Recombinant escherichia coli for producing D-psicose and preparation method thereof

Abstract

The invention discloses recombinant escherichia coli for producing D-psicose and a preparation method of the recombinant escherichia coli, and belongs to the field of genetic engineering. The technical problem to be solved in the invention is how to supply microorganisms for growth by using glycerol, acetic acid, fatty acid and the like as raw materials and prepare D-psicose by using glucose as a raw material. The invention provides recombinant Escherichia coli, which uses glycerol, acetic acid, fatty acid and the like as carbon sources to amplify (grow) the recombinant Escherichia coli, and uses D-glucose as a substrate to prepare D-psicose. According to the recombinant escherichia coli provided by the invention, glycerol, acetic acid, fatty acid and the like can be used as carbon sources for microbial growth, and D-psicose is synthesized by taking D-glucose as a raw material after the microbial growth reaches a certain biomass. Furthermore, a corresponding technical route for synthesizing the D-psicose is established.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and specifically relates to a recombinant Escherichia coli for producing D-psicose and a preparation method thereof. Background Art

[0002] D-allulose is a safe and healthy rare monosaccharide that can be widely used in the food, medicine and other industries. As a low-calorie sweetener, allulose is 70% as sweet as sucrose, but has only 0.3% of the energy of sucrose. D-allulose not only has moderate sweetness and extremely low calories, but also has multiple pharmacological effects such as lowering blood sugar response, reducing liver fat production, maintaining body weight, anti-inflammatory, neuroprotection and immunosuppression. Because of its special nutritional and biological functions, its preparation method has received more and more attention from researchers. The content of D-allulose in nature is extremely small, and it is generally found in wheat, fruit and various other foods. Therefore, it cannot be prepared by natural extraction.

[0003] Japan is the first country in the world to research and develop allulose. In 2012, Matsutani Chemical Industry of Japan has sold rare sugar syrup with a certain proportion of D-allulose nationwide, which is favored by consumers; in 2015, South Korea's CJ cooperated with Anderson of the United States to launch the AllSweet brand, mainly selling raw materials such as syrup and powdered sugar containing a certain proportion of D-allulose to North America and other places. At the same time, British Tate & Lyle Dolcia Prima low-calorie allulose syrup entered the market. Domestic research on allulose started late, and Jiangnan University was one of the earliest scientific research institutions to start research. Its research covers gene mining and strain development, enzyme immobilization, and separation and purification of D-allulose. In terms of the published technology, the enzymes and enzyme expression effects developed by the Jiangnan University team are not lower than the data published by large foreign companies, and are basically synchronized with the international advanced level. A number of related research and development patents have been applied for. However, the mainstream production method using diastereomerase on the market has the following defects: (1) the price of the fructose raw material used is relatively high; (2) due to the limitation of the equilibrium constant of diastereomerase, the raw material cannot be completely converted into the product, requiring a complex product separation process and expensive production equipment.

[0004] The main methods for producing D-psicose traditionally include chemical and biological methods. 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, and the sweetness of chemically synthesized sweeteners is often not pure enough, so they are not widely used.

[0005] The biosynthesis 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 the biotransformation method using enzymes as catalysts. Taking isomerase as an example, the thermal stability of the identified wild-type enzymes is generally low. In order to meet the needs of industrial applications, the directional mining and thermal stability analysis and transformation of isomerases with different properties have begun to be widely studied; at the same time, during use, the preservation of enzymes is an extremely important link. Being able to extend the storage life of enzymes and maintain stability is the basis for their commercial application. At present, the biocatalysts that have been discovered and characterized for the biosynthesis of D-psicose are D-psicose-3-epimerase (DPEase) and D-tagatose-3-epimerase, and in most cases, D-fructose is selected to produce D-psicose through the catalytic conversion of these two enzymes.

