The invention relates to a high-yield 2apos; recombinant escherichia coli of fucosyllactose as well as construction method and application of recombinant escherichia coli

Abstract

The invention relates to the technical field of bioengineering, in particular to recombinant Escherichia coli with high yield of 2 '-fucosyllactose as well as a construction method and application of the recombinant Escherichia coli. The recombinant Escherichia coli with high yield of 2 '-fucosyllactose is obtained through gene modification, the strain is adopted for fermentation production of 2'-fucosyllactose, the yield is greatly increased, and few by-products DFL exist in fermentation liquor.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and particularly relates to a recombinant Escherichia coli with high yield of 2'-fucosyllactose, a construction method and application thereof. BACKGROUND

[0002] Human milk oligosaccharides (HMOs) are a class of non-single structure, non-digestible carbohydrates in human milk, which play an important role in the health and growth of infants, and have been used as functional ingredients in infant formula. 2'-fucosyllactose (2'-FL) is a neutral fucosylated human milk oligosaccharide, accounting for about 31% (molar fraction) of HMOs, with a content of 0.06-3.93 g / L, and is the most abundant oligosaccharide in human milk. 2'-FL can regulate the composition of intestinal microbial community, promote the growth of beneficial bacteria, and regulate immune response, and has been approved by the US Food and Drug Administration (FDA) for the production of infant formula.

[0003] The main synthesis methods of 2'-FL include chemical synthesis, whole cell synthesis and enzyme catalysis synthesis. However, there are many difficulties in the actual production process of chemical synthesis or enzyme synthesis, such as stereochemical control, formation of specific linkage, availability of raw materials, etc. Compared with chemical synthesis and enzyme synthesis, the use of synthetic biology technology to synthesize through microbial metabolism is more economical and efficient. At present, the research is more on the use of Escherichia coli as a chassis cell, which has 2 complete pathways (de novo synthesis pathway and salvage pathway) for metabolic production of guanine 5'-diphospho-β-L-fucose (5'-diphospho-β-L-fucose, GDP-L-fucose). By constructing key enzyme genes in the metabolic synthesis pathway and overexpressing exogenous α-1,2-fucosyltransferase, 2'-FL is produced by fermentation.

[0004] Li et al. divided 2'-FL into GDP-L-fucose synthesis module, lactose fucosylation module and cofactor regeneration module, and obtained 22.3 g / L of 2'-FL by balancing the synthesis pathway through different plasmid combinations. Chen et al. obtained 112.56 g / L of 2'-FL by mutating α-1,2-fucosyltransferase. Xu et al. obtained 134.71 g / L of 2'-FL by knocking out the pgi gene and using glycerol as the only carbon source.

[0005] However, the fermentation yield of the existing strains still needs to be improved after the key genes of the 2'-FL synthesis pathway are modified, mainly because the supply of GDP-L-fucose precursor is insufficient, resulting in low 2'-FL conversion rate. In actual production, the production of 2'-FL is often accompanied by the generation of by-product lactose difucotetraose (DFL), and the high content of DFL will increase the cost of industrial production. The generation of this by-product is less concerned in current research. SUMMARY

[0006] The present application aims to overcome the deficiencies in the prior art and provide a recombinant Escherichia coli with high yield of 2'-fucosyllactose.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a recombinant Escherichia coli with high yield of 2'-fucosyllactose, which is obtained by modifying the following genes based on Escherichia coli as a starting strain: (1) integrating manC and manB at the ugd site; (2) integrating gmd and wcaG at the rhaA site; (3) integrating wbgL at at least one of the frwA, ybeQ, mbhA, hlyE, yjiV, ydeU, and yjgx sites.

