The invention relates to a method for efficiently producing 3apos; recombinant escherichia coli of-sialic acid lactose and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to recombinant escherichia coli for efficiently producing 3 '-sialyllactose and application of the recombinant escherichia coli. According to the method, a 3 '-SL de novo synthesis route is built in escherichia coli, acetic acid and lactic acid metabolism related genes are knocked out, efficient production of 3'-SL is achieved, and no acetic acid and lactic acid by-products are generated. The exogenous gene is introduced into the genome of the escherichia coli through the CRISPR-Cas9 technology, the strain is stable, the risk of plasmid loss does not exist, antibiotics do not need to be used, and the production cost is greatly reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a recombinant Escherichia coli for efficiently producing 3'-sialyllactose and application thereof. BACKGROUND

[0002] 3'-sialyllactose (3'-SL) as a key human milk oligosaccharide (HMO) plays an important role in infant cognitive and neural development, immune regulation and bone health. Enzymatic synthesis of 3'-SL requires the addition of CTP and other substrates, which is costly and not suitable for industrial production. Biosynthesis, as a new emerging synthetic biology technology, does not require the addition of expensive CTP and Neu5Ac, and can synthesize 3'-SL from scratch in microorganisms using glucose as a carbon source, greatly reducing the cost of industrialization. The team of Chen Xiangsong from the Institute of Plasma Physics, Chinese Academy of Sciences, reconstructed Escherichia coli, and the 3'-SL yield in a 5 L bioreactor was 56.8 g / L, providing a basis for industrial production.

[0003] However, most of the current 3'-sialyllactose-producing strains are plasmid-type strains, which require the addition of antibiotics during fermentation and are difficult to completely remove during purification. The production cost of 3'-SL is high, the process is complex, there are many by-products, and the cycle is long, which is not conducive to industrialization. SUMMARY

[0004] The present application aims to overcome the deficiencies in the prior art and provides a recombinant Escherichia coli for efficiently producing 3'-sialyllactose, which can achieve efficient production of 3'-SL and does not generate acetic acid and lactic acid by-products.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows: In a first aspect, the present application provides a recombinant Escherichia coli for efficiently producing 3'-sialyllactose, which is based on Escherichia coli as a starting strain and is genetically modified as follows: (1) knocking out L-arabinose isomerase gene araA, beta-galactosidase gene lacZ, UDP-glucose lipid carrier transferase gene wcaJ, sialidase gene nanA, sialate permease gene nanT, N-acetylmannosamine-6-phosphate epimerase gene nanE and N-acetylmannosamine kinase gene nanK; (2) inserting sialate synthase gene neuB, UDP-N-acetylglucosamine-2-epimerase gene neuC and CMP-sialate synthase gene neuA at rhaA site; (3) fusing pelB to the N-terminus of alpha2,3-sialyltransferase gene Vs16 and transposing it to lacA site to obtain the recombinant Escherichia coli for efficiently producing 3'-sialyllactose.

[0006] This invention involves gene modification of *Escherichia coli* BL21(DE3), knocking out the β-galactosidase gene (lacZ) in the lactose metabolism pathway, the UDP-glucose lipotransferase gene (wcaJ) in the salami acid synthesis pathway, and the sialidase gene (nanA), sialidase gene (nanT), N-acetylmnosamine-6-phosphate epimerase gene (nanE), and N-acetylmnosamine kinase gene (nanK) in the Neu5Ac metabolism pathway; and inserting the CMP-Neu5Ac synthesis pathway sialidase gene (neuB), UDP-N-acetylglucosamine-2-epimerase gene (neuC), and CMP-sialidase gene (neuA) to obtain a recombinant *E. coli* strain that efficiently produces 3'-sialic acid lactose.

[0007] Preferably, the Escherichia coli is Escherichia coli BL21(DE3).

[0008] Preferably, the gene modification further includes knocking out the global regulatory factor rpoS.

[0009] This invention enhances 3'-SL production and reduces acetic acid production by editing the global regulatory factor rpoS.

[0010] Preferably, the gene modification further includes knocking out at least one of the following: the response regulatory protein gene arcA, the transcription regulatory protein gene iclR, and the pyruvate oxidase poxB.

[0011] This invention blocks the production of acetic acid by further knocking out the genes arcA, iclR, and poxB in the acetic acid metabolism pathway.

[0012] More preferably, the genetic modification further includes knocking out the response regulatory protein gene arcA, the transcription regulatory protein gene iclR, and the pyruvate oxidase poxB.

[0013] Preferably, the genetic modification further includes knocking out at least one of the genome stability maintenance protein A gene mgsA and the lactate dehydrogenase gene ldhA.

[0014] This invention completely blocks lactic acid production by further knocking out the lactic acid metabolism pathway genes mgsA and ldhA.