[0006] Fatty acids are low-priced, easily available green organic raw materials. Their energy density is higher than that of raw materials such as glucose (D-glucose). They can also provide more reducing power for biosynthesis, thereby improving the conversion rate of biomanufacturing. They are one of the ideal raw materials for biomanufacturing and can be obtained at a low price from crude oil processing products, waste cooking oil and other sources. Glycerol and acetic acid are by-products of chemical products with a wide range of sources and low prices. Using glycerol or acetic acid as a carbon source to ferment and cultivate strains is conducive to solving the problem of competing for food with people. Glucose, as a raw material for the industrial production of fructose, is cheaper than fructose. Establishing a route for synthesizing D-psicose using glycerol, acetic acid, fatty acids and the like as raw materials for microbial growth and glucose as a raw material has great economic and social value. Summary of the invention

[0007] The technical problem to be solved by the present application is: how to utilize raw materials such as glycerol, acetic acid, fatty acids, etc. to supply microbial growth and prepare D-psicose as raw material using D-glucose.

[0008] The present application provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli has the following characteristics:

[0009] A1), substances containing glucose isomerase gene and / or regulating the expression of glucose isomerase gene;

[0010] A2), substances containing hexokinakinase gene and / or regulating the expression of hexokinakinase gene;

[0011] A3), a substance containing a UDP-glucose pyrophosphorylase gene and / or regulating the expression of a UDP-glucose pyrophosphorylase gene;

[0012] A4), a substance containing an NDP-hexose-3-epimerase gene and / or regulating the expression of an NDP-hexose-3-epimerase gene;

[0013] A5), substances containing NDP glycohydrolase gene and / or regulating the expression of NDP glycohydrolase gene;

[0014] A6), containing substances that reduce the expression of glucokinase gene and / or reduce the activity or content of glucokinase;

[0015] A7), containing substances that enhance the expression of nucleoside diphosphate kinase genes and / or enhance the activity or content of nucleoside diphosphate kinases;

[0016] A8), containing substances that enhance the expression of uridylate kinase gene and / or enhance the activity or content of uridylate kinase gene;

[0017] A9), containing substances that enhance uridine kinase gene expression and / or enhance uridine kinase activity or content;

[0018] A10), containing substances that enhance the expression of inorganic pyrophosphatase genes and / or enhance the activity or content of inorganic pyrophosphatase;

[0019] A11), substances containing acid phosphatase gene and / or regulating the expression of acid phosphatase gene;

[0020] A12), substances containing glucose transport cofactor genes and / or regulating the expression of glucose transport cofactor genes;

[0021] A13), does not contain the G subunit gene of the phosphotransferase system;

[0022] A14), does not contain the galactose repressor gene;

[0023] A15), does not contain 6-phosphate glucose dehydrogenase gene;

[0024] A16) does not contain the lipid A biosynthesis myristoyltransferase gene.

[0025] Furthermore, in the recombinant Escherichia coli, the substances described in A1)-A5) can be promoters.

[0026] Furthermore, the substance described in A1)-A5) can be a promoter trc promoter. The nucleotide sequence of the promoter trc promoter is 193-266 of SEQ ID NO.7.

[0027] Furthermore, in the recombinant Escherichia coli, the substance described in A6) is introduced into the promoter site of the glucose kinase gene of the recipient bacteria.

[0028] Furthermore, in the recombinant Escherichia coli, the substance described in A6) can be a constitutive weak promoter P 113 .

[0029] Furthermore, in the recombinant Escherichia coli, the substances described in A7)-A10) are respectively introduced into the promoter sites of the nucleoside diphosphate kinase gene, enhanced uridylate kinase gene, uridine kinase gene or inorganic pyrophosphatase gene of the recipient bacteria.

[0030] Furthermore, in the recombinant Escherichia coli, the substances described in A11)-A12) are respectively combined with the acid phosphatase gene or the glucose transport auxiliary factor gene to form an expression cassette and introduced into the recipient bacteria.

[0031] Furthermore, in the recombinant Escherichia coli, the introduction site of the acid phosphatase gene and / or the substance regulating the expression of the acid phosphatase gene is the G subunit gene site of the phosphotransferase system of the recipient bacteria.

[0032] Furthermore, in the recombinant Escherichia coli, the introduction site of the glucose transport cofactor gene and / or the substance regulating the expression of the glucose transport cofactor gene is the galactose repressor gene site of the recipient bacteria.

