A recombinant escherichia coli and its application in simultaneous production of d-mannose, d-psicose and d-tagatose

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a recombinant Escherichia coli and application of the recombinant Escherichia coli in simultaneous production of D-mannose, D-psicose and D-tagatose. Specifically, the application provides a recombinant Escherichia coli, the strain of which simultaneously expresses five key enzymes, can use cheap glucose as a substrate, and can simultaneously synthesize D-mannose, D-psicose and D-tagatose in a single reaction system through a multistage enzyme cascade reaction, so that a one-pot three-sugar production mode is realized. In the technical scheme, PPGK can use polyphosphate to replace ATP to provide a phosphate group, so that the addition of an expensive auxiliary factor is avoided, and the production cost is significantly reduced. Meanwhile, the three products are simultaneously generated in a single system, so that subsequent separation and purification steps are simplified, and the application has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant Escherichia coli and its application in the simultaneous production of D-mannose, D-allulose and D-tagatose. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] D-mannose, D-allulose, and D-tagatose are all rare functional sugars derived from natural sources, with broad application prospects in functional foods, precision medicine, and other fields. However, these three sugars are extremely rare in nature, and the cost of extracting them directly from natural raw materials is extremely high, making large-scale production difficult.

[0004] Currently, D-tagatose is mainly obtained through the conversion of D-galactose catalyzed by L-arabinose isomerase. Although the conversion rate is relatively high (up to about 79.7%), the cost of D-galactose substrate is expensive, making it unsuitable for industrial production. D-allulose is mainly produced by the fructose-to-fructose conversion catalyzed by D-allulose-3-epimerase, but the reaction is equilibrium-limited (product / substrate ratio of about 20%-35%), requiring additional high-concentration fructose separation and purification processes. The enzymatic synthesis of D-mannose is also limited by reaction equilibrium (conversion rate of about 20%-40%) and requires chromatographic separation or crystallization purification. Therefore, the high raw material costs, expensive product and by-product separation costs, and relatively low product yields of the production methods for D-mannose, D-tagatose, and D-allulose greatly limit their application. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides a recombinant *Escherichia coli* and its application in the simultaneous production of D-mannose, D-allulose, and D-tagatose. This invention is based on the above research findings.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a recombinant *Escherichia coli* strain that simultaneously overexpresses polyphosphate-dependent glucokinase (PPGK), glucose-6-phosphate isomerase (PGI), D-tagatose 1,6-bisphosphate aldolase (GatZ), glucose-6-phosphate isomerase / phosphomannose isomerase (PGI / PMI), and phosphate-tagatose phosphatase (TPP).

[0007] A second aspect of the invention provides the use of the recombinant Escherichia coli in the simultaneous production of D-mannose, D-allulose and D-tagatose.

[0008] A third aspect of the present invention provides a method for simultaneously producing D-mannose, D-allulose, and D-tagatose from glucose, the method comprising: using glucose and polyphosphate as substrates, employing the recombinant Escherichia coli as a catalyst, and carrying out a cascade enzyme-catalyzed reaction in a single reaction system.

[0009] The beneficial technical effects of one or more of the above technical solutions are as follows: The above-described technical solution provides a recombinant *E. coli* strain that simultaneously expresses five key enzymes. Using inexpensive glucose as a substrate, this strain can simultaneously synthesize D-mannose, D-allulose, and D-tagatose in a single reaction system via a multi-stage enzyme cascade reaction, achieving a one-pot production mode for three sugars. The PPGK in this solution utilizes polyphosphates to replace ATP in providing phosphate groups, avoiding the addition of expensive cofactors and significantly reducing production costs. Furthermore, the simultaneous generation of the three products in a single system simplifies subsequent separation and purification steps, demonstrating promising prospects for industrial application. Attached Figure Description

[0010] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0011] Figure 1 The image shows the exogenous plasmid pRSFDuet-PGI-PPGK-TPP in Example 1 of this invention.

