RNA polymerase β subunit mutant and its application

Abstract

The present invention belongs to the field of genetic engineering technology, specifically relating to a mutant of the RNA polymerase β subunit and its application. The present invention obtains an RpoB mutant through gene editing technology. By changing the 560th amino acid of RpoB from proline to leucine, the strain significantly increases the number of viable cells during fermentation and improves its tolerance to the late-stage fermentation tank environment. This mutant is then applied to Escherichia coli producing human milk oligosaccharides, ultimately significantly increasing human milk oligosaccharide production.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to an RNA polymerase beta subunit mutant and an application thereof. Background Art

[0002] Human milk oligosaccharides (HMOs), a crucial oligosaccharide in breast milk, have multiple benefits. They can act as prebiotics, maintain a balanced intestinal microbial environment, regulate immune defense, and promote brain development and cognitive development in infants. Because of their significant impact on infant growth and development, they can be added as a functional ingredient to infant formula and have a broad market prospect. Based on structural differences, there are over 200 types of HMOs, including lacto- N -trisaccharide (Lacto-N-Triose, LNT II), lactoyl- N -Lacto-N-Neotetraose (LNnT), lactose- N HMOs are mainly composed of lactose (Lacto-N-tetraose, LNT), 3'-sialyllactose (3'-Sialyllactose, 3'-SL), and 6'-sialyllactose (6'-Sialyllactose, 6'-SL). Currently, HMOs are produced by chemical synthesis, enzymatic catalysis, and microbial fermentation.

[0003] Escherichia coli has become a typical model strain in microbial fermentation due to its advantages such as clear background, simple culture method and short growth cycle, relatively clear metabolic pathway and mature genetic manipulation technology. It is used to synthesize various HMOs. In E. coli, when LNnT is synthesized, glucose or glycerol is used as the carbon source and lactose is used as the substrate. Glucose-6-phosphate (Glc-6-P) is synthesized in pgi Glucose-6-phosphate isomerase is catalyzed by the gene encoding glucose-6-phosphate to convert it into fructose-6-phosphate (F6P), which is then converted into glmS Encoded glutamine-fructose-6-phosphate aminotransferase, glmM Encoded phosphoglucosamine mutase, glmU The enzyme N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase encodes the urea-6-phosphate glycosides, which are converted into uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc). In the synthesis pathway of another precursor, uridine diphosphate-galactose (UDP-Gal), Glc-6-P is converted into urea-6-phosphate glycosides (UDP-GlcNAc). pgm Encoded phosphoglucomutase and galE UDP-glucose-4-isomerase is converted into UDP-Gal. lgtAUnder the catalysis of the encoded β-1,3-N-acetylglucosamine aminotransferase, lactose reacts with UDP-GlcNAc to generate the intermediate LNT II, ​​which is then lgtB Under the catalysis of the encoded β-1,4-galactosyltransferase, it combines with UDP-Gal to generate LNnT. wbgO LNT is produced under the catalysis of the encoded β-1,3-galactosyltransferase. During the synthesis of sialylated human milk oligosaccharides, Escherichia coli uses the UDP-GlcNAc synthesis pathway to synthesize cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) catalyzed by UDP-N-acetylglucosamine-2-epimerase NeuC, N-acetylneuraminic acid synthase NeuB, and N-acetylneuraminic acid cytidylyltransferase NeuA. CMP-Neu5Ac is then combined with lactose under the catalysis of α2,3-sialyltransferase α2,3-SiaT and α2,6-sialyltransferase α2,6-SiaT to produce 3'-SL and 6'-SL, respectively.

[0004] rpoB The gene encodes the β subunit of the Escherichia coli RNA polymerase. RpoB has a "flexible flap" element that interacts with the σ factor and participates in the transcription of various RNA polymerase holoenzyme complexes. It has been reported that RpoB mutants can alter the strain's sensitivity to antibiotics and accelerate the transcription rate of certain genes; increase the growth rate of ribosome synthesis and alter intracellular resource allocation; cause changes in the cell membrane, enhance the strain's ability to utilize specific metabolites, and improve the strain's environmental tolerance (Jeje O, Ewunkem AJ, Jeffers-Francis LK, et al. Serving two masters: effect of Escherichia coli dual resistance on antibiotic susceptibility. Antibiotics(Basel) 12: 603[EB / OL].(2023), (Yang KB, Cameranesi M, Gowder M, et al. High-resolution landscape of an antibiotic binding site[J]. Nature, 2023, 622(7981): 180-187.).