[0033] Furthermore, in the recombinant Escherichia coli, the substances described in A7)-A12) can be constitutive promoter P CPA1 .

[0034] Furthermore, the recombinant Escherichia coli also has the following characteristics:

[0035] A17), substances containing acetyl-CoA synthetase gene and / or regulating the expression of acetyl-CoA synthetase gene;

[0036] A18) does not contain the pyruvate oxidase gene.

[0037] Furthermore, in the recombinant Escherichia coli, the substance described in A17) can be a constitutive promoter PCPA1 .

[0038] Furthermore, in the recombinant Escherichia coli, the introduction site of the acetyl-CoA synthetase gene and / or the substance regulating the expression of the acetyl-CoA synthetase gene is the pyruvate oxidase gene of the recipient bacteria.

[0039] Furthermore, the recombinant Escherichia coli also has the following characteristics:

[0040] A19), containing substances that enhance the expression of acyl-CoA synthase genes and / or enhance the activity or content of acyl-CoA synthase;

[0041] A20), containing substances that enhance the gene expression of long-chain fatty acid transport proteins and / or enhance the activity or content of long-chain fatty acid transport proteins;

[0042] A21), containing substances that enhance NAD(P) transhydrogenase gene expression and / or enhance NAD(P) transhydrogenase activity or content;

[0043] A22), substances containing genes that enhance the expression of the short-chain fatty acid degradation regulatory gene cluster atoS and atoC and / or enhance the activity or content of the short-chain fatty acid degradation regulatory gene cluster atoS and atoC;

[0044] A23), does not contain fatty acid degradation transcription factor genes;

[0045] A24), does not contain β-ketoacyl-ACP synthase II gene;

[0046] A25), does not contain β-ketoacyl-ACP synthase III gene;

[0047] A26) does not contain the isocitrate lyase inhibitor gene.

[0048] Furthermore, in the recombinant Escherichia coli, the substances described in A19)-A22) are respectively introduced into the promoter sites of the acyl-CoA synthase gene, long-chain fatty acid transporter gene, NAD(P) transhydrogenase gene, and short-chain fatty acid degradation regulatory gene cluster atoS and atoC genes described in A19)-A22).

[0049] Furthermore, in the recombinant Escherichia coli, the substances described in A19)-A22) can be constitutive promoter P CPA1 .

[0050] The present application also provides a method for preparing the recombinant Escherichia coli, which may include the following steps:

[0051] M1), introducing glucose isomerase gene and / or substance regulating glucose isomerase gene expression into recipient bacteria;

[0052] M2), introducing the hexokinokinase gene and / or a substance regulating the expression of the hexokinokinase gene into the recipient bacteria;

[0053] M3), introducing UDP-glucose pyrophosphorylase gene and / or substance regulating UDP-glucose pyrophosphorylase gene expression into the recipient bacteria;

[0054] M4), introducing NDP-hexose-3-epimerase gene and / or a substance regulating the expression of NDP-hexose-3-epimerase gene into the recipient bacteria;

[0055] M5), introducing NDP glycohydrolase gene and / or substance regulating the expression of NDP glycohydrolase gene into the recipient bacteria;

[0056] M6), introducing a substance that reduces the expression of the glucokinase gene and / or reduces the activity or content of glucokinase into a recipient bacterium, wherein the recipient bacterium contains a glucokinase gene;

[0057] M7), introducing a substance that enhances the expression of a nucleoside diphosphate kinase gene and / or enhances the activity or content of a nucleoside diphosphate kinase into a recipient bacterium, wherein the recipient bacterium contains a nucleoside diphosphate kinase gene;

[0058] M8), introducing a substance that enhances uridylate kinase gene expression and / or enhances uridylate kinase activity or content into a recipient bacterium, wherein the recipient bacterium contains a uridylate kinase gene;

[0059] M9), introducing a substance that enhances uridine kinase gene expression and / or enhances uridine kinase activity or content into a recipient bacterium, wherein the recipient bacterium contains a uridine kinase gene;

