Recombinant escherichia coli and method for producing difucosyllactose

Abstract

The embodiment of the invention discloses recombinant escherichia coli and a method for producing difucosyllactose, and belongs to the technical field of gene bioengineering. According to the recombinant escherichia coli, escherichia coli is used as a host cell, and CBGW, wbgL and futM1 are integrated. Wherein the expression of the alpha-1, 3-fucosyltransferase is controlled by a promoter with a temperature-sensitive element. According to the recombinant escherichia coli provided by the invention, the expression opportunity of alpha-1, 3-fucosyltransferase can be regulated and controlled through temperature control in the fermentation process of the recombinant escherichia coli, so that the yield of DFL is increased, the residue of by-products is reduced, and the yield and purity of DFL are ensured.

Description

Technical Field

[0001] This application relates to the field of genetic bioengineering technology, specifically to a recombinant Escherichia coli and method for producing difucosylated lactose. Background Technology

[0002] Difucosyllactose (DFL) is a difucosylated oligosaccharide and a key component of human motility organic compounds (HMOs). It consists of a lactose core linked to two fucose residues. Difucosyllactose can regulate gut microbiota and promote the proliferation of beneficial bacteria; it can also bind to viruses, bacteria, and toxins, preventing their infection of cells and thus enhancing the body's resistance. Given the scarcity of DFL in breast milk, the cumbersome nature of its chemical synthesis, and the high cost of its enzyme synthesis, the industrial production of DFL currently relies primarily on microbial synthesis. Biosynthesis requires only inexpensive carbon sources and renewable intracellular donors as raw materials, achieving high economic output with relatively low environmental costs, thus possessing broader application prospects.

[0003] Within microorganisms, the precursors for DFL synthesis include intracellularly synthesized GDP-L-fucose and exogenously added lactose as a mediator. The synthesis process is as follows: first, fructose-6-phosphate is synthesized in the gene... manA , manB , manC , gmd and wcaG Under the action of β-galactosyltransferase, GDP-L-fucose is synthesized. Extracellular lactose is transported into the cell by β-galactosyltransferase LacY, and then reacts with GDP-L-fucose to synthesize 2'-FL. 2'-FL is then converted to DFL by α-1-3-fucosyltransferase. However, in the process of microbial synthesis of DFL, there is a tendency for intermediate product residues and a large number of byproducts, which affect the yield and purity of DFL. Summary of the Invention

[0004] This application provides a recombinant Escherichia coli and a method for producing difucosylated lactose, which can increase DFL yield, reduce by-product residues, and ensure the yield and purity of DFL.

[0005] According to a first aspect of this application, a recombinant *Escherichia coli* is provided, which integrates a gene encoding a GDP-fucose synthase cluster using *E. coli* as the host cell. CBGW Gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 ; The expression of α-1,3-fucosyltransferase is controlled by a promoter with a temperature-sensitive element; the gene encoding the GDP-fucosyl synthase cluster... CBGWGene encoding phosphomannose mutant enzyme ManB Gene encoding mannose-1-phosphate guanylate transferase ManC Gene encoding GDP-D-mannose-4,6-dehydrase Gmd Gene encoding GDP-L-fucose synthase WcaG composition.

[0006] Optionally, genes encoding the GDP-fucose synthase cluster CBGW Gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 The copy number ratio in recombinant Escherichia coli was 3:(2-5):1.

[0007] Optionally, genes encoding the GDP-fucose synthase cluster CBGW Gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 The copy number ratio is 3:3:1 or 3:2:1.

[0008] Optionally, genes encoding the GDP-fucose synthase cluster CBGW and the gene encoding α-1,2-fucosyltransferase wbgL The starters for all of them are T7 starters.

[0009] Optionally, a promoter with a temperature-sensitive element is used to manipulate the expression of α-1,3-fucosyltransferase at a temperature of 37°C–42°C.

[0010] Optionally, the thermosensitive element is a temperature-sensitive mutant of the cl protein, clts857, and the nucleotide sequence of the thermosensitive element is shown in SEQ ID NO.1.

[0011] Optionally, recombinant *E. coli*, using *E. coli* as the host cell, knocks out the gene encoding β-galactosidase. lacZ Gene encoding L-fucose isomerase fucI The gene encoding L-fucokinase fucK and the gene encoding UDP-glucose lipotransferase wcaJ .

[0012] According to a second aspect of this application, a method for producing difucosylated lactose is also provided, using lactose as a substrate and recombinant Escherichia coli as described above as a fermentation strain to produce difucosylated lactose.

