A group of Escherichia coli that synthesize D-allulose, their construction methods and applications

Abstract

This invention provides a group of recombinant *E. coli* strains capable of synthesizing D-allulose, wherein the recombinant *E. coli* strains include the following modifications: (a) introduction and expression of dTDP-glucose-3-epimerase gene, NDP sugar hydrolase gene, allosugar-1 phosphate-isomerase gene, and acid phosphatase gene; (b) enhanced expression of glucose-1-phosphate thymidine transferase gene, glucose-1-phosphate thymidine transferase 1 gene, and glucose-1-phosphate thymidine transferase 2 gene. This disclosure produces recombinant *E. coli* strains capable of high-yield D-allulose production, increasing the yield of the target compound while reducing the yield of by-products, thus solving a bottleneck problem in industrial production and enabling large-scale production of D-allulose.

Description

Technical Field

[0001] This invention relates to the fields of gene editing, gene recombination editing microbial strains, synthetic biology, and whole-cell microbial catalysis technology. Specifically, it relates to a recombinant strain for synthesizing D-allulose and its preparation method. Background Technology

[0002] D-Allulose is a safe and healthy rare monosaccharide with wide applications in the food, beverage, and pharmaceutical industries. As a low-calorie sweetener, allulose is 70% as sweet as sucrose but contains only 0.3% of its energy. Besides its moderate sweetness and extremely low calorie content, D-Allulose possesses various pharmacological effects, including lowering glycemic response, reducing liver fat production, maintaining weight, anti-inflammation, neuroprotection, and immunosuppression. Due to its unique nutritional and biological functions, its preparation methods are increasingly attracting research attention. D-Allulose is extremely rare in nature, generally found in wheat, fruits, and various other foods, therefore it cannot be prepared through natural extraction.

[0003] Japan is one of the earliest countries in the world to research and develop allulose. In 2012, Matsutani Chemical Industry in Japan launched a rare sugar syrup containing a certain proportion of D-allulose nationwide, which was well received by consumers. In 2015, South Korea's CJ and the United States' Anderson collaborated to launch the AllSweet brand, mainly selling raw materials such as syrup and sugar powder containing a certain proportion of D-allulose to North America and other regions. Around the same time, DolciaPrima low-calorie allulose syrup from the United Kingdom entered the market. Research on allulose in China started relatively late. Jiangnan University was one of the earliest research institutions to begin research, covering gene mining and strain development, enzyme immobilization, and the isolation and purification of D-allulose. Judging from the published technologies, the enzymes developed by the Jiangnan University team and their expression effects are no less than those published by major foreign companies, and are basically in sync with the international advanced level. Several related research and development patents have been applied for. However, the current mainstream method of production using epimerase has the following drawbacks: (1) the price of fructose raw material is relatively high; (2) due to the limitation of the equilibrium constant of epimerase, the raw material cannot be completely converted into the product.

[0004] Traditional methods for producing D-allulose mainly include chemical and enzymatic methods. Bilik et al. found that D-fructose can be converted to D-allulose in acidic aqueous solutions under the catalysis of molybdate ions. In 1997, Donald et al. prepared D-allulose via chemical synthesis from 1,2:4,5-di-O-isopropylidene-β-D-fructose. Additionally, D-allulose can also be synthesized by boiling ethanol and triethylamine. However, further research has revealed that chemical synthesis suffers from complex purification steps, generates chemical waste and worthless byproducts, and often results in less pure sweetness in chemically synthesized sweeteners, thus limiting its widespread application.

[0005] Enzymatic conversion utilizes abundant natural raw materials and biological enzymes as catalysts to prepare allulose, which not only reduces industrial production costs but also aligns with current green and environmentally friendly production principles. Currently, the main method involves using D-allulose-3-epimerase (D-psicose3-epimerase, DPEase) or D-tagatose-3-epimerase to synthesize D-allulose from fructose. However, due to the limitations of natural enzyme catalysis, enzymatic conversion cannot convert all fructose into D-allulose. Complex separation and purification processes are required to obtain high-purity D-allulose, limiting production costs and product quality. Patent CN202410459375.8 discloses a method for producing D-allulose from glucose using whole-cell microbial catalysis. This method constructs a D-allulose synthesis pathway in microbial strains, enabling high-efficiency conversion of glucose into D-allulose. This method solves the problem of low conversion efficiency in enzymatic conversion and avoids complex separation and purification processes. Meanwhile, this method uses glucose as a raw material, which is less expensive than fructose. However, due to the low catalytic efficiency of key reactions in the strain, it is necessary to further improve the efficiency of the D-allulose metabolic pathway in the strain. Summary of the Invention

