Escherichia coli for forming D-psicose as well as construction method and application of escherichia coli

Abstract

The invention provides a group of recombinant Escherichia coli for forming D-psicose. The recombinant Escherichia coli comprises the following multiple modifications: (a) introducing and expressing a dTDP-glucose-3-epimerase gene, an NDP glycohydrolase gene, an allose-1 phosphate-isomerase gene and an acid phosphatase gene; and (b) enhancing the expression of the glucose phosphate mutase gene, the expression of the glucose 1-phosphate thymine transferase 1 gene and the expression of the glucose 1-phosphate thymine transferase 2 gene. The recombinant Escherichia coli capable of producing D-psicose at high yield is prepared, the yield of by-products is reduced while the yield of a target object is increased, the bottleneck problem in industrial production is solved, and the recombinant Escherichia coli can be used for mass production of D-psicose.

Description

Technical Field

[0001] The present invention relates to the technical fields of gene editing, genetically recombined edited microbial strains, synthetic biology, and whole-cell microbial catalysis. Specifically, the present invention relates to a recombinant strain for synthesizing D-allulose and a method for preparing the same. Background Art

[0002] D-allulose is a safe and healthy rare monosaccharide that can be widely used in industries such as food and beverage, and medicine. As a low-calorie sweetener, the sweetness of allulose is 70% of that of sucrose, but it only has 0.3% of the energy of sucrose. Allulose not only has a moderate sweetness and extremely low calories, but also has various pharmacological effects such as reducing blood glucose response, reducing hepatic fat production, maintaining body weight, anti-inflammation, neuroprotection, and immunosuppression. Because of its special nutritional and biological functions, its preparation method has attracted more and more attention from researchers. The content of allulose in nature is extremely small and generally exists in wheat, fruits, and various other foods. Therefore, it is impossible to complete the preparation by natural extraction.

[0003] Japan is the first country in the world to research and develop allulose. In 2012, Matsutani Chemical Industry Co., Ltd. in Japan had already sold rare sugar syrup containing a certain proportion of D-allulose nationwide, which was favored by consumers. In 2015, CJ in South Korea cooperated with Anderson in the United States and launched the product under the brand name AllSweet, mainly selling raw materials such as syrup and powdered sugar containing a certain proportion of allulose to North America and other places. During the same period, Tate & Lyle's DolciaPrima low-calorie allulose syrup entered the market. The research on allulose in China started relatively late. Jiangnan University is one of the earliest scientific research institutions to conduct research, and its research covers gene mining and strain development, enzyme immobilization, and separation and purification of D-allulose. Judging from the published technologies, the enzymes developed by the Jiangnan University team and the expression effects of the enzymes are not lower than the data published by foreign large companies and are basically synchronized with the international advanced level. A number of related R & D patents have been applied for. However, the current mainstream method of producing allulose using epimerase has the following defects: (1) The price of the fructose raw material used is relatively high; (2) Limited by the equilibrium constant of epimerase, the raw materials cannot be completely converted into products.

[0004] The main traditional methods for producing D-psicose include chemical method and enzymatic conversion method. Bilik et al. found that in an acidic aqueous solution, D-fructose can be converted into D-psicose under the catalytic action of molybdate ions. In 1997, Donald et al. prepared D-psicose by chemical synthesis of 1,2:4,5-di-O-isopropyl-β-D-pyranose. In addition, D-psicose can also be synthesized by boiling ethanol and triethylamine. With in-depth research, it was found that chemical synthesis has complex purification steps, chemical waste and worthless by-products. At the same time, the sweetness of chemically synthesized sweeteners is often not pure enough, so they are not widely used.