[0015] More preferably, the genetic modification further includes knocking out the genome stability maintenance protein A gene mgsA and the lactate dehydrogenase gene ldhA.

[0016] Preferably, the genetic modification further includes knocking out the pyruvate kinase gene pykA and / or inserting the ppsA gene at the xylB site.

[0017] The application inhibits the PEP metabolic pathway by knocking out pykA, strengthens the PEP synthesis pathway by overexpressing ppsA, increases the precursor PEP supply for 3'-SL generation, thereby improving the 3'-SL yield.

[0018] More preferably, the genetic modification further comprises knocking out the pyruvate kinase gene pykA and inserting the ppsA gene at the xylB site.

[0019] Preferably, the genetic modification further comprises at least one of inserting the pyrG gene at the hlyE site, inserting the cmk gene at the ugd site, and inserting the ndk gene at the yjgX site.

[0020] The application strengthens the CTP synthesis pathway by overexpressing pyrG, improves the supply of CTP, and improves the 3'-SL yield.

[0021] More preferably, the genetic modification further comprises inserting the pyrG gene at the hlyE site, inserting the cmk gene at the ugd site, and inserting the ndk gene at the yjgX site.

[0022] Preferably, the genetic modification further comprises inserting at least one of the transporters ompG, mdtM-ompF at the yjiV site.

[0023] The application improves the 3'-SL yield by promoting the efflux of 3'-SL through transporters.

[0024] More preferably, the genetic modification further comprises inserting at least one of the transporters mdtM-ompF at the yjiV site.

[0025] Preferably, the genetic modification further comprises knocking out at least one of the N-acetylglucosamine-6-phosphate deacetylase gene nagA, the glucosamine-6-phosphate deaminase gene nagB, the 6-phosphomannose isomerase gene manA, the mannitol-1-phosphate dehydrogenase gene mtlD, the phosphofructokinase B gene pfkB, the UDP-N-acetylglucosamine transferase gene wecB, and the glucose-6-phosphate dehydrogenase gene zwf.

[0026] The application improves the supply of the precursor CMP-Neu5Ac by knocking out nagAB in the GlcNAc-6-P metabolic pathway, manA, mtlD, and pfkB in the fructose-6-phosphate metabolic pathway, wecB in the UDP-GlcNAc metabolic pathway, and zwf in the glucose-6-phosphate metabolic pathway, thereby improving the 3'-SL yield.

[0027] More preferably, the genetic modification further comprises knocking out the phosphofructokinase B gene pfkB.

[0028] In a second aspect, the present application provides the use of the recombinant E. coli for efficiently producing 3'-sialyllactose in the production of 3'-sialyllactose.

[0029] In a third aspect, the present application provides a method for producing 3'-sialyllactose, which utilizes the recombinant E. coli for efficiently producing 3'-sialyllactose to ferment 3'-sialyllactose.

[0030] Preferably, the recombinant E. coli can be used to routinely ferment 3'-sialyllactose.

[0031] Preferably, fed-batch fermentation culture can be used in large-scale production.

[0032] The present application has the following advantages: The present application introduces an exogenous gene into the genome of E. coli by CRISPR-Cas9 technology, which is stable and has no risk of plasmid loss, and does not require the use of antibiotics, thereby greatly reducing the production cost.

[0033] The present application knocks out the acetic acid and lactic acid metabolic pathways, and no acetic acid and lactic acid are generated in the by-products, which facilitates purification and reduces the consumption of ammonia water for pH adjustment during fermentation, thereby reducing the cost.

[0034] The recombinant E. coli constructed by the present application has a high yield and high productivity of 3'-SL, which is the highest level reported so far. DETAILED DESCRIPTION

[0035] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described in combination with specific embodiments.

[0036] 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.

[0037] In the construction process of the present application, pEcgRNA containing sgRNA sequence and N20 specific sequence is used for targeted gene editing; the knockout vector used in the gene knockout technology is pEcCas vector containing Cas9 and lambda-Red recombinase; pSPIN containing N32 specific sequence is used for targeted gene editing, and the transposition vector used in the gene transposition technology is pSPIN vector containing N32 specific sequence.

[0038] The specific sequences and primer sequences used in the present application are shown in Table 1.

