CN117737060A - Non-coding RNA CsrC mutant, genetically engineered bacterium and application - Google Patents

Non-coding RNA CsrC mutant, genetically engineered bacterium and application Download PDF

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CN117737060A
CN117737060A CN202311640429.2A CN202311640429A CN117737060A CN 117737060 A CN117737060 A CN 117737060A CN 202311640429 A CN202311640429 A CN 202311640429A CN 117737060 A CN117737060 A CN 117737060A
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csrc
sialyllactose
fucosyllactose
coding rna
genetically engineered
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宗剑飞
李庆刚
李玉
肖卫华
路福平
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Shandong Synthetic Vision Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a non-coding RNA CsrC mutant, a genetic engineering bacterium and application. The non-coding RNA CsrC mutant is obtained on the basis of wild-type non-coding RNA CsrC of Escherichia coli with Gene ID 2847776, and at least the 144 th a is mutated to t. The wild non-coding RNA CsrC in the escherichia coli genetic engineering bacteria for producing fucosyllactose and/or sialyllactose is mutated, so that after the 144 th a of the non-coding RNA CsrC is mutated to t, the yield of the fucosyllactose and/or sialyllactose of the obtained escherichia coli genetic engineering bacteria can be further improved, thereby better meeting the market demand.

Description

Non-coding RNA CsrC mutant, genetically engineered bacterium and application
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a non-coding RNA CsrC mutant, a genetic engineering bacterium and application.
Background
Breast milk oligosaccharides are beneficial components present in breast milk that contribute to infant development, and include fucosyllactoses such as 2'-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL), and sialyllactoses such as 3'-sialyllactose (3' -sialyllactose,3 '-SL), 6' -sialyllactose (6 '-sialyllactose,6' -SL). At present, a microbial fermentation method is mainly adopted to produce fucosyllactose and sialyllactose, wherein escherichia coli is a common engineering strain for producing fucosyllactose and sialyllactose.
In the aspect of microbial fermentation production of fucosyllactose, related regulatory genes have been intensively studied in the industry, and patent document CN112501106A, CN115786220A and the like have disclosed various escherichia coli genetically engineered bacteria for improving fucosyllactose yield and related genes, such as beta-galactosidase coding geneslacZUDP-glucose lipid carrier transferase coding genewcaJRegulatory genes in lactose lac operon sequencelacICoding gene of L-fucose isomerasefucIL-fucoidan encoding genefucK、L-fucoidan-1-phosphate aldolase encoding genefucAGDP-fucose synthase encoding genewcaG、GDP-mannose-4, 6-dehydratase coding genegmd、β-Coding gene of galactosidaselacY、Phosphomannose isomerase coding genemanA、Coding gene of phosphomannose mutasemanB、Sugar efflux transporter A coding genesetA、Mannose-1-phosphate guanine transferase coding genemanC、L-arabinose isomerase coding genearaA、Rhamnose isomerase coding generhaA、2' -fucosyllactose encoding genefutC、L-fucose kinase/GDP-L-fucose pyrophosphorylase encoding genefkpEtc.
In the microbial fermentative production of sialyllactose, N-acetylneuraminic acid lyase NanA, N-acetylneuraminic acid H have been found + Cotransporter NanT, N-acetylmannosamine-6-phosphate 2-pimelic acidThe enzymes NanE, N-acetylmannosamine kinase NanK, UDP-GlcNAc epimerase NeuC, N-acetylneuraminic acid synthase NeuB, N-acetylneuraminic acid cytidylyltransferase NeuA, sialyltransferase SiaT and the like are important genes affecting the fermentation yield of sialyllactose of E.coli (Zhu YY, et al Recent progress on health effects and biosynthesis of two key sialylated human milk oligosaccharides, 3'-sialyllactose and' -sialylactose [ J ]]. Biotechnology Advances, 2023, 62:108058.）。
The research provides a good basis for the fermentation production and industrialization of fucosyllactose and sialyllactose. However, changes in metabolic flux during fermentation production of microorganisms affect physiological properties of microorganisms, thereby affecting fucosyllactose and sialyllactose yields. CsrC is a novel non-coding RNA component of a carbon storage regulation system of Escherichia coli, is a non-coding RNA molecule containing 246 bases, and 1 CsrC binds to 9 CsrA proteins in Escherichia coli (Chan E et al Endonuclease IV ofEscherichia coli is Induced by Paraquat[J]PNAS, 1987, 84 (10): 3189-3193.). CsrA is an important regulatory protein for carbon metabolism of microorganisms. After CsrC binds to CsrA, the regulatory function of CsrA on the regulated gene is reduced (Thomas Weilbacher et al.A novel sRNA component of the carbon storage regulatory system of)Escherichia coli[J]Mol Microbiol, 2003, 48 (3): 657-670.). Based on the function of non-coding RNA CsrC, it has been used to increase the fermentation yield of amino acids, such as the technology disclosed in patent literature such as CN111770993A, US8759042B2, wherein the technology of introducing exogenous non-coding RNA CsrC into host cells or shortening non-coding RNA CsrC nucleotide sequence is involved.