[0008] The present application has found that the integration of the mannose-1-phosphate guanylyltransferase gene manC and the phosphomannomutase gene manB at the ugd site, the integration of the GDP-mannose-6-dehydrogenase gene gmd and the GDP-fucose synthetase gene wcaG at the rhaA site, and the integration of 1 copy of the alpha-1,2 fucosyltransferase gene wbgL at the frwA site, the mbhA site, and the ybeQ site, respectively, significantly improve the production of 2'-fucosyllactose by the obtained recombinant Escherichia coli. The fermentation of 2'-fucosyllactose by the recombinant Escherichia coli of the present application produces 180.72 g / L of 2'-FL after 90 h of culture, and the content of the by-product DFL in the fermentation broth is 3.89 g / L.

[0009] Preferably, the starting strain is BL21Star(DE3)ΔlacZΔwcaJΔfucIK, which is obtained by knocking out the lacZ, wcaj, and fucIK genes in Escherichia coli BL21(DE3).

[0010] Preferably, the copy number of wbgL integrated in step (3) is 1-6.

[0011] Preferably, the genetic modification further includes knocking out the pfkA gene.

[0012] The present application prevents the flow of Fructose-6-P to Fructose-1,6-2P by knocking out the phosphofructokinase-1 gene pfkA, increases the synthesis of GDP-L-fucose, and further increases the yield of 2'-FL.

[0013] Preferably, it further comprises any one of the following genetic modifications: (a) integrating the nadk or icd gene at the hlyE site; (b) integrating the maeB or sthA gene at the ilvG site; (c) integrating the pntAB gene at the xylB site.

[0014] The present application overexpresses the NADPH and its precursor NADP+ metabolism related genes NAD kinase gene nadK, isocitrate NAD+ dehydrogenase gene Icd, malic enzyme gene MaeB, soluble pyridine nucleotide transhydrogenase encoding gene sthA, and membrane-bound pyridine nucleotide transhydrogenase encoding gene pntAB in E. coli, realizes the overexpression of GDP-L-fucose, and finally finds that overexpression of the maeB gene can effectively increase the synthesis of GDP-L-fucose.

[0015] More preferably, the genetic modification further comprises integrating maeB at the ilvG site.

[0016] Preferably, the genetic modification further comprises knocking out the zwf gene.

[0017] The present application prevents the flow of Glucose-6-P to 6-P-Gluconate by knocking out the glucose-6-phosphate dehydrogenase gene zwf, increases the synthesis of GDP-L-fucose, and further increases the yield of 2'-FL.

[0018] Preferably, the genetic modification further comprises integrating the setA gene at the ykgh site.

[0019] The recombinant bacteria of the present application integrate the sugar efflux transporter SetA at the ykgh site, improve the output rate of 2'-FL, eliminate the inhibition of intracellular product accumulation on the synthesis process, and further realize a substantial increase in fermentation yield.

[0020] In a second aspect, the present application provides a construction method of the recombinant E. coli with high yield of 2'-fucosyllactose, comprising the following steps: (i) integrating the target gene in the E. coli genome using a transposition system; (ii) knocking out the pfkA gene and the zwf gene using the improved CRISPR-Cas9 technology to obtain the recombinant E. coli with high yield of 2'-fucosyllactose.

[0021] In a third aspect, the present application provides the use of the recombinant E. coli with high yield of 2'-fucosyllactose in the production of 2'-fucosyllactose.

[0022] In a fourth aspect, the present application provides a method for producing 2'-fucosyllactose, which utilizes the recombinant E. coli with high yield of 2'-fucosyllactose to ferment 2'-fucosyllactose.

[0023] The present application has the following advantages: The present application increases the supply of precursor GDP-L-fucose precursor by overexpressing mannose-1-phosphate guanylyltransferase gene manC, phosphomannomutase gene manB, GDP-mannose-6-dehydrogenase gene gmd, GDP-fucose synthetase gene wcaG, malate dehydrogenase gene maeB and knocking out phosphofructokinase-1 gene pfkA and glucose-6-phosphate dehydrogenase gene zwf, greatly improves the conversion rate of 2'-FL. Meanwhile, the sugar transporter SetA is overexpressed to improve the output rate of 2'-FL, and the inhibition of intracellular product accumulation on the synthesis process is eliminated, finally realizing the great improvement of fermentation yield, and the ratio of byproduct DFL / 2'-FL in the fermentation broth is less than 2% (2'-FL published regulatory product quality standard requires, difucosyllactose, w / %≤2.0). DETAILED DESCRIPTION

[0024] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described in combination with specific examples.