[0005] The enzymatic conversion method uses natural raw materials that exist in large quantities in nature and uses biological enzymes as catalysts to prepare allulose, which is not only conducive to reducing the cost of industrial production, but also in line with the current green and environmentally friendly production principles. At present, the main method is to synthesize D-psicose-3-epimerase (DPEase) or D-tagatose-3-epimerase using fructose as raw material. However, due to the limitations of natural enzyme catalytic reactions, the enzymatic conversion method cannot convert all fructose into D-psicose. A complex separation and purification process is required to obtain high-purity D-psicose, which limits production costs and product quality. Patent CN202410459375.8 discloses a method for producing D-psicose using glucose as raw material and using microbial whole-cell catalysis. This method can efficiently convert glucose raw materials into D-psicose by constructing a D-psicose synthesis pathway in a microbial strain. This method can solve the problem of low conversion efficiency of the enzymatic conversion method and avoid complex separation and purification processes. At the same time, this method uses glucose as a raw material, which is cheaper than fructose. However, due to the low catalytic efficiency of the key reaction in the strain, the efficiency of the D-psicose metabolic pathway in the strain needs to be further improved. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a group of stable strains, and to prepare D-psicose from glucose as a raw material through whole-cell catalysis, with high conversion rate, low cost and few side reactions. The present invention discloses a new D-psicose synthesis strategy, and solves the problem of metabolic intermediate accumulation through integration with other D-psicose synthesis pathways through a multi-pathway synergistic strategy. Thus, the efficiency of the technical route for synthesizing D-psicose using glucose as a raw material is improved, which has great economic and social value.

[0007] According to an exemplary embodiment of the present disclosure, the recombinant Escherichia coli is achieved by performing the following multiple transformations in the recipient bacteria:

[0008] (a): Introduce and express the gene gteC encoding dTDP-glucose-3-epimerase, the gene nshA encoding NDP sugar hydrolase, the gene api encoding allose-1-phosphate isomerase, and the gene Aapp encoding acid phosphatase;

[0009] (b): Enhance the expression of the gene pgm encoding phosphoglucomutase, the gene rfbA encoding glucose-1-phosphate thymidylyltransferase 1, and the gene rffH encoding glucose-1-phosphate thymidylyltransferase 2;

[0010] (c): Knock out the gene ldhA encoding D-lactate dehydrogenase and the gene pflB encoding pyruvate-formate lyase.

[0011] The above modifications (a) and (c) can specifically be to knock out the gene ldhA encoding D-lactate dehydrogenase and replace it with the expression cassette of the gene gteC encoding dTDP-glucose-3-epimerase and the gene nshA encoding NDP sugar hydrolase, knock out the gene pflB encoding pyruvate-formate lyase and replace it with the expression cassette of the gene api encoding allose-1-phosphate isomerase and the gene Aapp encoding acid phosphatase.

[0012] The above modification (b) can specifically be: replacing the promoters of the gene pgm encoding phosphoglucomutase, the gene rfbA encoding glucose-1-phosphate thymidylyltransferase 1, and the gene rffH encoding glucose-1-phosphate thymidylyltransferase 2 in the recipient strain with the constitutive promoter P119.

[0013] The above-mentioned recipient strain can specifically be Escherichia coli MG1655, Escherichia coli AS10 (CGMCC No. 27687), Escherichia coli AS10.11 (CGMCC No. 34051).

[0014] Further, the nucleotide sequence of the P119 is SEQ ID NO.2.

[0015] Further, the nucleotide sequence of the gene gteC encoding dTDP-glucose-3-epimerase is SEQ ID NO.5, and the amino acid sequence of the protein encoded by it is SEQ ID NO.6.

[0016] Further, the nucleotide sequence of the gene nshA encoding NDP sugar hydrolase is SEQ ID NO.7, and the amino acid sequence of the protein encoded by it is SEQ ID NO.8.

[0017] Further, the nucleotide sequence of the gene api encoding allose-1-phosphate isomerase is SEQ ID NO.9, and the amino acid sequence of the protein encoded by it is SEQ ID NO.10.

[0018] Furthermore, the nucleotide sequence of the acid phosphatase gene Aapp is SEQ ID NO.11, and the amino acid sequence of the protein encoded thereby is SEQ ID NO.12.

[0019] Another aspect of the present invention provides the use of the recombinant Escherichia coli according to any one of the foregoing in the preparation of D-allulose products by whole-cell catalysis.

[0020] The above product may be a bacterial agent containing the recombinant bacterium or / and a culture of the recombinant bacterium.

[0021] The term "culture" generally refers to liquid or solid products (i.e., fermentation products) with a microbial population after artificial inoculation and cultivation. That is, a product obtained by growing and / or amplifying microorganisms, which may be a pure biological culture of the microorganisms or may contain a certain amount of culture medium, metabolites or other components generated during the cultivation process.

[0022] Another aspect of the present invention provides any one of the following methods:

[0023] A method for producing or increasing the yield of D-allulose, comprising the following steps:

[0024] Culturing the bacterial cells in a growth medium, collecting the bacterial cells after centrifugation of the obtained culture, and then culturing after adding a transformation medium to obtain a culture solution containing a high concentration of D-allulose.

