Escherichia coli with high yield of 2 '-fucosyllactose as well as construction method and application of escherichia coli

Abstract

The invention relates to Escherichia coli with high yield of 2 '-fucosyllactose as well as a construction method and application of the Escherichia coli, and belongs to the technical field of genetic engineering. Alpha-1, 2-fucosyltransferase from neisseria is heterologously expressed in starting escherichia coli, a GDP-L-fucose synthesis route key enzyme coding gene cluster, a transcriptional regulation factor protein coding gene, a beta-galactoside permease coding gene and a sugar efflux transporter A coding gene are overexpressed, and a GDP-L-fucose synthesis route key enzyme coding gene cluster, a transcriptional regulation factor protein coding gene, a beta-galactoside permease coding gene and a glycosyl efflux transporter A coding gene are expressed. A beta-galactosidase coding gene, a UDP-glucose lipid carrier transferase coding gene, a GDP-mannosyl hydrolase coding gene and a galactoside O-acetyltransferase coding gene are knocked out, a recombinant escherichia coli strain with both yield and production efficiency is constructed, and in a fermentation system with glycerol as a carbon source, the recombinant escherichia coli strain with high yield and high production efficiency is obtained. The yield of the 2 '-fucosyllactose reaches 134.71 g / L, the production efficiency reaches 3.21 g / L.h, the industrial application potential is shown, and efficient production of the 2'-fucosyllactose is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to an Escherichia coli with high 2'-fucosyllactose production, and a construction method and application thereof. Background Art

[0002] Human milk oligosaccharides (HMO) are a very important type of nutrient in breast milk. Most of them are composed of 3 to 6 sugar groups, and more than 200 types have been discovered so far. Representative HMO components include 2'-fucosyllactose (2'-FL, CAS number: 41263-94-9), 3-fucosyllactose (3-FL), 6'-sialyllactose (6'-SL), lactose-N-neotetraose (LNnT), etc. Among them, 2'-fucosyllactose (2'-FL) has received widespread attention in the market because it helps to improve the immunity of infants and young children, regulates intestinal flora, and promotes brain development. In recent years, food companies such as Abbott and Nestle have added 2'-FL to infant formula to increase the added value of products, so the demand for 2'-FL has increased year by year. Achieving efficient synthesis of 2'-FL will help promote the low-cost industrial production of HMOs and ultimately achieve the market promotion of related products.

[0003] 2'-FL can be synthesized by chemical method, enzymatic method and microbial fermentation method. Among them, microbial fermentation method is more suitable for large-scale industrial production due to its advantages of high yield, low raw material price and green process. 2'-FL can be synthesized by fermentation of a variety of microorganisms, including recombinant Escherichia coli, Bacillus subtilis, Yarrowia lipolytica, Saccharomyces cerevisiae and other microbial strains. At present, the research on recombinant E. coli is relatively sufficient. For example, Chen et al. used recombinant E. coli as a production strain and fermented glycerol as a carbon source for 105 hours to obtain 112.56 g / L of 2'-FL, with a production efficiency of 1.10 g / L·h (Bioresource Technology, 2023); Zhu et al. used recombinant E. coli as a production strain and fermented glycerol as a carbon source for 83 hours to obtain 94.7 g / L of 2'-FL, with a production efficiency of 1.14 g / L·h (Journal of Agricultural and Food Chemistry, 2023); Sun et al. used recombinant E. coli as a production strain and fermented glycerol as a carbon source for 36 hours to obtain 61.06 g / L of 2'-FL, with a production efficiency of 1.70 g / L·h (Bioresource Technology, 2023). The above research results show that recombinant E. coli can synthesize 2'-FL with high efficiency. However, as a nutritional enhancer for infants and young children that will soon be widely used around the world, 2'-FL will have a huge demand in the future, and therefore requires low cost to be widely promoted. The current synthetic output still has room for improvement. For technology developers, further improving the existing recombinant E. coli production strains, effectively promoting the fermentation synthesis of 2'-FL, and further improving production efficiency to reduce production costs are technical issues that need to be solved urgently. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an Escherichia coli with high 2'-fucosyllactose production, and a construction method and application thereof. The β-galactosidase encoding gene, the UDP-glucose lipid carrier transferase encoding gene, the GDP-mannosyl hydrolase encoding gene and the galactoside O-acetyltransferase encoding gene are knocked out in the starting Escherichia coli by using an editing method, and the source of the α-1,2-fucosyltransferase is screened by using a strong promoter α-1,2-fucosyltransferase, a GDP-L-fucose synthesis pathway key enzyme encoding gene cluster, a transcriptional regulatory factor protein encoding gene, a β-galactoside permease encoding gene and a sugar efflux transporter A encoding gene. A recombinant Escherichia coli with both high yield and production efficiency is successfully constructed and can be applied to the industrial production of 2'-fucosyllactose.