[0025] The experimental methods used in the present application are conventional methods unless otherwise specified, and the materials, reagents, etc. used are conventional commercially available products unless otherwise specified, and can be obtained through commercial channels.

[0026] In the following examples and comparative examples, the plasmid transformation method used in the competent cells is chemical transformation unless otherwise specified; the resistance plate used is a kanamycin resistance plate; and the positive transformants selected are identified by PCR detection and sequencing of the cloned fragments.

[0027] In the following examples and comparative examples, the cloning refers to the single-fragment one-step cloning kit without ligase dependence unless otherwise specified, which is operated according to the kit instructions, and the single-fragment one-step cloning kit without ligase dependence is provided by Nanjing Novizen Biotech Co., Ltd. with the item number C112-02.

[0028] The recombinant E. coli of the present application can be fermented by conventional fermentation methods.

[0029] The starting strain used in the application is Escherichia coli I, and the construction method is disclosed in the invention patent CN119081982A. The beta-galactosidase lacZ, fucose isomerase / mannuronic acid kinase gene cluster fucIK and phosphotransferase wcaJ gene in Escherichia coli BL21(DE3) are knocked out by using Crispr-Cas9 technology, and the application is named BL21Star(DE3)AlacZAwcaJAfucIK.

[0030] The manC gene, the manB gene, the gmd gene and the wcaG gene are each independently derived from Escherichia coli BL21Star(DE3), and the nucleotide sequences are shown in SEQ ID NO. 1-4 in sequence.

[0031] The wbgL gene is derived from Escherichia coli E. coli O16, and the nucleotide sequence is shown in SEQ ID NO. 5.

[0032] The setA gene is derived from Escherichia coli BL21Star(DE3), and the sequence is shown in SEQ ID NO. 6.

[0033] The pEcCas vector, the pSPIN plasmid, the pETDuet-1 plasmid and the pEcgRNA plasmid are provided by addgene.

[0034] The pCDF-CBGW and the pETDuet-wbgL are synthesized by Beijing Qianke Biotechnology Co., Ltd.: the CBGW gene sequence is synthesized by taking the pCDFDuet-1 plasmid as a cloning vector to obtain the pCDF CBGW plasmid; and the wbgL gene sequence is synthesized by taking the pETDueT-1 plasmid as a cloning vector to obtain the pETDuet-wbgL plasmid.

[0035] The genes involved in the modification of the application and the information thereof are shown in Table 1.

[0036] Table 1 Example 1: An embodiment of the recombinant Escherichia coli for high yield of 2'-fucosyllactose according to the application.

[0037] 1. BL21star(DE3)AlacZwcaJAfucIK is used as a starting strain, manC and manB are integrated at the ugd site to obtain the strain LJ3-01.

[0038] According to the pCDF-CBGW plasmid design primer, the expression cassette containing T7 promoter manC and manB was obtained by PCR, and integrated (using the INTEGRATE system for genomic integration) into the ugd site of the strain BL21star (DE3) AlacZwcaJAlcuk genome to obtain the strain LJ3-01.

[0039] The specific construction method includes the following steps: First, the pSPIN plasmid containing the ugd-N32 specific sequence was cloned using T4 DNA ligase to obtain pSPIN-ugd.

[0040] Subsequently, the expression cassette fragment containing manC-manB was cloned into pSPIN-ugd to obtain plasmid pSPIN-ugd-manC-manB, which was transformed into BL21star (DE3) AlacZwcaJAlcuk, and positive transformant selection was performed on LB agar containing kanamycin. The correctly sequenced bacterial clones were passaged to remove the plasmid to obtain the strain LJ3-01.

