A method for synthesizing D-allulose from D-glucose and D-xylose in Escherichia coli

Abstract

This invention provides a method for synthesizing D-allulose from *Escherichia coli* using D-glucose and D-xylose fermentation. *E. coli* is used as the host cell, and the gene is overexpressed. galP ,Gene glk ,Gene because Genes with SUMO tags alsE and gene a6 PP Construct a pathway for the synthesis of D-allulose using D-glucose and D-xylose as substrates; by knocking out the gene ptsG Knockout gene pfkA、pfkB Silent genes zwf overexpression of genes gdh1、gdh2 Knockout gene crazy Knockout gene fryA The synthesis pathway of D-allulose was optimized; finally, the optimal ratio of mixed sugar fermentation was explored, so as to realize the efficient synthesis of D-allulose by Escherichia coli using mixed sugar fermentation of D-glucose and D-xylose.

Description

Technical Field

[0001] This invention belongs to the field of Escherichia coli metabolic processes, specifically relating to a method for Escherichia coli to synthesize D-allulose using D-glucose and D-xylose. Background Technology

[0002] D-Allulose is the C-3 epimer of D-fructose, with the molecular formula C6H10H2O. 12 O6, with a molecular weight of 180.16, is D-allulose. It has almost zero calories and a sweetness 70% that of sucrose. As a food additive, it is not easily absorbed by the human body, making it a low-energy sweetener and considered an ideal substitute for sucrose. Furthermore, D-allulose possesses various physiological functions, including lowering blood sugar, reducing fat accumulation, preventing obesity, protecting nerves, preventing atherosclerosis, anti-oxidation, and anti-inflammation, showing broad market prospects in the food, beverage, pharmaceutical, and health care industries.

[0003] D-Allulose is extremely rare in nature, and its production currently involves chemical synthesis and biosynthesis. Chemical synthesis is no longer the primary method for industrial D-allulose production due to its complex purification process, significant chemical pollution, and numerous byproducts. Biosynthesis, on the other hand, offers advantages such as specificity, simple product purification, mild conditions, and no pollution. Biosynthesis includes enzymatic catalysis and microbial fermentation. Enzymatic catalysis suffers from drawbacks such as high enzyme prices, a labor-intensive purification process, and limitations to simple one- or two-step reactions. In contrast, the main advantage of microbial fermentation for D-allulose production is that enzyme production and product synthesis can be completed in a single bioreactor, simultaneously addressing issues such as poor enzyme heat resistance, low conversion rates, and high costs. Summary of the Invention

[0004] This invention addresses the aforementioned problems by providing a method for synthesizing D-allulose from D-glucose and D-xylose in *E. coli*. By overexpressing five genes—galP, glk, pgi, sumoalsE, and a6PP—a biosynthetic pathway from D-glucose and D-xylose to D-allulose is constructed in *E. coli*. Then, genes ptsG, pfkA, and pfkB are knocked out, gene zwf is silenced, and genes gdh1 and gdh2 are overexpressed. Further knockout of genes galE and fryA is performed. Finally, the optimal ratio of mixed sugars in fermentation is investigated to increase the yield of D-allulose.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for synthesizing D-allulose from D-glucose and D-xylose in *E. coli* involves using *E. coli* JM109(DE3) as the chassis host bacterium. The method involves introducing genes galP, glk, pgi, sumoalsE, and a6PP to construct a pathway for D-allulose synthesis using D-glucose and D-xylose as substrates in *E. coli*. Further steps include knocking out gene ptsG to achieve mixed sugar utilization; knocking out genes pfkA and pfkB to block the flow of fructose-6-phosphate, an important precursor of D-allulose, to the glycolytic pathway; and silencing gene zwf to regulate... By controlling the carbon flux of the glycolysis and pentose phosphate pathways, more carbon sources were diverted to the D-allulose synthesis pathway. Genes gdh1 and gdh2 were overexpressed to construct a transhydrogenase cycle to optimize cofactor supply. Furthermore, gene galE was knocked out to prevent the conversion of D-allulose to D-sorbose; gene fryA was knocked out to prevent the transport of D-allulose from the extracellular space into the intracellular space, thereby increasing D-allulose production. Finally, the concentration ratio of D-glucose and D-xylose was adjusted to explore the optimal ratio for mixed-sugar fermentation, enabling *E. coli* to efficiently synthesize D-allulose using D-glucose and D-xylose.