[0039] Table 1 Example 1 Using the improved CRISPR-Cas9 technology, the following genetic modifications were made to the starting strain E. coli Bl21 (DE3): 1. Construction of E. coli BL21 (DE3) ΔaraA, ΔwcaJ, ΔlacZ, ΔnanA TEK, rhaA::neuBCA: (1) Knockout of araA, lacZ, wcaJ, nanA, nanT, nanE, nanK: (1.1) Amplify the upstream and downstream homologous arms using primers araA-up-F, araA-up-R and araA-down-F, araA-down-R, respectively, then use primers araA-up-F and araA-down-R to amplify the donor fragment with the upstream and downstream homologous arms as templates, use pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing to knockout araA, and use araA-DF and araA-DR for verification; (1.2) Amplify the upstream and downstream homologous arms using primers lacZ-up-F, lacZ-up-R and lacZ-down-F, lacZ-down-R, respectively, then use primers lacZ-up-F and lacZ-down-R to amplify the donor fragment with the upstream and downstream homologous arms as templates, use pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing to knockout lacZ, and use lacZ-DF and lacZ-DR for verification; (1.3) Amplify the upstream and downstream homologous arms using primers wcaJ-up-F, wcaJ-up-R and wcaJ-down-F, wcaJ-down-R, respectively, then use primers wcaJ-up-F and wcaJ-down-R to amplify the donor fragment with the upstream and downstream homologous arms as templates, use pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing to knockout wcaJ, and use wcaJ-DF and wcaJ-DR for verification; (1.4) The upstream and downstream homologous arms are amplified respectively by using nanATEK-up-F, nanATEK-up-R and nanATEK-down-F, nanATEK-down-R as primers, and the donor fragment is amplified by using primers nanATEK-up-F and nanATEK-down-R as primers and the upstream and downstream homologous arms as templates, and the nanATEK is knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, and nanATEK-DF and nanATEK-DR are used for verification; 2. Inserting neuB, neuC and neuA at the rhaA site: (2.1) pET-neuBCA is donated by Nanjing University of Technology; (2.2) Inserting neuBCA gene at the rhaA site: The rhaA-N32 specific sequence is cloned into the pSPIN plasmid to obtain the pSPIN-rhaA (N32) plasmid, and the complete neuBCA expression cassette is cloned into the pSPIN-rhaA (N32) by using primers T7-F and T7-R with the pET-neuBCA in step (2.1) as the template, and the transformants are verified by using primers pSPIN-DF and pSPIN-DR, and the positive transformants are screened to obtain the pSPIN-rhaA-neuBCA plasmid; the plasmid is transformed into the target strain, and the transformants are verified by using primers rhaA-DF and rhaA-DR, and the positive transformants are screened to remove the plasmid to obtain the strain 1.

[0040] 3. The strain 1 is used as the chassis strain, and pelB, InfB, MBP, NT, GST, NusA and Halo Tag are fused to the N-terminus of Vs16 respectively, and are transposed to the lacA site to construct strains 2-8 respectively: (3.1) The alpha-1, 3-fucosyltransferase Vs16, pelB-Vs16, InfB-Vs16, MBP-Vs16, NT-Vs16, GST-Vs16, NusA-Vs16 and Halo Tag-Vs16 are synthesized by Huada Gene and subcloned into the vector pETDuet-1 to construct the plasmid pET-(Fusion Tag)-Vs16 containing the complete alpha-1, 3-fucosyltransferase coding sequence expression cassette.

[0041] (3.2) Inserting (Fusion Tag)-Vs16 gene at the lacA site: The lacA-N32 specific sequence was cloned into the pSPIN plasmid to obtain the pSPIN-lacA (N32) plasmid, the complete (Fusion Tag)-Vs16 expression cassette obtained in step (3.1) was cloned into the pSPIN-lacA (N32) using primers T7-F and T7-R and the template pET-(Fusion Tag)-Vs16, the transformants were verified using primers pSPIN-DF and pSPIN-DR, and the positive transformants were screened to obtain the pSPIN-lacA-(Fusion Tag)-Vs16 plasmid; the plasmid was transformed into the target strain, the transformants were verified using primers lacA-DF and lacA-DR, the positive transformants were screened to remove the plasmid, and strains 2-8 were constructed.

[0042] 4. Fermentative production of 3'-SL The genetically engineered bacteria (strain 1-8) for producing 3'-SL obtained above were subjected to fed-batch fermentation culture in a 5 L tank (conventional fermentation was sufficient), and the fermentation conditions were as follows: (1) liquid volume: 2 L; inoculation amount: 160 mL; fermentation time: 72 h; (2) control index: pH = 6.8; fermentation temperature 37℃, the temperature was adjusted to 29.5℃ after feeding; IPTG and lactose were induced after 4 h of feeding; (3) aeration rate: 2.5 L / min; (4) rotation speed: initial rotation speed 200 rpm; (5) dissolved oxygen control: before feeding, DO was associated with rotation speed, DO was set to 30-40%, and the rotation speed range was set to 200-1000 rpm.

[0043] (6) carbon source uniform feeding: speed was 18 g glucose feeding solution / h; (7) lactose: was added in batches, 70 g of lactose was added each time.

