Genetically engineered bacterium for producing lacto-n-tetraose, preparation method therefor, and use thereof

Abstract

Provided are a genetically engineered bacterium for producing lacto-N-tetraose, a preparation method therefor, and use thereof. The genetically engineered bacterium overexpresses an exogenous transporter gene BmSet. The genetically engineered bacterium can enable lacto-N-tetraose to be quickly and efficiently excreted out of cells, promote the increase in the fermentation yield of lacto-N-tetraose, and increase the concentration of lacto-N-tetraose in extracellular media, thereby having good industrial application prospects.

Description

Genetically engineered bacteria for producing lactose-n-tetrasaccharide, and preparation method and application thereof

[0001] Related applications

[0002] The present disclosure claims priority to the Chinese patent application No. 202410831869.4, filed on June 26, 2024, and entitled "Genetically engineered bacteria for producing lactose-n-tetrasaccharide, and preparation method and application thereof", the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of genetic engineering, in particular to genetically engineered bacteria for producing lactose-n-tetrasaccharide, and preparation method and application thereof. BACKGROUND

[0004] Human milk oligosaccharides (HMOs) are a group of structurally diverse non-conjugated polysaccharides, which are the second most abundant component in human milk after lactose and lipids, and belong to the third largest nutrient. Lactose-n-tetrasaccharide (LNT) is the core structure of HMOs, accounting for 6% (w / w) of total HMOs. In addition, LNT is the core structure of fucosylation and sialylation, and its fucosylated and / or sialylated derivatives account for more than 30% (w / w) of total HMOs. Therefore, the synthesis of LNT is the key to the synthesis of various HMOs.

[0005] Studies have shown that LNT has the effects of increasing the abundance of intestinal probiotics, preventing pathogen adhesion, enhancing intestinal barrier, immune regulation, and anti-virus. With the continuous verification of the biological value of LNT as a bioactive component of breast milk, it has been approved by different countries and regions as a new food raw material to be added to infant formula and other foods, and has high commercial value.

[0006] At present, the main method to realize large-scale production of LNT is microbial synthesis fermentation of metabolic engineering bacteria. The synthesis process mainly includes using β-1,3-N-acetylglucosamine transferase to catalyze UDP-GlcNAc and lactose to synthesize lactose-n-triose, and then lactose-n-triose reacts with UDP-gal under the action of β-1,3-galactosyltransferase to generate LNT. However, in the actual fermentation process, LNT with a tetrasaccharide structure is difficult to be transferred from the intracellular to the extracellular medium. With the extension of fermentation time, intracellular accumulation of LNT will cause cell swelling and rupture, and premature termination of fermentation, which limits the increase of yield and also causes problems such as difficulty in separation and purification of downstream products. At present, there are few reports on the extracellular discharge of LNT, so it is necessary to seek appropriate ways and methods to promote the extracellular discharge of LNT. SUMMARY

[0007] Problems to be solved by the application

[0008] In view of the above problems existing in the prior art, the present application aims to provide a genetically engineered strain capable of efficiently transporting lacto-N-tetraose.

[0009] Solution for solving the problem

[0010] The present application provides a genetically engineered strain for producing lacto-N-tetraose, which overexpresses an exogenous transport protein gene BmSet.

[0011] Preferably, the BmSet is derived from Bacillus megaterium.

[0012] Preferably, the amino acid sequence of the BmSet has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared with SEQ ID NO. 4.

[0013] Preferably, the amino acid sequence of the BmSet is as shown in SEQ ID NO. 4.

[0014] Preferably, the genetically engineered strain further expresses an exogenous beta-1, 3-N-acetylglucosamine transferase gene lgtA, an exogenous beta-1, 3-galactosyltransferase gene wcf and an exogenous uridine diphosphate glucose-4-epimerase gene galE.

[0015] Preferably, the lgtA is derived from Neisseria meningitidis, the wcf is derived from Citrobacter freundii, and the galE is derived from Escherichia coli.