[0006] Furthermore, the above-mentioned method for synthesizing D-allulose from D-glucose and D-xylose by Escherichia coli includes the following steps: 1) Using Escherichia coli JM109(DE3) as the chassis host bacterium, the genes galP, glk, pgi, sumoalsE and a6PP were overexpressed to obtain recombinant E. coli (galP, glk, pgi, sumoalsE, a6PP); 2) Based on E. coli (galP, glk, pgi, sumoalsE, a6PP), the ptsG gene was knocked out to obtain recombinant E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG); 3) Based on E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG), the genes pfkA and pfkB were knocked out sequentially to obtain recombinant E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB); 4) Based on E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB), the zwf gene was silenced and the gdh1 and gdh2 genes were overexpressed to obtain recombinant E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB); 5) Based on E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB), the genes galE and fryA were knocked out sequentially to obtain recombinant E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA); 6) Using D-glucose and D-xylose at different mass concentrations as substrates, E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) was fed with the mixture to produce D-allulose, and the optimal ratio for mixed sugar fermentation was investigated.

[0007] In step 1) above, the nucleotide sequence of gene galP is shown in SEQ ID NO.1; the nucleotide sequence of gene glk is shown in SEQ ID NO.2; the nucleotide sequence of gene pgi is shown in SEQ ID NO.3; the nucleotide sequence of gene sumoalsE is shown in SEQ ID NO.4; and the nucleotide sequence of gene a6PP is shown in SEQ ID NO.5.

[0008] In step 2) above, the nucleotide sequence of the gene ptsG is shown in SEQ ID NO.6.

[0009] In step 3) above, the nucleotide sequence of gene pfkA is shown in SEQ ID NO.7; the nucleotide sequence of gene pfkB is shown in SEQ ID NO.8.

[0010] In step 4) above, the nucleotide sequence of gene zwf is shown in SEQ ID NO.9; the nucleotide sequence of gene gdh1 is shown in SEQ ID NO.11; and the nucleotide sequence of gene gdh2 is shown in SEQ ID NO.12.

[0011] In step 5) above, the nucleotide sequence of gene galE is shown in SEQ ID NO.13; the nucleotide sequence of gene fryA is shown in SEQ ID NO.14.

[0012] In step 6) above, the fermentation method is as follows: E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) is inoculated into a solution containing 50 μg / mL Kan RAntibiotics and 30 μg / mL Cm R After culturing the antibiotic in LB liquid medium at 37°C and 220 rpm for 14–16 h, it was then transferred to a medium containing 50 μg / mL Kans. R Antibiotics, 30 μg / mL Cm R In LB liquid medium containing antibiotics, D-glucose, and D-xylose, cultured at 37°C and 220 rpm until OD 600 When the pH value reaches 0.6–0.8, add IPTG to a final concentration of 0.2 mM and ferment at 37°C and 220 rpm. The optimal ratio for mixed sugar fermentation is: a total mass concentration of 8 g / L for D-glucose and D-xylose, and a mass concentration ratio of 1:2 for D-glucose and D-xylose.

[0013] The above-mentioned method for synthesizing D-allulose from D-glucose and D-xylose by Escherichia coli is applied in the production of D-allulose.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention successfully constructed a pathway for the synthesis of D-allulose from D-glucose and D-xylose in Escherichia coli. By rationally regulating carbon flux through gene knockout, gene silencing, and optimization of cofactor supply, a recombinant strain, E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA), was obtained. This recombinant strain can effectively co-utilize inexpensive D-glucose and D-xylose to produce D-allulose, achieving a yield of 1.95 g / L. Attached Figure Description

[0015] Figure 1 Diagram of the cell factory in Escherichia coli synthesizing D-allulose.

[0016] Figure 2 A is a schematic diagram of the construction of pRSFDuet-galP-glk-pgi; B is a schematic diagram of the construction of pACYCDuet-sumoalsE-a6PP; C is a schematic diagram of the construction of pETDuet-aszwf-gdh1-gdh2.

[0017] Figure 3 Image A shows the fermentation products of E. coli JM109(DE3)-pRSFDuet-pACYCDuet; Image B shows the fermentation products of E. coli (galP,glk,pgi,sumoalsE,a6PP); Image C shows the fermentation products of E. coli (galP,glk,pgi,sumoalsE,a6PP,ΔptsG).

[0018] Figure 4 Image A shows the fermentation products of E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA); Image B shows the fermentation products of E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB); Image C shows the fermentation products of E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, ΔptsG, ΔpfkA, ΔpfkB).

[0019] Figure 5 Image A shows the fermentation products of E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB); Image B shows the fermentation products of E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE); Image C shows the fermentation products of E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA).