[0044] The formula of the culture medium was as follows: Fermentation medium (g / L): Base medium 1: 10 g of glycerol, 4 g of ammonium sulfate, 9.2 g of potassium phosphate dibasic trihydrate, 8.2 g of potassium phosphate monobasic, 0.3 g of citric acid, 6 g of proteose peptone, 2 g of yeast powder, and 15 mL / 100 L of antifoam 204 (Araldite) were weighed, dissolved in tap water, mixed uniformly, the pH was adjusted to 6.8, and sterilized at 121℃.

[0045] Base medium 2: 6 g of magnesium sulfate heptahydrate, 0.06 g of anhydrous calcium chloride, and 0.03015 g of vitamin B1 were dissolved in 50 mL of tap water, and filtered through a 0.22 μm membrane.

[0046] Seed medium 3: Take 30 mL trace element solution (Ferric ammonium citrate 5.6 g; Zinc sulfate heptahydrate 0.9 g; CoCl2·6H2O 0.2 g; Manganese chloride tetrahydrate 1.0 g; CuCl2·2H2O 0.10 g; Boric acid 0.2 g; Na2MoO4·2H2O 0.2 g; Dissolved in 1000 mL tap water), 0.22 μm membrane filtration.

[0047] Seed medium 4: Take 0.15 g IPTG dissolved in 50 mL tap water, 0.22 μm membrane filtration.

[0048] Feed medium 1: Take 480 g glucose, 720 g glycerol, 6.75 g magnesium sulfate heptahydrate and 5 mL trace element solution, make up to 1.5 L, sterilize at 121 ℃.

[0049] Feed medium 2: 140 g lactose plus tap water to 400 g, sterilize at 115 ℃. pH adjustor: 28% ammonia water, 0.22 μm membrane filtration.

[0050] The above seed medium 1, 2, 3 are added together before inoculation, and seed medium 4 is added at the time of induction; Feed medium 1, 2 are added according to the actual detection.

[0051] The strain is fermented in a 5 L tank, Vs16 (strain 1) without a fusion tag is used as a control strain, and a liquid chromatograph (Thermo high-performance liquid chromatograph) is used to detect the yield of acetic acid, lactic acid and 3'-SL after 72 h of fermentation.

[0052] Detection conditions: Detector: DAD detector, 210 nm.

[0053] 3'-SL detection: Column: Tskgel Amide-80 (4.6 x 250 mm, 5 μm); mobile phase: 70% acetonitrile, 30% 10 mM ammonium formate aqueous solution, pH = 4.5; flow rate: 1 mL / min; column temperature: 40 ℃; elution mode: isocratic elution.

[0054] Acetic acid and lactic acid detection: Column: Aminex HPX-87H (7.8 x 300 mm, 5 μm); mobile phase: 12 mM sulfuric acid aqueous solution; flow rate: 0.5 mL / min; column temperature: 65 ℃; elution mode: isocratic elution.

[0055] The results are shown in Table 2.

[0056] Table 2: Comparison of yields of different fusion tags The fusion tags pelB, InfB, MBP, NT, GST, NusA, Halo Tag and Vs16 fusion were used, wherein the pelB fusion protein had the most significant effect on the production of 3'-SL of the strain, and the 3'-SL yield was 43.43 g / L, which was increased by 24.6% compared with that of the strain 1. Therefore, the strain 2 was selected for the next modification.

[0057] Example 2: The strain 2 was used as a starting strain, and rpoS was knocked out to obtain the strain 9.

[0058] The construction process is as follows: The strain 2 was used as a starting strain, and the improved CRISPR-Cas9 technology was used for rpoS knockout: the upstream and downstream homologous arms were amplified by using rpoS-up-F, rpoS-up-R and rpoS-down-F, rpoS-down-R as primers, respectively, and then the donor fragment was amplified by PCR using the upstream and downstream homologous arms as templates and using rpoS-up-F and rpoS-down-R as primers, the rpoS was knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, rpoS-DF and rpoS-DR were used for verification, and the strain 9 was constructed.

[0059] The fermentation conditions of Example 1 were used for fermentation for detection, and the strain 2 was used as a control. The fermentation results are shown in Table 3.

[0060] Table 3 The 3'-SL yield of the strain 9 was 55.75 g / L, which was increased by 28.3% compared with that of the strain 2; and the acetic acid yield was 8.56 g / L, which was decreased by 51.9% compared with that of the strain 2. This shows that, by editing the global regulatory factor rpoS, the 3'-SL yield is increased and the generation of acetic acid is reduced.

[0061] Example 3: The strain 9 was used as a starting strain, and arcA was knocked out to obtain the strain 10; on the basis of the strain 10, iclR was knocked out to obtain the strain 11; and on the basis of the strain 11, poxB was knocked out to obtain the strain 12.

