ºC for 15 s, 72 ºC for 1 min 30 s); 16 ºC for 5 min. After 10 cycles, 2.5 μL of each primer was added and another 30 cycles were performed.

[0037] The primer sequences are shown in SEQ ID No.5 and SEQ ID No.8.

[0038] The PCR product was purified by gel recovery using a gel recovery kit (Vazyme, catalog number DC301-01).

[0039] (5) Site-specific integration into the E. coli genome The obtained genomic integration DNA fragment was electroporated into the prepared electrocompetent cells, and cultured in SOC medium containing 0.5 mM IPTG at 37°C for 12 h, then spread on a solid LB plate containing chloramphenicol and cultured at 37°C until a single colony grew. Positive clones were screened and the resistance gene was removed after correct sequencing to obtain an engineered strain.

[0040] Example 2 Fermentation production of high-yield colanic acid engineered bacteria 1. Strain activation and fermentation culture The engineered strain with correct sequencing was streaked at -80℃ on LBG solid medium and cultured in a 30℃ incubator for 24 h. A single colony was taken and cultured in 20 mL LBG liquid medium at 25℃ and shaker at 200 rpm for 12 h. It was inoculated into 50 mL LBG liquid medium at 2% inoculation volume and cultured at 25℃ and shaker at 200 rpm for 48 h. Samples were taken at 16 h, 24 h, and 48 h and OD was measured. 600， The results are as follows Figure 3 shown.

[0041] 2. Extraction of colanic acid Take 500 μL of fermentation broth and boil it in water for 20 min. After cooling to room temperature, centrifuge at 13000 rpm for 15 min to obtain the fermentation supernatant. Add 5 mL of pre-cooled anhydrous ethanol, precipitate overnight, centrifuge for 5 min, remove the supernatant, and dry in a 55°C oven to obtain crude colanic acid.

[0042] Preparation of glucuronic acid standard curve The colanic acid synthesis of the strain in the liquid culture medium was quantitatively determined according to the amount of glucuronic acid in the colanic acid molecule. The D-glucuronic acid standard was prepared into solutions with concentrations of 0, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, and 0.5 g / L. 100 μL of D-glucuronic acid standard solution of different concentrations was mixed with 500 μL of 0.475 g / L sodium tetraborate sulfuric acid solution, boiled in a 100℃ water bath for 5 min, cooled to room temperature, 10 μL of 1.5 g / L m-hydroxybiphenyl was added, and the mixture was ultrasonically debubbled for 15 min. 150 μL of the sample was taken and detected using an enzyme reader at a wavelength of 526 nm. The glucuronic acid standard curve was drawn according to the glucuronic acid concentration and absorbance as y=1.332x+0.07914, R 2 =0.9936.

[0043] 4. Colanic acid concentration detection Take 100 μL of colanic acid crude extract sample and mix it with 500 μL of 0.475 g / L sodium tetraborate sulfuric acid solution, boil it in 100℃ water bath for 5 min, cool it to room temperature, add 10 μL of 1.5 g / L m-hydroxybiphenyl, remove bubbles by ultrasonic mixing for 15 min, take 150 μL and use microplate reader to detect at 526 nm wavelength. The results are as follows: Figure 4 shown.

[0044] Example 3 Transcriptome Analysis of High-Colanic Acid Producing Engineering Bacteria 1. Transcription level of high-colanic acid-producing engineered bacteria Single colonies of strains MG1655 and MG1655ΔyeiE were picked and transferred to 20 mL LB medium and cultured at 37°C for 12 h. Then, they were transferred to 250 mL shake flasks containing 100 mL shake flask fermentation medium at a 1% (v / v) inoculation ratio and cultured continuously in a shaker at 200 rpm and 37°C. When MG1655 and MG1655ΔyeiE grew to mid-exponential phase, the cells were collected for transcriptome analysis, which was performed at Ouyi Biotechnology Co., Ltd. (Shanghai, China). The results showed that the constructed high-yield colanic acid engineered bacteria enhanced the colanic acid synthesis-related gene cluster at the transcriptional level. manA , manB , gD , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya, wxya Expressions such as Figure 5 shown.

[0045] The above results show that the high-yield colanic acid engineered strain MG1655ΔyeiE prepared by the present invention has a yield of 1.5 times higher than that of the original strain when producing colanic acid, reaching 0.971 g / L, which effectively improves the yield of colanic acid in microbial synthesis, and the method is simple.

[0046] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for knocking out regulatory factor genes in Escherichia coli yei A method for increasing the production of colanic acid, characterized in that The following steps are involved: (1) Using the pKD3 plasmid as a template, PCR amplification was performed to obtain the FRT-chloramphenicol resistance gene-FRT fragment; yei 500 bp of upstream and downstream homology arms were obtained by PCR amplification. After the above-mentioned fragments were purified using a gel recovery kit, the three purified DNA fragments were used as templates and fused by PCR amplification using the upstream primer of the upstream fragment and the downstream primer of the downstream fragment to obtain the upstream-chloramphenicol-downstream genomic site-specific integration fusion fragment, and the fusion fragment was purified and recovered.

2. (2) The thermosensitive plasmid pTKRed was transferred into Escherichia coli and cultured in a 30°C constant temperature incubator until a single colony was grown. During the culture process, 25 μg / mL spectinomycin was added to maintain the stability of the pTKRed plasmid, induce the expression of the recombinase, and prepare electrocompetent cells.

3. (3) The genomic integration DNA fragment obtained in step (1) is electroporated into the electrocompetent cells prepared in step (2), and the cells are cultured in SOC medium containing 0.5 mM IPTG at 37°C for 12 h. The cells are spread on a solid LB plate containing chloramphenicol and cultured at 37°C until a single colony grows. Positive clones are screened and the resistance gene is removed after sequencing is correct to obtain an engineered strain.

4. (4) The engineered strain was cultured and activated in LBG liquid medium, and then transferred to 50 mL LBG liquid medium for culture and fermentation production of colanic acid.

5. The preparation method according to claim 1, characterized in that: The knockout gene yei From Escherichia coli K-12 MG1655.

6. The preparation method according to claim 1, characterized in that: The knockout gene yei The nucleotide sequence is shown as SEQ ID No.

1.

7. The preparation method according to claim 1, characterized in that: The knockout yei The key gene has one of the following nucleotide sequences: (1) yei The nucleotide sequence of Up is shown in SEQ ID NO.2; (2) The nucleotide sequence of CM is shown in SEQ ID NO.3; (3) yei The Down nucleotide sequence is shown in SEQ ID NO.

4.

8. The preparation method according to claim 1, characterized in that: In step (4), the engineered bacteria were cultured in LBG liquid medium at 25°C for 12 h for activation, and 2% of the inoculum was transferred to 50 mL LBG liquid medium and cultured at 25°C for 48 h for colanic acid fermentation production.

9. A high-yield colanic acid producing strain obtained by using the Red homologous recombination system preparation method according to claims 1-4.

10. A high-yield colanic acid producing strain according to claim 6, wherein the colanic acid yield reaches 0.971 g / L, which is 1.5 times that of the original strain.

11. A method for enhancing the colanic acid synthesis-related gene cluster at the transcriptional level using the high-yield colanic acid production strain according to claim 6 manA , manB , gD , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya , wxya expression.

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