[0041] 2. Take strain LJ3-01 as the starting strain, integrate gmd and wcaG at rhaA site to obtain strain LJ3-02.

[0042] According to the pCDF-CBGW plasmid design primer, the expression cassette containing T7 promoter gmd and wcaG was obtained by PCR, and the same integration method as step 1 was used to integrate it into the rhaA site of the strain LJ3-01 genome to obtain the strain LJ3-02.

[0043] 3. Take strain LJ3-02 as the starting strain, integrate wbgL at frwA site to obtain strain LJ3-03.

[0044] According to the pETDuet-wbgL plasmid design primer, the expression cassette containing T7 promoter wbgL was obtained by PCR, and the same integration method as step 1 was used to integrate it into the frwA site of the strain LJ3-02 genome to obtain the strain LJ3-03.

[0045] 4. The expression cassette fragments of mbhA, ybeQ, hlyE, ydeU, yjgX, yjiV were cloned into the pSPIN plasmid to obtain the multi-copy integration plasmid pSPIN-AQEUXV (AQEUXV refers to mbhA, ybeQ, hlyE, ydeU, yjgX, yjiV 6 sites).

[0046] 5. Take strain LJ3-02 as the starting strain, use pSPIN-AQEUXV for transposition to obtain strains with 2, 3, 4, 5, and 6 copies of wbgL, respectively: LJ3-04 (2 copies): wbgL was integrated into frwA and yjgx sites, respectively; LJ3-05 (3 copies): wbgL was integrated into frwA, mbhA and ybeQ sites, respectively; LJ3-06 (4 copies): wbgL was integrated into frwA, mbhA, ybeQ and ydeU sites, respectively; LJ3-07 (5 copies): wbgL was integrated into frwA, mbhA, hlyE, yjiV and ydeU sites, respectively; LJ3-08 (6 copies): wbgL was integrated into frwA, mbhA, hlyE, yjiV, ydeU and yjgx sites, respectively.

[0047] 6. The strains LJ3-03, LJ3-04, LJ3-05, LJ3-06, LJ3-07 and LJ3-08 were subjected to conventional shake flask fermentation and the 2’-FL yield was detected.

[0048] Method: (5.1) Seed culture: the recombinant E. coli was activated, a single colony was picked and inoculated into seed culture medium, and cultured at 35-39°C and 200-250 rpm for 5-7 h; (5.2) Fermentation culture: the seed culture was inoculated into a 500 mL conical flask containing 100 mL of batch fermentation medium at an inoculation amount of 1%, 1 mL of trace elements, 5 g / L of glucose, 1 g / L of MgSO4·7H2O, 20 mg / L of vitamin B1 were added, and the culture was incubated at 35-39°C until OD 600 =0.6-0.8, 0.2 mmol / L of IPTG was added for induction culture at 35-39°C, and the fermentation broth was obtained.

[0049] The formula of the conventional shake flask fermentation medium was: Na2HPO4·12H2O 17.9 g / L, KH2PO4 3.0 g / L, NH4Cl 2.0 g / L, (NH4)2HPO4 1.0 g / L, trisodium citrate dihydrate 2.2 g / L, CaCl2·2H2O 0.015 g / L, Triton-X 100 0.3 mL, yeast extract 2.0 g / L, glycerol 30 g / L, tryptone 15 g / L, and the pH was adjusted to 7.0.

[0050] The formula of the trace elements is: sodium salt of nitrilotriacetic acid 13.74 g / L, ferric ammonium citrate 5.6 g / L, zinc sulfate heptahydrate 0.9 g / L, CoCl2·6H2O 0.2 g / L, manganese chloride tetrahydrate 1.0 g / L, CuCl2·2H2O 0.10 g / L, boric acid 0.2 g / L, Na2MoO4·2H2O 0.2 g / L.

