Method for synthesizing D-psicose from D-glucose and D-xylose by escherichia coli

Abstract

The invention provides a method for synthesizing D-psicose through fermentation of escherichia coli by using D-glucose and D-xylose. The method comprises the following steps: by taking escherichia coli as a host cell, over-expressing a galactose permease gene galP, a glucokinase gene glk, a 6-phosphate glucose isomerase gene pgi, a D-psicose-6-phosphate epimerase gene alsE with an SUMO tag and a D-psicose-6-phosphate phosphatase gene a6PP, and constructing a path for synthesizing D-psicose by taking D-glucose and D-xylose as substrates; the synthetic route of D-psicose is optimized by knocking out a D-glucose phosphorylation transport pathway gene ptsG, knocking out D-fructose-6-phosphokinase genes pfkA and pfkB, silencing a glucose-6-phosphate dehydrogenase gene zwf, overexpressing glutamate transhydrogenase genes gdh1 and gdh2, knocking out a UDP-glucose-4-epimerase gene galE and knocking out a transport protein key gene fryA in D-psicose; and finally, the optimal proportion of mixed sugar fermentation is explored, so that efficient synthesis of D-psicose from escherichia coli through mixed sugar fermentation of D-glucose and D-xylose is realized.

Description

Technical Field

[0001] The invention belongs to the field of Escherichia coli metabolic process, and specifically relates to a method for synthesizing D-psicose by using D-glucose and D-xylose in Escherichia coli. Background Art

[0002] D-psicose is the C-3 diastereomer of D-fructose, and its molecular formula is C6H 12 O6, molecular weight is 180.16. The calories of D-psicose are almost zero, and its sweetness is 70% of that of sucrose. When used as a food additive, it is not easily absorbed by the human body. It is a low-energy sweetener and is considered to be an ideal substitute for sucrose. In addition, D-psicose has multiple physiological functions such as lowering blood sugar, reducing fat accumulation, preventing obesity, protecting nerves, resisting atherosclerosis, anti-oxidation, and anti-inflammation, showing broad market prospects in the fields of food, beverages, medicine and health care.

[0003] D-psicose is very rare in nature. Currently, there are two methods for producing D-psicose: chemical synthesis and biosynthesis. Chemical synthesis is no longer the main method for industrial preparation of D-psicose due to complex product purification steps, serious chemical pollution and many by-products. The biosynthesis method has the advantages of specificity, simple product purification, mild conditions and no pollution. The biosynthesis method includes enzyme catalysis and microbial fermentation. Among them, the enzyme catalysis method has the problems of high enzyme price, large workload in the refining process, and limited to one or two simple reactions. In contrast, the main advantage of the microbial fermentation method for producing D-psicose is that the production of enzymes and the synthesis of products can be completed in one bioreactor, while solving the problems of poor enzyme heat resistance, low conversion rate and high cost. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for synthesizing D-psicose by using D-glucose and D-xylose in Escherichia coli. The biosynthetic pathway from D-glucose and D-xylose to D-psicose is constructed in Escherichia coli by overexpressing five genes, namely, galP, glk, pgi, sumoalsE and a6PP, and then knocking out genes ptsG, pfkA and pfkB, silencing gene zwf, overexpressing two genes, namely, gdh1 and gdh2, further knocking out genes galE and fryA, and finally exploring the optimal ratio of mixed sugar fermentation, thereby increasing the yield of D-psicose.

[0005] To achieve the above object, the present invention adopts the following technical solution: A method for synthesizing D-psicose by using D-glucose and D-xylose by Escherichia coli, which uses E. coli JM109 (DE3) as a chassis host bacterium, introduces genes galP, glk, pgi, sumoalsE, and a6PP, constructs a pathway for synthesizing D-psicose by using D-glucose and D-xylose as substrates in Escherichia coli; further knocks out the gene ptsG to achieve mixed sugar co-utilization; further knocks out the genes pfkA and pfkB to block the flow of fructose-6-phosphate, an important precursor of D-psicose, to the glycolysis pathway; further silences the gene zwf to regulate The carbon flux of the glycolysis pathway and the pentose phosphate pathway was controlled so that more carbon sources flowed to the D-psicose synthesis pathway, and the genes gdh1 and gdh2 were overexpressed to construct a transhydrogenase cycle to optimize the cofactor supply; the gene galE was further knocked out to prevent the conversion of D-psicose into D-sorbose; the gene fryA was further knocked out to prevent D-psicose from being transported from the extracellular space into the intracellular space, thereby increasing the yield of D-psicose; finally, the concentration ratio of D-glucose and D-xylose was adjusted to explore the optimal ratio of mixed sugar fermentation, so as to enable Escherichia coli to efficiently synthesize D-psicose using D-glucose and D-xylose.