[0016] Preferably, the amino acid sequence of the lgtA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared with SEQ ID NO. 1, and preferably, the amino acid sequence of the lgtA is as shown in SEQ ID NO. 1.

[0017] Preferably, the amino acid sequence of the wcf has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 2, preferably the amino acid sequence of the wcf is as shown in SEQ ID NO. 2.

[0018] Preferably, the amino acid sequence of the galE has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 3, preferably the amino acid sequence of the galE is as shown in SEQ ID NO. 3.

[0019] The present application also provides a method for preparing the genetically engineered bacteria, comprising the following steps:

[0020] (1) constructing a plasmid expressing the lgtA, wcf and galE;

[0021] (2) introducing the plasmid expressing the lgtA, wcf and galE into an Escherichia coli starting strain to construct a chassis strain;

[0022] (3) constructing a plasmid expressing the BmSet;

[0023] (4) introducing the plasmid expressing the BmSet into the chassis strain to construct the genetically engineered bacteria.

[0024] The present application also provides a biological agent comprising the genetically engineered bacteria.

[0025] The present application also provides an application of the genetically engineered bacteria or the biological agent in producing lacto-N-tetraose.

[0026] The present application also provides a method for producing lacto-N-tetraose, which uses the genetically engineered bacteria or the biological agent to ferment lacto-N-tetraose with glucose as a carbon source and lactose as a substrate.

[0027] Effects of the present application

[0028] The genetically engineered bacteria of the present application can rapidly and efficiently discharge lacto-N-tetraose outside the cell, promote the increase of lacto-N-tetraose fermentation yield, and increase the lacto-N-tetraose concentration in the extracellular medium, thus having a good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a plasmid map of the expression plasmid pAC-wcf-galE-lgtA.

[0030] Figure 2 is a plasmid map of the expression plasmid pCO-BmSet.

[0031] Figure 3 is the yield of lacto-N-tetraose of BmSet overexpression strain. DETAILED DESCRIPTION

[0032] In order to make the technical solutions and beneficial effects of the present application more obvious and easy to understand, the following will be described in detail by way of listing specific embodiments. The drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meaning as the technical and scientific terms in the technical field to which the present application belongs.

[0033] In certain embodiments, the LB liquid medium used in the present application comprises peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0034] In certain embodiments, the LB solid medium used in the present application comprises peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 20 g / L.

[0035] In certain embodiments, the glucose medium used in the present application comprises glucose 15.07 g / L, citric acid 1.7 g / L, KH2PO4 13.5 g / L, (NH4)2PO4 4 g / L, MgSO4·7H2O 1.4 g / L, trace metal solution 10 mL / L (ferric citrate 10 g / L, ZnSO4·7H2O 2.25 g / L, CuSO4·5H2O 1.0 g / L, MnSO4·H2O 0.35 g / L, Na2B4O7·2H2O 0.23 g / L, (NH4)6Mo7O 24 0.11 g / L, CaCl2·2H2O 2.0 g / L).

[0036] The present application provides a genetically engineered bacterium for producing lacto-N-tetraose, which overexpresses an exogenous transporter gene BmSet.

[0037] In certain embodiments, the BmSet is derived from Bacillus megaterium.

[0038] In some embodiments, the amino acid sequence of the BmSet has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, compared to SEQ ID NO. 4.

[0039] In some embodiments, the amino acid sequence of the BmSet is as shown in SEQ ID NO. 4.

[0040] SEQ ID No. 4:

[0041] In some embodiments, the genetically engineered bacterium further expresses an exogenous β-1, 3-N-acetylglucosaminyltransferase gene lgtA.

[0042] In some embodiments, the genetically engineered bacterium further expresses an exogenous β-1, 3-galactosyltransferase gene wcf.

[0043] In some embodiments, the genetically engineered bacterium further expresses an exogenous uridine diphosphate glucose-4-epimerase gene galE.

[0044] In some embodiments, the lgtA is derived from Neisseria meningitidis.

[0045] In some embodiments, the wcf is derived from Citrobacter freundii.