[0006] The technical problem this invention aims to solve is to provide a stable strain of bacteria that, using glucose as a raw material, can prepare D-allulose with high conversion rate, low cost, and few side reactions through whole-cell catalysis. This invention discloses a novel D-allulose synthesis strategy and, by integrating it with other D-allulose synthesis pathways, solves the problem of metabolic intermediate accumulation through a multi-pathway synergistic strategy. This improves the efficiency of the glucose-based D-allulose synthesis route, possessing significant economic and social value.

[0007] According to an exemplary embodiment of this disclosure, the recombinant Escherichia coli is achieved by performing the following modifications on the recipient bacteria:

[0008] (a): Import and expression of the gene gteC encoding dTDP-glucose-3-epimerase, the gene nshA encoding NDP sugar hydrolase, the gene api encoding allosugar-1 phosphate-isomerase, and the gene Aapp encoding acid phosphatase.

[0009] (b): Enhance the expression of the gene pgm encoding glucose phosphate mutase, the gene rfbA encoding glucose 1-phosphate thymidine transferase 1, and the gene rffH encoding glucose 1-phosphate thymidine transferase 2.

[0010] (c): Knockout of the gene ldhA encoding D-lactose dehydrogenase and the gene pflB encoding pyruvate-formate lyase.

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

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

[0013] The recipient strains mentioned above may specifically be Escherichia coli MG1655, Escherichia coli AS10 (CGMCC No. 27687), and Escherichia coli AS10.11 (CGMCC No. 34051).

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

[0015] Furthermore, the nucleotide sequence of the gene gteC for the dTDP-glucose-3-epimerase is SEQ ID NO.5, and the amino acid sequence of the protein it encodes is SEQ ID NO.6.

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

[0017] Furthermore, the nucleotide sequence of the allosugar-1 phosphate-isomerase gene api is SEQ ID NO.9, and the amino acid sequence of the protein it encodes 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 it encodes is SEQ ID NO.12.

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

[0020] The above-mentioned products may be bacterial agents containing the recombinant bacteria or / and cultures of the recombinant bacteria.

[0021] The term "culture" refers to a liquid or solid product (i.e., fermentation product) that has grown a microbial community after artificial inoculation and cultivation. It is a product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process.

[0022] In another aspect, the present invention provides any of the following methods:

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

[0024] The bacterial cells were cultured in a growth medium. The obtained culture was collected by centrifugation and then added to a transformation medium for further culture to obtain a culture medium containing a high concentration of D-allulose.

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

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

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

[0028] In this invention, the NCBI Reference Sequence of the glucose phosphate mutase gene pgm is NC_000913.3 (713558..715198(+)), and the NCBI Reference Sequence of the amino acid sequence of the protein it encodes is NP_415214.1 (2022.3.9).

[0029] In this invention, the NCBI Reference Sequence of the glucose-1-phosphate thymidine transferase 1 gene rfbA is NC_000913.3 (2110138..2111019 (-)) (2022.3.9), and the NCBI Reference Sequence of the amino acid sequence of the protein it encodes is NP_416543.1 (2022.3.9).

[0030] In this invention, the NCBI Reference Sequence number of the glucose-1-phosphate thymidine transferase 2 gene rffH is NC_000913.3 (3973608..3974489(+)) (2022.3.9), and the NCBI Reference Sequence number of the amino acid sequence of the protein it encodes is NP_418236.1 (2022.3.9).

[0031] In this invention, the NCBI Reference Sequence of the D-lactose dehydrogenase gene ldhA is NC_000913.3 (1441854..1442843 (+)) (2022.3.9), and the NCBI Reference Sequence of the amino acid sequence of the protein it encodes is NP_415898.1 (2022.3.9).