[0005] The first object of the present invention is to provide an Escherichia coli with high 2'-fucosyllactose production, wherein the transformation of the Escherichia coli comprises:

[0006] (1) heterologously expressing the α-1,2-fucosyltransferase encoding gene FutC from Neisseria, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0007] (2) Overexpression of the GDP-L-fucose synthesis pathway key enzyme encoding gene cluster, transcriptional regulatory factor protein encoding gene, β-galactoside permease encoding gene LacY and sugar efflux transporter A encoding gene SetA;

[0008] The GDP-L-fucose synthesis pathway key enzyme encoding gene clusters include α-D-mannose 1-phosphoguanylate transferase, phosphomannose mutase encoding gene clusters manC-manB, and GDP-mannose 6-dehydrogenase, GDP-L-fucose synthase encoding gene clusters gmd-wcaG, and the transcriptional regulatory factor protein encoding genes include rcsA and rcsB;

[0009] (3) Knockout the β-galactosidase encoding gene LacZ, the UDP-glucose lipid carrier transferase encoding gene wcaJ, the GDP-mannosyl hydrolase encoding gene nudD, and the galactoside O-acetyltransferase encoding gene LacA.

[0010] Furthermore, the nucleotide sequence of manB is shown in SEQ ID NO.2, the nucleotide sequence of manC is shown in SEQ ID NO.3, the nucleotide sequence of gmd is shown in SEQ ID NO.4, the nucleotide sequence of wcaG is shown in SEQ ID NO.5, the nucleotide sequence of rcsA is shown in SEQ ID NO.6, the nucleotide sequence of rcsB is shown in SEQ ID NO.7, the nucleotide sequence of LacY is shown in SEQ ID NO.8, and the nucleotide sequence of SetA is shown in SEQ ID NO.9.

[0011] Furthermore, the nucleotide sequence of LacZ is shown in SEQ ID NO.10, the nucleotide sequence of wcaJ is shown in SEQ ID NO.11, the nucleotide sequence of nudD is shown in SEQ ID NO.12, and the nucleotide sequence of LacA is shown in SEQ ID NO.13.

[0012] Furthermore, the transformation of the Escherichia coli also includes knocking out the 6-phosphoglucose isomerase encoding gene pgi.

[0013] Furthermore, the nucleotide sequence of the pgi is shown in SEQ ID NO.14.

[0014] Furthermore, the SD sequences of the gene clusters manC-manB and gmd-wcaG were replaced, and the replaced SD sequences are shown in SEQ ID NO.15.

[0015] Furthermore, the Escherichia coli expresses the LacY gene at the nudD gene site and the SetA gene at the LacA gene site.

[0016] Furthermore, the GDP-L-fucose synthesis pathway key enzyme encoding gene cluster, β-galactoside permease encoding gene LacY and sugar efflux transporter A encoding gene SetA are overexpressed by Trc promoter.

[0017] Furthermore, the transcriptional regulatory factor protein encoding gene is overexpressed by a T7 promoter.

[0018] A second object of the present invention is to provide a microbial preparation comprising the above-mentioned Escherichia coli.

[0019] The third object of the present invention is to provide a method for constructing the above-mentioned Escherichia coli, comprising the following steps:

[0020] Step S1, knocking out the LacZ gene, the wcaJ gene and the pgi gene in the starting Escherichia coli, knocking in the lacY gene at the nudD gene site, and knocking in the setA gene at the lacA gene site, to obtain a modified Escherichia coli;

[0021] Step S2, constructing a first recombinant plasmid, including a Trc promoter, a manC-manB gene cluster after replacing the SD sequence, and a gmd-wcaG gene cluster after replacing the SD sequence;

[0022] Step S3, constructing a second recombinant plasmid, including T7 promoter, rcsA gene, rcsB gene and FutC gene;

[0023] Step S4, transferring the first recombinant plasmid and the second recombinant plasmid into the transformed Escherichia coli to obtain the Escherichia coli with high 2'-fucosyllactose production.

[0024] The fourth object of the present invention is to provide use of the above-mentioned Escherichia coli or the above-mentioned microbial preparation in producing 2'-fucosyllactose.

[0025] The fifth object of the present invention is to provide a method for producing 2'-fucosyllactose, wherein the fermentation system uses glycerol as a carbon source and is added with the above-mentioned Escherichia coli or the above-mentioned microbial preparation for production.

[0026] In one embodiment of the present invention, the seed solution of the above-mentioned Escherichia coli is inoculated into a fermenter containing 30 g / L glycerol, 10 g / L lactose and 0.2 mM IPTG, the ventilation volume is controlled at 1-2 vvm, the rotation speed is 200-900 rpm, and after the initial glycerol is exhausted, a feed solution containing 500 g / L glycerol and 10 g / L magnesium sulfate solution is manually added, and ammonia water is maintained at pH = 7.0. The whole process is cultured at 30°C for not less than 42 hours.

[0027] Beneficial effects of the present invention:

[0028] The present invention transforms a starting strain of Escherichia coli to express key enzyme genes in the 2'-fucosyllactose synthesis pathway, knocks out genes that hinder the synthesis of 2'-fucosyllactose, knocks in genes that express sugar transporters, and further improves the expression level of the target gene by replacing the SD sequence in the gene cluster. The recombinant Escherichia coli constructed has a 2'-fucosyllactose yield of 134.71 g / L and a production efficiency of 3.21 g / L·h in a fermentation system with glycerol as a carbon source, showing potential for industrial application and realizing efficient production of 2'-fucosyllactose. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0030] Figure 1 Schematic diagram of the synthesis route of 2'-fucosyllactose of the present invention;

[0031] Figure 2 is the evolutionary tree of α-1,2-fucosyltransferase in Example 2 of the present invention;

[0032] Figure 3 is a primary sequence alignment of α-1,2-fucosyltransferase in Example 2 of the present invention, wherein Motif is a conserved sequence;