[0013] Optionally, a method for producing difucosyl lactose includes: The recombinant Escherichia coli seed culture was added to the fermentation medium and cultured at a temperature of 35℃-40℃ until any point in the late logarithmic phase to the first 1 / 3 of the stationary phase to obtain the first culture medium. Add an inducer and supplement lactose to the first culture medium and culture at 28℃-32℃ until the lactose is exhausted. Then adjust the temperature to 35℃-40℃ to enable recombinant Escherichia coli to express α-1,3-fucosyltransferase. Culture for 80h-140h to obtain the second culture medium. The second culture medium was separated and purified to obtain difucosyl lactose.

[0014] Optionally, the temperature is adjusted to 35℃-40℃ to enable recombinant Escherichia coli to express α-1,3-fucosyltransferase, and cultured for 80h-140h, including: Adjust the temperature to 35℃-40℃ to enable recombinant Escherichia coli to express α-1,3-fucosyltransferase, maintain the lactose concentration at 0.2g / L-0.8g / L, and culture for 80h-140h.

[0015] The recombinant Escherichia coli provided in this application integrates a gene encoding the GDP-fucose synthase cluster. CBGW Gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 This method can promote the synthesis of intermediates such as GDP-fucose and 2'-FL, as well as DFL. By manipulating the expression of α-1,3-fucosyltransferase with a promoter equipped with a temperature-sensitive element, the expression timing of α-1,3-fucosyltransferase can be regulated by controlling the temperature during the fermentation of recombinant E. coli. This reduces the reaction of α-1,3-fucosyltransferase synthesizing 3-FL byproducts using lactose as a substrate, reduces the generation of byproducts during DFL synthesis, increases DFL yield, reduces byproduct residue, and ensures the yield and purity of DFL. Detailed Implementation

[0016] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0017] This application provides a recombinant *Escherichia coli* and a method for producing difucosylated lactose. Detailed descriptions are provided below. It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". The terms first, second, third, etc., are used merely as illustrative and do not impose numerical requirements or establish an order. Various embodiments of the present invention may exist in a range format; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description specifically discloses all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that a range description from 1 to 6 specifically discloses sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the above ranges, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0018] Firstly, a recombinant *E. coli* strain is provided, which integrates a gene encoding the GDP-fucose synthase cluster using *E. coli* as the host cell. CBGW Gene encoding α-1,2-fucosyltransferase wbgL (NCBI sequence number: WP_000022655.1) and the gene encoding α-1,3-fucosyltransferase. futM1 (NCBI accession number: MBO5664686.1). The expression of α-1,3-fucosyltransferase is controlled by a promoter with a temperature-sensitive element; the gene encoding the GDP-fucose synthase cluster... CBGW Gene encoding phosphomannose mutant enzyme ManB (NCBI sequence number: WP_001350528.1), encoding the mannose-1-phosphate guanylate transferase gene. ManC (NCBI accession number: WP_000079274.1) Encoding the GDP-D-mannose-4,6-dehydrase gene Gmd (NCBI accession number: EFB3410420.1), encoding the GDP-L-fucose synthase gene. WcaG (NCBI serial number: WP_000043654.1) Composed of.

[0019] The recombinant Escherichia coli provided in this application embodiment integrates a gene encoding the GDP-fucose synthase cluster. CBGW Gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1This method can promote the synthesis of GDP-fucose, intermediates such as 2'-FL, and DFL, respectively. By manipulating the expression of α-1,3-fucosyltransferase with a promoter equipped with a temperature-sensitive element, the expression timing of α-1,3-fucosyltransferase can be regulated by temperature control during the fermentation of recombinant E. coli. This reduces the reaction of α-1,3-fucosyltransferase synthesizing 3-FL byproducts using lactose as a substrate, thus reducing the generation of byproducts during DFL synthesis, increasing DFL yield, reducing byproduct residues, and ensuring the yield and purity of DFL.

[0020] Specifically, by integrating genes encoding the GDP-fucose synthase cluster CBGW It can enhance the synthesis of GDP-fucose by integrating a gene that catalyzes the formation of α-1,2-glycosidic bonds between GDP-fucose and lactose to generate 2'-fucosyllactose (2'-FL), an important intermediate in DFL synthesis. futM1 It can catalyze the formation of α-1,3-glycosidic bonds between GDP-fucose and 2'-FL or lactose, generating DFL or other fucosylated products (e.g., 3-fucosylated lactose 3-FL).