[0012] Figure 2 This is the spectrum of the exogenous plasmid pETDuet-GatZ-PMI in Example 1 of the present invention.

[0013] Figure 3 This is a nucleic acid gel image used in Example 2 of this invention to verify whether the plasmid pETDuet-GatZ-PGI / PMI was successfully transformed into E. coli; where M is the marker and I is the recombinant strain. E. coli Transformant of ATG-2, 2 is control plasmid pETDuet-GatZ-PGI / PMI.

[0014] Figure 4 This is a nucleic acid gel image used in Example 2 of this invention to verify whether the plasmid pRSFDuet-PGI-PPGK-TPP was successfully transformed into E. coli; where M is the marker and 3 is the recombinant strain. E. coli Transformant of ATG-2, 4 is control plasmid pRSFDuet-PGI-PPGK-TPP.

[0015] Figure 5 The recombinant strain in Example 3 of this invention E. coli SDS-PAGE analysis of five enzymes expressed in ATG-2; where M is the protein marker, S is the crude enzyme solution sample in supernatant after lysis and centrifugation, and P is the crude enzyme solution sample in precipitate after lysis and centrifugation.

[0016] Figure 6 To verify the recombinant strain in Example 4 of this invention E. coli High-performance liquid chromatography (HPLC) chromatograms of whether ATG-2 can simultaneously produce D-mannose, D-allulose, and D-tagatose using D-glucose as a substrate. Detailed Implementation

[0017] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] In a typical embodiment of the present invention, a recombinant Escherichia coli is provided, wherein the recombinant Escherichia coli simultaneously overexpresses polyphosphate-dependent glucokinase (PPGK), glucose-6-phosphate isomerase (PGI), D-tagatose 1,6-bisphosphate aldolase (GatZ), glucose-6-phosphate isomerase / Phosphomannose isomerase (PGI / PMI), and phosphate-tagatose phosphatase (TPP).

[0020] The functions of the five enzymes are as follows: (1) Polyphosphate-dependent glucokinase (PPGK) can catalyze the conversion of glucose to glucose-6-phosphate using glucose and polyphosphate as substrates. The phosphodiester bond in the polyphosphate provides energy, and no additional ATP is required. (2) Glucose-6-phosphate isomerase (PGI) can isomerize glucose-6-phosphate to fructose-6-phosphate; (3) D-tagatose 1,6-bisphosphate aldolase (GatZ) can convert fructose-6-phosphate into tagatose-6-phosphate and allulose-6-phosphate; (4) Glucose phosphate isomerase / mannose phosphate isomerase difunctional enzyme (PGI / PMI), which can convert glucose-6-phosphate to mannose-6-phosphate; (5) 6-phosphate tagatose phosphatase (TPP) can dephosphorylate mannose-6-phosphate, tagatose-6-phosphate and allulose-6-phosphate to generate D-mannose, D-tagatose and D-allulose, respectively.

[0021] In another specific embodiment of the present invention, the recombinant Escherichia coli is constructed by means of: using wild-type Escherichia coli as the starting strain, and introducing the encoding genes of polyphosphate-dependent glucokinase (PPGK), glucose-6-phosphate isomerase (PGI), D-tagatose 1,6-bisphosphate aldolase (GatZ), glucose phosphate isomerase / mannose phosphate isomerase difunctional enzyme (PGI / PMI) and 6-phosphate tagatose phosphatase (TPP) into the starting strain.

[0022] The wild-type Escherichia coli can be Escherichia coli BL21(DE3).

[0023] Furthermore, the encoding genes of the aforementioned enzymes can be introduced into the starting strain using a recombinant expression vector. Further, the recombinant expression vector includes a first recombinant expression vector and a second recombinant expression vector. The first recombinant expression vector contains the encoding genes for GatZ and PGI / PMI, and the second recombinant expression vector contains the encoding genes for PGI, PPGK, and TPP. Even further, the recombinant expression vector can be a recombinant plasmid. The plasmid backbone of the first recombinant expression vector can be pETDuet, therefore the first recombinant expression vector is pETDuet-GatZ-PGI / PMI. The plasmid backbone of the second recombinant expression vector can be pRSFDuet, therefore the second recombinant expression vector is pRSFDuet-PGI-PPGK-TPP.