However, no report is made about the relation between the expression of non-coding RNA CsrC and the fermentation production of breast milk oligosaccharide, and no report is made about the improvement of the production level of related industrial strains by the non-coding RNA CsrC point mutant, and how to improve the fermentation yield of the breast milk oligosaccharide of escherichia coli by the point mutation of the gene of the non-coding RNA CsrC is still lack of research.
Disclosure of Invention
In order to solve the technical problems, the invention obtains the non-coding RNA CsrC mutant capable of improving the yield of fucosyllactose and/or sialyllactose through gene editing, and can be applied to the production of fucosyllactose and/or sialyllactose.
To achieve the above object, one of the technical solutions of the present invention provides a non-coding RNA CsrC mutant, the nucleotide sequence of which corresponds to the wild-type non-coding RNA CsrC of E.coli K12 MG1655 (Gene ID: 2847776), the 144 th a of which is mutated to t, i.e. has the nucleotide sequence shown in SEQ ID NO. 1; or the nucleotide sequence of the non-coding RNA CsrC mutant corresponds to the nucleotide sequence shown in SEQ ID NO.1, at least 144 th base is t, and has at least 90% homology with the nucleotide sequence shown in SEQ ID NO. 1. Compared with RNA transcribed from the original non-coding CsrC gene, the binding capacity of the mutant RNA and the transcription regulating factor CsrA is changed, so that the metabolic regulation of the CsrA on microorganisms is changed, and the fermentation yield of fucosyllactose and sialyllactose is improved.
The second technical scheme provided by the invention is to provide a genetic engineering bacterium for producing fucosyllactose and/or sialyllactose, wherein the genetic engineering bacterium is escherichia coli genetic engineering bacterium, and the genome of the escherichia coli genetic engineering bacterium carries the non-coding RNA CsrC mutant nucleotide sequence. Wherein the fucosyllactose can be any one of 2' -fucosyllactose and 3-fucosyllactose; sialyllactose is any one of 3'-sialyllactose and 6' -sialyllactose. The "genetically engineered bacterium producing fucosyllactose and/or sialyllactose" refers to a genetically engineered bacterium which has the ability to produce fucosyllactose and/or sialyllactose by itself even if it does not carry the non-coding RNA CsrC mutant nucleotide sequence.
The third technical scheme provided by the invention is to provide the application of the non-coding RNA CsrC mutant, in particular to the application in the production of fucosyllactose and sialyllactose, namely the application in the preparation of fucosyllactose and/or sialyllactose by fermentation of escherichia coli. Wherein the fucosyllactose can be any one of 2' -fucosyllactose and 3-fucosyllactose; sialyllactose is any one of 3'-sialyllactose and 6' -sialyllactose.
Advantageous effects
The invention obtains a non-coding RNA CsrC mutant by a gene editing technology, and can further improve the yield of fucosyllactose and sialyllactose by applying the non-coding RNA CsrC mutant to escherichia coli for producing fucosyllactose and/or sialyllactose.
The non-coding RNA CsrC mutant for improving the yield of fucosyllactose and/or sialyllactose is simpler to prepare compared with CsrC RNA gene editing fragments obtained by techniques such as insertion induction genes and the like; in particular to a non-coding RNA CsrB mutant prepared by single point mutation, which is simpler to prepare.
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FIG. 1 is a diagram showing the PCR verification of the first step homologous recombination colony of the strain W5; m is Marker,1, 2 are PCR verification strip;
FIG. 2 is a diagram showing PCR verification of a second step homologous recombination colony of the strain W5; m is Marker,1, 2 are PCR verification bands.
Detailed Description
The invention is further described below by means of specific embodiments. Unless otherwise indicated, the technical means, materials, etc. to which the following embodiments relate may be known to those skilled in the art, and appropriate ones may be selected among known means and materials capable of solving the respective technical problems. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
The non-coding RNA CsrC mutant is obtained by mutating the 144 th a into t on the basis of wild non-coding RNA CsrC (Gene ID is 2847776) of escherichia coli K12 MG1655, and the nucleotide sequence of the obtained non-coding RNA CsrC mutant is shown as SEQ ID NO. 1.