[0062] 1. The strain 9 was used as a starting strain, and the improved CRISPR-Cas9 technology was used for arcA knockout: The upstream and downstream homology arms are respectively amplified by using primers arcA-up-F and arcA-up-R and primers arcA-down-F and arcA-down-R, and then the donor fragment is amplified by using primers arcA-up-F and arcA-down-R and taking the upstream and downstream homology arms as templates, arcA is knocked out by using pEcgRNA containing an sgRNA sequence and an N20 specific sequence for targeted gene editing, and arcA-DF and arcA-DR are used for verification, so as to obtain strain 10.

[0063] 2. On the basis of strain 10, iclR is knocked out by using improved CRISPR-Cas9 technology: The upstream and downstream homology arms are respectively amplified by using primers icIR-up-F and icIR-up-R and primers icIR-down-F and icIR-down-R, and then the donor fragment is amplified by using primers icIR-up-F and icIR-down-R and taking the upstream and downstream homology arms as templates, icIR is knocked out by using pEcgRNA containing an sgRNA sequence and an N20 specific sequence for targeted gene editing, and icIR-DF and icIR-DR are used for verification, so as to obtain strain 11.

[0064] 3. On the basis of strain 11, poxB is knocked out by using improved CRISPR-Cas9 technology: The upstream and downstream homology arms are respectively amplified by using primers poxB-up-F and poxB-up-R and primers poxB-down-F and poxB-down-R, and then the donor fragment is amplified by using primers poxB-up-F and poxB-down-R and taking the upstream and downstream homology arms as templates, poxB is knocked out by using pEcgRNA containing an sgRNA sequence and an N20 specific sequence for targeted gene editing, and poxB-DF and poxB-DR are used for verification, so as to obtain strain 12.

[0065] Fermentation is carried out under the fermentation conditions of Example 1, and strain 9 is used as a control. The fermentation results are shown in Table 4.

[0066] Table 4 Among them, the yield of 3'-SL of strain 12 is 57.30 g / L, which has no obvious difference with strain 9; the yield of acetic acid is 0 g / L, which is reduced by 100% compared with strain 9. By further knocking out the acetic acid metabolic genes arcA, iclR and poxB, the generation of acetic acid is blocked.

[0067] Example 4: Strain 12 was used as the starting strain, mgsA was knocked out to construct strain 13; ldhA was knocked out on the basis of strain 13 to construct strain 14.

[0068] 1. Strain 12 was used as the starting strain, and mgsA was knocked out by using the improved CRISPR-Cas9 technology: The upstream and downstream homologous arms were amplified by using primers mgsA-up-F and mgsA-up-R, respectively, and then the donor fragment was amplified by using primers mgsA-up-F and mgsA-down-R as primers and the upstream and downstream homologous arms as templates, mgsA was knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, and strain 13 was constructed by using mgsA-DF and mgsA-DR for verification.

[0069] 2. On the basis of strain 13, ldhA was knocked out by using the improved CRISPR-Cas9 technology: The upstream and downstream homologous arms were amplified by using primers ldhA-up-F and ldhA-up-R, respectively, and then the donor fragment was amplified by using primers ldhA-up-F and ldhA-down-R as primers and the upstream and downstream homologous arms as templates, ldhA was knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, and strain 14 was constructed by using ldhA-DF and ldhA-DR for verification.

[0070] Fermentation was carried out under the fermentation conditions of Example 1, and strain 12 was used as a control. The fermentation results are shown in Table 5.

[0071] Table 5 Among them, the yield of 3'-SL of strain 14 is 57.31 g / L, which has no obvious difference with strain 12; the yield of lactic acid is 0 g / L, which is reduced by 100% compared with strain 12. By further knocking out the lactic acid metabolism genes mgsA and ldhA, the generation of lactic acid is completely blocked.

[0072] Example 5: Strain 14 was used as the starting strain, pykA was knocked out to construct strain 15; ppsA was overexpressed on the basis of strain 15 to construct strain 16.

[0073] 1. Strain 14 was used as the starting strain, and pykA was knocked out: The upstream and downstream homology arms were amplified by using primers pykA-up-F and pykA-up-R and pykA-down-F and pykA-down-R, respectively, and the donor fragment was amplified by using primers pykA-up-F and pykA-down-R and the upstream and downstream homology arms as templates. The pykA gene was knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, and verified by using pykA-DF and pykA-DR, to construct strain 15.

[0074] 2. Overexpressing ppsA based on strain 15: (2.1) Constructing pET-ppsA plasmid (containing a complete ppsA expression cassette), the process is as follows: The vector pETDuet-1 was linearized by using NcoI and KpnI restriction endonucleases, and the primers ppsA-F and ppsA-R were designed to amplify the target fragment ppsA by PCR using the E. coli B121 (DE3) genome as the template; The target fragment and the linearized vector were connected by homologous recombination using the LightNing® DNA Assembly Mix Plus kit, transformed into E. coli DH5α, and the pET-ppsA plasmid containing the complete ppsA expression cassette was constructed.