[0024] In another specific embodiment of the present invention, the recombinant Escherichia coli is provided for use in the simultaneous production of D-mannose, D-allulose and D-tagatose.

[0025] In another specific embodiment of the present invention, a method for simultaneously producing D-mannose, D-allulose and D-tagatose from glucose is provided. The method includes: using glucose and polyphosphate as substrates, and employing the recombinant Escherichia coli as a catalyst, carrying out a cascade enzyme catalytic reaction in a single reaction system.

[0026] Specifically, the catalytic pathway is as follows: (1) Under the catalysis of PPGK, glucose reacts with polyphosphate to generate glucose-6-phosphate; (2) Glucose-6-phosphate is isomerizes to fructose-6-phosphate under PGI catalysis; at the same time, glucose-6-phosphate isomerizes to mannose-6-phosphate under PGI / PMI catalysis; (3) Fructose-6-phosphate is converted into tagatose-6-phosphate and allulose-6-phosphate under GatZ catalysis; (4) Mannose-6-phosphate, tagatose-6-phosphate and allulose-6-phosphate undergo dephosphorylation reaction under TPP catalysis to generate D-mannose, D-tagatose and D-allulose, respectively.

[0027] In some embodiments of the present invention, the polyphosphate is sodium hexametaphosphate.

[0028] In some embodiments of the present invention, Mg is also added to the reaction system. 2+ .

[0029] In some embodiments of the present invention, the reaction temperature is 60-70°C (preferably 65°C) and the reaction time is 10-30 h (preferably 20 h).

[0030] In some embodiments of the present invention, the initial concentration of glucose in the reaction system is 50-200 g / L (preferably 100 g / L).

[0031] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials, reagents, instruments, and methods used in the following examples are all conventional materials, reagents, instruments, and methods in the art and are commercially available. The culture medium involved in the following examples is LB liquid medium, whose composition is: 5.0 g·L⁻¹ yeast extract. -1 10.0 g·L peptone -1 and NaCl 10.0 g·L -1 .

[0032] The detection methods involved in the following examples are as follows: high performance liquid chromatography (HPLC) detection, Ca 2+ The chromatographic column was pure water, and the flow rate was 0.6 mL / min. The column temperature was 80℃. The detector was a differential refractive index detector.