[0033] Figure 4 The 2'-fucosyllactose yield after shake flask fermentation of different α-1,2-fucosyltransferases screened in Example 3 of the present invention;

[0034] Figure 5 Comparison of 2'-fucosyllactose fermentation yields of different recombinant strains constructed in Example 5 of the present invention;

[0035] Figure 6 The fermentation yields of 2'-fucosyllactose of different recombinant strains and the changes in the growth conditions of the recombinant strains constructed in Example 6 of the present invention;

[0036] Figure 7This is the HPLC high performance liquid chromatography result of 2'-fucosyllactose in the fermentation broth of the recombinant strain BL21-ΔZWP-YS-SD-NsFutC in Example 6 of the present invention. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0038] The CRISPR / Cas9 technology used in the experiment refers to previous research reports [Li, Q. et al. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichiacoli. Acta Biochim. Biophys. Sin. 2021, 53(5), 620-627].

[0039] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are performed according to the kit instructions.

[0040] Routine operation methods such as colony PCR, agarose gel nucleic acid electrophoresis, heat shock transformation, electrotransformation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to conventional molecular experiments.

[0041] The sequencing of plasmids and DNA fragments was completed by Anhui General Bioengineering Company.

[0042] The culture medium and detection method used are as follows:

[0043] LB liquid medium: peptone 10g / L, yeast extract powder 5g / L, sodium chloride 10g / L;

[0044] LB solid medium: peptone 10g / L, yeast extract powder 5g / L, sodium chloride 10g / L, agar powder 20g / L;

[0045] Fermentation medium: glycerol 20g / L, yeast extract powder 1g / L, citric acid 2g / L, sodium sulfate 0.02g / L, ammonium sulfate 2.5g / L, magnesium sulfate 2g / L, dipotassium hydrogen phosphate 7.5g / L, ferrous sulfate 7.56mg / L, cobalt chloride 4mg / L, zinc sulfate 6.4mg / L, copper sulfate 0.6mg / L;

[0046] Feed solution A: 500 g / L glycerol, 10 g / L magnesium sulfate;

[0047] Feed solution B: lactose 200 g / L.

[0048] HPLC detection conditions: The HPLC instrument was Agilent 1200, the chromatographic column was an amide bonded column (HPLCONE-5AMIDE, 4.6mm ID*250mm, Suzhou Suyan Pharmaceutical Technology Co., Ltd.), the detector was a differential refractometer, the mobile phase was acetonitrile:water = 64:36 (v / v), the flow rate was 1.0 mL / min, and the injection volume was 5 μL.

[0049] Example 1: Gene Editing of Escherichia coli BL21 (DE3) Strain

[0050] 1. Construction steps of BL21-ΔZ strain, the primers involved are shown in Table 1:

[0051] (1) Construction of knockout plasmid pEcgRNA-lacZ-KO

[0052] According to the sequence characteristics of the target gene LacZ (SEQ ID NO.10), the N20 sequence was designed on the CHOPCHOP website, and the N20 fragment was obtained by gradient annealing with primers lacZ-N20-F / R, and the gRNA plasmid was constructed with the original pEcgRNA plasmid by the Golden Gate connection method; the Escherichia coli MG1655 genome was specifically amplified using primers up-lacZ-F / R and dn-lacZ-F / R to obtain the upstream and downstream homology arm fragments of lacZ, and the pEcgRNA plasmid with N20 was linearized using primers pgRNA-ZF / R primers. Finally, the homology arms and the linearized N20 plasmid were connected by the Golden gate connection method and coated on an LB plate containing spectinomycin (50 μg / mL). After overnight culture, the plasmid pEcgRNA-lacZ-KO was obtained, and sequencing verification was performed to ensure that the sequences in the constructed plasmid were consistent with the target sequence.

[0053] (2) Preparation of Escherichia coli BL21 (DE3) containing pEcCas for electroporation

[0054] The pEcCas plasmid (10 μg) was mixed with 100 μL of commercial BL21 (DE3) competent cells and placed in an ice bath for 30 minutes, followed by heat shock in a 42°C water bath for 1 minute. After the heat shock, the cells were immediately placed on ice for 2 minutes, and 1 mL of antibiotic-free LB was added to recover at 37°C for 1 hour. 100 μL of the supernatant was spread on a kanamycin (50 μg / mL) resistant plate, and the transformant BL21-pEcCas containing the pEcCas plasmid was picked after overnight culture. BL21-pEcCas was inoculated with 2% inoculum into 100 mL of LB liquid medium containing 10 mM arabinose and 50 μg / mL kanamycin and cultured for two hours. After washing three times with 10% (v / v) glycerol, the cells were aliquoted and stored in a -80°C ultra-low temperature refrigerator for later use.

[0055] (3) Gene editing and screening of strains

[0056] Take 100 μL of BL21-pEcCas electroporated competent cells and mix with 10 μg of knockout plasmid pEcgRNA-lacZ-KO and transfer to a pre-cooled 1 mm electroporation cup. After electric shock at 1.8 kV and 5 mS, quickly add 1 mL of non-resistant LB liquid culture medium and place it in a 37 ° C shaker for recovery for 1 hour. After the recovery, centrifuge at 6000 rpm for 2 minutes, aspirate 1 mL of supernatant and use the remaining liquid to resuspend the bacterial mud and apply it to a double-resistant LB plate containing spectinomycin (50 μg / mL) and kanamycin (50 μg / mL). Culture in a 37 ° C incubator overnight, pick a single colony and inoculate it into a single-resistant LB liquid culture medium containing 10 mM rhamnose and 50 μg / mL kanamycin, and use primers Verify-lacZ-F / R for verification.