[0025] In a specific embodiment of the present invention, the growth medium may be as follows:

[0026] Dissolve the following components in water and reach the corresponding final concentrations: Na2HPO4: 25 mM, KH2PO4: 25 mM, NH4Cl: 50 mM, Na2SO4: 5 mM, MgSO4: 2 mM, glucose: 1% (g / 100 mL), glycerol: 1% (g / 100 mL), yeast powder: 0.5% (g / 100 mL), trace elements: 50 μM FeCl3, 20 μM CaCl2, 10 μM MnCl2, 10 μM ZnSO4, 2 μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3;

[0027] The transformation medium may be as follows: Dissolve the following components in water and reach the corresponding final concentrations: Na2HPO4: 25 mM, KH2PO4: 25 mM, MgCl2: 5 mM, D-glucose: 20% (g / 100 mL).

[0028] In the present invention, the NCBI reference sequence number of the phosphoglucomutase gene pgm is NC_000913.3 (713558..715198 (+)), and the amino acid reference sequence number of the protein encoded by it is NP_415214.1 (March 9, 2022).

[0029] In the present invention, the NCBI reference sequence number of the glucose-1-phosphate thymidylyltransferase 1 gene rfbA is NC_000913.3 (2110138..2111019 (-)) (March 9, 2022), and the amino acid reference sequence number of the protein encoded by it is NP_416543.1 (March 9, 2022).

[0030] In the present invention, the NCBI reference sequence number of the glucose-1-phosphate thymidylyltransferase 2 gene rffH is NC_000913.3 (3973608..3974489 (+)) (March 9, 2022), and the amino acid reference sequence number of the protein encoded by it is NP_418236.1 (March 9, 2022).

[0031] In the present invention, the NCBI reference sequence number of the D-lactate dehydrogenase gene ldhA is NC_000913.3 (1441854..1442843 (+)) (March 9, 2022), and the amino acid reference sequence number of the protein encoded by it is NP_415898.1 (March 9, 2022).

[0032] In the present invention, the NCBI reference sequence number of the pyruvate formate-lyase gene pflB is NC_000913.3 (951272..953554 (+)) (March 9, 2022), and the amino acid reference sequence number of the protein encoded by it is NP_415423.1 (March 9, 2022).

[0033] The beneficial technical effects obtained by the present disclosure are as follows:

[0034] The present disclosure has prepared recombinant Escherichia coli that can highly produce the above-mentioned D-psicose, which not only increases the yield of the target product but also reduces the yield of by-products, solves the bottleneck problem in industrial production, and can be used for the large-scale production of related D-psicose substances industries. Depository Note

[0035] (1) Name of the strain: Escherichia coli

[0036] Latin name: Escherichia coli

[0037] Strain number: AS10.21

[0038] Depository institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms

[0039] Abbreviation of the depository institution: CGMCC

[0040] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0041] Date of deposit: April 1, 2025

[0042] Registration number in the depository center: CGMCC No. 34052

[0043] (2) Name of the strain: Escherichia coli

[0044] Latin name: Escherichia coli

[0045] Strain number: AS10.22

[0046] Depository institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms

[0047] Abbreviation of the depository institution: CGMCC

[0048] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0049] Date of deposit: April 1, 2025

[0050] Registration number in the depository center: CGMCC No. 34053

[0051] (3) Name of the strain: Escherichia coli

[0052] Latin name: Escherichia coli

[0053] Strain number: AS10.23

[0054] Depository institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms

[0055] Abbreviation of the depositary institution: CGMCC

[0056] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0057] Deposit date: April 1, 2025

[0058] Registration number of the depositary center: CGMCC No. 34054

[0059] (4)Name of the bacterial strain: Escherichia coli

[0060] Latin name: Escherichia coli

[0061] Strain number: AS10

[0062] Depositary institution: China General Microbiological Culture Collection Center

[0063] Abbreviation of the depositary institution: CGMCC

[0064] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0065] Deposit date: June 25, 2023

[0066] Registration number of the depositary center: CGMCC No. 27687

[0067] (5)Name of the bacterial strain: Escherichia coli

[0068] Latin name: Escherichia coli

[0069] Strain number: AS10.11

[0070] Depositary institution: China General Microbiological Culture Collection Center

[0071] Abbreviation of the depositary institution: CGMCC

[0072] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0073] Deposit date: April 1, 2025

[0074] Registration number of the depositary center: CGMCC No. 34051 Description of the drawings

[0075] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings required for the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0076] Figure 1 Standard curve prepared for D - psicose standard.