[0020] Figure 6 : Exploring the optimal ratio of D-glucose to D-xylose in mixed sugar fermentation. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to the embodiments. It should be noted that the embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0022] The metabolic diagram of the recombinant strain E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) synthesizing D-allulose from D-glucose and D-xylose in this invention is shown below. Figure 1 As shown; a schematic diagram of recombinant plasmid construction is shown below. Figure 2 As shown.

[0023] The nucleotide sequences of the galactose permease gene galP in this invention are shown in SEQ ID NO.1; the nucleotide sequence of the glucokinase gene glk is shown in SEQ ID NO.2; the nucleotide sequence of the glucose-6-phosphate isomerase gene pgi is shown in SEQ ID NO.3; the nucleotide sequence of the D-allulose-6-phosphate epimerase gene alsE (i.e., sumoalsE gene) with the fusion protein tag SUMO is shown in SEQ ID NO.4; the nucleotide sequence of the D-allulose-6-phosphate phosphatase gene a6PP is shown in SEQ ID NO.5; the nucleotide sequence of the glucose transporter gene ptsG is shown in SEQ ID NO.6; the nucleotide sequence of the D-fructose-6-phosphate kinase gene pfkA is shown in SEQ ID NO.7; the nucleotide sequence of the D-fructose-6-phosphate kinase gene pfkB is shown in SEQ ID NO.8; the nucleotide sequence of the glucose-6-phosphate dehydrogenase gene zwf is shown in SEQ ID NO.9; and the antisense RNA gene targeting the glucose-6-phosphate dehydrogenase gene zwf (i.e., antisense RNA gene) is also shown in SEQ ID NO.9. The nucleotide sequence of the zwf gene (also known as the aszwf gene) is shown in SEQ ID NO.10; the nucleotide sequence of the gdh1 glutamate transhydrogenase gene is shown in SEQ ID NO.11; the nucleotide sequence of the gdh2 glutamate transhydrogenase gene is shown in SEQ ID NO.12; the nucleotide sequence of the galE UDP-glucose-4-epimerase gene is shown in SEQ ID NO.13; and the nucleotide sequence of the fryA key gene for D-allulose internal transporter is shown in SEQ ID NO.14.

[0024] Example 1: LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, the remainder is water.

[0025] Genes galP, glk, and pgi were ligated into plasmid pRSFDuet-1 to obtain the recombinant plasmid pRSFDuet-galP-glk-pgi; genes sumoalsE and a6PP were ligated into plasmid pACYCDuet-1 to obtain the recombinant plasmid pACYCDuet-sumoalE-a6PP. Plasmids pRSFDuet-1 and pACYCDuet-1 were simultaneously transformed into E. coli JM109(DE3) via electroporation to obtain the recombinant strain E. coli JM109(DE3)-pRSFDuet-pACYCDuet. The recombinant plasmids pRSFDuet-galP-glk-pgi and pACYCDuet-sumoalE-a6PP were simultaneously transformed into E. coli JM109(DE3) via electroporation to obtain the recombinant strain E. coli(galP,glk,pgi,sumoalsE,a6PP). Based on E. coli(galP,glk,pgi,sumoalsE,a6PP), the ptsG gene was knocked out using the λ-Red homologous recombination method to obtain the recombinant strain E. coli(galP,glk,pgi,sumoalsE,a6PP,ΔptsG).

[0026] Single colonies of the above recombinant bacteria were inoculated into 4 mL of solution containing 50 μg / mL Kansin. R Antibiotics and 30 μg / mL Cm R After culturing the antibiotic in LB liquid medium at 37°C and 220 rpm for 14–16 h, 1 mL of the culture medium was transferred to 50 mL of medium containing 50 μg / mL Kansin. R Antibiotics, 30 μg / mL Cm R In LB liquid medium containing antibiotics, 4 g / L D-glucose, and 4 g / L D-xylose, cultured at 37°C and 220 rpm until OD500. 600 When the protein expression value reached 0.6–0.8, IPTG at a final concentration of 0.2 mM was added to induce protein expression. Fermentation was carried out at 37°C and 220 rpm for a total fermentation time of 72 h. Samples were taken every 12 h during the fermentation period, and the OD was measured. 600 Values ​​were determined. High-performance liquid chromatography (HPLC) and a refractive index detector were used for quantitative detection of fermentation products. D-glucose, D-xylose, and D-alulose were analyzed using a Sugar-Pak™ I column (6.5 × 300 mm). The mobile phase was Wahaha purified water, which required pre-filtration and sonication for 1 h. The flow rate was 0.5 mL / min, the column temperature was 85℃, the injection volume was 10 μL, and the sample retention time was 20 min. The analytical results are as follows: Figure 3As shown, no D-allulose was detected in the fermentation products of E. coli JM109(DE3)-pRSFDuet-pACYCDuet, indicating a carbon catabolite repression (CCR) effect from the perspective of sugar consumption. D-allulose was produced in the fermentation products of E. coli (galP,glk,pgi,sumoalsE,a6PP) at a yield of 0.07 g / L, also exhibiting a CCR effect. However, E. coli (galP,glk,pgi,sumoalsE,a6PP,ΔptsG) eliminated the CCR effect, achieving co-utilization of mixed sugars through fermentation, increasing the D-allulose yield to 0.31 g / L. Therefore, the feasibility of the proposed method can be demonstrated.