[0021] Based on previous research, the applicant found that adding lactose as a precursor is essential for the synthesis of DFL. However, α-1-3-fucosyltransferase can synthesize 3-FL using lactose as a substrate, inevitably leaving 3-FL as a byproduct in the fermentation broth. This affects the yield and purity of DFL. In the presence of 3-FL, due to the small molecular weight difference between 3-FL and DFL, efficient purification using only ultrafiltration (UF) or nanofiltration (NF) membranes is difficult, significantly increasing the difficulty and cost of downstream purification processes.

[0022] In the recombinant Escherichia coli provided in this application embodiment, the expression of α-1,3-fucosyltransferase is manipulated by a promoter with a temperature-sensitive element. This allows the recombinant E. coli to control the expression timing of α-1,3-fucosyltransferase during fermentation by adjusting the fermentation temperature, thereby reducing the synthesis process of α-1,3-fucosyltransferase using lactose as a substrate to synthesize 3-FL, thus reducing the generation of by-products, allowing more substrate to be converted into DFL, ensuring DFL yield and purity, and reducing the difficulty of subsequent purification processes.

[0023] In some embodiments, genes encoding GDP-fucose synthase clusters CBGW Gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 The copy number ratio in recombinant Escherichia coli was 3:(2-5):1.

[0024] By recombinant E. coli genes encoding the GDP-fucose synthase cluster CBGW Gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 The copy number ratio is 3:(2-5):1, which can reduce the residue of byproducts during DFL synthesis, improve the conversion rate of the substrate lactose, and enhance the yield and purity of DFL.

[0025] In some embodiments, genes encoding GDP-fucose synthase clusters CBGW Gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 The copy number ratio is 3:3:1 or 3:2:1.

[0026] By making CBGW , wbgL and futM1 The copy number ratio of 3:3:1 or 3:2:1 enables recombinant E. coli to continuously increase DFL production during fermentation, delay the arrival of the plateau phase, improve the conversion rate of precursor substances, and reduce by-product residues.

[0027] In some embodiments, genes encoding GDP-fucose synthase clusters CBGW and the gene encoding α-1,2-fucosyltransferase wbgL The starters for all of them are T7 starters.

[0028] The T7 promoter exhibits high specificity and efficient transcriptional activity, enhancing the expression efficiency of target genes. Furthermore, the T7 promoter can communicate via isopropyl β-transferase. d 1 Inducers such as thiogalactopyranoside (IPTG) precisely regulate the expression of target genes and flexibly adjust the expression level of target genes.

[0029] In some embodiments, a promoter with a temperature-sensitive element is used to manipulate the expression of α-1,3-fucosyltransferase at a temperature of 37°C-42°C.

[0030] The expression of α-1,3-fucosyltransferase was manipulated by a promoter with a temperature-sensitive element at a temperature of 37℃-42℃, which can match the fermentation temperature of recombinant Escherichia coli, regulate the expression of α-1,3-fucosyltransferase, and ensure the synthesis rate of DFL.

[0031] In some embodiments, the thermosensitive element is a temperature-sensitive mutant of the cl protein, clts857, and the nucleotide sequence of the thermosensitive element is shown in SEQ ID NO.1.

[0032] clts857 remains active at around 30℃, inhibiting promoter transcription and thus preventing α-1,3-fucosyltransferase expression. However, at temperatures between 37℃ and 42℃, clts857 is inactivated, relieving the inhibition of the promoter and allowing α-1,3-fucosyltransferase expression, thus ensuring the precision of temperature regulation.

[0033]

[0034] In some embodiments, recombinant Escherichia coli, using Escherichia coli as the host cell, knocks out the gene encoding β-galactosidase. lacZ (NCBI sequence number: ACT42197.1), encoding L-fucose isomerase gene fucI (NCBI sequence number: ACT44469.1), encoding the L-fucokinase gene fucK (NCBI sequence number: ACT44470.2) and the gene encoding UDP-glucose lipotransferase wcaJ (NCBI serial number: ACT43804.1).

[0035] The recombinant Escherichia coli provided in this application embodiment is obtained by knocking out the gene encoding β-galactosidase. lacZ Gene encoding L-fucose isomerase fucI The gene encoding L-fucokinase fucK and the gene encoding UDP-glucose lipotransferase wcaJ It can prevent lactose from being hydrolyzed, reduce the generation of by-products, and improve the production efficiency and substrate conversion rate of difucosyl lactose (DFL).