[0033] Example 1: Construction of pETDuet-GatZ-PGI / PMI and pRSFDuet-PGI-PPGK-TPP First, construct the exogenous plasmid pETDue-GatZ-PGI / PMI (expressing the target gene). gatZ , pgi / pmi ) and pRSFDue-PGI-PPGK-TPP (expressing the target gene) pgi , ppgk , tpp To express five genes from different sources, ppgk The nucleotide sequence is shown in SEQ ID NO.1. pgi / pmi The nucleotide sequence is shown in SEQ ID NO.2. pgi The nucleotide sequence is shown in SEQ ID NO.3. gatZ The nucleotide sequence is shown in SEQ ID NO.4. tpp The nucleotide sequence is shown in SEQ ID NO.5. The specific construction process of the dual plasmid is as follows: Construction process of pETDuet-GatZ-PGI / PMI: Using plasmid pETDuet as a template, primers pETDuet2-RBS-F and PET-R were designed to amplify and obtain the target DNA fragment pETDuet of the assembled plasmid; using the synthesized plasmid pet28a-GatZ as a template, primers GatZ-PET-F and GatZ-PET-R were designed to amplify and obtain the target DNA fragment GatZ-PET of the assembled plasmid; using plasmid pETDuet as a template, primers T7-GatZ-F and T7-GatZ-R were designed to amplify and obtain the target DNA fragment T7-GatZ of the assembled plasmid; using the synthesized plasmid pet28a-GatZ as a template, primers pet28a-GatZ-F and T7-GatZ-R were designed to amplify and obtain the target DNA fragment T7-GatZ of the assembled plasmid; using the synthesized plasmid pet28a-GatZ-PGI / PMI as a template, primers pet28a-GatZ-PGI / PMI-PGI / ... Using 28a-PGI / PMI (a 6×His tag fused to the start codon of this gene, with the sequence catcaccaccaccaccat) as a template, primers PMI-pETRBS-F and PMI-pETRBS-R were designed to amplify the target DNA fragment PMI-PET of the assembled plasmid. The three fragments GatZ-PET, T7-GatZ, and PMI-PET were then fused by PCR to obtain the target fragment GatZ-T7-PMI. Finally, the GatZ-T7-PMI fragment and the amplified vector backbone pETDuet were homologously recombinated to construct the plasmid pETDuet-GatZ-PGI / PMI. Validation primers YZ-PET-F and YZ-PET-R were designed for specific amplification by PCR followed by electrophoresis verification. The correct band size was 2847 bp. After electrophoresis verification, the band size was confirmed to be correct. Sequencing was then performed by a sequencing company, and the sequence was confirmed to be correct, proving the successful construction of the plasmid pETDuet-GatZ-PGI / PMI.

[0034] The construction process of pRSFDuet-PGI-PPGK-TPP is as follows: Using pRSFDuet as a template, primers pRSF-F and pRSF-R are designed to amplify and obtain the target DNA fragment pRSFDuet of the assembled plasmid; using the synthesized plasmid pet28a-PGI as a template, primers PGI-PRSF-F and PGI-PRSF-R are designed to amplify and obtain the target DNA fragment PGI-pRSF of the assembled plasmid; using plasmid pRSFDuet as a template, primers T7-PGI-F and T7-PPGK-R are designed to amplify and obtain the target DNA fragment of the assembled plasmid. The target DNA fragment T7-PGI was amplified using the synthesized plasmid pet28a-PPGK as a template. Primers PPGK-T7-F and PPGK-T7-R were designed to amplify the target DNA fragment PPGK-pRSF in the assembled plasmid. The three fragments PGI-pRSF, T7-PGI, and PPGK-pRSF were then fused by PCR to obtain the target fragment PGI-T7-PPGK. Finally, the fragment PGI-T7-PPGK was homologously recombinated with the amplified vector backbone pRSFDuet to construct the plasmid pRSFDuet-PGI-PPGK. Validation primers YZ-PRSF-F and YZ-PRSF-R were designed and used for specific amplification by PCR followed by electrophoresis verification. The correct band size was 2682 bp. After electrophoresis verification, the band size was confirmed to be correct. The plasmid was then sent to a sequencing company for sequencing verification. The sequence was correct, confirming the successful construction of the plasmid pRSFDuet-PGI-PPGK. Using the constructed plasmid pRSFDuet-PGI-PPGK as a template, the target DNA fragment pRSFDuet-PGI-PPGK of the assembled plasmid was amplified using primers pRSF-F and PPGK-T7-R. Using the synthesized plasmid pet28a-TPP as a template, primers TPP-T7-F and TPP-PRSF-R were designed to amplify the target DNA fragment TPP-pRSF of the assembled plasmid. Using the plasmid pRSFDuet as a template, primers T7-PPGK-F and T7-TPP-R were designed to amplify the target DNA fragment PPGK-T7 of the assembled plasmid. The two fragments PPGK-T7 and TPP-pRSF were fused by PCR to obtain the target fragment T7-TPP. Finally, the fragment T7-TPP and the amplified vector backbone pRSFDuet-PGI-PPGK were homologously recombinated to construct the plasmid pRSFDuet-PGI-PPGK-TPP. The primers YZ-PPGK-F and YZ-PRSF-R were designed and validated. After specific amplification by PCR, electrophoresis was performed to verify the correct band size, which was 1382 bp. After verification by electrophoresis, the band size was confirmed to be correct. The sample was then sent to a sequencing company for sequencing verification. The sequence was confirmed to be correct, proving that the plasmid pRSFDuet-PGI-PPGK-TPP was successfully constructed.The biosynthetic pathways involve gene names, sizes, functions, and sources, as shown in Table 1. The primer sequences for amplifying the coding gene fragments are shown in Table 2. The primer sequences for plasmid detection are shown in Table 3.