[0027] Example 2: Flux regulation of the EMP and PP pathways was employed to enhance D-allulose synthesis. Based on *E. coli* (galP, glk, pgi, sumoalsE, a6PP, ΔptsG), the genes pfkA and pfkB were sequentially knocked out using λ-Red homologous recombination to block the flow of fructose-6-phosphate, an important precursor of D-allulose, to the EMP pathway, resulting in recombinant strains *E. coli* (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA) and *E. coli* (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB). The gene aszwf was ligated into plasmid pETDuet-1 to obtain the recombinant plasmid pETDuet-aszwf. The recombinant plasmid pETDuet-aszwf was then transformed into E. coli (galP,glk,pgi,sumoalsE,a6PP,ΔptsG,ΔpfkA,ΔpfkB) by electroporation to silence the zwf gene, reduce the flux of glucose-6-phosphate to the PP pathway, maximize its flow to the glycolysis pathway to synthesize fructose-6-phosphate, reduce carbon source loss, and enhance the synthesis of D-allulose, resulting in the recombinant strain E. coli (galP,glk,pgi,sumoalsE,a6PP,aszwf,ΔptsG,ΔpfkA,ΔpfkB).

[0028] The obtained recombinant bacteria were fermented and verified according to the method in Example 1. Figure 4 As shown, knocking out genes pfkA and pfkB and silencing gene zwf increased the production of D-allulose from 0.31 g / L to 1.0 g / L.

[0029] Example 3: Genes aszwf, gdh1, and gdh2 were ligated into plasmid pETDuet-1 to obtain the recombinant plasmid pETDuet-aszwf-gdh1-gdh2. The recombinant plasmid pETDuet-aszwf-gdh1-gdh2 was then transformed into *E. coli* (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB) via electroporation to construct a transhydrogenase cycle and optimize cofactor supply, resulting in recombinant *E. coli* (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB). Then, based on *E. coli* (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB), λ-... The galE gene was knocked out using the Red homologous recombination method to prevent the further conversion of D-allulose to D-sorbose, resulting in the recombinant strain E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE). Further, the fryA gene was knocked out using the λ-Red homologous recombination method on top of E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE) to prevent the translocation of D-allulose from the extracellular space into the intracellular space, resulting in the recombinant strain E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA).

[0030] The obtained recombinant bacteria were fermented and verified according to the method in Example 1. Figure 5 As shown, the yield of D-allulose in E. coli (galP,glk,pgi,sumoalsE,a6PP,aszwf,gdh1,gdh2,ΔptsG,ΔpfkA,ΔpfkB,ΔgalE,ΔfryA) was increased to 1.34 g / L.

[0031] E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) was fermented and verified according to the method in Example 1, except that the D-glucose:D-xylose mass concentration ratio was adjusted to 3:1, 2:1, 1:1, 1:2, and 1:3 while maintaining the total mass concentration of substrate (i.e., D-glucose + D-xylose) in the culture medium at 8 g / L, to explore the optimal D-glucose:D-xylose ratio for mixed sugar fermentation. Figure 6 As shown, when the mass concentration ratio of D-glucose to D-xylose is 1:2, the yield of D-allulose in E. coli (galP,glk,pgi,sumoalsE,a6PP,aszwf,gdh1,gdh2,ΔptsG,ΔpfkA,ΔpfkB,ΔgalE,ΔfryA) is the highest, at 1.95 g / L.

[0032] The above descriptions are all preferred embodiments of the present invention. All equivalent changes and modifications made in accordance with the scope of the claims of the present invention shall fall within the scope of the present invention.