[0036] Specifically, β-galactosidase hydrolyzes lactose into glucose and galactose, and lactose is an important precursor for DFL synthesis. Knocking out the gene encoding β-galactosidase... lacZ This prevents lactose from being hydrolyzed, allowing it to be used more for DFL synthesis and improving lactose utilization. L-fucose isomerase catalyzes the isomerization of L-fucose, and in DFL synthesis, it's crucial to ensure L-fucose is converted via a specific metabolic pathway. Knocking out the gene encoding L-fucose isomerase... fucI It can prevent L-fucose isomerization. L-fucokinase can catalyze the phosphorylation of L-fucose; knocking out the gene encoding L-fucokinase... fucK It can block the phosphorylation of L-fucose, preventing its utilization by other pathways. UDP-glucose lipotransferase plays an important role in cell wall synthesis and consumes GDP-fucose (a precursor to DFL). Knocking out the gene encoding UDP-glucose lipotransferase... wcaJ It can block the consumption pathway of GDP-fucose, thereby increasing the accumulation of GDP-fucose in cells and providing more precursor substances for DFL synthesis.

[0037] It should be noted that the recombinant *E. coli* provided in this application embodiment employs conventional techniques in the art for both gene knockout and gene integration. For example, gene knockout can be performed through homologous recombination, the CRISPR / Cas9 system, or RNA interference. Gene integration can be performed through homologous recombination integration, CRISPR / Cas9 system-mediated homologous targeted repair, transposon system integration, integrase-mediated site-specific integration, or viral vector-mediated integration, which will not be elaborated further here. Techniques not described in detail in this application embodiment are all commonly used techniques in the art; please refer to *Molecular Biology Experiment Handbook* (Ma Wenli, People's Military Medical Publishing House), *Molecular Biology Experiments (Second Edition)* (Zhejiang University Press), and *Cell Biology Experiments* (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0038] In some embodiments, the host cell includes Escherichia coli BL21(DE3).

[0039] Escherichia coli BL21(DE3) is a lysogen that integrates the DE3 region of λ phage into its chromosome. This region contains the T7 RNA polymerase gene, which enables the induction of target gene expression in the presence of an inducer. Escherichia coli BL21(DE3) is suitable for high-density fermentation, compatible with multiple expression vectors, has a clear genetic background, and is easy to perform gene knockout and integration operations, giving it significant advantages.

[0040] Secondly, embodiments of this application also provide a method for producing difucosylated lactose, using lactose as a substrate and recombinant Escherichia coli as described above as a fermentation strain to produce difucosylated lactose.

[0041] The method for producing difucosylated lactose provided in this application has all the beneficial effects of recombinant Escherichia coli as described above, and will not be repeated here.

[0042] In some embodiments, a method for producing difucosyl lactose includes: The recombinant Escherichia coli seed culture was added to the fermentation medium and cultured at a temperature of 35℃-40℃ until any point in the late logarithmic phase to the first 1 / 3 of the stationary phase to obtain the first culture medium. Add an inducer and supplement lactose to the first culture medium and culture at 28℃-32℃ until the lactose is exhausted. Then adjust the temperature to 35℃-40℃ to enable recombinant Escherichia coli to express α-1,3-fucosyltransferase. Culture for 80h-140h to obtain the second culture medium. The second culture medium was separated and purified to obtain difucosyl lactose.

[0043] The first culture medium can be added to the fermentation system at any point during the late logarithmic growth phase to the first third of the stationary phase. In practice, it is preferable to add it to the fermentation system during the first sixth of the stationary phase, and more preferably when it enters the stationary phase. Those skilled in the art can determine the time of the stationary phase by fermentation time, OD600, dissolved oxygen, and carbon source consumption. For example, fermentation in the fermenter can be completed after 18 hours; the OD600 of the fermentation broth can reach 140; or the dissolved oxygen curve can be observed, showing a rapid decrease in dissolved oxygen followed by stabilization after carbon source replenishment.

[0044] By culturing at 35℃-40℃, recombinant *E. coli* can achieve a high proliferation rate, ensuring a sufficient number of recombinant *E. coli* bacteria in the first culture medium. Then, an inducer is added to induce the expression of GDP-fucose synthase clusters and α-1,2-fucosyltransferase in the recombinant *E. coli*. Lactose is supplemented, and the culture is maintained at 28℃-32℃ until lactose is depleted. At this stage, since α-1,3-fucosyltransferase is not expressed, the side reaction of α-1,3-fucosyltransferase synthesizing 3-FL using lactose as a substrate is avoided. Lactose is converted to 2'-FL under the action of α-1,2-fucosyltransferase. The temperature is then adjusted to 35℃-40℃ to allow α-1,3-fucosyltransferase expression, enabling it to synthesize DFL using 2'-FL and GDP-fucose as precursors. The method for producing difucosyl lactose provided in this application can reduce the occurrence of side reactions, thereby reducing by-product residues and increasing the yield and purity of DFL.