[0035] Table 1. Gene names, sizes, functions, and sources involved in biosynthetic pathways.

[0036] Table 2 Primer sequences for fragment amplification

[0037] Table 3 Primer sequences for plasmid detection

[0038] Example 2 Recombinant strain E. coli Construction of ATG-2 Plasmids pETDuet-GatZ-PGI / PMI and pRSFDuet-PGI-PPGK-TPP were simultaneously transformed into *E. coli* BL21(DE3) competent cells. The cells were plated on LB agar plates containing 50 mg / L kanamycin and 100 mg / L ampicillin and cultured overnight. Positive strains with successful dual-plasmid transformation were screened. Single colonies were picked and amplified specifically by PCR, followed by electrophoresis for verification. After successful verification, the cells were sent to a sequencing company for sequencing confirmation. Based on the sequencing results, the successfully constructed dual-plasmid expression strains were preserved. The specific procedures are as follows: Preparation of competent cells from the recipient strain *Escherichia coli* BL21(DE3): *E. coli* BL21(DE3) in glycerol cryovials were streaked onto antibiotic-free LB agar to activate the cells. Single colonies with good morphology were selected and incubated overnight at 37°C and 220 rpm in 5 mL of antibiotic-free LB liquid medium. 1% of the bacterial culture was then transferred to 50 mL of LB liquid medium and cultured until the bacterial OD value reached 90%. 600 Between 0.4 and 0.6. Collect bacterial cells by centrifugation at 8000 rpm at 4°C, resuspend the precipitate in pre-cooled 0.1 mol / L CaCl2 aqueous solution, and then place on ice for 50 min; centrifuge at 5000 rpm at 4°C for 10 min, discard the supernatant, resuspend the precipitate in pre-cooled 0.1 mol / L CaCl2 aqueous solution containing 15% glycerol, aliquot 100 μL into each centrifuge tube, and store at -80°C.

[0039] Transformation: The material stored at -80 ℃... E. coliBL21(DE3) competent cells were placed on ice for 10 min, then plasmids pRSFDuet-PGI-PPGK-TPP and pETDuet-GatZ-PGI / PMI were added in a clean bench, and the cells were incubated on ice for 30 min, then incubated in a 42°C water bath for 90 s, followed by an ice bath for 2 min. 500 µL of LB medium was added, and the cells were cultured at 37°C and 200 rpm for 45 min. The cells were then plated on LB agar plates containing 50 mg / L kanamycin and 100 mg / L ampicillin resistance, and cultured at 37°C for 12–16 h. Single colonies were picked and amplified specifically by PCR, followed by electrophoresis verification.

[0040] Recombinant strain verification: Specific amplification was performed by PCR, followed by electrophoresis to verify whether the two plasmids were successfully transformed into BL21(DE3) competent cells. Specific amplification was performed by PCR using YZ-PET-F and YZ-PET-R primers, followed by electrophoresis to verify whether the plasmid pETDuet-GatZ-PGI / PMI was successfully transformed. The correct band size was 2847 bp. Electrophoresis confirmed the correct band size. The nucleic acid gel results are as follows. Figure 3 As shown. Specific amplification was performed by PCR using YZ-PRSF-F and YZ-PRSF-R primers, followed by electrophoresis to verify successful transformation of the plasmid pRSFDuet-PGI-PPGK-TPP. The correct band size was 3496 bp. Electrophoresis confirmed the correct band size. The nucleic acid gel results are shown below. Figure 4 As shown, after verification, the sample was sent to a sequencing company for sequencing verification. The sequence was correct, thus proving that the plasmids pETDuet-GatZ-PGI / PMI and pRSFDuet-PGI-PPGK-TPP were successfully transformed into BL21(DE3) competent cells, and the recombinant strain was successfully constructed. This strain was named recombinant Escherichia coli. E. coli ATG-2.