Claims

1. A method for synthesizing D-allulose from Escherichia coli using D-glucose and D-xylose, characterized in that: by E. coli JM109 (DE3) As a chassis host bacterium, through the introduction of genes galP, glk, pgi, sumoalsE, a6PP A pathway for the synthesis of D-allulose using D-glucose and D-xylose as substrates was constructed in E. coli; the gene was then knocked out. ptsG To achieve the co-utilization of mixed sugars; further gene knockout pfkA, pfkB By blocking the flow of fructose-6-phosphate, an important precursor of D-allulose, to the glycolytic pathway; further silencing genes. zwf By regulating carbon flux in the glycolysis and pentose phosphate pathways, more carbon sources are diverted to the D-allulose synthesis pathway, and the gene is overexpressed. gdh1, gdh2 To construct a transhydrogenase cycle to optimize cofactor supply; further gene knockout galE To prevent the conversion of D-allulose to D-sorbose; further gene knockout fryA To prevent D-allulose from being transported from the extracellular space into the intracellular space, thereby increasing the yield of D-allulose; finally, the concentration ratio of D-glucose and D-xylose was adjusted to explore the optimal ratio for mixed sugar fermentation, so as to realize the efficient synthesis of D-allulose by Escherichia coli using D-glucose and D-xylose. Among them, genes gdh1 The nucleotide sequence is shown in SEQ ID NO.11; gene gdh2 The nucleotide sequence is shown in SEQ ID NO. 12; The optimal ratio for mixed sugar fermentation is: a total mass concentration of 8 g / L for D-glucose and D-xylose, and a mass concentration ratio of 1:2 for D-glucose and D-xylose.

2. The method according to claim 1, characterized in that: Includes the following steps: 1) With Escherichia coli E. coli JM109 (DE3) As a chassis host bacterium, it overexpresses genes. galP, glk, pgi, sumoalsE and a6PP Recombinant bacteria were obtained. E.coli (galP, glk, pgi, sumoalsE, a6PP) ; 2) In E.coli (galP, glk, pgi, sumoalsE, a6PP) Based on this, knock out genes ptsG Recombinant bacteria were obtained. E.coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG) ; 3) In E.coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG) Based on this, knock out genes sequentially. pfkA, pfkB Recombinant bacteria were obtained. E.coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, Δ pfkB) ; 4) In E.coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB) Based on this, silent genes zwf and overexpress gene gdh1 and gdh2 Recombinant bacteria were obtained. E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB) ; 5) In E.coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, Δ pfkA, ΔpfkB) Based on this, knock out genes sequentially. galE、fryA Recombinant bacteria were obtained. E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) ; 6) Using D-glucose and D-xylose at different mass concentration ratios as substrates for supply E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) Fermentation production of D-allulose: exploring the optimal ratio of mixed sugars for fermentation.

3. The method according to claim 2, characterized in that: In step 1), genes galP The nucleotide sequence is shown in SEQ ID NO.1; gene glk The nucleotide sequence is shown in SEQ ID NO.2; gene pgi The nucleotide sequence is shown in SEQ ID NO. 3; gene sumoalsE The nucleotide sequence is shown in SEQ ID NO.4; gene a6PP The nucleotide sequence is shown in SEQ ID NO.

5.

4. The method according to claim 2, characterized in that: In step 2), genes ptsG The nucleotide sequence is shown in SEQ ID NO.

6.

5. The method according to claim 2, characterized in that: In step 3), genes pfkA The nucleotide sequence is shown in SEQ ID NO.7; gene pfkB The nucleotide sequence is shown in SEQ ID NO.

8.

6. The method according to claim 2, characterized in that: In step 4), genes zwf The nucleotide sequence is shown in SEQ ID NO.

9.

7. The method according to claim 2, characterized in that: In step 5), the gene galE The nucleotide sequence is shown in SEQ ID NO. 13; gene fryA The nucleotide sequence is shown in SEQ ID NO.

14.

8. The method according to claim 2, characterized in that: In step 6), the fermentation method is as follows: E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) Inoculated with 50 μg / mL Kan R Antibiotics and 30 μg / mL Cm R After culturing the antibiotic in LB liquid medium at 37°C and 220 rpm for 14–16 h, it was then transferred to a medium containing 50 μg / mL Kans. R Antibiotics, 30 μg / mL Cm R In LB liquid medium containing antibiotics, D-glucose, and D-xylose, cultured at 37°C and 220 rpm until OD 600 When the pH reaches 0.6~0.8, add IPTG to a final concentration of 0.2mM and ferment at 37℃ and 220rpm.

9. The use of the method according to any one of claims 1 to 8 in the production of D-allulose.

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