[0045] In some embodiments, the fermentation medium is formulated as follows: 8 g / L-12 g / L carbon source, 3 g / L-5 g / L ammonium sulfate, 9 g / L-9.5 g / L dipotassium hydrogen phosphate, 8 g / L-8.5 g / L potassium dihydrogen phosphate, 0.2 g / L-0.4 g / L citric acid, 5 g / L-7 g / L tryptone, 1 g / L-3 g / L yeast extract, 8 mL / L-12 mL / L thiamine, 1 g / L-3 g / L magnesium sulfate heptahydrate, 0.01 g / L-0.03 g / L calcium sulfate, and 8 mL / L-12 mL / L trace element solution.

[0046] By including the above-mentioned substances in the fermentation medium, a rich nutrient supply can be provided for recombinant Escherichia coli, which helps to maintain the stability of the acid-base buffer system and provides a stable environment for the amplification of recombinant Escherichia coli and the synthesis of DFL.

[0047] In some embodiments, the carbon source includes glycerol. Glycerol is a high-energy-density carbon source that can provide sufficient energy for the growth of recombinant Escherichia coli, reduce metabolic inhibition, and promote precursor synthesis.

[0048] In some embodiments, recombinant Escherichia coli seed culture is added to a fermentation medium and cultured at a temperature of 35°C-40°C until any point in the late logarithmic phase to the first third of the stationary phase, including: The recombinant Escherichia coli seed culture was added to the fermentation medium, and cultured at a pH of 6.5-7, a dissolved oxygen content of 30%-50%, and a culture temperature of 35℃-40℃. Once the carbon source in the fermentation medium is depleted, the feed solution is replenished at a feed rate of 3 g / L / h to 5 g / L / h. The culture is continued until any point in the late logarithmic growth phase to the first third of the stationary phase, yielding the first culture medium. The feed solution is formulated with 750 g / L to 850 g / L of carbon source.

[0049] Maintaining high temperatures can increase the reproduction rate of recombinant E. coli, while a suitable pH range and dissolved oxygen concentration also provide a favorable environment for its growth and reproduction. Fed-coated culture can maintain the continuous and rapid growth of recombinant E. coli.

[0050] In some embodiments, the temperature is adjusted to 35℃-40℃ to enable recombinant Escherichia coli to express α-1,3-fucosyltransferase, and cultured for 80h-140h, including: Adjust the temperature to 35℃-40℃ to enable recombinant Escherichia coli to express α-1,3-fucosyltransferase, maintain the lactose concentration at 0.2g / L-0.8g / L, and culture for 80h-140h.

[0051] In other words, maintaining a low lactose concentration at this stage can further increase DFL production and reduce byproduct residues.

[0052] In some embodiments, the concentration of the inducer is 0.2 mM-0.3 mM.

[0053] By maintaining the concentration of the inducer within the aforementioned range, it is possible to induce the expression of the integrated gene in recombinant Escherichia coli, ensure the expression level, reduce the negative impact on cell growth, and guarantee the stability of the fermentation process.

[0054] In some embodiments, the inducer includes isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0055] IPTG, as an inducer, can specifically bind to the target gene in recombinant Escherichia coli, precisely regulate gene expression, and is not affected by metabolic pathways, thus exhibiting excellent induction effects and increasing expression levels.

[0056] The embodiments of this application are further illustrated below with reference to specific examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to the conditions recommended by the manufacturer.

[0057] Example 1-1 Build CBGW , wbgL and futM1 The recombinant Escherichia coli with a copy number ratio of 3:2:1, among which, CBGW , wbgL The promoter is the T7 promoter. futM1 The promoter is a temperature-sensitive promoter, clts. pR pL, specifically E. coli star BL21(DE3)△l acZ △ fucIK △ wcaj △ lacA: :T7- wbgL △ wzxC ::T7- CBGW △ intQ ::T7- CBGW △ caiB ::T7- CBGW △ydeU::clts pR pL- futM1 △ yjiv ::T7- wbgL The strain was named PL-321. Gene knockout was performed using the CRISPR / Cas9 system, and gene integration was performed using the transposon system.