[0051] (5.3) The fermentation broth was boiled in a water bath for 5 min to kill the strain, and then centrifuged at 16000xg for 5 min. The obtained supernatant was filtered using a 0.22 μm filter, and the obtained filtrate was used for detection of 2'-FL.

[0052] A Shim-pack Scepter Diol-HILIC-120 (4.6 x 250 mm, 5 μm) chromatographic column, a high-performance liquid chromatograph (LC-16, Shimadzu, Japan), and a Shimadzu differential refractometer detector (RID-20A) were used to detect 2'-FL in the sample. 0-2.0 g / L gradient concentrations of 2'-FL standard were used to draw a standard curve with 2'-FL concentration as the abscissa and the liquid chromatograph peak area as the ordinate. The results are shown in Table 2.

[0053] Table 2 The results show that LJ3-05 integrated with 3 copies of wbgL exhibits the highest 2'-FL yield, reaching 7.21 g / L in shake flask culture.

[0054] The primers designed in this example are shown in Table 3.

[0055] Table 3 Example 2: An embodiment of the recombinant Escherichia coli for high-yield 2'-fucosyllactose.

[0056] LJ3-05 was used as the starting strain, and the pfkA gene was knocked out using the improved CRISPR-Cas9 technology to obtain strain LJ3-09. The knockout vector used was pEcCas vector containing Cas9 and lambda-Red recombinase, and pEcgRNA containing sgRNA sequence and N20 specific sequence was used for targeted gene editing.

[0057] According to the fermentation culture method of Example 1, routine shake flask fermentation was carried out, and the 2'-FL yield of strain LJ3-09 was detected to be 8.96 g / L.

[0058] The primers designed in this example are shown in Table 4.

[0059] Table 4 Example 3 An embodiment of the recombinant Escherichia coli with high yield of 2'-fucosyllactose according to the present application.

[0060] 1. Using LJ3-09 as the starting strain, the nadk and icd genes were integrated at the hlyE site (for specific methods, refer to Example 1, the same below), to obtain strains LJ3-10 and LJ3-11.

[0061] 2. Using LJ3-09 as the starting strain, the maeB and sthA genes were integrated at the ilvG site, to obtain strains LJ3-12 and LJ3-13.

[0062] 3. Using LJ3-09 as the starting strain, the pntAB gene was integrated at the xylB site, to obtain strain LJ3-14.

[0063] Conventional shake flask fermentation was performed according to the fermentation culture method of Example 1, and the yield of 2'-FL was detected. The results are shown in Table 5.

[0064] Table 5 The results show that the LJ3-12 strain overexpressing the maeB gene has the highest yield, and the yield of shake flask fermentation is 9.59 g / L.

[0065] The primers designed in this example are shown in Table 6.

[0066] Table 6 Example 4 An embodiment of the recombinant Escherichia coli with high yield of 2'-fucosyllactose according to the present application.

[0067] Using LJ3-12 as the starting strain, the zwf gene was knocked out using the improved CRISPR-Cas9 technology, to obtain strain LJ3-15. The knockout vector used was the pEcCas vector containing Cas9 and λ-Red recombinase, and the pEcgRNA containing the sgRNA sequence and the N20 specific sequence was used for targeted gene editing.

[0068] According to the fermentation culture method of Example 1, conventional shake flask fermentation was performed, and the yield of 2'-FL of the LJ3-15 strain was detected to be 10.54 g / L.

[0069] The primers designed in this example are shown in Table 7.

[0070] Table 7 Example 5 An embodiment of the recombinant E. coli with high yield of 2'-fucosyllactose according to the present application.

[0071] LJ3-16 was obtained by integrating setA gene at ykgh site with LJ3-15 as the starting strain.

[0072] The setA gene expression cassette regulated by T7 promoter was obtained by amplifying the primer with LJ3-01 bacterial genome and pCDF vector as templates, and was integrated at ykgh site of LJ3-15 strain by the same integration technology as in step 1 to obtain strain LJ3-16.

