CN117736280A - SecY protein mutant, genetically engineered bacterium and application - Google Patents

SecY protein mutant, genetically engineered bacterium and application Download PDF

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CN117736280A
CN117736280A CN202311640459.3A CN202311640459A CN117736280A CN 117736280 A CN117736280 A CN 117736280A CN 202311640459 A CN202311640459 A CN 202311640459A CN 117736280 A CN117736280 A CN 117736280A
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sialyllactose
fucosyllactose
secy
genetically engineered
engineered bacterium
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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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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 SecY protein mutant, a genetic engineering bacterium and application. The SecY protein mutant is obtained by mutating at least leucine at position 375 into proline based on a wild-type protein transporter SecY with Gene ID 947799. The invention further provides a coding gene for the SecY protein mutant. The encoding gene of the SecY protein mutant is applied to escherichia coli genetic engineering bacteria for producing fucosyllactose and/or sialyllactose, so that the yield of fucosyllactose and/or sialyllactose can be further improved.

Description

SecY protein mutant, genetically engineered bacterium and application
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a protein transporter SecY mutant, a genetic engineering bacterium and application.
Background
Breast milk contains beneficial components such as 2' -fucosyllactose (2 ' -FL), 3-fucosyllactose (3-FL), 3' -sialyllactose (3 ' -sialyllactose,3' -SL), 6-sialyllactose (6 ' -sialyllactose,6' -SL), and the like, and such components are called breast milk oligosaccharides, and are ingredients of general interest in the field of infant formulas and the like at present. The production method is mainly adopted at present by a microbiological fermentation method (scientific consensus of the Chinese food science and technology society, breast milk oligosaccharides (HMOs) [ J ]. Chinese food science and newspaper, 2023, 23 (6): 452-457.) which is suitable for industrialization and is friendly to the environment.
Coli is a common strain for the fermentative production of fucosyllactose (including 2' -FL, 3-FL, etc.), sialyllactose (including 3' -SL, 6' -SL). There have been many studies on the main genes, enzymes and functions of the fermentation production of fucosyllactose and sialyllactose by E.coli, such as the gene encoding beta-galactosidase associated with the fermentation production of fucosyllactoselacZUDP-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. (CN 115786220A, CN112501106a etc.); n-acetylneuraminic acid lyase NanA, N-acetylneuraminic acid H+ homotransporter NanT, N-acetylmannosamine related to sialyllactose fermentation production2-heptyl6-phosphate enzyme NanE, N-acetylmannosamine kinase NanK, UDP-GlcNAc epimerase NeuC, N-acetylneuraminic acid synthase NeuB, N-acetylneuraminic acid cytidylyltransferase NeuA, sialyltransferase SiaT and the like (Zhu YY, et al Recent progress on health effects and biosynthesis of two key sialylated human milk oligosaccharides, 3'-sialyllactose and 6' -sialylactose [ J ]]. Biotechnology Advances, 2023, 62:108058.）。
The protein transporter encoded by the secY gene is the central subunit of the SecYEG protein translocase complex, a protein transport channel formed by 10 transmembrane segments. In the previous studies of the inventors, a strain having improved lysine tolerance and yield was obtained by screening lysine-producing strains, and the SecY protein in the strain had a point mutation (CN 112251391A) which did not cause improvement in the yields of fucosyllactose and sialyllactose. The construction of xylose to xylitol metabolic pathway by over-expressing SecY protein encoding gene has also been studied, overcoming catabolite repression effect of e.coli (CN 113293121 a). However, research on the relationship between SecY protein and the production of fucosyl lactose and sialyl lactose by escherichia coli fermentation is still lacking, and reports on the application of SecY protein mutants and related genes to the production of fucosyl lactose and sialyl lactose by escherichia coli fermentation are also lacking.
Disclosure of Invention
In order to solve the technical problems, the invention utilizes a gene editing technology to edit the coding gene of the escherichia coli protein transporter SecY, so as to obtain a SecY mutant capable of improving the yields of fucosyllactose and sialyllactose and apply the SecY mutant to the production of fucosyllactose and sialyllactose.
One of the technical schemes of the invention aims at providing a protein transporter SecY mutant which is obtained on the basis of a wild type protein transporter SecY (Gene ID: 947799). The amino acid sequence of the SecY protein mutant is shown as SEQ ID NO. 1; or the amino acid sequence of the SecY protein mutant corresponds to the amino acid sequence shown in SEQ ID NO.1, at least 375 th amino acid is proline, and has at least 90% homology with the amino acid sequence shown in SEQ ID NO. 1.
The second technical scheme of the invention aims at providing a coding gene of a protein transporter SecY mutant of which the coding amino acid sequence is shown as SEQ ID NO.1, and the coding gene has a nucleotide sequence shown as SEQ ID NO. 2.
The coding gene and the coded expressed protein transporter SecY mutant can enhance the tolerance of the strain in the fermentation process, thereby further improving the yields of fucosyllactose and sialyllactose.
The third 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 a nucleotide fragment shown as SEQ ID NO.2 or expresses the SecY protein mutant. The term "genetically engineered bacterium producing fucosyllactose and/or sialyllactose" as used herein refers to a genetically engineered bacterium having the ability to produce fucosyllactose and/or sialyllactose by itself by fermentation even when the SecY protein mutant is not expressed.
It has been revealed that when the aforementioned SecY protein mutant is expressed by a genetically engineered bacterium having the ability to produce fucosyllactose and/or sialyllactose by a technique such as gene editing, the production of fucosyllactose and/or sialyllactose can be further improved. Therefore, the fourth technical scheme provided by the invention is to provide the application of the protein transporter SecY mutant and the coding gene, wherein the SecY protein mutant or the coding gene is used for improving the yield of fucosyllactose and/or sialyllactose prepared by escherichia coli fermentation.
Advantageous effects