[0060] M10), introducing a substance that enhances the inorganic pyrophosphatase gene and / or enhances the activity or content of inorganic pyrophosphatase into a recipient bacterium, wherein the recipient bacterium contains an inorganic pyrophosphatase gene;

[0061] M11), introducing acid phosphatase gene and / or substance regulating the expression of acid phosphatase gene into the recipient bacteria;

[0062] M12), introducing glucose transport cofactor genes and / or substances regulating the expression of glucose transport cofactor genes into the recipient bacteria;

[0063] M13), knocking out the G subunit gene of the phosphosugar transferase system in the recipient bacteria, wherein the recipient bacteria contains the G subunit gene of the phosphosugar transferase system;

[0064] M14), knocking out the galactose repressor gene in the recipient bacteria, wherein the recipient bacteria contains the galactose repressor gene;

[0065] M15), knocking out the 6-phosphate glucose dehydrogenase gene in the recipient bacteria, wherein the recipient bacteria contains the 6-phosphate glucose dehydrogenase gene;

[0066] M16), knocking out the lipid A biosynthesis myristoyltransferase gene in the recipient bacteria, wherein the recipient bacteria contains the lipid A biosynthesis myristoyltransferase gene.

[0067] Furthermore, in the method described above, the introduction site of the acid phosphatase gene and / or the substance regulating the expression of the acid phosphatase gene described in M11) is located at the G subunit gene of the phosphoglycotransferase system described in M13).

[0068] Furthermore, in the method described above, the introduction site of the glucose transport cofactor gene and / or the substance regulating the expression of the glucose transport cofactor gene described in M12) is located at the galactose repressor gene described in M14).

[0069] Furthermore, in the method described above, the substance described in M6) is introduced into the promoter site of the glucokinase gene of the recipient bacteria.

[0070] Furthermore, in the method described above, the substance described in M6) may be a constitutive weak promoter P 113 .

[0071] Furthermore, in the method described above, the substances described in M7)-M10) are respectively introduced into the promoter sites of the nucleoside diphosphate kinase gene, enhanced uridylate kinase gene, uridine kinase gene, and inorganic pyrophosphatase gene of the recipient bacteria.

[0072] Furthermore, in the method described above, the substances described in M11)-M12) are respectively combined with the acid phosphatase gene or the glucose transport auxiliary factor gene to form an expression cassette and introduced into the recipient bacteria.

[0073] Furthermore, in the method described above, the substance described in M7)-M12) can be a constitutive strong promoter P CPA1 .

[0074] Furthermore, the steps described in M1)-M5) can be achieved by introducing into the recipient bacteria a recombinant plasmid containing the glucose isomerase gene, hexoketose kinase gene, UDP-glucose pyrophosphorylase gene, NDP-hexose-3-epimerase gene, NDP sugar hydrolase gene and / or the substances described in M1)-M5). In some embodiments of the present application, the recombinant plasmid is pTrc-BHASR, and the map of the recombinant expression vector is as follows Figure 1 , whose nucleotide sequence is SEQ ID NO.7.

[0075] Furthermore, the method further comprises the following steps:

[0076] M17, introducing acetyl-CoA synthetase gene and / or substance regulating the expression of acetyl-CoA synthetase gene into the recipient bacteria;

[0077] M18), knocking out the pyruvate oxidase gene in the recipient bacteria, wherein the recipient bacteria contains the pyruvate oxidase gene;

[0078] Furthermore, in the method, the introduction site of the acetyl-CoA synthetase gene and / or the substance regulating the expression of the acetyl-CoA synthetase gene is located at the pyruvate oxidase gene.

[0079] Furthermore, in the method described above, the substance described in M17) can be a constitutive strong promoter P CPA1 .