The invention will be further explained by means of specific embodiments.
EXAMPLE 1 construction of Strain W5
Based on the E.coli TKYW1 described in patent document CN115786220A, a non-coding RNA CsrC gene on the genome was mutated, and the 144 th a was mutated to t, to construct a strain W5.
The construction of E.coli TKYW1 was briefly described based on the content of patent document CN115786220A, and the E.coli TKYW1 construction method was introduced into this example. Wherein the Escherichia coli TKYW1 in CN115786220A is Escherichia coli W2% described in patent document CN112501106AE.coli K12 MG1655△lacIZ::Ptrc-wcaG-gmd-lacy,△adhE::Ptrc-manB-manA) To construct the original strain, the UDP-glucose lipid carrier transferase coding gene on the genome of the original strain is knocked outwcaJAnd GDP-mannose hydrolase encoding genenudDObtained. Construction of E.coli TKYW1wcaJIs 946583;nudDis 946559.
The wild-type non-coding RNA CsrC nucleotide sequence Gene ID of E.coli K12 MG1655 was 2847776. Based on the strain TKYW1, the CRISPR/Cas9 technology is utilized to mutate a non-coding RNA CsrC gene on a genome, the 144 th site a is mutated to t, the nucleotide sequence of the obtained non-coding RNA CsrC mutant is shown as SEQ ID NO.1, and the constructed strain is named as W5. The CRISPR/Cas9 technology used in the experiments was referred to earlier studies report (Zhao D, et al, CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency, sci Rep, 2017, 7 (1): 16624). The specific construction method of the strain W5 is as follows:
1. construction of homologous recombination fragments
The MG1655 wild strain stored in the laboratory is used as a template, and primer pairs CsrC-up-F/R and CsrC-down-F/R in the table 1 are respectively used as primers, and the upstream homology arm and the downstream homology arm of homologous recombination are obtained through PCR amplification. PCR obtained by constructing the strain TKYW1cat-Fragments of the N20 sequence are used as templates, and primer pairs Cs are usedPCR amplification is carried out by taking rC-cat-F/R as a primer to obtain a novel primer withcat-Fragments of the N20 sequence. The homologous arm above and downstream, new withcat-Fragments of N20 sequence, 3 fragments as templates, were subjected to overlap PCR using the primers CsrC-up-F and CsrC-down-R to obtain homologous recombination fragments containingCsrCMutation of genes, i.e. wild typeCsrCThe 144 th base a of the gene is mutated into t.
2. First step homologous recombination
The pCAGO plasmid was transformed into the strain TKYW1 using a conventional plasmid transformation method to obtain strain TKYW1 (pCAGO). TKYW1 (pCAGO) was competent in preparation of the strain by using LB medium containing 1% glucose and 0.1. 0.1 mM% IPTG (isopropyl-. Beta. -D-thiogalactoside), the homologous recombinant fragment was introduced by electrotransformation, and the transformed strain was spread on LB plates containing 100 mg/L ampicillin and 25 mg/L chloramphenicol, and 1% glucose, and cultured at 30 ℃. And selecting the transformant to perform colony PCR verification, and if the recombination is correct, obtaining the homologous recombination strain in the first step, wherein the size of the stripe is about 2700 bp, and the verification result is shown in figure 1, and the stripe is correct.
3. Second step homologous recombination
The grown monoclonal was subjected to colony PCR validation. If the recombination is correct, the band size is about 1700 bp, the verification result is shown in figure 2, the band is correct, the PCR product of the band is sequenced, and the sequencing result is correct, so that the homologous recombination strain of the second step is obtained. The second homologous recombination strain is further cultivated at 37 ℃ to lose pCAGO plasmid therein, thereby obtaining the recombinant strain with the functions ofCsrCThe strain with the gene mutation (SEQ ID NO. 1) was designated as W5.
TABLE 1 constructionCsrCPrimers for gene mutant strains
Example 2 construction of plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ist
Construction of plasmid pTrc99a-P Using plasmid pTrc99a as template J23119 -neuB-neuC-P trc -neuA-ist. P involved in construction of the plasmid trc The nucleotide sequence of the promoter is shown as SEQ ID NO. 2; n-acetylneuraminic acid synthaseneuBNucleotide sequence of gene and UDP-N-acetylglucosamine 2-differential enzymeneuCNucleotide sequence of gene and N-acyl neuraminic acid cytidylyltransferase geneneuAShares a GenBank accession number with the nucleotide sequence of (A): AF400048.1; sialyltransferase geneistIs 61281137.