[0075] (2.2) Inserting the ppsA gene at the xylB site: The xylB-N32 specific sequence was cloned into the pSPIN plasmid to obtain the pSPIN-xylB (N32) plasmid, and the complete ppsA expression cassette obtained in step (1) was cloned into pSPIN-xylB (N32) using primers T7-F and T7-R and the pET-ppsA as the template. The transformants were verified by using primers pSPIN-DF and pSPIN-DR, and the positive transformants were selected to obtain the pSPIN-xylB-ppsA plasmid. The plasmid was transformed into the target strain, and the transformants were verified by using primers xylB-DF and xylB-DR, and the positive transformants were selected to remove the plasmid, and strain 16 was constructed.

[0076] The fermentation conditions of Example 1 were used for fermentation detection, and strain 14 was used as a control. The fermentation results are shown in Table 6.

[0077] Table 6 The 3'-SL yield of the strain 16 is 70.37 g / L, which is increased by 25.7% than that of the strain 14. The PEP metabolic pathway is inhibited by knocking out pykA, the PEP synthesis pathway is strengthened by overexpressing ppsA, the precursor PEP supply for 3'-SL generation is increased, and thus the 3'-SL yield is increased.

[0078] Example 6 The strain 16 is used as a starting strain, pyrG is overexpressed to construct the strain 17, cmk is overexpressed on the basis of the strain 17 to construct the strain 18, and ndk is overexpressed on the basis of the strain 18 to construct the strain 19.

[0079] 1. The strain 16 is used as a starting strain, and pyrG is overexpressed. (1.1) The primers pyrG-F and pyrG-R are designed to obtain the target fragment pyrG, and the pET-pyrG plasmid containing the complete pyrG expression cassette is constructed according to the construction process of step (2.1) in Example 5; (1.2) The pyrG gene is inserted into the hlyE site. The hlyE-N32 specific sequence is cloned into the pSPIN plasmid to obtain the pSPIN-hlyE (N32) plasmid, the complete pyrG expression cassette obtained in step (1) is cloned into the pSPIN-hlyE (N32) using the primers T7-F and T7-R and the pET-pyrG as a template, the transformants are verified using the primers pSPIN-DF and pSPIN-DR, the positive transformants are screened to obtain the pSPIN-hlyE-pyrG plasmid, the plasmid is transformed into the target strain, the transformants are verified using the primers hlyE-DF and hlyE-DR, the positive transformants are screened to remove the plasmid, and the strain 17 is constructed; 2. cmk is overexpressed on the basis of the strain 17. (2.1) The primers cmk-F and cmk-R are designed to obtain the target fragment cmk, and the pET-cmk plasmid containing the complete cmk expression cassette is constructed according to the construction process of step (2.1) in Example 5; (2.2) The cmk gene is inserted into the ugd site. The ugd-N32 specific sequence is cloned into the pSPIN plasmid to obtain the pSPIN-ugd (N32) plasmid, the complete cmk expression cassette obtained in step (1) is cloned into the pSPIN-ugd (N32) using primers T7-F and T7-R and the template of pET-cmk, the transformants are verified using primers pSPIN-DF and pSPIN-DR, the positive transformants are screened to obtain the pSPIN-ugd-cmk plasmid; the plasmid is transformed into the target strain, the transformants are verified using primers ugd-DF and ugd-DR, the positive transformants are screened to remove the plasmid, and the strain 18 is constructed.

[0080] 3. Overexpressing ndk based on the strain 18: (3.1) The primers ndk-F and ndk-R are designed to obtain the target fragment ndk, and the pET-ndk plasmid containing the complete ndk expression cassette is constructed according to the construction process of step (2.1) in Example 5; (3.2) Inserting the ndk gene at the yjgX site: The yjgX-N32 specific sequence is cloned into the pSPIN plasmid to obtain the pSPIN-yjgX (N32) plasmid, the complete ndk expression cassette obtained in step (1) is cloned into the pSPIN-yjgX (N32) using primers T7-F and T7-R and the template of pET-ndk, the transformants are verified using primers pSPIN-DF and pSPIN-DR, the positive transformants are screened to obtain the pSPIN-yjgX-ndk plasmid; the plasmid is transformed into the target strain, the transformants are verified using primers yjgX-DF and yjgX-DR, the positive transformants are screened to remove the plasmid, and the strain 19 is constructed.

[0081] The fermentation is carried out according to the fermentation conditions of Example 1, and strain 16 is used as a control. The fermentation results are shown in Table 7.