[0080] Furthermore, the method further comprises the following steps:

[0081] M19), introducing a substance that enhances the expression of acyl-CoA synthase gene and / or enhances the activity or content of acyl-CoA synthase into a recipient bacterium, wherein the recipient bacterium contains an acyl-CoA synthase gene;

[0082] M20), introducing a substance that enhances the expression of a long-chain fatty acid transport protein gene and / or enhances the activity or content of a long-chain fatty acid transport protein into a recipient bacterium, wherein the recipient bacterium contains a long-chain fatty acid transport protein gene;

[0083] M21), introducing into the recipient bacteria a substance that enhances the expression of the NAD(P) transhydrogenase gene and / or enhances the activity or content of the NAD(P) transhydrogenase, wherein the recipient bacteria contains the NAD(P) transhydrogenase gene;

[0084] M22), introducing substances that enhance the short-chain fatty acid degradation regulatory gene cluster atoS and atoC genes and / or enhance the activity or content of the short-chain fatty acid degradation regulatory gene cluster atoS and atoC into the recipient bacteria, wherein the recipient bacteria contain the short-chain fatty acid degradation regulatory gene cluster atoS and atoC genes;

[0085] M23), knocking out the fatty acid degradation transcription factor gene in the recipient bacteria, wherein the recipient bacteria contains the fatty acid degradation transcription factor gene;

[0086] M24), knocking out the β-ketoacyl-ACP synthase II gene in the recipient bacteria, wherein the recipient bacteria contains the β-ketoacyl-ACP synthase II gene;

[0087] M25), knocking out the β-ketoacyl-ACP synthase III gene in the recipient bacteria, wherein the recipient bacteria contains the β-ketoacyl-ACP synthase III gene;

[0088] M26), knocking out the isocitrate lyase inhibitor gene in the recipient bacteria, wherein the recipient bacteria contains the isocitrate lyase inhibitor gene.

[0089] Further, in the method described above, the P in M19)-M22) CPA1 The promoters are introduced into the recipient bacteria at the positions of the acyl-CoA synthase gene promoter, the long-chain fatty acid transporter gene promoter, the NAD(P) transhydrogenase gene promoter, and the short-chain fatty acid degradation regulatory gene cluster gene atoS and atoC promoters, respectively.

[0090] Furthermore, in the method described above, the substance described in M19)-M22) is a constitutive strong promoter P CPA1 .

[0091] The present application also provides the recombinant Escherichia coli prepared by the above method.

[0092] The present application also provides the use of the recombinant Escherichia coli and / or the method described in C1) or C2),

[0093] C1) Application in the production of D-psicose;

[0094] C1) Application in the preparation of products for producing D-psicose.

[0095] The present application also provides a composition, which is D1) or D2),

[0096] D1), containing the recombinant Escherichia coli;

[0097] D2), a culture containing the recombinant Escherichia coli.

[0098] The active ingredient of the above composition may be the recombinant Escherichia coli and / or a metabolite of the recombinant Escherichia coli and / or a culture of the recombinant Escherichia coli.

[0099] The above-mentioned culture can be a substance obtained by culturing the recombinant Escherichia coli in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the recombinant Escherichia coli and a substance secreted into a liquid culture medium, or a solid fermentation product containing the recombinant Escherichia coli and a substance secreted into a solid culture medium).

[0100] In the above, the metabolite can be a product obtained by removing the recombinant Escherichia coli from the culture, such as culturing the recombinant Escherichia coli in a liquid fermentation medium, collecting the fermentation broth (containing the recombinant Escherichia coli and substances secreted into the liquid culture medium), removing the recombinant Escherichia coli from the fermentation broth, and collecting the remaining components of the fermentation broth to obtain the metabolite of the recombinant Escherichia coli.

[0101] The active ingredients of the above composition may also contain other biological ingredients or non-biological ingredients. Those skilled in the art may determine the other active ingredients of the above composition according to the effects of the composition.

[0102] The above composition may be the culture. The above composition may also be a bacterial agent.

[0103] The above-mentioned bacterial agent refers to a live bacterial preparation made by expanding the target microorganisms and using a carrier as an adsorbent to adsorb the fermentation liquid or solid fermentation product of the bacteria.

[0104] The above-mentioned bacterial agents may be in various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.

[0105] The bacterial agent may further include a carrier as required, and the carrier may be a solid carrier or a liquid carrier.