[0005] During the industrial high-level production of human milk oligosaccharides, a large amount of metabolic intermediates and end products accumulate in the fermentation environment. The strain has poor tolerance to the fermentation environment, and its growth and production capacity are subject to certain restrictions.rpoB The gene mutation is expected to improve the strain's tolerance to the fermentation environment and further increase the production of human milk oligosaccharides. Summary of the Invention

[0006] To address the above technical issues, the present invention utilizes gene editing technology to edit the genes of Escherichia coli. Through gene editing, a production strain capable of increasing the production of human milk oligosaccharides was obtained.

[0007] One of the technical solutions provided by the present invention is an RNA polymerase β subunit mutant, which is obtained by mutating Pro at position 560 to Leu based on the wild-type RNA polymerase β subunit (RpoB, Gene ID: 948488; SEQ ID NO. 4). The amino acid sequence of the RNA polymerase β subunit mutant is shown in SEQ ID NO. 1.

[0008] The present invention also provides a gene encoding the above-mentioned RNA polymerase β subunit mutant;

[0009] Furthermore, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2.

[0010] The second technical solution provided by the present invention is the application of the above-mentioned RNA polymerase β subunit mutant, especially in the production of human milk oligosaccharides;

[0011] Furthermore, when the RNA polymerase β subunit in the human milk oligosaccharide production strain is mutated to the mutant shown in SEQ ID NO.1, the strain's tolerance to the fermentation environment can be improved, further increasing the production of human milk oligosaccharides.

[0012] Furthermore, the human milk oligosaccharides include but are not limited to: lactoyl- N -trisaccharide (Lacto-N-Triose, LNTII), lactoyl- N -Lacto-N-Neotetraose (LNnT), lactose- N -tetraose (Lacto-N-tetraose, LNT), 3'-sialyllactose (3'-Sialyllactose, 3'-SL) and 6'-sialyllactose (6'-Sialyllactose, 6'-SL), etc.

[0013] The third technical solution provided by the present invention is a method for increasing the production of human milk oligosaccharides, wherein the method is to mutate the RNA polymerase β subunit encoding gene in the human milk oligosaccharide production strain to the sequence shown in SEQ ID NO.2;

[0014] Furthermore, the human milk oligosaccharides include but are not limited to: lactoyl- N -trisaccharide (Lacto-N-Triose, LNT II), lactoyl- N -Lacto-N-Neotetraose (LNnT), lactose- N -tetraose (Lacto-N-tetraose, LNT), 3'-sialyllactose (3'-Sialyllactose, 3'-SL) and 6'-sialyllactose (6'-Sialyllactose, 6'-SL).

[0015] A fourth technical solution provided by the present invention is a strain for producing human milk oligosaccharides, wherein the amino acid sequence of the RNA polymerase β subunit in the strain is shown in SEQ ID NO.1;

[0016] Furthermore, the strain uses Escherichia coli K12 MG1655 as a host and knocks out the host lactose operon sequence. lacZ , and P trc Promoter overexpression lacY , and the amino acid sequence of the RNA polymerase β subunit in the strain is shown in SEQ ID NO.1; on this basis, the strain also contains a human milk oligosaccharide production pathway;

[0017] Furthermore, the human milk oligosaccharides include but are not limited to: lactoyl- N -trisaccharide (Lacto-N-Triose, LNT II), lactoyl- N -Lacto-N-Neotetraose (LNnT), lactose- N -Lacto-N-tetraose (LNT), 3'-sialyllactose (3'-Sialyllactose, 3'-SL) and 6'-sialyllactose (6'-Sialyllactose, 6'-SL);

[0018] Furthermore, the human milk oligosaccharide production pathway is any one of the following plasmids:

[0019] pTrc99a-P trc - lgtA 、pTrc99a-P trc - lgtB - lgtA, pTrc99a-P trc - wbgO - lgtA, pTrc99a-P J23119 - neuB - neuC -P trc -neuA - ist and pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 .