[0082] Table 7 Among them, the 3'-SL yield of strain 17 is 76.68 g / L, which is increased by 9.0% compared with strain 16. The supply of CTP is improved by overexpressing pyrG and strengthening the CTP synthesis pathway, and the 3'-SL yield is improved.

[0083] Example 7: Strains 20-27 are constructed by transposition of nec, ompG, scrY, fred, yberC, mdfA-cymA, mdtM-ompF and mdtL-ompG, respectively, based on strain 17 as a starting strain.

[0084] 1. pET-transporter plasmid (containing complete nec, ompG, scrY, fred, yberC, mdfA-cymA, mdtM-ompF, mdtL-ompG expression cassettes respectively) was synthesized by Huada Gene and subcloned into vector pETDuet-1 (purchased from Huada Gene) to construct pET-transporter plasmid containing complete transporter protein expression cassettes.

[0085] 2. Each transporter protein (nec, ompG, scrY, fred, yberC, mdfA-cymA, mdtM-ompF, mdtL-ompG) gene was inserted into yjiV site respectively: The hlyE-N32 specific sequence was cloned into the pSPIN plasmid to obtain the pSPIN-yjiV (N32) plasmid, and the complete transporter protein expression cassettes obtained in step (1) were cloned into the pSPIN-yjiV (N32) using primers T7-F and T7-R with pET-transporter as a template, and the transformants were verified using primers pSPIN-DF and pSPIN-DR, and positive transformants were screened to obtain the pSPIN-yjiV-transporter plasmid; the plasmid was transformed into the target strain, and the transformants were verified using primers yjiV-DF and yjiV-DR, and positive transformants were screened to remove the plasmid, and strains 20-27 were constructed respectively.

[0086] The fermentation conditions of Example 1 were used for fermentation for detection, and strain 17 was used as a control. The fermentation results are shown in Table 8.

[0087] Table 8: Comparison of the effects of inserting each transporter protein Among them, the 3'-SL yield of strain 26 was 81.98 g / L, which was increased by 6.9% compared with strain 17. The present application promotes the efflux of 3'-SL through the transporter protein, thereby improving the yield of 3'-SL.

[0088] Example 8: Strains 28-33 were constructed by knocking out nagAB, manA, mtlD, pfkB, wecB and zwf respectively to improve the supply of precursor substance CMP-Neu5Ac, with strain 26 as the starting strain.

[0089] 1. Strain 26 was used as the starting strain, and nagAB was knocked out: The upstream and downstream homologous arms were amplified with nagAB-up-F, nagAB-up-R and nagAB-down-F, nagAB-down-R as primers, respectively, and the donor fragment was amplified with primers nagAB-up-F and nagAB-down-R as primers and the upstream and downstream homologous arms as templates, nagAB was knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, nagAB-DF and nagAB-DR were used for verification, and strain 28 was constructed.

[0090] 2. Strain 26 was used as the starting strain, and manA was knocked out. The upstream and downstream homologous arms were amplified with manA-up-F, manA-up-R and manA-down-F, manA-down-R as primers, respectively, and the donor fragment was amplified with primers manA-up-F and manA-down-R as primers and the upstream and downstream homologous arms as templates, manA was knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, manA-DF and manA-DR were used for verification, and strain 29 was constructed.

[0091] 3. Strain 26 was used as the starting strain, and mtlD was knocked out. The upstream and downstream homologous arms were amplified with mtlD-up-F, mtlD-up-R and mtlD-down-F, mtlD-down-R as primers, respectively, and the donor fragment was amplified with primers mtlD-up-F and mtlD-down-R as primers and the upstream and downstream homologous arms as templates, mtlD was knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, mtlD-DF and mtlD-DR were used for verification, and strain 30 was constructed.

[0092] 4. Strain 26 was used as the starting strain, and pfkB was knocked out. The upstream and downstream homologous arms were amplified with pfkB-up-F, pfkB-up-R and pfkB-down-F, pfkB-down-R as primers, respectively, and the donor fragment was amplified with primers pfkB-up-F and pfkB-down-R as primers and the upstream and downstream homologous arms as templates, pfkB was knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, pfkB-DF and pfkB-DR were used for verification, and strain 31 was constructed.

[0093] 5. Strain 26 was used as the starting strain, and wecB was knocked out. The upstream and downstream homology arms are respectively amplified by primers wecB-up-F and wecB-up-R and wecB-down-F and wecB-down-R, and then the donor fragment is amplified by fusion PCR with primers wecB-up-F and wecB-down-R and the upstream and downstream homology arms as templates, and wecB is knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, and verification is performed by using wecB-DF and wecB-DR, and strain 32 is constructed.