[0032] In this invention, the NCBI Reference Sequence number of the pyruvate-formate lyase gene pflB is NC_000913.3 (951272..953554(+)) (2022.3.9), and the NCBI Reference Sequence number of the amino acid sequence of the protein it encodes is NP_415423.1 (2022.3.9).

[0033] The beneficial technical effects achieved by this disclosure are as follows:

[0034] This disclosure describes the preparation of a recombinant Escherichia coli capable of high-yielding the aforementioned D-allulose, which increases the yield of the target substance while reducing the yield of by-products, thus solving a bottleneck problem in industrial production and can be used for the large-scale production of related D-allulose substances.

[0035] Preservation Instructions

[0036] (1) Bacterial species name: Escherichia coli

[0037] Latin name: Escherichia coli

[0038] Strain number: AS10.21

[0039] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0040] Collection institution abbreviation: CGMCC

[0041] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0042] Deposit date: April 1, 2025

[0043] CGMCC Registration Number: CGMCC No. 34052

[0044] (2) Bacterial species name: Escherichia coli

[0045] Latin name: Escherichia coli

[0046] Strain number: AS10.22

[0047] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0048] Collection institution abbreviation: CGMCC

[0049] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0050] Deposit date: April 1, 2025

[0051] CGMCC Registration Number: CGMCC No. 34053

[0052] (3) Bacterial species name: Escherichia coli

[0053] Latin name: Escherichia coli

[0054] Strain number: AS10.23

[0055] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0056] Collection institution abbreviation: CGMCC

[0057] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0058] Deposit date: April 1, 2025

[0059] CGMCC Registration Number: CGMCC No. 34054

[0060] (4) Bacterial species name: Escherichia coli

[0061] Latin name: Escherichia coli

[0062] Strain number: AS10

[0063] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0064] Collection institution abbreviation: CGMCC

[0065] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0066] Deposit date: June 25, 2023

[0067] CGMCC Registration Number: CGMCC No. 27687

[0068] (5) Bacterial species name: Escherichia coli

[0069] Latin name: Escherichia coli

[0070] Strain number: AS10.11

[0071] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0072] Collection institution abbreviation: CGMCC

[0073] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0074] Deposit date: April 1, 2025

[0075] CGMCC Registration Number: CGMCC No. 34051 Attached Figure Description

[0076] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0077] Figure 1 A standard curve was prepared for D-allulose standards.

[0078] Figure 2 This is an HPLC peak diagram of a representative reaction solution sample. Detailed Implementation

[0079] The present disclosure will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0080] The liquid LB medium (pH 7.0) in the following examples contains 1 g / 100 mL NaCl, 1 g / 100 mL tryptone, 0.5 g / 100 mL yeast extract, and the remainder is water. The solid medium is obtained by adding agarose to the liquid medium.

[0081] The *E. coli* MG1655 (CGSC#: 6300) and plasmid pKD46 (CGSC#: 7739) used in the following examples were products of the *E. coli* Genetic Collection Center (CGSC) at Yale University, USA. The pKD46 plasmid contains the temperature-sensitive origin of replication oriR101, which replicates normally at 30°C but is automatically lost at temperatures above 37°C. *E. coli* carrying the pKD46 plasmid, when cultured at 30°C with arabinose induction, exhibits high expression of Gam, Exo, and Beta. Once exogenous dsDNA is electroporated into the cells, homologous recombination with the genomic target sequence can occur. It also carries an ampicillin resistance gene as a selection marker. The gene sequence of the pSCre plasmid is shown in SEQ ID NO. 23, and can be obtained by the public through gene synthesis. E. coli carrying the pSCre plasmid, when cultured at 30°C, can express the recombinase (Cre) gene, causing recombination between the lox66 and lox71 sequences on the chromosome, thereby eliminating the DNA sequence located between the lox66 and lox71 sequences. In this patent, the kanamycin resistance gene between the lox66 and lox71 sequences, as shown in SEQ ID NO. 1, can be eliminated. E. coli carrying the pSCre plasmid will automatically lose the pSCre plasmid when cultured at 42°C. The streptomycin resistance gene is used as a selection marker. E. coli AS10 (CGMCC No. 27687) and AS10.11 (CGMCC No. 34051) are both E. coli capable of producing D-allulose and are deposited at the China General Microbiological Culture Collection Center. The public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials are only for repeating the experiments of this invention and cannot be used for other purposes.