[0106] The present application also provides a method for preparing D-psicose, comprising using the recombinant Escherichia coli and / or the composition to prepare D-psicose using D-glucose as a substrate.

[0107] Furthermore, the method includes culturing the recombinant Escherichia coli in a growth medium to grow the recombinant Escherichia coli cells, wherein the growth medium contains any one, two, three or four of glycerol, acetic acid, fatty acids, triglycerides and glucose.

[0108] In some embodiments of the present application, the growth medium may be any one of the following E1)-E4):

[0109] E1), Na2HPO4: 25mM, KH2PO4: 25mM, NH4Cl: 50mM, Na2SO4: 5mM, MgSO4: 2mM, glycerol: 3g / 100mL, yeast powder: 0.5g / 100mL, trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μMZnSO4, CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3 each 2μM, and the rest is water.

[0110] E2), Na2HPO4: 25mM, KH2PO4: 25mM, NH4Cl: 50mM, Na2SO4: 5mM, MgSO4: 2mM, sodium acetate: 2g / 100mL, yeast powder: 0.5g / 100mL, trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μMZnSO4, CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3 each 2μM, and the rest is water.

[0111] E3), 25mM Na2HPO4, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 0.5g / 100mL glycerol, 0.5g / 100mL yeast powder, 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM CoCl2, 2μM NiCl2, 2μM Na2MO4, 2μM Na2SeO3 and 2μM H3BO3, and the rest is water.

[0112] E4), add palmitic acid and polyoxyethylene ether Brij58 emulsifier to E3) until the concentration of palmitic acid is 0.5 g / 100 mL and the concentration of polyoxyethylene ether Brij58 emulsifier is 0.2 g / 100 mL.

[0113] In some embodiments of the present application, the components of the conversion medium for preparing D-psicose are: Na2HPO4: 100 mM, NaH2PO4: 100 mM, MgCl2: 5 mM, D-glucose: 15 g / 100 mL, and the rest is water.

[0114] In the present application, the glucose transport cofactor is derived from Zymomonas mobilis.

[0115] In the present application, the glucose isomerase gene may be derived from Bacillus sp.

[0116] In the present application, the hexokinokinase gene may be derived from Homo sapiens

[0117] In the present application, the UDP-glucose pyrophosphorylase gene may be derived from Arabidopsis thaliana

[0118] In the present application, the NDP-hexose-3-epimerase gene may be derived from Streptomyces spp.

[0119] In the present application, the NDP glycohydrolase gene may be derived from rat (Rattus norvegicus)

[0120] In the present application, the acid phosphatase gene may be derived from Aspergillus sp.

[0121] In the present application, the amino acid sequence of the glucose isomerase is SEQ ID No. 8. The nucleotide sequence of the glucose isomerase gene is SEQ ID No. 7, positions 267-1604.

[0122] In the present application, the amino acid sequence of the hexokinases is SEQ ID No. 9. The nucleotide sequence of the hexokinases gene is SEQ ID No. 7, positions 1620-2516.

[0123] In the present application, the amino acid sequence of the UDP-glucose pyrophosphorylase is SEQ ID No. 10. The nucleotide sequence of the UDP-glucose pyrophosphorylase gene is SEQ ID No. 7, positions 2532-3941.

[0124] In the present application, the amino acid sequence of the NDP-hexose-3-epimerase is SEQ ID No. 11. The nucleotide sequence of the NDP-hexose-3-epimerase gene is SEQ ID No. 7, positions 4123-4743.

[0125] In the present application, the amino acid sequence of the NDP glycohydrolase is SEQ ID No. 12. The nucleotide sequence of the NDP glycohydrolase gene is SEQ ID No. 7, positions 4759-5646.

[0126] In the present application, the NCBI reference sequence number of the amino acid sequence of the glucokinase is NP_416889.1 (09-MAR-2022). The nucleotide sequence of the glucokinase gene (NCBI Gene ID: 946858) is the 2508461st to 2509426th positions in the reference sequence number NC_000913.3 (09-MAR-2022). The promoter sequence of the glucokinase gene is the 2509427th to 2509522th positions in the reference sequence number NC_000913.3 (09-MAR-2022).