[0057] (4) Plasmid loss

[0058] The successfully verified colony was inoculated into LB medium containing 10mM rhamnose and kanamycin for overnight culture, and then streaked onto a kanamycin plate to isolate a single clone, which was then inoculated into liquid LB medium containing spectinomycin (50μg / mL) and kanamycin (50μg / mL), respectively. If it grew normally in the medium containing kanamycin, but did not grow in the dual-resistance medium containing kanamycin and spectinomycin, the pEcgRNA plasmid was successfully lost; the clone with successful pEcgRNA loss was inoculated into LB medium containing 5g / L glucose and cultured overnight at 37°C, and the fermentation broth was streaked onto a plate containing 5g / L glucose and 50g / L sucrose to isolate a single clone, and the grown single clone was inoculated into LB medium without resistance and containing kanamycin, respectively. It grew normally in the medium without resistance, but did not grow in the culture medium containing kanamycin, which proved that the pEcCas plasmid was successfully lost, and the recombinant strain BL21-ΔZ with the lacZ gene knocked out was obtained.

[0059] 2. Construction of BL21-ΔZW strain

[0060] Using strain BL21-ΔZ as the chassis strain, the wcaJ gene (nucleotide sequence as shown in SEQ ID NO.11) was knocked out using the above method to obtain strain BL21-ΔZW. The primers involved are shown in Table 1.

[0061] 3. Construction of BL21-ΔZW-Y strain

[0062] Using strain BL21-ΔZW as the chassis strain, the P was knocked into the nudD gene (nucleotide sequence as shown in SEQ ID NO.12) using the above method. trc -LacY gene expression cassette fragment to obtain strain BL21-ΔZW-Y.

[0063] Compared with the construction of knockout plasmid, knock-in plasmid also needs to add the expression box fragment of the target gene between the upstream and downstream homology arm fragments. The detailed steps are as follows, and the primers involved are shown in Table 1:

[0064] (1)P trc - Acquisition of LacY gene expression cassette

[0065] The genome of Escherichia coli MG1655 was used as a template, and the gene lacY (nucleotide sequence as shown in SEQ ID NO.8) was amplified by PCR using primers LacY-F / R, respectively. The amplified fragments were gel purified and recovered, and the recovered LacY gene fragments were connected with the ptrc99a plasmid fragments that had been double-digested with EcoRI and HindIII. The vector fragment and the target gene fragment were mixed in a molar ratio of 1:2, and DNA recombinase (Novozymes) and buffer were added and connected at 37°C for 30 minutes. The ligation products were transformed into Escherichia coli DH5α competent cells and screened on ampicillin (100 μg / mL) plates to obtain the recombinant plasmid ptrc99a-lacY. The primers P trc -LacY-nudD-F / R was used to amplify the plasmid ptrc99a-LacY by PCR and purified to obtain P trc -LacY gene expression cassette fragment;

[0066] (2) Construction of knock-in plasmid pEcgRNA-nudD-KI-LacY

[0067] First, the knockout plasmid pEcgRNA-nudD-KO was constructed using the above method, and the knockout plasmid was linearized using primers pKO-nudD-F / R and then cleaved with purified P trc-LacY expression box DNA fragment ligation, the vector fragment and the target gene fragment were mixed at a molar ratio of 1:2 and DNA recombinase (Novozymes) and buffer were added and ligated at 37°C for 30 minutes. The ligation product was transformed into Escherichia coli DH5α competent cells and screened on spectinomycin plates (50 μg / mL) to obtain positive transformants; positive transformants were cultured in LB liquid medium and plasmids were extracted to obtain the knock-in plasmid pEcgRNA-nudD-KI-LacY;

[0068] 4. Construction of BL21-ΔZW-YS strain

[0069] Using strain BL21-ΔZW-Y as the chassis strain, the above method was used to knock in P at the LacA gene (nucleotide sequence as shown in SEQ ID NO.13) site. trc -setA (the nucleotide sequence of the setA gene is shown in SEQ ID NO.9) gene expression cassette to obtain strain BL21-ΔZW-YS;

[0070] The primers involved are shown in Table 1.

[0071] Table 1 Primers used for gene editing in Escherichia coli

[0072]

[0073]

[0074]

[0075] Example 2: Construction of recombinant plasmid

[0076] Using Escherichia coli MG1655 genomic DNA as a template, PCR technology was used to obtain key enzyme genes in the synthesis pathway of the important intermediate GDP-L-fucose. The gene cluster included genes encoding α-D-mannose 1-phosphoguanylate transferase and phosphomannose mutase (manC-manB) (the nucleotide sequence of manB is shown in SEQ ID NO.2, and the nucleotide sequence of manC is shown in SEQ ID NO.3), as well as genes encoding genes of the gene cluster GDP-mannose 6-dehydrogenase and GDP-L-fucose synthase (gmd-wcaG) (the nucleotide sequence of gmd is shown in SEQ ID NO.4, and the nucleotide sequence of wcaG is shown in SEQ ID NO.5), which were cloned into the ptrc99a vector to obtain the ptrc99a-CBGW-WT recombinant plasmid for use.