[0077] Figure 2 HPLC peak pattern of a representative reaction solution sample. Specific embodiments

[0078] The following further describes the present disclosure in detail in combination with specific embodiments. The provided embodiments are only for clarifying the present disclosure, rather than limiting the scope of the present disclosure. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0079] The following liquid LB medium (pH 7.0) in the embodiments: containing 1 g / 100 mL NaCl, 1 g / 100 mL tryptone, 0.5 g / 100 mL yeast powder, and the balance is water. The solid medium is obtained by adding agarose to the liquid medium.

[0080] The Escherichia coli MG1655 (CGSC#: 6300) and plasmid pKD46 (CGSC#: 7739) in the following embodiments are products of the Escherichia coli Genetic Stock Center CGSC of Yale University, USA. Plasmid pKD46: contains a temperature-sensitive replication origin oriR101, can replicate normally at 30°C, and will be automatically lost when cultured at temperatures above 37°C; Escherichia coli carrying the pKD46 plasmid is cultured at 30°C, and Gam, Exo, and Beta are highly expressed after induction with arabinose. Once foreign dsDNA is electrotransformed into the cell, it can undergo homologous recombination with the genomic target sequence; carries an ampicillin resistance gene as a selection marker. The gene sequence of plasmid pSCre is shown in SEQ ID NO.23, and the public can obtain it by gene synthesis: Escherichia coli carrying the pSCre plasmid can express the recombinase (Cre) gene when cultured at 30°C, causing recombination between the lox66 sequence and the lox71 sequence on the chromosome, thereby eliminating the DNA sequence between the lox66 sequence and the lox71 sequence. In this patent, the kanamycin resistance gene between the lox66 sequence and the lox71 sequence in SEQ ID NO.1 can be eliminated; Escherichia coli carrying the pSCre plasmid will automatically lose the pSCre plasmid when cultured at 42°C; carries a streptomycin resistance gene as a selection marker. Escherichia coli AS10 (CGMCC No. 27687) and AS10.11 (CGMCC No. 34051) are both Escherichia coli capable of producing D-allulose and are preserved in the China General Microbiological Culture Collection Center. The public can obtain the above biological materials from the applicant, and the obtained above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0081] Table 1. List of sequence fragments

[0082]

[0083] Table 2. Primers for Example 1

[0084]

[0085] Example 1. Construction of recombinant Escherichia coli AS10.21

[0086] The preparation method of the relevant strains is obtained according to the following steps (1)-(2):

[0087] (1)Starting from Escherichia coli MG1655, replace the promoter of the phosphoglucomutase gene pgm in this strain with the P119 promoter to obtain strain AS100C1.

[0088] The specific steps are as follows:

[0089] (1-a)Preparation of the targeting fragment AS-C1

[0090] Genes were synthesized (by GenScript) with the following DNA fragments: from 5' to 3', it consists of pgmup (fragment 13), lox66-Kan-lox71 (fragment 1), P119 promoter (fragment 2), and pgmdown (fragment 14) in sequence. pgm-1 / pgm-2 were used as primers, and PCR amplification was performed using the synthesized DNA fragment as a template to obtain the targeting fragment AS-C1.

[0091] (1-b)Preparation of the host bacterium containing plasmid pKD46

[0092] The pKD46 plasmid (derived from the E. coli Genetic Stock Center CGSC at Yale University, USA) was transformed into Escherichia coli MG1655 by the calcium chloride transformation method. After overnight culture at 30 °C on an LB plate containing ampicillin, clones were selected to obtain the recombinant Escherichia coli MG1655 / pKD46 containing the plasmid pKD46. After induction with arabinose, MG1655 / pKD46 expressed three recombinant proteins of λ phage, and the host bacterium acquired the ability of homologous recombination. Then, competent cells of MG1655 / pKD46 were prepared by washing with 10% glycerol.