[0041] Example 3: Enzyme Preparation The correct recombinant E. coli E. coli ATG-2 was streaked onto LB agar plates containing 50 mg / L kanamycin and 100 mg / L ampicillin resistance, and the culture was incubated at 37 °C for 12–16 h to activate the bacterial strain. Single colonies were picked from the plates and inoculated into test tubes containing 5 mL of resistant LB liquid medium, and incubated overnight (approximately 16 h) at 37 °C and 200 rpm.

[0042] Take 5 mL of the overnight cultured seed culture and transfer it to a shake flask containing 500 mL of LB liquid medium with antibiotic resistance. Incubate at 37 °C and 200 rpm until OD reaches the target. 600Once the concentration reaches approximately 0.4-0.6 (about 3 h), add 50 μL of 1 M IPTG solution to the shake flask to bring the final IPTG concentration to 0.1 mM. Induce overnight at 25 ℃ and 180 rpm.

[0043] The overnight cultured bacterial cells were centrifuged at 7000 rpm for 10 min to collect the cells. Bacterial sludge was weighed and resuspended at a ratio of 0.2 g of bacterial cells to 1 mL of reaction buffer. The bacterial suspension was then disrupted using an ultrasonic disruptor, sonicating for 3 seconds, pausing for 2 seconds, for a total of 3 minutes (the time can be adjusted according to the volume of the ultrasonic device). After centrifugation, the supernatant obtained was the crude enzyme solution of the recombinant strain. The supernatant and precipitate were diluted 10-fold and analyzed by SDS-PAGE. Figure 5 As shown, the target bands of the five enzymes are approximately 45 kDa for GatZ, approximately 38 kDa for PGI / PMI, approximately 53 kDa for PGI, approximately 29 kDa for PPGK, and approximately 24 kDa for TPP. The gel image shows that all five enzymes are soluble and expressed.

[0044] Example 4 Recombinant Escherichia coli E. coli ATG-2 simultaneously produces D-mannose, D-allulose, and D-tagatose from glucose. The reaction system involved in the examples had a D-glucose concentration of 100 g / L and a final sodium hexametaphosphate concentration of 20 mM. 50 μL, 100 μL, 150 μL, and 200 μL of the crude enzyme solution prepared in Example 3 were added, respectively. Then, 20 μL of a 500 mM MgCl2 solution was added to the reaction system to make the Mg... 2+ The final concentration was 10 mM. Finally, 50 mM PB 7.0 buffer was added to bring the reaction volume to 1 mL.