[0077] The gene cluster manC-manB was PCR amplified using primers manC-manB-F / R, and the amplified fragment was gel-cleaved and purified. The original SD sequence between the two genes in the manC-manB gene cluster was replaced using primers SD-manB-F and manB-R (the replaced SD sequence is shown in SEQ ID NO.15), and the amplified fragment was gel-cleaved and purified to obtain the DNA fragment manC-SD-manB; the gmd-wcaG gene cluster was PCR amplified using primers gmd-wcaG-F / R in Table 2, and the amplified fragment was gel-cleaved and purified and used as a template. The original SD sequence between the two genes in the gmd-wcaG cluster was replaced using primers SD-wcaG-F and wcaG-R (the replaced SD sequence is shown in SEQ ID NO.15), and the amplified fragment was gel-cleaved and purified to obtain the DNA fragment gmd-SD-wcaG. The vector was linearized and amplified using primers 99a-F / R, and the linearized vector fragment was recovered by gel-cleaving. The linearized plasmid vector ptrc99a, manC-SD-manB fragment, and gmd-SD-wcaG fragment were mixed at a molar ratio of 1:1:1, and BsaI enzyme, T4 DNA ligase, and 10×T4 buffer were added, and then incubated at 37°C for 1 min and 16°C for 1 min. The above reaction was repeated for 15 cycles and then incubated at 60°C for 5 min before the reaction was terminated. The ligation product was transformed into Escherichia coli DH5α competent cells and screened on LB plates containing ampicillin (100 μg / mL), and the recombinant plasmid ptrc99a-CBGW-SD was finally obtained for standby use.

[0078] Using the genomic DNA of Escherichia coli MG1655 as a template, the rcsA-rcsB gene cluster encoding the transcriptional regulatory factor protein was PCR amplified using primers rcsAB-F / R (the rscA nucleotide sequence is shown in SEQ ID NO.6, and the rscB nucleotide sequence is shown in SEQ ID NO.7). The amplified product was excised from the gel to recover the rcsA-rcsB fragment, and the pRSFDuet-1 vector was double-digested with NdeI and bglII and ligated with the rcsA-rcsB fragment at a molar ratio of 1:2. The ligation was performed using DNA recombinase (Novozyme Biotechnology Co., Ltd., product number C112). The ligation product was transformed into Escherichia coli DH5α competent cells and screened on LB plates containing kanamycin (50 μg / mL) to obtain the recombinant plasmid pRSFDuet-AB.

[0079] With reference to the α-1,2-fucosyltransferase (α-1,2-fucosyltransferase, α-1,2-FT, EC 2.4.1.69) reported in the literature that can be used for 2'-FL synthesis, HpFutC from Helicobacter pylori26695, WbgL from Escherichia coli (accession number: ADN43847.1), SAMT from Azospirillum lipoferum (accession number: SMH41196.1), BKHT from Helicobacter sp.11S02629-2 (accession number: PAF50342.1), WcfB from Bacteroides fragilis NCTC9343 (accession number: CAH06753.1) and WcfB from Thermosynechococcus sp.NK55a were used as the reference. Te2FT (accession number: BAC08546.1) was used as a reference to mine more efficient α-1,2-FTs. Using the above sequence as a probe, α-1,2-FTs from different sources were selected as candidates through the HMMER online server search. The ClustalW website was used to align the known and candidate α-1,2-FTS amino acid sequences, and the sequence alignment results were then imported into the ESPript website to generate the sequence alignment result map. MEGA 4.0 program was used to perform multiple sequence alignment of α-1,2-FTs, and a phylogenetic tree was constructed based on the maximum likelihood method of the JTT matrix model, in which positions containing alignment gaps and missing data were partially deleted. The statistical confidence of the inferred phylogenetic relationship was evaluated by performing 1000 bootstrap replicates. Finally, five α-1,2-fucosyltransferases from different sources were selected as candidates through the HMMER online server search, namely NsFutC from Neisseria sp. (accession number: RTL12957.1), HmFutC from Helicobacter mustelae (accession number: WP_115056173.1), BaFutC from Bacteroides sp. (accession number: WP_055173642.1), SeFutC from Salmonella enterica (accession number: WP_023214330.1), and SeFutC from Flavobacterium limi's FlFutC (accession number: WP_163394409.1), the phylogenetic tree constructed is as follows Figure 2 As shown, the primary sequence comparison is Figure 3As shown. General Bio was commissioned to synthesize 9 candidate α-1,2-fucosyltransferase encoding genes optimized according to the codon preference of Escherichia coli and ligated between the EcoRV and XhoI sites of the pRSFDuet-AB plasmid to obtain the recombinant plasmids pRSFDuet-AB-NsFutC, pRSFDuet-AB-HpFutC, pRSFDuet-AB-BKHT, pRSFDuet-AB-SAMT, pRSFDuet-AB-WbgL, pRSFDuet-AB-HmFutC, pRSFDuet-AB-BaFutC, pRSFDuet-AB-SeFutC, and pRSFDuet-AB-FlFutC. Taking NsFutC from Neisseria sp. as an example, the α-1,2-fucosyltransferase gene NsFutC from Neisseria sp. (the nucleotide sequence after codon optimization is shown in SEQ ID NO.1) was PCR amplified using primers fucT-F / R, the amplified fragment was gel-cut and purified, the recovered NsFutC fragment was connected with the pRSFDuet-AB plasmid fragment double-digested with EcoRV and XhoI, the vector fragment and the target gene fragment were mixed in a molar ratio of 1:2, and after adding Novozymes one-step cloning enzyme, the connection was carried out at 37°C for 30 minutes. The connection product was transformed into Escherichia coli DH5α competent cells and spread on kanamycin (50 μg / mL) plates to screen for positive transformants. The positive transformants were cultured and the plasmid was extracted to obtain the recombinant plasmid pRSFDuet-AB-NsFutC.