[0082] Table 1. List of Sequence Fragments

[0083]

[0084] Table 2 Primers for Example 1

[0085]

[0086] Example 1: Construction of recombinant Escherichia coli AS10.21

[0087] The relevant strains were prepared according to the following steps (1)-(2):

[0088] (1) Starting from Escherichia coli MG1655, the promoter of the glucose phosphate mutase gene pgm in this strain was replaced with the P119 promoter to obtain strain AS100C1.

[0089] The specific steps are as follows:

[0090] (1-a) Preparation of the target fragment AS-C1

[0091] The synthesized DNA fragment (GenScript) is as follows: from 5' to 3', it consists of the following components: pgmup (fragment 13), lox66-Kan-lox71 (fragment 1), P119 promoter (fragment 2), and pgmdown (fragment 14). Using pgm-1 / pgm-2 as primers, PCR amplification was performed using the synthesized DNA fragment as a template to obtain the targeted fragment AS-C1.

[0092] (1-b) Preparation of host bacteria containing pKD46 plasmid

[0093] The pKD46 plasmid (derived from the Yale University Genetic Collection of E. coli, CGSC) was transformed into *E. coli* MG1655 using the calcium chloride conversion method. After overnight incubation on LB agar plates containing ampicillin at 30°C, clones were selected to obtain recombinant *E. coli* MG1655 / pKD46 containing plasmid pKD46. Upon induction with arabinose, MG1655 / pKD46 expressed three recombinant proteins of λ phage, thus endowing the host bacterium with homologous recombination capabilities. Competent MG1655 / pKD46 cells were then prepared by washing with 10% glycerol.

[0094] (1-c) Homologous recombination

[0095] The target fragment AS-C1 prepared in (1-a) was electroporated into MG1655 / pKD46 competent cells prepared in (1-b). Cells were incubated overnight at 37°C on LB plates containing kanamycin (50 µg / ml). Genomic DNA was extracted from selected clones, and PCR amplification was performed using pgm-3 / Kan-R primers. A positive result was indicated by the amplification of a target band of approximately 1000 bp. The positive clone was named AS100C1-kan. Sequencing analysis showed that the AS-C1 fragment was present in the genome of AS100C1-kan. AS100C1-kan was cultured overnight at 42°C to eliminate the temperature-sensitive plasmid pKD46.

[0096] (1-d) Elimination of resistance

[0097] The pSCre plasmid was transformed into the AS100C1-kan strain, which had pKD46 eliminated, using the calcium chloride transformation method. The strain was incubated overnight at 30°C on LB agar plates containing 50 mg / L streptomycin and 0.2% L arabinose. The kanamycin resistance fragment was eliminated using the Cre recombinase on the pSCre plasmid. The strain was then incubated overnight at 42°C to eliminate the temperature-sensitive pSCre plasmid. The resulting strain was named AS100C1.

[0098] (2) Obtaining strains AS100C2, AS100C3, AS100C4, and AS10.21

[0099] Using the same method as in Example 1 (1), starting from AS100C1, the promoter of the glucose 1-phosphate thymidine transferase 1 gene rfbA in this strain was replaced with the P119 promoter to obtain strain AS100C2.

[0100] Starting with AS100C2, the promoter of the glucose-1-phosphate thymidine transferase 2 gene rffH in this strain was replaced with the P119 promoter to obtain strain AS100C3.

[0101] Starting with AS100C3, the gene ldhA encoding D-lactate dehydrogenase was knocked out and replaced with the expression cassettes of the gene gteC encoding dTDP-glucose-3-epimerase and the gene nshA encoding NDP sugar hydrolase, thus obtaining strain AS100C4.

[0102] Starting with AS100C4, the gene pflB encoding pyruvate-formate lyase was knocked out and replaced with the expression cassettes of allosugar-1 phosphate-isomerase gene api and acid phosphatase gene Aapp, resulting in strain AS10.21.

[0103] The difference between each strain construction process and Example 1 (1) is that different starting strains, different targeting fragments, and different primers were used. The specific experimental materials used and the information of the obtained strains are shown in Table 3.