[0058] Examples 1-2 Using the same method, construct CBGW , wbgL and futM1 The recombinant E. coli with a copy number ratio of 3:3:1, compared to the one in Example 1 with the knockout... nagB And added at that site wbgL The gene was named PL-331.

[0059] Comparative Example 1-1 Using the same method, construct CBGW , wbgL and futM1 The recombinant Escherichia coli with a copy number ratio of 3:2:1, compared to Example 1, will... futM1 The promoter was replaced with the T7 promoter, named M1-321.

[0060] Comparative Examples 1-2 Using the same method, construct CBGW , wbgL and futM1 The recombinant Escherichia coli with a copy number ratio of 3:3:1, compared to Example 2, will... futM1 The promoter was replaced with the T7 promoter, named M1-331.

[0061] Shake-flask fermentation experiments were conducted using strains PL-321, PL-331, M1-321, and M1-331 constructed in Examples 1-1, 1-2, 1-1, and 1-2, respectively.

[0062] The culture medium for shake-flask fermentation consisted of: 30g glycerol, 17.9g disodium hydrogen phosphate dodecahydrate, 3.1g potassium dihydrogen phosphate, 2.0g ammonium chloride, 1.0g diammonium hydrogen phosphate, 1.7g citric acid, 15mg calcium chloride, 2g yeast extract, 15g Oxoid tryptone, 2.2g trisodium citrate dihydrate, 1g magnesium sulfate heptahydrate, 0.3mL Triton X-100, 10mg vitamin B1, and 10mL trace element solution.

[0063] The process conditions for shake-flask fermentation are as follows: (1) PL-321, PL-331, M1-321 and M1-331 were cultured in LB liquid medium (5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride) at 37°C overnight with shaking to obtain seed culture; (2) Take 1 mL of seed culture and inoculate it into a 500 mL shake flask containing 100 mL of shake flask fermentation medium. Incubate at 37 °C with shaking until OD reaches 100 mL. 600 The temperature was increased to 0.6-0.8 (after 5 hours of incubation), and then 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) and 6 g / L lactose were added for induction. The temperature was adjusted according to the values ​​shown in Table 1, and the mixture was incubated with shaking for 60 hours.

[0064] Table 1

[0065] The yield of DFL and the residual amount of FL in different fermentation broths were tested respectively. The test methods were as follows: (1) Take 1 mL of fermentation broth and boil it in a water bath for 10 min to kill the strain, then centrifuge it at 12000g for 10 min. The supernatant obtained was filtered through a 0.22 μm filter and the filtrate was used to detect FL and DFL. (2) Using a Shim-pack Scepter Diol-HILIC column (Shimadzu, Japan) and a RID-20A refractive index detector (Shimadzu), FL and DFL in the test sample were detected by high performance liquid chromatography (HPLC). The contents of lactose, FL and DFL in the test sample were calculated according to the external standard method. The chromatographic conditions were: mobile phase acetonitrile / water (75:25, v / v), flow rate 1.0 mL / min, column temperature 40℃, and injection volume 100 μL. (3) DFL standards with gradient concentrations of 0-2.0 g / L were taken, and a standard curve was plotted with DFL concentration as the abscissa and HPLC peak area as the ordinate. The test results are shown in Tables 2 and 3: Table 2

[0066] As shown in Table 2, when PL-321 from Example 1-1 was used as the fermentation strain, the residual amount of FL was lower than when M1-321 was used as the fermentation strain. This indicates that the recombinant *E. coli* provided in this application can manipulate the expression timing of α-1,3-fucosyltransferase through a temperature-sensitive element, which helps to reduce the byproduct residues during DFL synthesis. The comparison of AG in Table 2 shows that the temperature regulation process of 37℃-30℃-37℃ helps to further increase DFL yield and reduce FL residue. The comparison of OP in Table 2 shows that, since strain M1-321 does not contain a temperature-sensitive promoter, temperature control during fermentation did not produce a significant difference in DFL yield and FL residue, and DFL yield decreased.