[0020] Beneficial effects:

[0021] The present invention obtains an RpoB mutant through gene editing technology. After the 560th amino acid of RpoB is changed from proline to leucine, the number of viable cells of the strain during fermentation is greatly increased, and its tolerance to the late fermentation tank environment is improved. It is applied to Escherichia coli for the production of human milk oligosaccharides, ultimately significantly increasing the production of human milk oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 for rpoB The first step is colony PCR verification of homologous recombination.

[0023] Figure 2 for rpoB The second step is PCR verification of homologous recombination colonies. DETAILED DESCRIPTION

[0024] The present invention will be further described below by specific embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following embodiments may be well known to those skilled in the art, and appropriate ones may be selected from the known means and materials that can solve the corresponding technical problems. In addition, the embodiments are to be understood as illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, various changes or modifications made to the material composition and dosage in these embodiments also fall within the scope of protection of the present invention.

[0025] The RNA polymerase β subunit mutant involved in the present invention is based on the wild-type RNA polymerase β subunit (RpoB, Gene ID: 948488), in which the Pro at position 560 is mutated to Leu. The amino acid sequence of the mutant is shown in SEQ ID NO.1:

[0026]

[0027] The nucleotide sequence of the RNA polymerase β subunit mutant is shown in SEQ ID NO.2:

[0028]

[0029] The present invention will be further explained below through specific embodiments.

[0030] Example 1 Construction of strain A1

[0031] Escherichia coli K12 MG1655 ( Escherichia coli K12 MG1655) was constructed as the starting strain, and CRISPR / Cas9 technology (Zhao D, et al. CRISPR / Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. SciRep7, 16624) was used to knock out the lactose operon sequence of the starting strain. lacZ , in the original lacZ P trc Promoter overexpression lacY , named strain A0. The specific construction process of this strain is detailed in Example 1 of patent CN 119464168 A.

[0032] Strain A0 carrying the RpoB mutant encoding gene (SEQ ID NO. 2) was named strain A1, and its specific construction method was as follows:

[0033] 1. Construction of homologous recombination fragments

[0034] Using wild-type strain MG1655 as template, primers listed in Table 1 were used to construct homologous recombination fragments. Using rpoB-up-f / r and rpoB-down-f / r as primers, PCR amplification was performed to obtain the upstream and downstream homology arms of homologous recombination. cat 、 cat The promoter and N20 sequence vector were used as templates, and rpoB-cat-f / r was amplified by PCR using primers to obtain cat - The fragment of N20 sequence. The upper and downstream homology arms, with cat - The fragment of N20 sequence (SEQID NO.3) was used as template, and overlapping PCR was performed using primers rpoB-up-f and rpoB-down-r to obtain a homologous recombinant fragment containing rpoB The mutation site of the gene.

[0035] 2. First step: homologous recombination

[0036] The pCAGO plasmid was transformed into strain A0 using conventional plasmid transformation methods to obtain strain A0 (pCAGO). Prepare the A0 (pCAGO) competent medium using LB medium containing 1% (m / v) glucose and 0.1 mM IPTG. The homologous recombination fragment obtained in step 1 was introduced using electroporation. The transformed bacterial solution was spread on LB plates containing 100 mg / L ampicillin, 25 mg / L chloramphenicol, and 1% glucose and cultured at 30°C. Transformants were selected for colony PCR verification (verification primers: rpoB-yz-f / r). If the recombination was successful, the band size was approximately 3013 bp, and the verification results were as follows: Figure 1 As shown, the bands are correct, that is, the first homologous recombination is successful, and the correct transformants are picked for the second step of homologous recombination.

[0037] 3. Second step homologous recombination

[0038] The strain that has been successfully recombined is inoculated into an LB test tube containing 100 μg / mL AMP and 0.1 mM IPTG, and cultured on a shaker at 30°C for more than 6 hours to induce the expression of the CRISPR / Cas9 system and λ-red protein to complete the second recombination. Three areas are drawn on the LB plate containing ampicillin to isolate single colonies. The isolated single colonies are picked and spotted on the chloramphenicol-resistant LB plate and the ampicillin-resistant LB plate respectively. The single colonies that do not grow on the chloramphenicol medium but grow on the ampicillin medium are selected and verified by colony PCR (verification primers are: rpoB-yz-f / r). If the recombination is correct, the band size is about 2078 bp, and the verification result is as follows. Figure 2 The band was correct, and the PCR product of the band was sequenced. The sequencing result was correct, and the second-step homologous recombination strain was obtained. The second-step homologous recombination strain was further cultured at 37 ° C to lose the pCAGO plasmid, thereby obtaining a strain with rpoB The strain with the mutation (SEQ ID NO. 2) was named A1.