[0006] Furthermore, the method for synthesizing D-psicose by using D-glucose and D-xylose in Escherichia coli comprises the following steps: 1) Using E. coli JM109 (DE3) as the chassis host bacteria, overexpressing the genes galP, glk, pgi, sumoalsE and a6PP to obtain recombinant bacteria E. coli (galP, glk, pgi, sumoalsE, a6PP); 2) Based on E. coli (galP, glk, pgi, sumoalsE, a6PP), the gene ptsG was knocked out to obtain the recombinant bacteria 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 in sequence to obtain the recombinant bacteria E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB); 4) On the basis of E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB), the gene zwf was silenced and the genes gdh1 and gdh2 were overexpressed to obtain the recombinant bacteria 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 in sequence to obtain the recombinant bacteria E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA); 6) Different mass concentration ratios of D-glucose and D-xylose were used as substrates to feed E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) to produce D-psicose by fermentation, and the optimal ratio of mixed sugar fermentation was explored.

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

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

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

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

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

[0012] In the above step 6), the fermentation method is: inoculate E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) in a solution containing 50 μg / mL Kan RAntibiotics and 30 μg / mL Cm R The culture medium was incubated at 37°C and 220 rpm for 14-16 h in LB liquid medium containing antibiotics, and then transferred to a medium containing 50 μg / mL Kan R Antibiotics, 30 μg / mL Cm R The culture was carried out in LB liquid medium containing antibiotics, D-glucose and D-xylose at 37°C and 220 rpm until OD 600 When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.2 mM and fermented at 37 °C and 220 rpm; The optimum ratio of mixed sugar fermentation is: the total mass concentration of D-glucose and D-xylose is 8 g / L, and the mass concentration ratio of D-glucose and D-xylose is 1:2.

[0013] The method for synthesizing D-psicose by using D-glucose and D-xylose by Escherichia coli is used in producing D-psicose.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention successfully constructs a pathway for synthesizing D-psicose from D-glucose and D-xylose in Escherichia coli, and rationally regulates carbon flux through methods such as gene knockout, gene silencing, and optimization of cofactor supply, thereby obtaining recombinant bacteria E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA). The recombinant bacteria can effectively utilize cheap D-glucose and D-xylose to produce D-psicose, and the D-psicose yield reaches 1.95 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 :Diagram of the cell factory in which Escherichia coli synthesizes D-psicose.

[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 : A is a picture of the fermentation products of E. coli JM109 (DE3) -pRSFDuet-pACYCDuet; B is a picture of the fermentation products of E. coli (galP, glk, pgi, sumoalsE, a6PP); C is a picture of the fermentation products of E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG).

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

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

[0020] Figure 6 :To explore the optimal ratio of D-glucose:D-xylose in mixed sugar fermentation. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with an embodiment. It should be pointed out that the embodiment is only used to explain the present invention rather than 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) of the present invention for synthesizing D-psicose from D-glucose and D-xylose is shown in FIG. Figure 1 As shown; the schematic diagram of recombinant plasmid construction is shown Figure 2 shown.

[0023] The nucleotide sequence of the galactose permease gene galP in the present invention is shown in SEQ ID NO.1; the nucleotide sequence of the glucose kinase gene glk is shown in SEQ ID NO.2; the nucleotide sequence of the 6-phosphoglucose isomerase gene pgi is shown in SEQ ID NO.3; the nucleotide sequence of the D-psicose-6-phosphate epimerase gene alsE (i.e., sumoalsE gene) with a fusion protein tag SUMO is shown in SEQ ID NO.4; the nucleotide sequence of the D-psicose-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 shown in SEQ ID NO. 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 glutamate transhydrogenase gene gdh1 is shown in SEQ ID NO.11; the nucleotide sequence of the glutamate transhydrogenase gene gdh2 is shown in SEQ ID NO.12; the nucleotide sequence of the UDP-glucose-4-isomerase gene galE is shown in SEQ ID NO.13; and the nucleotide sequence of the D-psicose transporter key gene fryA is shown in SEQ ID NO.14.

[0024] Embodiment 1: LB liquid culture medium: 5 g / L yeast powder, 10 g / L tryptone, 10 g / L sodium chloride, and the rest is water.