[0046] In some embodiments, the galE is derived from Escherichia coli.

[0047] In some embodiments, the amino acid sequence of the lgtA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, compared to SEQ ID NO. 1.

[0048] In some embodiments, the amino acid sequence of the lgtA is as shown in SEQ ID NO. 1.

[0049] SEQ ID No. 1:

[0050] In some embodiments, the amino acid sequence of the wcf has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO. 2.

[0051] In some embodiments, the amino acid sequence of the wcf is as set forth in SEQ ID NO. 2.

[0052] SEQ ID No. 2:

[0053] In some embodiments, the amino acid sequence of the galE has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO. 3.

[0054] In some embodiments, the amino acid sequence of the galE is as set forth in SEQ ID NO. 3.

[0055] SEQ ID No. 3:

[0056] The present application also provides a method for preparing the genetically engineered bacterium, comprising the following steps:

[0057] (1) constructing a plasmid expressing the lgtA, wcf and galE;

[0058] (2) introducing the plasmid expressing the lgtA, wcf and galE into an E. coli starting strain to construct a chassis strain;

[0059] (3) constructing a plasmid expressing the BmSet;

[0060] (4) introducing the plasmid expressing the BmSet into the chassis strain to construct the genetically engineered bacterium.

[0061] In some embodiments, the plasmid expressing the lgtA, wcf and galE in step (2) is a plasmid in which the lgtA, wcf and galE are linked in series.

[0062] In some embodiments, the starting strain in step (2) is E. coli.

[0063] In some embodiments, the starting strain in step (2) is E. coli BL21(DE3).

[0064] The present application also provides a biological agent comprising the genetically engineered bacteria.

[0065] The present application also provides an application of the genetically engineered bacteria or the biological agent in producing lacto-N-tetraose.

[0066] The present application also provides a method for producing lacto-N-tetraose, which uses the genetically engineered bacteria or the biological agent to ferment lacto-N-tetraose with glucose as a carbon source and lactose as a substrate.

[0067] Example 1: Construction of chassis strain

[0068] 1. According to the literature reports and the NCBI database query, the genes encoding β-1, 3-N-acetylglucosamine transferase from Neisseria meningitidis (Genbank ID: AAC44084.1) and β-1, 3-galactosyltransferase from Citrobacter freundii (Genbank ID: WP_187258760.1) were obtained, and were named as genes lgtA and wcf, respectively, according to the codon preference of E. coli. The lgtA and wcf genes were synthesized by Suzhou Jinyuzhi Company (the amino acid sequence of lgtA is shown in SEQ ID No. 1, and the amino acid sequence of wcf is shown in SEQ ID No. 2), and the E. coli MG1655 genome was extracted to obtain the gene galE encoding uridine diphosphate glucose-4-epimerase (the amino acid sequence is shown in SEQ ID No. 3).

[0069] SEQ ID No. 1:

[0070] SEQ ID No. 2:

[0071] SEQ ID No. 3:

[0072] 2. Design primers pAC-HP-F / R, lgtA-F / R, wcf-F / R and galE-F / R (primer sequences are shown in Table 1), respectively, to perform PCR reaction with expression vector pACYCDuet-1, synthetic gene lgtA, synthetic gene wcf, and E. coli MG1655 genome as templates. Obtain pACYCDuet-1 linear vector, lgtA gene fragment, wcf gene fragment, and galE gene fragment, determine the size of the amplified fragment by gel electrophoresis, then recover the linear fragment for seamless cloning, assemble the fragment and the vector, and transform the seamless cloning reaction liquid into E. coli DH5α competent cells by heat shock, spread on a chloramphenicol-resistant LB solid plate, and incubate at 37°C overnight to screen positive clones for sequencing. After correct sequencing, expand the culture and extract the plasmid to obtain the expression plasmid pAC-wcf-galE-lgtA. The plasmid map is shown in Figure 1.