[0067] Table 3

[0068] As shown in Table 3, when PL-331 from Examples 1-2 was used as the fermentation strain, the residual amount of FL was lower than when M1-331 was used as the fermentation strain. This indicates that the recombinant *E. coli* provided in this application can manipulate the expression timing of α-1,3-fucosyltransferase through a temperature-sensitive element, which helps to reduce byproduct residues during DFL synthesis. The comparison of HN in Table 3 shows that the temperature regulation process of 37℃-30℃-37℃ helps to further increase DFL yield and reduce FL residue. The comparison of QR in Table 3 shows that, since the M1-331 strain does not contain a temperature-sensitive promoter, no significant difference in DFL yield and FL residue was observed through temperature control during fermentation.

[0069] Fermentation experiments were conducted in fermenters using strains PL-321, PL-331, M1-321, and M1-331 constructed in Examples 1-1, 1-2, and Comparative Examples 1-1 and 1-2, respectively. Example 2-1 (1) Seed culture preparation: ① Recombinant Escherichia coli PL-321 was cultured on LB solid medium for 16 h for plate activation; ② Single colonies were concentrated in LB liquid medium and cultured for 12 h to obtain primary seed culture; ③ Primary seed culture was inoculated into LB liquid medium at an inoculation rate of 1% and cultured for 6 h to obtain secondary seed culture; (2) Transfer 150 mL of secondary seed culture into a 5 L fermenter containing 2.5 L of fermentation medium, add 28% (v / v) NH4OH to adjust the pH to 6.8, control the stirring speed to maintain dissolved oxygen at 30%-50%, the culture temperature is 37℃, the aeration rate is 2 vvm, and the stirring speed is 900 rpm; the fermentation medium formula is: 10 g / L glycerol, 4.0 g / L (NH4)2SO4, 9.2 g / L K2HPO4, 8.2 g / L KH2PO4, 0.3 g / L citric acid, 6.0 g / L tryptone, 2.0 g / L yeast extract, 10 mg / L thiamine, 2.0 g / L MgSO4·7H2O, 0.02 g / L CaCl2 and 10 mL / L trace element solution; (3) After the initial glycerol in the fermentation medium is depleted, feed solution is added to the fermenter at a constant rate of 4 g / L / h, and fermentation continues until the OD reaches 100%. 600 The culture medium was obtained when the pH reached 0.6; the feed solution was formulated as follows: 800 g / L glycerol. (4) Add IPTG to the first culture medium to a concentration of 0.2 mM. At the same time, add 140 g of lactose and culture at 30 °C for 55 h until the lactose is exhausted. Then adjust the fermentation temperature to 37 °C and continue fermentation for 120 h to obtain the second culture medium.

[0070] Example 2-2 The difference between this embodiment and embodiment 2-1 is that the strain is PL-331, while all other conditions are the same as in embodiment 2-1.

[0071] Example 2-3 The difference between this embodiment and embodiment 2-1 is that in step (4), after adjusting the fermentation temperature to 37°C, the lactose concentration is kept at 0.3 g / L and fermentation continues for 120 h. All other conditions are the same as in embodiment 2-1.

[0072] Comparative Example 2-1 The difference between this comparative example and Example 2-1 is that recombinant Escherichia coli M1-321 was used to replace recombinant Escherichia coli PL-321 in Example 2-1, while all other conditions remained the same as in Example 2-1.

[0073] Comparative Example 2-2 The main difference between this comparative example and comparative example 2-1 is that the strain was replaced with M1-331, while the other conditions remained the same as those in comparative example 2-1.

[0074] The yield of DFL and the residual amount of FL in different fermentation broths were tested respectively. The test method was as follows: (1) Take 1 mL of the second culture medium with different fermentation times, boil it in a water bath for 10 min to kill the strain, and then centrifuge it at 12000g for 10 min. The supernatant obtained was filtered through a 0.22 μm filter, and the filtrate was used to detect FL and DFL. (2) Using a Shim-pack Scepter Diol-HILIC column (Shimadzu, Japan) and a RID-20A refractive index detector (Shimadzu), FL and DFL in the test sample were detected by high performance liquid chromatography (HPLC). The contents of lactose, FL and DFL in the test sample were calculated according to the external standard method. The chromatographic conditions were: mobile phase acetonitrile / water (75:25, v / v), flow rate 1.0 mL / min, column temperature 40℃, and injection volume 100 μL. (3) Take DFL standards with gradient concentrations of 0-2.0 g / L, and plot a standard curve with DFL concentration as the abscissa and HPLC peak area as the ordinate. The test results are shown in Table 4: Table 4

[0075] As shown in Table 5, the recombinant *E. coli* provided in this application can utilize a temperature-sensitive element to manipulate the expression timing of α-1,3-fucosyltransferase, reducing the generation of byproducts during DFL synthesis, thereby increasing DFL yield and FL residue compared to the comparative example. Specifically, a comparison between Examples 2-1 and 2-3 shows that after α-1,3-fucosyltransferase expression, controlling the lactose content in the fermentation broth to a low level helps to further increase DFL yield and reduce FL residue.