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

[0026] The single colonies of the above recombinant bacteria were inoculated into 4 mL of 50 μg / mL Kan R Antibiotics and 30 μg / mL Cm R After culturing in LB liquid medium containing antibiotics at 37°C and 220 rpm for 14-16 h, 1 mL of the culture medium was transferred to 50 mL of LB liquid medium containing 50 μg / mL Kan R Antibiotics, 30 μg / mL Cm R The culture was carried out in LB liquid medium containing antibiotics, 4 g / L D-glucose and 4 g / L D-xylose at 37 °C and 220 rpm until OD 600 When the value reached 0.6-0.8, IPTG with a final concentration of 0.2 mM was added to induce protein expression. The fermentation was carried out at 37 °C and 220 rpm for a total of 72 h. Samples were taken every 12 h during the fermentation and the OD was measured. 600 The fermentation products were quantitatively detected using high performance liquid chromatography and a refractive index detector. D-glucose, D-xylose, and D-psicose were analyzed using a Sugar-PakTMⅠ column (6.5×300mm). The mobile phase was Wahaha purified water, which was pre-membrane-passed and ultrasonicated for 1h. The flow rate was 0.5mL / min, the column temperature was 85℃, the injection volume was 10μL, and the sample retention time was 20min. The analysis results are shown in Figure 3As shown, D-psicose was not detected in the fermentation product of E. coli JM109 (DE3) -pRSFDuet-pACYCDuet, and there was a carbon catabolite repression (CCR) effect from the perspective of sugar consumption; D-psicose was produced in the fermentation product of E. coli (galP, glk, pgi, sumoalsE, a6PP), with a yield of 0.07 g / L, and there was also a CCR effect; while E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG) eliminated the CCR effect, achieved mixed sugar fermentation and co-utilization, and the D-psicose yield was increased to 0.31 g / L. Therefore, it can be proved that the path of the present invention is feasible.

[0027] Embodiment 2: Flux regulation of the EMP and PP pathways was performed to enhance D-psicose synthesis. Based on E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG), the genes pfkA and pfkB were knocked out in sequence using the λ-Red homologous recombination method to block the flow of fructose-6-phosphate, an important precursor of D-psicose, to the EMP pathway, and the recombinant strains E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA) and E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB) were obtained, respectively. The gene aszwf was connected to the plasmid pETDuet-1 to obtain the recombinant plasmid pETDuet-aszwf; the recombinant plasmid pETDuet-aszwf was transformed into E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB) by electroporation to silence the gene zwf, reduce the flux of glucose-6-phosphate to the PP pathway, make it flow to the glycolysis pathway to the greatest extent to synthesize fructose-6-phosphate, reduce the loss of carbon source, and enhance the synthesis of D-psicose, so as to obtain the recombinant bacteria 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, after knocking out the genes pfkA and pfkB and silencing the gene zwf, the production of D-psicose increased from 0.31 g / L to 1.0 g / L.

[0029] Embodiment 3: The genes aszwf, gdh1, and gdh2 were connected to the plasmid pETDuet-1 to obtain the recombinant plasmid pETDuet-aszwf-gdh1-gdh2; the recombinant plasmid pETDuet-aszwf-gdh1-gdh2 was transformed into E. coli (galP, glk, pgi, sumoalsE, a6PP, ΔptsG, ΔpfkA, ΔpfkB) by electroporation to construct a transhydrogenase cycle to optimize the cofactor supply and obtain the recombinant bacteria 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) was used. The galE gene was knocked out by the λ-Red homologous recombination method to prevent the further conversion of D-psicose into D-sorbose, and the recombinant bacteria E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE) were obtained. The fryA gene was knocked out by the λ-Red homologous recombination method based on E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE) to prevent D-psicose from being transported from the extracellular into the intracellular, and the recombinant strain E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) was obtained.

[0030] The obtained recombinant bacteria were fermented and verified according to the method in Example 1. Figure 5 As shown, the production of D-psicose 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 according to the method in Example 1, except that the mass concentration ratio of D-glucose:D-xylose was adjusted to 3:1, 2:1, 1:1, 1:2, 1:3 respectively under the condition of 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 ratio of D-glucose:D-xylose in mixed sugar fermentation. Figure 6 As shown, when the mass concentration ratio of D-glucose:D-xylose was 1:2, the production of D-psicose of E. coli (galP, glk, pgi, sumoalsE, a6PP, aszwf, gdh1, gdh2, ΔptsG, ΔpfkA, ΔpfkB, ΔgalE, ΔfryA) was the highest, which was 1.95 g / L.