PCR amplification was performed using the plasmid pTrc99a as a template and the primers 99a-p119-F/R in Table 2 as primers to obtain linear vectors pTrc99a-p119; PCR amplification is carried out by taking neuB-F/R as a primer to obtain a linear gene fragmentneuBThe method comprises the steps of carrying out a first treatment on the surface of the PCR amplification is carried out by taking neuC-F/R as a primer to obtain a linear gene fragmentneuCThe method comprises the steps of carrying out a first treatment on the surface of the PCR amplification is carried out by taking ptrc-neuA-F/R as a primer to obtain a linear gene fragment P trc The method comprises the steps of carrying out a first treatment on the surface of the PCR amplification is carried out by taking neuA-F/R as a primer to obtain a linear gene fragmentneuAThe method comprises the steps of carrying out a first treatment on the surface of the To be used foristPCR amplification is carried out by taking F/R as a primer to obtain linear gene fragmentsist。Purifying and recovering the linear vector and the linear gene fragment obtained by the PCR, and using ClonExpress cube ® II recombinant ligation kit (Novain Biotechnology Co., ltd.) was ligated, transformed into E.coli DH 5. Alpha. Competent cells, cultured on LB plate containing 100 mg/L ampicillin, and transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid designated plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ist。
TABLE 2 construction of plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-istPrimers used
Example 3 construction of plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ST6
With plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-istConstruction of plasmid pTrc99a-P for template J23119 -neuB-neuC-P trc -neuA-ST6. Sialyltransferase involved in construction of the plasmidST6Is AB293985.1.
With plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-istPCR amplification was performed using the bst-AB-supported-F/R in Table 3 as a primer to obtain the linear vector pTrc99a-ST6; PCR amplification is carried out by taking bst-AB-F/R as a primer to obtain a linear gene fragmentST6The method comprises the steps of carrying out a first treatment on the surface of the Purifying and recovering the linear vector and the linear gene fragment obtained by the PCR, and using ClonExpress cube ® II recombinant ligation kit (Novain Biotechnology Co., ltd.) was ligated, transformed into E.coli DH 5. Alpha. Competent cells, cultured on LB plate containing 100 mg/L ampicillin, and transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid designated plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA- ST6。
TABLE 3 construction of plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ST6Primers used
Example 4 construction of fucosyllactose and sialyllactose producing Strain and fermentation test
Plasmid pTrc99a-P was transformed by electrotransformation trc -futC-manC、pTrc99a-P trc -futA-manC、pTrc99a-P J23119 -neuB-neuC-P trc -neuA-istAnd pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ST6TKYW1 and W5 were introduced, respectively, to construct 2' -fucosyllactose-producing strain F1 (TKYW 1 (pTrc 99 a-P) trc -futC-manC) F2 (W5 (pTrc 99 a-P) trc -futC-manC) 3-fucoseGlycosyllactose producing strain F3 (TKYW 1 (pTrc 99 a-P) trc -futA-manC) F4 (W5 (pTrc 99 a-P) trc -futA-manC) 3' -sialyllactose-producing Strain F5 (TKYW 1 (pTrc 99 a-P) J23119 -neuB-neuC-P trc -neuA-ist) F6 (W5 (pTrc 99 a-P) J23119 -neuB-neuC-P trc -neuA-ist) 6' -sialyllactose-producing strain F7 (TKYW 1 (pTrc 99 a-P) J23119 -neuB-neuC-P trc -neuA-ST6) And F8 (W5 (pTrc 99 a-P) J23119 -neuB-neuC-P trc -neuA-ST6) And the fermentation production level of the above strain was tested. Wherein plasmid pTrc99a-P trc -futC-manC、pTrc99a-P trc -futA-manCThe specific construction procedure of (a) is described in examples 3 and 4 of patent document CN116334025 a.
The culture medium used was:
LB medium: naCl 10 g/L, yeast powder 5 g/L, peptone 10 g/L and pH 7.0.