[0127] In the present application, the NCBI reference sequence number of the amino acid sequence of the nucleoside diphosphate kinase is NP_417013.1 (09-MAR-2022). The nucleotide sequence of the nucleoside diphosphate kinase gene (NCBI GeneID: 945611) is the 2644433rd to 2644864th positions in the reference sequence number NC_000913.3 (09-MAR-2022). The promoter sequence of the nucleoside diphosphate kinase gene is the 2644865th to 2644993rd positions in the reference sequence number NC_000913.3 (09-MAR-2022).

[0128] In the present application, the NCBI reference sequence number of the amino acid sequence of the uridylate kinase is NP_414713.1 (09-MAR-2022). The nucleotide sequence of the uridylate kinase gene (NCBI GeneID: 944989) is the 191855 to 192580 positions in the reference sequence number NC_000913.3 (09-MAR-2022). The promoter sequence of the uridylate kinase gene is the 191752 to 191854 positions in the reference sequence number NC_000913.3 (09-MAR-2022).

[0129] In the present application, the NCBI reference sequence number of the amino acid sequence of the uridine kinase is NP_416570.2 (09-MAR-2022). The nucleotide sequence of the uridine kinase gene (NCBI GeneID: 946597) is the 2142307 to 2142948 positions in the reference sequence number NC_000913.3 (09-MAR-2022). The promoter sequence of the uridine kinase gene is the 2142949 to 2143088 positions in the reference sequence number NC_000913.3 (09-MAR-2022).

[0130] In the present application, the NCBI reference sequence number of the amino acid sequence of the inorganic pyrophosphatase is NP_418647.1 (09-MAR-2022). The nucleotide sequence of the inorganic pyrophosphatase gene (NCBI GeneID: 948748) is the 4449122 to 4449652 positions in the reference sequence number NC_000913.3 (09-MAR-2022). The promoter sequence of the inorganic pyrophosphatase gene is the 4449653 to 4449806 positions in the reference sequence number NC_000913.3 (09-MAR-2022).

[0131] In the present application, the amino acid sequence of the acid phosphatase is SEQ ID No. 6. The nucleotide sequence of the acid phosphatase gene is SEQ ID No. 5.

[0132] In the present application, the NCBI reference sequence number of the amino acid sequence of the glucose transport cofactor (derived from Zymomonas mobilis) is AAA27691.1 (BCT 26-APR-1993). The nucleotide sequence of the glucose transport cofactor gene is the 368681 to 370102 (+) positions in the NCBI reference sequence number NC_006526.2.

[0133] In the present application, the NCBI reference sequence number of the amino acid sequence of the phosphotransferase system G subunit is NP_415619.1 (09-MAR-2022). The nucleotide sequence of the phosphotransferase system G subunit gene (NCBI Gene ID: 945651) is the 1157869th to 1159302th positions in the reference sequence number NC_000913.3 (09-MAR-2022).

[0134] In the present application, the NCBI reference sequence number of the amino acid sequence of the galactose repressor is NP_417314.1 (09-MAR-2022). The nucleotide sequence of the galactose repressor gene (NCBI Gene ID: 947314) is the 2976599 to 2977630 positions in the reference sequence number NC_000913.3 (09-MAR-2022).

[0135] In the present application, the NCBI reference sequence number of the amino acid sequence of the 6-phosphate glucose dehydrogenase is AAC74922.1 (17-NOV-2022). The nucleotide sequence of the 6-phosphate glucose dehydrogenase gene (NCBI Gene ID: 946370) is the 1934839 to 1936314 positions in the reference sequence number NC_000913.3 (09-MAR-2022). The NCBI reference sequence number of the amino acid sequence of the 6-phosphate glucose dehydrogenase corresponding to Gene ID: 946370 is NP_416366.1 (09-MAR-2022).

[0136] In the present application, the NCBI reference sequence number of the amino acid sequence of the lipid A biosynthesis myristoyltransferase is NP_416369.1 (09-MAR-2022). The nucleotide sequence of the lipid A biosynthesis myristoyltransferase gene (NCBI GeneID: 945143) is respectively from position 1939222 to position 1940193 in NC_000913.3 (09-MAR-2022).