[0073] 1. The fermentation culture method according to example 1 was used for conventional shake flask fermentation, and the 2'-FL yield of strain LJ3-16 was 12.04 g / L after 2'FL fermentation.

[0074] 2. Fed-batch fermentation experiment: The recombinant E. coli was cultured in 1 L shake flask containing 150 mL LB medium for 6 h to obtain seed liquid; pH=6.8, temperature: 37 ℃, initial rotation speed 200 rpm, DO associated with rotation speed, rotation speed range set to 200-900 rpm, carbon source fed at a constant speed, and lactose solution was added in batches according to the design in the experiment.

[0075] The fermentation medium used in the fed-batch fermentation experiment contained 12.5 g / L initial glycerol, 5.0 g / L (NH4)2SO4, 11.5 g / L K2HPO4, 10.25 g / L KH2PO4, 0.375 g / L citric acid, 7.5 g / L tryptone, 2.5 g / L yeast extract, 15 mg / L thiamine, 3 g / L MgSO4·7H2O, 0.03 g / L CaCl2, and 15 mL / L trace element solution.

[0076] After detection, the strain LJ3-16 produced 180.72 g / L of 2'-FL and 3.89 g / L of DFL in 90 h, and the ratio of byproduct DFL / 2'-FL in the fermentation broth was 2.15%.

[0077] The primers designed in this example are shown in Table 8.

[0078] Table 8 Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A recombinant *Escherichia coli* strain that produces high levels of 2'-fucosylated lactose, characterized in that... Using Escherichia coli as the starting strain, the following genetic modifications were performed: (1) Integration of manC and manB at the ugd site; (2) Integration of gmd and wcaG at the rhaA site; (3) Integrating wbgL at at least one of frwA, ybeQ, mbhA, hlyE, yjiV, ydeU, and yjgx.

2. The recombinant *Escherichia coli* strain with high 2'-fucosylated lactose production as described in claim 1, characterized in that, The starting strain was BL21Star(DE3)ΔlacZΔwcaJΔfucIK, obtained by knocking out the lacZ, wcaj and fucIK genes in Escherichia coli BL21(DE3).

3. The recombinant *Escherichia coli* strain with high 2'-fucosylated lactose production as described in claim 1, characterized in that... In step (3), the number of copies of wbgL integrated is 1-6.

4. The recombinant *Escherichia coli* strain with high 2'-fucosylated lactose production as described in claim 1, characterized in that... The genetic modification also includes knocking out the pfkA gene.

5. The recombinant *Escherichia coli* strain with high 2'-fucosylated lactose production as described in claim 4, characterized in that, This also includes any of the following genetic modifications: (a) The hlyE site integrates the nadk or icd gene; (b) Integration of the maeB or sthA gene at the ilvG site; (c) Integration of the pntAB gene at the xylB site.

6. The recombinant *Escherichia coli* strain with high 2'-fucosylated lactose production as described in claim 5, characterized in that, The genetic modification also includes knocking out the zwf gene.

7. The recombinant *Escherichia coli* strain with high 2'-fucosylated lactose production as described in claim 6, characterized in that, The genetic modification also includes the integration of the setA gene at the ykgh site.

8. A method for constructing a recombinant *Escherichia coli* strain that produces high levels of 2'-fucosylated lactose as described in any one of claims 1-7, characterized in that... Includes the following steps: (i) Integrating the target gene into the Escherichia coli genome using a transposon system; (ii) The pfkA and zwf genes were knocked out using a modified CRISPR-Cas9 technology to obtain the recombinant Escherichia coli that produces high levels of 2'-fucosylated lactose.

9. The use of the recombinant Escherichia coli with high 2'-fucosylated lactose production as described in any one of claims 1-7 in the production of 2'-fucosylated lactose.

10. A method for producing 2'-fucosylated lactose, characterized in that, 2'-fucosylated lactose is produced by fermentation using recombinant Escherichia coli that produces high levels of 2'-fucosylated lactose as described in any one of claims 1-7.

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