[0039] Table 1 Primers used to construct strain A1

[0040]

[0041] Example 2 Construction of plasmid pTrc99a-P trc - lgtA

[0042] lgtA Gene encoding β-1,3- N -acetyl glucosamine transferase, plasmid pTrc99a was used as a template to construct plasmid pTrc99a-P trc - lgtAThe specific construction process of the plasmid is referred to Example 4 of patent CN 119464168A.

[0043] Example 3 Construction of plasmid pTrc99a-P trc - lgtB-lgtA

[0044] lgtB The gene encoding lipooligosaccharide biosynthesis protein is located in the plasmid pTrc99a-P trc - lgtA The plasmid pTrc99a-P was constructed based on trc - lgtB - lgtA The specific construction process of this plasmid is referred to Example 5 of patent CN 119464168A.

[0045] Example 4 Construction of plasmid pTrc99a-P trc - wbgO - lgtA

[0046] wbgO The gene encoding β-1,3-galactosyltransferase is located in the plasmid pTrc99a-P trc - lgtA The plasmid pTrc99a-P was constructed based on trc - wbgO - lgtA The specific construction process of this plasmid is referred to Example 6 of patent CN 119464168A.

[0047] Example 5 Construction of plasmid pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist

[0048] Plasmid pTrc99a was used as a template to construct plasmid pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist The specific construction process of this plasmid refers to Example 2 of patent CN117736280A.

[0049] Example 6 Construction of plasmid pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6

[0050] Plasmid pTrc99a-P J23119 - neuB - neuC -P trc - neuA - istThe plasmid pTrc99a-P was constructed as a template J23119 - neuB - neuC -P trc - neuA - ST6 The specific construction process of this plasmid refers to Example 3 of patent CN117736280A.

[0051] Example 7 Construction and fermentation testing of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL production strains

[0052] The plasmid pTrc99a-P was transformed into trc - lgtA 、pTrc99a-P trc - lgtB - lgtA, pTrc99a-P trc - wbgO - lgtA, pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ist and pTrc99a-P J23119 - neuB - neuC -P trc - neuA - ST6 Introduce A0 and A1 strains respectively and construct:

[0053] (1) LNT II production strain:

[0054] B1[A0 (pTrc99a-P trc - lgtA )];

[0055] B2[A1 (pTrc99a-P trc - lgtA )];

[0056] (2) LNnT production strains:

[0057] B3[A0(pTrc99a-P trc - lgtB - ​ )];

[0058] B4[A1(pTrc99a-P trc - ​ - ​ )];

[0059] (3) LNT production strains:

[0060] B5[A0(pTrc99a-P trc - ​ - ​ )];

[0061] B6[A1(pTrc99a-P trc - ​ - ​ )];

[0062] (4) 3'-SL production strain:

[0063] B7[A0(pTrc99a-P J23119 - ​ -P trc - ​ )];

[0064] B8[A1(pTrc99a-P J23119 - ​ -P trc - ​ )];

[0065] (5) 6'-SL production strain:

[0066] B9[A0(pTrc99a-P J23119 - ​ -P trc - ​ - ​ )];

[0067] B10[A1(pTrc99a-P J23119 - ​ -P trc - ​ - ​ )];

[0068] Table 2 Strains and plasmids used

[0069]

[0070] The production levels of the above strains were tested by fermentation. The culture medium used was:

[0071] LB medium (1 L): NaCl 10 g, yeast powder 5 g, peptone 10 g.

[0072] Fermentation medium (1 L): KH2PO4 3 g, yeast powder 8 g, (NH4)2SO4 4 g, citric acid 1.7 g, MgSO4·7H2O 2 g, thiamine 10 mg, MOPS 60 g, glycerol 10 g, lactose 5 g, 1 mL trace elements, ammonia water to adjust pH to 7.0.