[0093] (1-c)Homologous recombination

[0094] The targeting fragment AS-C1 prepared in (1-a) was electrotransformed into the competent cells of MG1655 / pKD46 prepared in (1-b), and cultured overnight at 37 °C on an LB plate containing kanamycin (concentration: 50 μg / ml). Clones were selected and genomic DNA was extracted. PCR amplification was performed using pgm-3 / Kan-R as primers for identification. An amplified target band of approximately 1000 bp was considered positive, and the positive clone was named AS100C1-kan. The sequencing analysis results showed that the genome of AS100C1-kan contained the AS-C1 fragment. AS100C1-kan was cultured overnight at 42 °C to eliminate the temperature-sensitive plasmid pKD46.

[0095] (1-d)Elimination of resistance

[0096] The pSCre plasmid was transformed into the AS100C1-kan strain that had eliminated pKD46 by the calcium chloride transformation method. It was cultured overnight at 30 °C on an LB plate containing 50 mg / L streptomycin and 0.2% L-arabinose, and the Cre recombinase on the pSCre plasmid was used to eliminate the kanamycin resistance fragment. It was cultured overnight at 42 °C to eliminate the temperature-sensitive plasmid pSCre. The obtained strain was named AS100C1.

[0097] (2)Obtaining of strains AS100C2, AS100C3, AS100C4, and AS10.21

[0098] Using the exact same method as in Example 1(1), starting from AS100C1 in sequence, the promoter of the glucose-1-phosphate thymidylyltransferase 1 gene rfbA in this strain was replaced with the P119 promoter to obtain the AS100C2 strain;

[0099] Starting from AS100C2, the promoter of the glucose-1-phosphate thymidylyltransferase 2 gene rffH in this strain was replaced with the P119 promoter to obtain the AS100C3 strain.

[0100] Starting from AS100C3, the gene ldhA encoding D-lactate dehydrogenase in this strain was knocked out and replaced with the expression cassettes of the genes gteC for dTDP-glucose-3-epimerase and nshA for NDP sugar hydrolase to obtain the AS100C4 strain.

[0101] Starting from AS100C4, the gene pflB encoding pyruvate-formate lyase in this strain was knocked out and replaced with the expression cassettes of the genes api for allose-1-phosphate isomerase and Aapp for acid phosphatase to obtain the AS10.21 strain.

[0102] What was different from Example 1(1) in the construction process of each strain was the use of different starting strains, different targeting fragments, and different primers. The specific experimental materials used and the information of the obtained strains are shown in Table 3.

[0103] Table 3. Construction process of AS100C1 - AS100C4 and AS10.21

[0104]

[0105] The recombinant bacterium AS10.21 was deposited on April 1, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 34052, and the taxonomic name is Escherichia coli.

[0106] Example 2. Construction of Recombinant Escherichia coli AS10.22 and AS10.23

[0107] The construction processes of strains AS10.22 and AS10.23 are exactly the same as that of strain AS10.21 in Example 1. The difference is that the starting strain Escherichia coli MG1655 used in Example 1 is replaced with Escherichia coli AS10 (CGMCC NO.27687) to obtain AS10.22; the starting strain Escherichia coli MG1655 used in Example 1 is replaced with Escherichia coli AS10.11 (CGMCC NO.34051) to obtain AS10.23.

[0108] The recombinant strain AS10.22 was deposited on April 1, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No.34053, and the taxonomic name is Escherichia coli.

[0109] The recombinant strain AS10.23 was deposited on April 1, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No.34054, and the taxonomic name is Escherichia coli.

[0110] Example 3. Production of D-psicose using Escherichia coli AS10, AS10.11, AS10.21, AS10.22, and AS10.23

[0111] (1) The test bacteria are: Escherichia coli AS10, AS10.11, AS10.21, AS10.22, and AS10.23.

[0112] (2) Cultivation of bacteria and induction of enzymes:

[0113] The overnight-cultured strains of AS10, AS10.11, AS10.21, AS10.22, and AS10.23 were inoculated into a shake flask containing 200 mL of growth medium at an inoculation amount of 1%, cultured at 30 °C for 15 - 17 h, and the cells were collected by centrifugation at 8000 rpm for 10 min.

[0114] (3) Whole-cell catalysis of D-psicose

[0115] The collected bacterial cells mentioned above were separately resuspended in shake flasks containing 10 mL of transformation medium, and reacted at 37 °C with a rotation speed of 220 r / min. The start of the reaction was recorded as 0 h. The supernatant was taken by centrifugation at the 12th h, 18th h, and 24th h of the reaction respectively to detect the content of D-allulose. Three replicates were set for each reaction time.