[0076] In summary, the recombinant Escherichia coli provided in this application, by manipulating the expression of α-1,3-fucosyltransferase with a promoter equipped with a temperature-sensitive element, can regulate the expression timing of α-1,3-fucosyltransferase during the fermentation process of recombinant E. coli through temperature control. This reduces the reaction of α-1,3-fucosyltransferase synthesizing 3-FL byproducts using lactose as a substrate, reduces the generation of byproducts during DFL synthesis, increases DFL yield, reduces byproduct residues, and ensures the yield and purity of DFL.

[0077] The above provides a detailed description of a recombinant Escherichia coli and a method for producing difucosylated lactose provided in the embodiments of this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A recombinant Escherichia coli, characterized in that, Using Escherichia coli as the host cell, the gene encoding the GDP-fucose synthase cluster was integrated. CBGW Gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 ; The expression of the α-1,3-fucosyltransferase is controlled by a promoter with a temperature-sensitive element; the gene encoding the GDP-fucose synthase cluster... CBGW Gene encoding phosphomannose mutant enzyme ManB Gene encoding mannose-1-phosphate guanylate transferase ManC Gene encoding GDP-D-mannose-4,6-dehydrase Gmd Gene encoding GDP-L-fucose synthase WcaG composition.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The gene encoding the GDP-fucose synthase cluster CBGW The gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 The copy number ratio in the recombinant Escherichia coli was 3:(2-5):

1.

3. The recombinant Escherichia coli according to claim 1, characterized in that, The gene encoding the GDP-fucose synthase cluster CBGW The gene encoding α-1,2-fucosyltransferase wbgL and the gene encoding α-1,3-fucosyltransferase futM1 The copy number ratio is 3:3:1 or 3:2:

1.

4. The recombinant Escherichia coli according to claim 1, characterized in that, The gene encoding the GDP-fucose synthase cluster CBGW and the gene encoding α-1,2-fucosyltransferase wbgL The starters for all of them are T7 starters.

5. The recombinant Escherichia coli according to claim 1, characterized in that, The promoter with a temperature-sensitive element is used to manipulate the expression of α-1,3-fucosyltransferase at a temperature of 37℃-42℃.

6. The recombinant Escherichia coli according to claim 5, characterized in that, The temperature-sensitive element is a temperature-sensitive mutant of the cl protein, clts857, and the nucleotide sequence of the temperature-sensitive element is shown in SEQ ID NO.

1.

7. The recombinant Escherichia coli according to any one of claims 1-6, characterized in that, The recombinant *E. coli* strain, using *E. coli* as the host cell, had the gene encoding β-galactosidase knocked out. lacZ Gene encoding L-fucose isomerase fucI The gene encoding L-fucokinase fucK and the gene encoding UDP-glucose lipotransferase wcaJ .

8. A method for producing difucosyl lactose, characterized in that, Using lactose as a substrate and the recombinant Escherichia coli as described in any one of claims 1-7 as a fermentation strain, difucosylated lactose is produced by fermentation.

9. The method for producing difucosyl lactose according to claim 8, characterized in that, The method for producing difucosyl lactose includes: The recombinant Escherichia coli seed culture was added to the fermentation medium and cultured at a temperature of 35℃-40℃ until any point in the late logarithmic phase to the first 1 / 3 of the stationary phase to obtain the first culture medium. An inducer was added to the first culture medium, and lactose was added. The culture was carried out at a temperature of 28℃-32℃ until the lactose was exhausted. Then the temperature was adjusted to 35℃-40℃ to enable the recombinant Escherichia coli to express the α-1,3-fucosyltransferase. The culture was carried out for 80h-140h to obtain the second culture medium. The second culture medium was separated and purified to obtain difucosylated lactose.

10. The method for producing difucosyl lactose according to claim 9, characterized in that, The temperature was adjusted to 35℃-40℃ to enable the recombinant *E. coli* to express α-1,3-fucosyltransferase, and cultured for 80h-140h, including: The temperature was adjusted to 35℃-40℃ to enable the recombinant Escherichia coli to express α-1,3-fucosyltransferase, and the lactose concentration was maintained at 0.2g / L-0.8g / L for 80h-140h.

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