[0080] The primers involved in the above steps are shown in the following table:

[0081] Table 2 Primers used to construct recombinant plasmids

[0082]

[0083] Example 3: Comparison of α-1,2-fucosyltransferases from different sources

[0084] The E. coli BL21-ΔZW-YS obtained by CRISPR editing in Example 1 was prepared into competent cells, and the recombinant plasmid ptrc99a-CBGW-WT and pRSFDuet-AB-NsFutC (or pRSFDuet-AB-HpFutC, or pRSFDuet-AB-BKHT, or pRSFDuet-AB-SAMT, or pRSFDuet-AB-WbgL, or pRSFDuet-AB-HmFutC, or pRSFDuet-AB-BaFutC, or pRSFDuet-AB-SeFutC ... SFDuet-AB-FlFutC) were co-transformed into the competent cells of BL21-ΔZW-YS to obtain the recombinant Escherichia coli BL21-ΔZW-YS-NsFutC (or BL21-ΔZW-YS-HpFutC, BL21-ΔZW-YS-BKHT, BL21-ΔZW-YS-SAMT, BL21-ΔZW-YS-WbgL, BL21-ΔZW-YS-HmFutC, BL21-ΔZW-YS-BaFutC, BL21-ΔZW-YS-SeFutC, BL21-ΔZW-YS-FlFutC) capable of synthesizing 2'-fucosyllactose.

[0085] Taking BL21-ΔZW-YS-NsfutC as an example, the specific operation method is as follows:

[0086] (1) Preparation of competent cells of BL21-ΔZW-YS strain

[0087] The strain BL21-ΔZW-YS was inoculated into 5 mL of LB medium and cultured at 37°C with a shaker speed of 200 rpm for about 8 h. 2 mL of the cultured seed solution was transferred to 100 mL of LB medium and cultured at 37°C with a shaker speed of 200 rpm until the OD 600 At about 0.55, 100 mL of bacterial solution was transferred to two pre-cooled 50 ml centrifuge tubes and placed on ice for 30 minutes to cool the culture to 0°C. The bacteria were collected by centrifugation at 4000 rpm for 10 minutes at 4°C. All cells were resuspended in 10 ml of pre-cooled 0.1 mol / L CaCl2 by gentle blowing and transferred to a 50 ml centrifuge tube, ice bathed for 30 minutes, and centrifuged at 4000 rpm for 10 minutes (4°C) to recover the cells. All cells were resuspended in 4 mL of ice-cold 0.1 mol / L CaCl2 solution (containing 15% glycerol) for each cell pellet, and the cells were divided on ice and frozen at -80°C for later use, thus obtaining competent cells of strain BL21-ΔZW-YS.

[0088] (2) Co-transformation of ptrc99a-CBGW-WT and pRSFDuet-AB-NsFutC plasmids

[0089] The ptrc99a-CBGW-WT and pRSFDuet-AB-NsFutC plasmids (3 μg each) were mixed with 100 μL of BL21-ΔZW-YS competent cells and placed in an ice bath for 30 min, followed by heat shock in a 42°C water bath for 1 min, and immediately placed in an ice bath for 2 min. 1 mL of non-resistant LB medium was added to resuscitate at 37°C for 1 h, and 100 μL of the supernatant was spread on a double-resistant plate containing ampicillin (100 μg / mL) and kanamycin (50 μg / mL). The positive transformants containing the double plasmids were picked after overnight culture, which was the fermentation strain BL21-ΔZW-YS-NsFutC.

[0090] The recombinant strain was inoculated into 5 mL LB medium (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin) and cultured overnight at 37°C and 220 rpm for about 12 h. 1 mL of the cultured seed solution was transferred into a baffled shake flask containing 50 mL LB medium (containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and a final concentration of 30 g / L glycerol) and cultured at 37°C and 220 rpm until the bacterial solution OD 600 About 0.8, IPTG with a final concentration of 0.2mM and lactose with a final concentration of 10g / L were added to continue inducing fermentation for 72h, and lactose with a final concentration of 10g / L was added again at 24h and 48h after induction until the fermentation was completed. The fermentation broth was centrifuged at 12000rpm for 10min and the supernatant was collected. The sample was filtered through a 0.22μm membrane for HPLC determination. The fermentation results are shown in Figure 4 As shown, the yield of 2'-fucosyllactose of BL21-ΔZW-YS-NsFutC after 72h fermentation reached 13.82g / L, which is 1.18, 1.65, 1.33, and 1.42 times of HpFutC, WbgL, BKHT, and SAMT reported in the literature. Compared with α-1,2-fucosyltransferases from other sources, the yield of the recombinant strain expressing the HmFutC enzyme from Helicobacter mustelae is 8.41g / L, the yield of the recombinant strain expressing the BaFutC enzyme from Bacteroides sp. is 4.57g / L, the yield of the recombinant strain expressing the SeFutC enzyme from Salmonella enterica is 0.38g / L, and the yield of the recombinant strain expressing the FlFutC enzyme from Flavobacterium limi is 0.14g / L. Therefore, this example selects the NsFutC enzyme from Neisseria sp. for subsequent applications.