[0032] The above are all preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for synthesizing D-psicose by using D-glucose and D-xylose in Escherichia coli, characterized in that: by E. coli JM109 (DE3) is the chassis host strain, and the gene galP, glk, pgi, sumoalsE, a6PP Constructing a pathway for synthesizing D-psicose using D-glucose and D-xylose as substrates in Escherichia coli; further knocking out the gene ptsG To achieve mixed sugar utilization; further knock out the gene pfkA, pfkB Block fructose-6-phosphate, an important precursor of D-psicose, from flowing into the glycolysis pathway; further silence the gene wxya To regulate the carbon flux of the glycolysis pathway and the pentose phosphate pathway so that more carbon sources flow to the D-psicose synthesis pathway and overexpress the gene gdh1, gdh2 To construct the transhydrogenase cycle to optimize the cofactor supply; further knockout genes galE To prevent the conversion of D-psicose to D-sorbose; further knockout the gene fryA To prevent D-psicose from being transported from outside the cell into the cell, thereby increasing the yield of D-psicose; finally, the concentration ratio of D-glucose and D-xylose was adjusted to explore the optimal ratio of mixed sugar fermentation, so that Escherichia coli can efficiently synthesize D-psicose using D-glucose and D-xylose.

2. The method according to claim 1, characterized in that: The following steps are involved: 1) Escherichia coli E. coli JM109 (DE3) is the chassis host strain, overexpressing genes galP, glk, pgi, sumoalsE and 6P , and obtain recombinant bacteria E. coli ( galP , glk , pgi , sumoalsE , 6P ); 2) In E. coli ( galP , glk , pgi , sumoalsE , 6P ) based on the knockout gene ptsG , and obtain recombinant bacteria E. coli ( galP , glk , pgi , sumoalsE , 6P ,Δ ptsG ); 3) In E. coli ( galP , glk , pgi , sumoalsE , 6P ,Δ ptsG ) based on the knockout genes pfkA, pfkB , and obtain recombinant bacteria E. coli ( galP , glk , pgi , sumoalsE , 6P ,Δ ptsG ,Δ pkq ,Δ pkq ); 4) In E. coli ( galP , glk , pgi , sumoalsE , 6P ,Δ ptsG ,Δ pkq ,Δ pkq ) based on the silencing gene wxya , and overexpressed genes gdh1 and gdh2 , and obtain recombinant bacteria E. coli ( galP , glk , pgi , sumoalsE , 6P , wxya , gdh1 , gdh2 ,Δ ptsG , Δ pkq , Δ pkq ); 5) In E. coli ( galP , glk , pgi , sumoalsE , 6P , wxya , gdh1 , gdh2 ,Δ ptsG , Δ pkq , Δ pkq ) based on the knockout genes galE、fryA , and obtain recombinant bacteria E. coli ( galP , glk , pgi , sumoalsE , 6P , wxya , gdh1 , gdh2 ,Δ ptsG , Δ pkq , Δ pkq , Δ galE , Δ fryA ); 6) Using D-glucose and D-xylose at different mass concentration ratios as substrates E. coli ( galP , glk , pgi , sumoalsE , 6P , wxya , gdh1 , gdh2 ,Δ ptsG , Δ pkq , Δ pkq , Δ galE , Δ fryA ) fermentation to produce D-psicose and explore the optimal ratio of mixed sugar fermentation.

3. The method according to claim 2, characterized in that: In step 1), the gene galP The nucleotide sequence of gene is shown in SEQ ID NO.1; glk The nucleotide sequence of gene is shown in SEQ ID NO.2; pgi The nucleotide sequence of gene is shown in SEQ ID NO.3; sumoalsE The nucleotide sequence of gene a6 is shown in SEQ ID NO.4; PP The nucleotide sequence is shown as SEQ ID NO.

5.

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

6.

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

8.

6. The method according to claim 2, characterized in that: In step 4), the gene wxya The nucleotide sequence of gene is shown in SEQ ID NO.9; gdh1 The nucleotide sequence of gene is shown in SEQ ID NO.11; gdh2 The nucleotide sequence is shown in SEQ ID NO.

12.

7. The method according to claim 2, characterized in that: In step 5), the gene galE The nucleotide sequence of gene is shown in SEQ ID NO.13; 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: E. coli ( galP , glk , pgi , sumoalsE , 6P , wxya , gdh1 , gdh2 ,Δ ptsG , Δ pkq , Δ pkq , Δ galE , Δ fryA ) inoculated with 50 μg / mL Kan R Antibiotics and 30 μg / mL Cm R The culture medium was incubated at 37°C and 220 rpm for 14-16 h in LB liquid medium containing antibiotics, and then transferred to a medium containing 50 μg / mL Kan R Antibiotics, 30 μg / mL Cm R The culture was carried out in LB liquid medium containing antibiotics, D-glucose and D-xylose at 37°C and 220 rpm until OD 600 When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.2 mM and fermented at 37 °C and 220 rpm.

9. The method according to claim 8, characterized in that: The total mass concentration of D-glucose and D-xylose was 8 g / L, and the mass concentration ratio of D-glucose to D-xylose was 1:

2.

10. Use of the method according to any one of claims 1 to 9 in producing D-psicose.

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