[0104] Table 3. Construction process of AS100C1-AS100C4, AS10.21

[0105]

[0106] The recombinant strain AS10.21 was deposited on April 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 34052, and the strain is classified as Escherichia coli.

[0107] Example 2: Construction of recombinant Escherichia coli AS10.22 and AS10.23

[0108] The construction process of strains AS10.22 and AS10.23 is completely consistent with the construction process of strain AS10.21 in Example 1. The difference is that the starting strain Escherichia coli MG1655 used in Example 1 was replaced with Escherichia coli AS10 (CGMCC NO.27687) to obtain AS10.22; and the starting strain Escherichia coli MG1655 used in Example 1 was replaced with Escherichia coli AS10.11 (CGMCC NO.34051) to obtain AS10.23.

[0109] The recombinant strain AS10.22 was deposited on April 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 34053, and the strain is classified as Escherichia coli.

[0110] The recombinant strain AS10.23 was deposited on April 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 34054, and the strain is classified as Escherichia coli.

[0111] Example 3: Production of D-allulose using Escherichia coli AS10, AS10.11, AS10.21, AS10.22, and AS10.23

[0112] (1) The tested bacteria were: Escherichia coli AS10, AS10.11, AS10.21, AS10.22, and AS10.23.

[0113] (2) Culture of bacterial cells and induction of enzymes:

[0114] The AS10, AS10.11, AS10.21, AS10.22, and AS10.23 strains cultured overnight were inoculated at a rate of 1% into shake flasks containing 200 mL of growth medium and cultured at 30°C for 15–17 h. The bacterial cells were then collected by centrifugation at 8000 rpm for 10 min.

[0115] (3) Whole-cell catalysis of D-allulose

[0116] The collected bacterial cells were resuspended in shake flasks containing 10 mL of transformation medium and reacted at 37°C and 220 rpm. The reaction start time was recorded as 0 h. The supernatant was centrifuged at 12 h, 18 h, and 24 h to determine the D-allulose content. Three replicates were set for each reaction time.

[0117] The content of D-allulose was determined by HPLC using the external standard method. HPLC was performed using a Hi-Plex Ca column (300 mm × 7.7 mm, 8 μm); the mobile phase was ultrapure water, the flow rate was 0.5 mL / min; the column temperature was 78 ℃; the detector was a differential refractive index detector; and the temperature was 40 ℃. In the HPLC analysis, D-allulose (Shanghai Yuanye Biotechnology product (catalog number: S48276)) was used as the standard for qualitative analysis based on the retention time of the standard, and quantitative analysis was performed using the standard curve method (external standard method). Figure 1 The standard curve prepared for D-allulose standards was used, and the derived equation for the D-allulose concentration was: Y = 122616.73X - 6377.83, r² = 0.9999, r = 0.9999. In the equation, Y is the characteristic peak area, and X is the D-allulose concentration of the sample (g / L). Yield is defined as the concentration of D-allulose produced in the reaction solution; conversion rate is defined as the ratio of the estimated mass of D-allulose produced in the reaction solution to the mass of D-glucose raw material consumed.

[0118] Figure 2 The HPLC peak shape of a representative conversion solution sample is shown. The content of D-glucose was determined by HPLC using the external standard method. The HPLC detection method and conditions were the same as those for D-allulose detection described above. D-glucose (Beijing Mairuida product (catalog number: M043597)) was used as the standard for qualitative analysis based on the retention time of the standard, and quantitative analysis of D-glucose was performed using the standard curve method (external standard method). The results showed that strain AS10.21 had a significant advantage in the rate of D-allulose synthesis, achieving a yield of 78.4 ± 6.9 g / L after 12 hours. Furthermore, the multi-pathway strains AS10.22 and AS10.23, based on this synthesis strategy, further improved the conversion rate of D-allulose synthesis from the original strain, with a significantly higher conversion rate after 24 hours compared to the control strains AS10 and AS10.11. This demonstrates the effectiveness of the present invention.

[0119] Table 4. D-allulose yield of each strain

[0120]

Claims

1. A type of Escherichia coli Escherichia coli AS10.22, characterized in that After collecting the bacterial cells through fermentation, D-allulose can be prepared by whole-cell catalysis using glucose as a raw material. The preservation number is CGMCC No.34053.

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