[0045] The reaction system described in the above examples was placed in a constant-temperature reactor at 65°C for 20 hours. Afterward, samples were taken, centrifuged at 12000 rpm for 2 minutes, and the supernatant was filtered through a 0.22 μm filter membrane for high-performance liquid chromatography (HPLC). The conversion rates of the three sugars at different enzyme concentrations are shown in Table 4. The results show that when the enzyme concentration was 150 μL, the conversion rates of D-mannose, D-tagatose, and D-allulose were the highest. The HPLC chromatograms are attached. Figure 6 As shown in the figure. The liquid phase results indicate that, under the above reaction system and conditions, recombinant *E. coli*... E. coliATG-2 can simultaneously produce D-mannose, D-tagatose, and D-alulose from glucose, with conversion rates of approximately 22.15% for D-mannose, 11.56% for D-tagatose, and 59.88% for D-alulose. Analysis of SDS-PAGE results revealed that the expression levels of glucose phosphate isomerase / mannose phosphate isomerase difunctional enzymes PGI / PGM, which catalyze the conversion of glucose-6-phosphate to mannose-6-phosphate, were low, resulting in a relatively low conversion rate of mannose-6-phosphate. Conversely, the expression levels of D-tagatose 1,6-bisphosphate aldolase GatZ, which catalyzes the conversion of fructose-6-phosphate to tagatose-6-phosphate and allulose-6-phosphate, were relatively high, leading to a higher overall conversion rate of tagatose-6-phosphate and allulose-6-phosphate compared to mannose-6-phosphate. This enzyme exhibits substrate inconsistency, with a higher catalytic efficiency for the conversion of fructose-6-phosphate to allulose-6-phosphate than for the conversion of fructose-6-phosphate to tagatose-6-phosphate. Furthermore, the final step catalyzes the dephosphorylation reaction of mannose-6-phosphate, tagatose-6-phosphate, and allulose-6-phosphate to generate D-mannose, D-tagatose, and D-allulose. The phosphatase TPP for allulose-6-phosphate has a stronger affinity for allulose-6-phosphate, so the conversion rate of D-allulose is higher than that of D-mannose and D-tagatose.

[0046] Table 4. Conversion rates of D-mannose, D-tagatose, and D-allulose under different enzyme concentrations

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli simultaneously overexpresses polyphosphate-dependent glucokinase, glucose-6-phosphate isomerase, D-tagatose 1,6-bisphosphate aldolase, glucose phosphate isomerase / mannose phosphate isomerase binocular enzyme, and 6-phosphate tagatose phosphatase.

2. The recombinant Escherichia coli as described in claim 1, characterized in that, The method for constructing the recombinant Escherichia coli includes: using wild-type Escherichia coli as the starting strain, and introducing the encoding genes of polyphosphate-dependent glucokinase (PPGK), glucose-6-phosphate isomerase (PGI), D-tagatose 1,6-bisphosphate aldolase (GatZ), glucose phosphate isomerase / mannose phosphate isomerase difunctional enzyme (PGI / PMI), and 6-phosphate tagatose phosphatase (TPP) into the starting strain.

3. The recombinant Escherichia coli as described in claim 2, characterized in that, In the construction method, the encoding gene of the above enzyme is introduced into the starting strain using a recombinant expression vector; Furthermore, the recombinant expression vector includes a first recombinant expression vector and a second recombinant expression vector, wherein the first recombinant expression vector contains the coding genes for GatZ and PGI / PMI, and the second recombinant expression vector contains the coding genes for PGI, PPGK, and TPP; Furthermore, the recombinant expression vector is a recombinant plasmid, wherein the plasmid backbone of the first recombinant expression vector can be pETDuet; and the plasmid backbone of the second recombinant expression vector is pRSFDuet.

4. The recombinant Escherichia coli as described in claim 2, characterized in that, The wild-type Escherichia coli is Escherichia coli BL21(DE3).

5. The use of the recombinant Escherichia coli according to any one of claims 1-4 in the simultaneous production of D-mannose, D-allulose and D-tagatose.

6. A method for simultaneously producing D-mannose, D-allulose, and D-tagatose from glucose, characterized in that, The method comprises: using glucose and polyphosphate as substrates, and employing the recombinant Escherichia coli according to any one of claims 1-4 as a catalyst, to carry out a cascade enzyme catalytic reaction in a single reaction system.

7. The method as described in claim 6, characterized in that, The polyphosphate is sodium hexametaphosphate.

8. The method as described in claim 6, characterized in that, Mg was also added to the reaction system. 2+ .

9. The method as described in claim 6, characterized in that, The reaction temperature is 60-70℃, and the reaction time is 10-30h.

10. The method as described in claim 6, characterized in that, The initial concentration of glucose in the reaction system is 50-200 g / L.

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