[0073] Table 1: Primer sequences used for constructing pAC-wcf-galE-lgtA plasmid

[0074] 3. Use E. coli BL21(DE3) as the starting strain to prepare E. coli BL21(DE3) electrotransformation competent cells, and transform the plasmid pAC-wcf-galE-lgtA into E. coli BL21(DE3). Spread on a chloramphenicol-resistant LB solid plate, and incubate at 37°C overnight to screen positive clones. The correct positive transformant is the high-efficiency lacto-N-tetraose synthetic chassis strain LNT-01 ((E. coli BL21(DE3)-pAC-wcf-galE-lgtA)).

[0075] Example 2: Construction of a strain for high-efficiency transport of lacto-N-tetraose

[0076] 1. Obtain the gene encoding a sugar efflux transporter protein from Bacillus megaterium (Genbank ID: WP_013082411) by querying the NCBI database, and name it as gene BmSet (amino acid sequence is shown in SEQ ID No. 4). Optimize according to the codon preference of E. coli, and perform full sequence synthesis by Suzhou Jinyu Zhi Company.

[0077] SEQ ID No. 4:

[0078] 2. Design primers pCO-HP-F / R and BmSet-F / R (primer sequences are shown in Table 2), and perform PCR reaction with the expression vector pCOLADuet-1 and the synthetic gene BmSet as templates, respectively. Obtain the pCOLADuet-1 linear vector and the BmSet gene fragment, determine the size of the amplified fragment by gel electrophoresis, then recover the linear fragment for seamless cloning, assemble the fragment and the vector, and transform the seamless cloning reaction liquid into E. coli DH5a competent cells by heat shock, spread on ampicillin-resistant LB solid plates, and incubate at 37°C overnight to screen positive clones for sequencing. After correct sequencing, expand the culture and extract the plasmid to obtain the expression plasmid pCO-BmSet. The plasmid map is shown in Figure 2.

[0079] Table 2: Primer sequences used for constructing pCO-BmSet and pCO-MV plasmids

[0080] 3. Design primers pCO-MV-F / R (primer sequences are shown in Table 2), and perform PCR reaction with the expression vector pCOLADuet-1 as template, for constructing the blank control plasmid pCO-MV without the transporter BmSet, using the same construction method as that for plasmid pCO-BmSet.

[0081] 4. Prepare LNT-01 ((E. coli BL21(DE3)-pAC-wcf-galE-lgtA)) electrocompetent cells, and transform the blank control plasmid pCO-MV and the expression plasmid pCO-BmSet into LNT-01, respectively, spread on ampicillin and chloramphenicol-resistant LB solid plates, and incubate at 37°C overnight to screen positive clones. Verify the correct positive transformants as the control strain LNT-02 ((E. coli BL21(DE3)-pAC-wcf-galE-lgtA-pCO-MV)) containing the pCOLADuet-1 empty vector for efficient synthesis of lactose-N-tetrasaccharide, and the engineering strain LNT-03 ((E. coli BL21(DE3)-pAC-wcf-galE-lgtA-pCO-BmSet)) for efficient transport of lactose-N-tetrasaccharide by overexpression of the transporter BmSet gene through the pCOLADuet-1 plasmid.

[0082] Example 3: High-performance liquid chromatography for detecting lactose-N-tetrasaccharide content

[0083] The determination of lacto-N-tetraose content uses liquid chromatography HPLC (Agilent 1260 series, USA), the detector is a differential refractometer RID, the chromatographic column is Rezex™ ROA-Organic Acid H+(8%, 300mm x 7.8mm), the mobile phase is 10mM dilute sulfuric acid, the detection temperature is 82℃, and the flow rate is 0.6mL / min.

[0084] Sample preparation: 1mL of fermentation broth was centrifuged at 12000rpm for 5min at room temperature, and the supernatant was taken and then filtered with a water phase membrane with a pore size of 0.22μm, and the concentration of extracellular product was detected by HPLC. 1mL of fermentation broth was heated at 100℃ for 10min, centrifuged at 12000rpm for 5min at room temperature, and the supernatant was taken and then filtered with a water phase membrane with a pore size of 0.22μm, and the total concentration of product was detected by HPLC.