[0094] 6. Strain 26 is used as a starting strain, and zwf is knocked out. The upstream and downstream homology arms are respectively amplified by primers zwf-up-F and zwf-up-R and zwf-down-F and zwf-down-R, and then the donor fragment is amplified by fusion PCR with primers zwf-up-F and zwf-down-R and the upstream and downstream homology arms as templates, and zwf is knocked out by using pEcgRNA containing sgRNA sequence and N20 specific sequence for targeted gene editing, and verification is performed by using zwf-DF and zwf-DR, and strain 33 is constructed.

[0095] Fermentation is performed under the fermentation conditions of Example 1, and strain 26 is used as a control. The fermentation results are shown in Table 9.

[0096] Table 9 Among them, the 3'-SL yield of strain 31 at 72 h is 86.68 g / L, which is increased by 5.7% compared with strain 26. The GlcNAc-6-P metabolic pathway is knocked out by nagAB in the application; the fructose-6-phosphate metabolic pathway is knocked out by manA, mtlD and pfkB; the UDP-GlcNAc metabolic pathway is knocked out by wecB; and the glucose-6-phosphate metabolic pathway is knocked out by zwf. The supply of precursor substance CMP-Neu5Ac is improved, and the 3'-SL yield is improved.

[0097] In summary, the genetically engineered bacteria provided by the application have very significant effects and high application value.

[0098] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the application and not to limit the protection scope of the application. Although the 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 application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the application.

Claims

1. A recombinant Escherichia coli strain that efficiently produces 3'-sialic acid lactose, characterized in that, Using Escherichia coli as the starting strain, the following genetic modifications were performed: (1) Knock out L-arabinose isomerase gene araA, β-galactosidase gene lacZ, UDP-glucose lipotransferase gene wcaJ, sialidase gene nanA, sialic acid permease gene nanT, N-acetylmannosamine-6-phosphate epimerase gene nanE and N-acetylmannosamine kinase gene nanK. (2) Insert the sialic acid synthase gene neuB, the UDP-N-acetylglucosamine-2-epimerase gene neuC and the CMP-sialic acid synthase gene neuA into the rhaA site; (3) pelB was fused to the N-terminus of the α2,3-sialyltransferase gene Vs16 and transposed to the lacA site to obtain the recombinant Escherichia coli that efficiently produces 3'-sialyl lactose.

2. The recombinant Escherichia coli for efficient production of 3'-sialic acid lactose as described in claim 1, characterized in that, The genetic modification also includes knocking out the global regulatory factor rpoS.

3. The recombinant Escherichia coli for efficient production of 3'-sialic acid lactose as described in claim 2, characterized in that, The genetic modification also includes knocking out at least one of the following: the response regulatory protein gene arcA, the transcription regulatory protein gene iclR, and the pyruvate oxidase poxB.

4. The recombinant Escherichia coli for efficient production of 3'-sialic acid lactose as described in claim 3, characterized in that, The genetic modification also includes knocking out at least one of the genome stability maintenance protein A gene mgsA and the lactate dehydrogenase gene ldhA.

5. The recombinant Escherichia coli for efficient production of 3'-sialic acid lactose as described in claim 4, characterized in that, The genetic modification also includes knocking out the pyruvate kinase gene pykA and / or inserting the ppsA gene at the xylB site.

6. The recombinant Escherichia coli for efficient production of 3'-sialic acid lactose as described in claim 5, characterized in that, The genetic modification also includes at least one of inserting the pyrG gene at the hlyE site, inserting the cmk gene at the ugd site, and inserting the ndk gene at the yjgX site.

7. The recombinant Escherichia coli for efficient production of 3'-sialic acid lactose as described in claim 6, characterized in that, The genetic modification also includes inserting at least one of the transport proteins ompG and mdtM-ompF at the yjiV site.

8. The recombinant Escherichia coli for efficient production of 3'-sialic acid lactose as described in claim 7, characterized in that, The genetic modification also includes knocking out at least one of the following genes: N-acetylglucosamine-6-phosphate deacetylase gene nagA, glucosamine-6-phosphate deaminase gene nagB, 6-phosphate mannose isomerase gene manA, mannitol-1-phosphate dehydrogenase gene mtlD, phosphofructokinase B gene pfkB, UDP-N-acetylglucosamine transferase gene wecB, and glucose-6-phosphate dehydrogenase gene zwf.

9. The use of the recombinant Escherichia coli as described in any one of claims 1-8 for the efficient production of 3'-sialic acid lactose in the production of 3'-sialic acid lactose.

10. A method for producing 3'-sialic acid lactose, characterized in that, 3'-Sialyllactose is produced by fermentation using the recombinant Escherichia coli according to any one of claims 1-8 for efficient production of 3'-sialic acid lactose.

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