[0073] Trace elements (1 L): FeCl3·6H2O 25 g, MnCl2·4H2O 9.8 g, CoCl2·6H2O 1.6 g, CuCl2·H2O 1 g, H3BO3 1.9 g, ZnCl2 2.6 g, Na2M O O4·2H2O 1.1 g, Na2SeO31.5 g, NiSO4·6H2O 1.5 g.

[0074] The fermentation test process is:

[0075] Single colonies of the LNT II, ​​LNnT, LNT, 3'-SL, and 6'-SL production strains were picked and cultured overnight in LB liquid medium containing 50 mg / L ampicillin at 37°C and 220 rpm / min. The overnight culture was used as seed liquid and inoculated into a 24-well plate containing 1 mL of fermentation medium at a 2% (v / v) inoculation volume. The fermentation medium contained 50 mg / L ampicillin and 0.1 mmol / L IPTG. Fermentation was performed at 37°C and 800 rpm / min. Three parallel cultures were cultured for each strain. During the fermentation process, the bacterial growth (OD 600 ), LNT II, ​​LNnT, LNT, 3'-SL, and 6'-SL production. High-performance liquid chromatography (HPLC) was used to determine the concentrations of LNT II, ​​LNnT, LNT, 3'-SL, and 6'-SL in the samples. Standard curves for LNT II, ​​LNnT, LNT, 3'-SL, and 6'-SL were used to quantify the sample concentrations. The HPLC detection conditions for LNT II, ​​LNnT, LNT, 3'-SL, and 6'-SL were similar to those in Example 4 of CN117736280A. The results are shown in Tables 3 to 7:

[0076] Table 3 Test results of LNT II production by different strains

[0077]

[0078] Table 4 Test results of LNnT production by different strains

[0079]

[0080] Table 5 Test results of LNT production by different strains

[0081]

[0082] Table 6 Test results of 3'-SL production by different strains

[0083]

[0084] Table 7 Test results of 6'-SL production by different strains

[0085]

[0086] From the above results, it can be seen that after the 560th amino acid of RpoB was changed from proline to leucine, the number of viable cells of the strain during fermentation was greatly increased, its tolerance to the late fermentation tank environment was improved, and ultimately the production of human milk oligosaccharides was greatly increased. This work provides new ideas for the construction of strains that can efficiently synthesize human milk oligosaccharides.

[0087] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. An RNA polymerase β subunit mutant, characterized in that: The mutant is obtained by mutating Pro at position 560 to Leu on the basis of the wild-type RNA polymerase β subunit. The amino acid sequence of the RNA polymerase β subunit mutant is shown in SEQ ID NO.

1.

2. A gene encoding the RNA polymerase β subunit mutant according to claim 1.

3. The coding gene according to claim 2, wherein The nucleotide sequence is shown in SEQ ID NO.

2.

4. A strain for producing human milk oligosaccharides, characterized in that: The strain uses Escherichia coli K12 MG1655 as a host and knocks out the host lactose operon sequence. lacZ , overexpression lacY , and the amino acid sequence of the RNA polymerase β subunit in the strain is shown in SEQ ID NO.1; on this basis, the strain also contains a human milk oligosaccharide production pathway; The human milk oligosaccharide is: lactoyl- N -Trisaccharide, lactoyl- N - Neotetraose, lactose- N -tetrasaccharide, 3'-sialyllactose or 6'-sialyllactose; The human milk oligosaccharide production pathway is any one of the following plasmids: pTrc99a - P trc - lgtA 、pTrc99a - P trc - lgtB - lgtA, pTrc99a - P trc - wxya - lgtA, pTrc99a - P J23119 - neuB-neuC -P trc - neuA-ist or pTrc99a - P J23119 - neuB-neuC -P trc - neuA-ST6 ; Among them, pTrc99a-P trc - lgtA 、pTrc99a-P trc - lgtB - lgtA, pTrc99a-P trc - wxya - lgtA The plasmid was constructed according to patent CN 119464168A; pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ist 、pTrc99a-P J23119 - neuB-neuC -P trc - neuA-ST6 The plasmid was constructed according to patent CN117736280A.

5. Use of the strain according to claim 4 in the production of human milk oligosaccharides, characterized in that The human milk oligosaccharide is lactoyl- N -Trisaccharide, lactoyl- N - Neotetraose, lactose- N -tetrasaccharide, 3'-sialyllactose or 6'-sialyllactose.

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