[0085] LNT-02 and LNT-03 monoclonal antibodies were inoculated into glucose medium containing ampicillin and chloramphenicol resistance for small-scale fermentation test, the culture temperature was 37℃, the rotation speed was 220rpm, 0.2mM IPTG was added to induce the expression of target genes when the OD600 was about 0.6, and the concentration of 5g / L lactose was added as a substrate, and the culture temperature was reduced to 30℃, and the lacto-N-tetraose content was detected after 48h of induction and sampling.

[0086] The results are shown in Figure 3. The extracellular lacto-N-tetraose concentration of the control strain LNT-02 was 0.76g / L, and the total lacto-N-tetraose concentration was 2.53g / L. The extracellular lacto-N-tetraose concentration of the transporter BmSet overexpression strain LNT-03 was 2.65g / L, and the total lacto-N-tetraose concentration was 3.62g / L, which was 43.1% higher than that of the control strain, and the extracellular concentration was much higher than that of the control strain.

[0087] It should be understood that the above examples are all exemplary and are not intended to include all possible embodiments contained in the claims. Various modifications and changes can also be made on the basis of the above examples without departing from the scope of the disclosure. Similarly, any combination of the technical features of the above examples can also be made to form additional embodiments of the application that have not been explicitly described. Therefore, the above examples only express several embodiments of the application, and do not limit the protection scope of the patent of the application.

Claims

1. A genetically engineered bacterium that produces lactose-N-tetrasaccharide, characterized in that, The genetically engineered bacteria overexpress the exogenous transporter gene BmSet.

2. The genetically engineered bacterium according to claim 1, characterized in that, The BmSet is derived from Bacillus megaterium.

3. The genetically engineered bacteria according to claim 2, characterized in that, The amino acid sequence of the BmSet has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.

4. Preferably, the amino acid sequence of the BmSet is shown in SEQ ID NO.

4.

4. The genetically engineered bacteria according to claim 3, characterized in that, The genetically engineered bacteria also expressed the exogenous β-1,3-N-acetylglucosamine transferase gene lgtA, the exogenous β-1,3-galactosyltransferase gene wcf, and the exogenous uridine diphosphate glucose-4-epimerase gene galE.

5. The genetically engineered bacterium according to claim 4, characterized in that, The lgtA is derived from Neisseria meningitidis, the wcf is derived from Citrobacter freundii, and the galE is derived from Escherichia coli.

6. The genetically engineered bacterium according to claim 5, characterized in that, The amino acid sequence of the lgtA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.

1. Preferably, the amino acid sequence of the lgtA is as shown in SEQ ID NO.

1. Preferably, the amino acid sequence of the wcf has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.2; preferably, the amino acid sequence of the wcf is as shown in SEQ ID NO.

2. Preferably, the amino acid sequence of galE has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO.

3. Preferably, the amino acid sequence of galE is as shown in SEQ ID NO.

3.

7. A method for preparing genetically engineered bacteria according to any one of claims 1-6, characterized in that, The method includes the following steps: (1) Construct plasmids expressing the lgtA, wcf and galE; (2) Using Escherichia coli as the starting strain, the plasmids expressing lgtA, wcf and galE are introduced into the starting strain to construct a chassis strain; (3) Construct a plasmid expressing the BmSet; (4) The plasmid expressing BmSet is introduced into the chassis strain to construct the genetically engineered bacteria.

8. A biological agent, characterized in that, The biological agent includes genetically engineered bacteria according to any one of claims 1-6.

9. The use of a genetically engineered bacterium according to any one of claims 1-6, or a biological agent according to claim 8, in the production of lactose-N-tetrasaccharide.

10. A method for producing lactose-N-tetrasaccharide, characterized in that, The method uses glucose as a carbon source and lactose as a substrate to produce lactose-N-tetrasaccharide through fermentation using genetically engineered bacteria according to any one of claims 1-6 or biological agents according to claim 8.

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