[0091] Example 4: Beneficial effects of SD sequence replacement

[0092] The Escherichia coli BL21-ΔZW-YS prepared by CRISPR editing in Example 1 was prepared into competent cells, and the recombinant plasmid ptrc99a-CBGW-SD and the recombinant plasmid pRSFDuet-AB-NsFutC obtained in Example 2 were co-transformed into the competent cells of BL21-ΔZW-YS to obtain the recombinant Escherichia coli BL21-ΔZW-YS-SD-NsFutC capable of synthesizing 2'-fucosyllactose. According to the operating method in Example 3, the BL21-ΔZW-YS-SD-NsFutC strain obtained a yield of 18.41 g / L in shake flask culture, which was higher than the BL21-ΔZW-YS-NsFutC strain whose SD sequence was not optimized (i.e., the strain BL21-ΔZW-YS-NsFutC in Example 3, with a yield of 13.82 g / L).

[0093] Example 5: Beneficial effects of knocking out the pgi gene

[0094] The Escherichia coli BL21-ΔZWP-YS prepared by CRISPR editing in Example 1 was prepared into competent cells, and the recombinant plasmid ptrc99a-CBGW-SD and the recombinant plasmid pRSFDuet-AB-NsFutC obtained in Example 2 were co-transformed into the competent cells of BL21-ΔZWP-YS to obtain the recombinant Escherichia coli BL21-ΔZWP-YS-SD-NsFutC capable of synthesizing 2'-fucosyllactose. According to the operating method in Example 3, the BL21-ΔZWP-YS-SD-NsFutC strain obtained a yield of 25.85 g / L in shake flask culture, which was higher than the BL21-ΔZW-YS-SD-NsFutC strain without knocking out the pgi gene.

[0095] The specific operation method is as follows:

[0096] (1) Preparation of competent cells

[0097] The strain BL21-ΔZWP-YS was inoculated into 5 mL of LB medium and cultured at 37°C with a shaker speed of 200 rpm for about 8 h. 2 mL of the cultured seed solution was transferred to 100 mL of LB medium and cultured at 37°C with a shaker speed of 200 rpm until the OD 600At about 0.55, 100 mL of bacterial solution was transferred to two pre-cooled 50 ml centrifuge tubes and placed on ice for 30 minutes to cool the culture to 0°C. The bacteria were collected by centrifugation at 4000 rpm for 10 minutes at 4°C. All cells were resuspended in 10 ml of pre-cooled 0.1 mol / L CaCl2 by gentle blowing and transferred to a 50 ml centrifuge tube, ice bathed for 30 minutes, and centrifuged at 4000 rpm for 10 minutes (4°C) to recover the cells. All cells were resuspended in 4 mL of ice-cold 0.1 mol / L CaCl2 solution (containing 15% glycerol) for each cell pellet, and the cells were divided on ice and frozen at -80°C for later use, thus obtaining the competent cells of the strain BL21-ΔZWP-YS.

[0098] (2) Co-transformation of ptrc99a-CBGW-SD and pRSFDuet-AB-NsFutC plasmids

[0099] The ptrc99a-CBGW-SD and pRSFDuet-AB-NsFutC plasmids (3 μg each) were mixed with 100 μL of BL21-ΔZWP-YS competent cells and placed in an ice bath for 30 min, followed by heat shock in a 42°C water bath for 1 min, and immediately placed in an ice bath for 2 min. 1 mL of non-resistant LB medium was added to resuscitate at 37°C for 1 h, and 100 μL of the supernatant was spread on a double-resistant plate containing ampicillin (100 μg / mL) and kanamycin (50 μg / mL). The positive transformants containing the double plasmids were cultured overnight, which was the fermentation strain BL21-ΔZWP-YS-SD-NsFutC.

[0100] The recombinant strain was inoculated into 5 mL LB medium (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin) and cultured overnight at 37°C and 220 rpm for about 12 h. 1 mL of the cultured seed solution was transferred into a baffled shake flask containing 50 mL LB medium (containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and a final concentration of 30 g / L glycerol) and cultured at 37°C and 220 rpm until the bacterial solution OD 600 About 0.8, IPTG with a final concentration of 0.2mM and lactose of 10g / L were added to continue inducing fermentation for 72h, and lactose with a final concentration of 10g / L was added again at 24h and 48h after induction until the fermentation was completed. The fermentation broth was centrifuged at 12000rpm for 10min to collect the supernatant, and the sample was processed through a 0.22μm filter membrane for HPLC determination. The fermentation results showed that the yield of 2'-fucosyllactose in BL21-ΔZWP-YS-SD-NsFutC reached 25.85g / L after 72h fermentation.

[0101] The fermentation results are as follows Figure 5As shown, the production of 2'-fucosyllactose in the BL21-ΔZWP-YS-SD-NsFutC strain with the pgi gene knocked out was increased by 1.4 times compared with the recombinant strain BL21-ΔZW-YS-SD-NsFutC strain without the knockout gene, and the recombinant strain BL21-ΔZWP-YS-SD-NsFutC has greater potential for industrial scale-up application.