The protein transport protein SecY mutant is obtained through a gene editing technology, and is applied to escherichia coli for producing fucosyllactose and sialyllactose, so that the yield of fucosyllactose or sialyllactose is greatly improved.
The protein transporter SecY mutant and the coding gene for improving the yield of fucosyllactose and/or sialyllactose are point mutation, so that the acquisition mode is simpler and more convenient, and the gene expression is more stable.
Drawings
FIG. 1 is a diagram showing the PCR verification of the first step homologous recombination colony of the strain W3; m is Marker,1 is PCR verification strip;
FIG. 2 is a diagram showing PCR verification of a second step homologous recombination colony of the strain W3; m is Marker,1 is PCR verification band.
Detailed Description
The invention is further described below by means of specific embodiments. Unless otherwise indicated, the technical means, materials, and the like, to which the following embodiments relate, are known to those skilled in the art, and may be appropriately 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 protein transporter SecY mutant referred to below was obtained by mutating leucine at position 375 into proline based on wild-type protein transporter SecY (Gene ID: 947799), and had the amino acid sequence shown in SEQ ID NO. 1. The nucleotide sequence of the related protein transporter SecY mutant is shown as SEQ ID NO. 2. The invention will be further explained by means of specific embodiments.
EXAMPLE 1 construction of Strain W3
The wild type protein transporter SecY amino acid sequence of E.coli K12 MG1655 is QPA16999.1. Based on the escherichia coli TKYW1, the CRISPR/Cas9 gene editing technology is utilized to carry out gene editing on protein transporter SecY on genomesecYMutation is carried out, leucine at 375 th position of a translation protein is mutated into proline, and the amino acid sequence of a corresponding protein transporter SecY mutant is shown as SEQ ID NO:1, the constructed strain was designated as W3. CRISPR/Cas9 technology reference used in experimentsPrevious studies reported (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).
Wherein the escherichia coli TKYW1 is prepared by using escherichia coli K12 MG 1655%Escherichia coli K12 MG 1655) is constructed as an initial strain, and P in lactose lac operon sequence of the initial strain is knocked out lac Promoter sequences and regulatory geneslacIAndlacZin situlacZAfter the site with P trc Promoter overexpressionwcaG、gmdAndlacYfurther, ethanol dehydrogenase encoding geneadhEReplaced by P trc Over-expression of promotersmanAAndmanBknockout of UDP-glucose lipid carrier transferase coding gene on genomewcaJGDP-mannose hydrolase encoding genenudD，The specific construction of this strain is described in example 1 of CN115786220 a.
The specific construction method of the strain W3 is as follows:
1. construction of homologous recombination fragments
The wild strain of Escherichia coli K12 MG1655 stored in a laboratory is used as a template, and the primer pairs S-up-F/R and S-down-F/R in the table 1 are respectively used as primers to obtain the upstream and downstream homology arms of homologous recombination through PCR amplification. PCR obtained by constructing the strain TKYW1cat-The fragment of the N20 sequence is used as a template, and a primer pair S-cat-F/R is used as a primer for PCR amplification to obtain a novel primercat-Fragments of the N20 sequence. The homologous arm above and downstream, new withcat-Fragments of N20 sequence, 3 fragments as templates, and primers S-up-F and S-down-R were used for overlap PCR to obtain homologous recombination fragments containingsecYMutation of the gene, i.e., leucine at position 375 of the wild-type SecY protein, into proline.
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 ofsecYThe mutant strain (SEQ ID NO. 2) was designated as W3.
TABLE 1 constructionsecYPrimers 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. 3; 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 Uses neuC-F/R as primerPerforming PCR amplification to obtain 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 (Norwegian Biotechnology Co., ltd.)Ligating, transforming into E.coli DH5 alpha competent cells, culturing on LB plate containing 100 mg/L ampicillin, picking up transformant, performing colony PCR and sequencing verification to obtain correct recombinant plasmid named 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 W3 were introduced, respectively, to construct 2' -fucosyllactose-producing strain F1 (TKYW 1 (pTrc 99 a-P) trc -futC-manC) F2 (W3 (pTrc 99 a-P) trc -futC-manC) 3-fucosyllactose-producing strain F3 (TKYW 1 (pTrc 99 a-P) trc -futA-manC) F4 (W3 (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) And F6 (W3 (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 (W3 (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 example 3 and example 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 aqueous ammonia.
The fermentation test process comprises the following steps:
the single colonies of the fucosyllactose-producing strain and sialyllactose-producing strain were picked up, 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. Sample pair using fucosyllactose or sialyllactose standardThe concentration was quantified. The results are shown in tables 4 to 7:
from tables 4 to 7, it can be seen that mutation of leucine to proline at position 375 of SecY protein 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 SecY protein mutant is characterized in that the amino acid sequence of the SecY protein mutant is shown as SEQ ID NO. 1; or the amino acid sequence of the SecY protein mutant corresponds to the amino acid sequence shown in SEQ ID NO.1, at least 375 th amino acid is proline, and has at least 90% homology with the amino acid sequence shown in SEQ ID NO. 1.
2. A gene encoding the SecY protein mutant according to claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.
3. 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 a nucleotide fragment shown in SEQ ID No.2 or expresses the SecY protein mutant of claim 1.
4. The genetically engineered bacterium of claim 3, 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 3, wherein the genetically engineered bacterium of escherichia coli is a genetically engineered bacterium of escherichia coli producing 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 SecY protein mutant according to claim 1 or a coding gene according to claim 2 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.
CN202311640459.3A 2023-12-04 2023-12-04 SecY protein mutant, genetically engineered bacterium and application Pending CN117736280A (en)

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