[0116] The content of D-allulose was detected by HPLC using the external standard method. The HPLC used a Hi-Plex Ca chromatographic column (300 mm × 7.7 mm, 8 μm); the mobile phase was ultrapure water with a flow rate of 0.5 mL / min; the column temperature was 78 °C; the detector was a differential refractive index detector; the temperature was 40 °C. In the HPLC analysis, D-allulose (product of Shanghai Yuanye Bio-Technology Co., Ltd., catalog number: S48276) was used as the standard product for qualitative analysis according to the retention time of the standard product, and the standard curve method (external standard method) was used for quantitative analysis of D-allulose. Figure 1 For the standard curve prepared for the D-allulose standard product, the derived equation formula for the concentration of D-allulose: Y = 122616.73X - 6377.83, r2 = 0.9999, r = 0.9999. In the formula, Y is the characteristic peak area, and X is the concentration of D-allulose in the sample injected into the instrument (unit: g / L). Among them: The yield is defined as the concentration of D-allulose produced in the reaction solution; the conversion rate is defined as the ratio of the mass of D-allulose produced in the deduced reaction solution to the mass of D-glucose raw material consumed.

[0117] Figure 2 It is the HPLC peak pattern diagram of a representative sample of the transformation solution. The content of D-glucose was detected by HPLC using the external standard method. The HPLC detection method and conditions are the same as those of the above D-allulose detection method. Among them, D-glucose (product of Beijing Myrada Co., Ltd., catalog number: M043597) was used as the standard product for qualitative analysis according to the retention time of the standard product, and the standard curve method (external standard method) was used for quantitative analysis of D-glucose. The results show that the strain AS10.21 has obvious advantages in the synthesis rate of D-allulose, and the D-allulose yield can reach 78.4 ± 6.9 g / L at 12 hours. And the multi-path strains AS10.22 and AS10.23 based on this synthesis strategy can further improve the conversion rate of D-allulose synthesis of the original strain, and the conversion rates at 24 hours are significantly higher than those of the control strains AS10 and AS10.11 respectively. This proves the effect of the present invention.

[0118] Table 4 D-allulose yields of each strain

[0119]

Claims

1. A recombinant Escherichia coli for synthesizing D-psicose, wherein the recombinant Escherichia coli includes the following multiple modifications: Import and express the gene gteC encoding dTDP-glucose-3-epimerase, the gene nshA encoding NDP sugar hydrolase, the gene api encoding allose-1-phosphate isomerase, and the gene Aapp encoding acid phosphatase.

2. The recombinant Escherichia coli according to claim 1, wherein the recombinant Escherichia coli further includes the following modification: Enhance the expression of the gene pgm encoding phosphoglucomutase, the gene rfbA encoding glucose-1-phosphate thymidylyltransferase 1, and the gene rffH encoding glucose-1-phosphate thymidylyltransferase 2.

3. The recombinant Escherichia coli according to claims 1-2, wherein the recombinant Escherichia coli further includes the following modification: Knock out the gene ldhA encoding D-lactate dehydrogenase and the gene pflB encoding pyruvate-formate lyase.

4. Use of the recombinant Escherichia coli according to any one of claims 1 to 3 in the preparation of D-psicose by whole-cell catalysis.

5. An Escherichia coli, characterized in that After collecting the cells by fermentation, it can catalyze the preparation of D-psicose from glucose by whole-cell catalysis, and the preservation number is CGMCC No. 27687.

6. An Escherichia coli, characterized in that After collecting the cells by fermentation, it can catalyze the preparation of D-psicose from glucose by whole-cell catalysis, and the preservation number is CGMCC No. 34051.

7. An Escherichia coli, characterized in that After collecting the cells by fermentation, it can catalyze the preparation of D-psicose from glucose by whole-cell catalysis, and the preservation number is CGMCC No. 34052.

8. An Escherichia coli, characterized in that After collecting the cells by fermentation, it can catalyze the preparation of D-psicose from glucose by whole-cell catalysis, and the preservation number is CGMCC No. 34053.

9. An Escherichia coli, characterized in that After collecting the cells by fermentation, it can catalyze the preparation of D-psicose from glucose by whole-cell catalysis, and the preservation number is CGMCC No. 34054.

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