[0102] In this example, a recombinant Escherichia coli strain capable of efficiently synthesizing 2'-fucosyllactose was successfully obtained, laying a foundation for further fermentation verification.

[0103] Example 6: High-density fermentation of engineered strains to produce 2'-fucosyllactose

[0104] The BL21-ΔZWP-YS-SD-NsFutC strain was inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin) and cultured at 37°C with a shaker speed of 220 rpm for about 8 h. 2 mL of the cultured seed solution was transferred into 100 mL of LB medium (containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and a final concentration of 30 g / L glycerol) and cultured at 37°C with a shaker speed of 220 rpm. 220rpm culture for about 10h; 100mL seed liquid was transferred to a 5L automatic fermentation tank with a liquid volume of 2L, and IPTG with a final concentration of 0.2mM, lactose with a final concentration of 10g / L, glycerol with a final concentration of 30g / L, ampicillin with a final concentration of 100μg / mL, and kanamycin with a final concentration of 50μg / mL were added at the beginning of the fermentation. The whole process was fermented at 30°C, the ventilation was controlled to be 1-2vvm, the speed was 200-900rpm, the pH was 7.0±0.2 (automatic control by adding ammonia water), the feed liquid was 500g / L glycerol and 10g / L magnesium sulfate, and 200g / L lactose, the glycerol concentration in the culture medium was maintained at about 10g / L, and the lactose concentration was manually added when it was lower than 3g / L. The fermentation broth with different fermentation times was centrifuged at 12000rpm for 2min, the supernatant was collected and filtered with a 0.22μm filter membrane and detected by high performance liquid chromatography HPLC. The results are as follows Figure 6 As shown, after 42h of fermentation, the OD 600 The yield of 2'-fucosyllactose reached 94.6, the production of 2'-fucosyllactose reached 134.71 g / L, and the production efficiency of 2'-FL reached 3.21 g / L·h. The fermentation products were tested by high performance liquid chromatography. The results are as follows Figure 7 As shown, the final fermentation product of the BL21-ΔZWP-YS-SD-NsFutC strain in a 5 L fermenter was 2'-fucosyllactose.

[0105] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. An Escherichia coli with high 2'-fucosyllactose production, characterized in that: The transformation of the Escherichia coli includes: (1) heterologously expressing the α-1,2-fucosyltransferase encoding gene FutC from Neisseria, the nucleotide sequence of which is shown in SEQ ID NO.1; (2) Overexpression of the GDP-L-fucose synthesis pathway key enzyme encoding gene cluster, transcriptional regulatory factor protein encoding gene, β-galactoside permease encoding gene LacY and sugar efflux transporter A encoding gene SetA; The GDP-L-fucose synthesis pathway key enzyme encoding gene clusters include α-D-mannose 1-phosphoguanylate transferase, phosphomannose mutase encoding gene clusters manC-manB, and GDP-mannose 6-dehydrogenase, GDP-L-fucose synthase encoding gene clusters gmd-wcaG, and the transcriptional regulatory factor protein encoding genes include rcsA and rcsB; (3) Knockout the β-galactosidase encoding gene LacZ, the UDP-glucose lipid carrier transferase encoding gene wcaJ, the GDP-mannosyl hydrolase encoding gene nudD, and the galactoside O-acetyltransferase encoding gene LacA.

2. The Escherichia coli according to claim 1, characterized in that: The transformation of the Escherichia coli also includes knocking out the 6-phosphoglucose isomerase encoding gene pgi.

3. The Escherichia coli according to claim 1, characterized in that: The SD sequences of the gene clusters manC-manB and gmd-wcaG were replaced, and the replaced SD sequences are shown in SEQ ID NO.

15.

4. The Escherichia coli according to claim 1, characterized in that: The Escherichia coli expresses the LacY gene at the nudD gene site and expresses the setA gene at the LacA gene site.

5. The Escherichia coli according to claim 1, characterized in that: The GDP-L-fucose synthesis pathway key enzyme encoding gene cluster, β-galactosidase permease encoding gene LacY and sugar efflux transporter A encoding gene SetA are overexpressed by Trc promoter.

6. The Escherichia coli according to claim 1, characterized in that: The transcriptional regulatory factor protein encoding gene is overexpressed by the T7 promoter.

7. A microbial preparation comprising the Escherichia coli according to any one of claims 1 to 6.

8. The method for constructing Escherichia coli according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step S1, knocking out the LacZ gene, the wcaJ gene and the pgi gene in the starting Escherichia coli, knocking in the LacY gene at the nudD gene site, and knocking in the setA gene at the LacA gene site to obtain a modified Escherichia coli; Step S2, constructing a first recombinant plasmid, including a T7 promoter, an rcsA gene, an rcsB gene and a FutC gene; Step S3, constructing a second recombinant plasmid, including the Trc promoter, the manC-manB gene cluster after replacing the SD sequence, and the gmd-wcaG gene cluster after replacing the SD sequence; Step S4, transferring the first recombinant plasmid and the second recombinant plasmid into the transformed Escherichia coli to obtain the Escherichia coli with high 2'-fucosyllactose production.

9. Use of the Escherichia coli according to any one of claims 1 to 6 or the microbial preparation according to claim 7 in producing 2'-fucosyllactose.

10. A method for producing 2'-fucosyllactose, characterized in that: The fermentation system uses glycerol as a carbon source and adds the Escherichia coli described in any one of claims 1 to 6 or the microbial preparation described in claim 7 for production.

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