Fermentation medium: KH (KH) 2 PO 4 3 g/L, yeast powder 8 g/L, (NH) 4 ) 2 SO 4 4. 4 g/L, citric acid 1.7 g/L, mgSO 4 ·7H 2 O2 g/L, thiamine 10 mg/L, glycerol 10 g/L, lactose 5 g/L,1 ml/L trace elements (FeCl) 3 ·6H 2 O 25 g/L，MnCl 2 ·4H 2 O 9.8 g/L，CoCl 2 ·6H 2 O 1.6 g/L，CuCl 2 ·H 2 O 1 g/L，H 3 BO 3 1.9 g/L， ZnCl 2 2.6 g/L，Na 2 M O O 4 ·2H 2 O 1.1 g/L，Na 2 SeO 3 1.5 g/L，NiSO 4 ·6H 2 O1.5. 1.5 g/l), pH was adjusted to 7.2 with ammonia.
The fermentation test process comprises the following steps:
single colonies of fucosyllactose or sialyllactose producing strains were picked up, respectively, and cultured overnight at 37℃at 220 rpm/min in LB liquid medium containing 50 mg/L of ampicillin.The bacterial liquid cultured overnight is taken as seed liquid, the bacterial liquid is transferred into a 24-well plate containing 2 mL fermentation culture medium with the inoculum size of 1 percent, 50 mg/L of ampicillin and 0.1 mmol/L of IPTG are contained in the fermentation culture medium, and fermentation is carried out at 37 ℃ and 800 rpm/min. 3 samples were grown in parallel for each strain. During fermentation, the growth (OD) of the cells was measured 600 ) The concentration of fucosyllactose or sialyllactose in the sample was measured by HPLC using a chromatographic column of Carbohydrate ES 5u 250mm x 4.6mm, a detector of evaporative light detector and a mobile phase of 70% acetonitrile (acetonitrile: water), the flow rate is 0.8 mL/min, the column temperature is 30 ℃, and the sample injection amount is 5 mu L. The sample concentration was quantified using fucosyllactose or sialyllactose standards. The results are shown in tables 4 to 7:
as can be seen from tables 4 to 7, mutation of nucleotide a at position 144 of the non-coding RNA CsrC gene to t greatly increases the yield of fucosyllactose or sialyllactose.
TABLE 4 results of 2'-fucosyllactose (2' -FL) production test by different strains
TABLE 5 results of 3-fucosyllactose (3-FL) production test by different strains
TABLE 6 results of 3'-sialyllactose (3' -SL) production test by different strains
TABLE 7 results of 6'-sialyllactose (6' -SL) production test by different strains
Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims (10)

1. The non-coding RNA CsrC mutant is characterized in that the nucleotide sequence of the non-coding RNA CsrC mutant is shown as SEQ ID NO. 1; or the nucleotide sequence of the non-coding RNA CsrC mutant corresponds to the nucleotide sequence shown in SEQ ID NO.1, at least 144 th base is t, and has at least 90% homology with the nucleotide sequence shown in SEQ ID NO. 1.
2. A genetically engineered bacterium for producing fucosyllactose and/or sialyllactose, wherein the genetically engineered bacterium is an escherichia coli genetically engineered bacterium, and the genome of the escherichia coli genetically engineered bacterium carries the non-coding RNA CsrB mutant nucleotide sequence of claim 1.
3. The genetically engineered bacterium of claim 2, wherein the genome of the genetically engineered bacterium of escherichia coli carries a non-coding RNA CsrC mutant nucleotide sequence as set forth in SEQ ID No. 1.
4. The genetically engineered bacterium of claim 2, wherein the genetically engineered bacterium of escherichia coli is a genetically engineered bacterium of escherichia coli that produces fucosyllactose.
5. The genetically engineered bacterium of claim 4, wherein the fucosyllactose is any one of 2' -fucosyllactose and 3-fucosyllactose.
6. The genetically engineered bacterium of claim 2, wherein the genetically engineered bacterium of escherichia coli is a genetically engineered bacterium of escherichia coli that produces sialyllactose.
7. The genetically engineered bacterium of claim 6, wherein the sialyllactose is any one of 3'-sialyllactose and 6' -sialyllactose.
8. Use of a non-coding RNA CsrC mutant according to claim 1, for increasing the yield of fucosyllactose and/or sialyllactose produced by fermentation of e.
9. The use according to claim 8, wherein the fucosyllactose is any one of 2' -fucosyllactose and 3-fucosyllactose.
10. The use according to claim 8, wherein the sialyllactose is any one of 3'-sialyllactose and 6' -sialyllactose.
CN202311640429.2A 2023-12-04 2023-12-04 Non-coding RNA CsrC mutant, genetically engineered bacterium and application Pending CN117737060A (en)

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