[0137] In the present application, the NCBI reference sequence number of the acetyl-CoA synthetase is NP_418493.1 (09-MAR-2022). The nucleotide sequence of the acetyl-CoA synthetase gene (NCBI Gene ID 948572) is the 4285413 to 4287371 positions in the reference sequence number NC_000913.3 (09-MAR-2022).

[0138] In the present application, the NCBI reference sequence number of the amino acid sequence of the pyruvate oxidase is NP_415392.1 (09-MAR-2022). The nucleotide sequence of the pyruvate oxidase gene (NCBI Gene ID: 946132) is the 909331st to 911049th position in the reference sequence number NC_000913.3 (09-MAR-2022).

[0139] In the present application, the NCBI reference sequence number of the amino acid sequence of the acyl-CoA synthase is NP_416319.1 (09-MAR-2022). The nucleotide sequence of the acyl-CoA synthase gene (NCBI Gene ID: 946327) is shown in the NCBI reference sequence number NC_000913.3 (09-MAR-2022) from the 1888061st to the 1889746th position. The promoter sequence of the acyl-CoA synthase gene is shown in the NCBI reference sequence number NC_000913.3 (09-MAR-2022) from the 1889747th to the 1889867th position.

[0140] In the present application, the NCBI reference sequence number of the amino acid sequence of the long-chain fatty acid transport protein is NP_416846.2 (09-MAR-2022). The nucleotide sequence of the long-chain fatty acid transport protein gene (NCBI Gene ID: 946820) is the 2461306 to 2462646 positions in the NCBI reference sequence number NC_000913.3 (09-MAR-2022). The promoter sequence of the long-chain fatty acid transport protein gene is the 2461145 to 2461305 positions in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0141] In the present application, the NCBI reference sequence number of the amino acid sequence of the NAD(P) transhydrogenase is NP_418397.2 (09-MAR-2022). The nucleotide sequence of the NAD(P) transhydrogenase gene (NCBI Gene ID: 948461) is the 4159390th to 4160790th positions in the NCBI reference sequence number NC_000913.3 (09-MAR-2022). The promoter sequence of the NAD(P) transhydrogenase gene is the 4160791st to 4160862nd positions in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0142] In the present application, the nucleotide sequences of the short-chain fatty acid degradation regulatory gene cluster genes atoS and atoC (NCBI Gene ID: 949011 and 947444) are respectively positions 2320043 to 2321869 and positions 2321866 to 2323251 in NC_000913.3 (09-MAR-2022). The NCBI reference sequence numbers of the amino acid sequences of the proteins encoded by the short-chain fatty acid degradation regulatory gene clusters atoS and atoC are NP_416723.1 (09-MAR-2022) and NP_416724.1 (09-MAR-2022), respectively. The promoter sequences of the short-chain fatty acid degradation regulatory gene clusters atoS and atoC genes are positions 2320000 to 2320042 in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0143] In the present application, the NCBI reference sequence number of the amino acid sequence of the fatty acid degradation transcription factor is NP_415705.1 (09-MAR-2022). The nucleotide sequence of the fatty acid degradation transcription factor gene (NCBI Gene ID: 948652) is the 1234938 to 1235657 positions in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0144] In the present application, the NCBI reference sequence number of the amino acid sequence of the β-ketoacyl-ACP synthase II is NP_415613.1 (09-MAR-2022). The nucleotide sequence of the β-ketoacyl-ACP synthase II gene (NCBI Gene ID: 946665) is respectively from position 1151939 to position 1153180 in NC_000913.3 (09-MAR-2022).

[0145] In the present application, the NCBI reference sequence number of the amino acid sequence of the β-ketoacyl-ACP synthase III is NP_415609.1 (09-MAR-2022). The nucleotide sequence of the β-ketoacyl-ACP synthase III gene (NCBI Gene ID: 946003) is respectively from position 1148759 to position 1149712 in NC_000913.3 (09-MAR-2022).

[0146] In the present application, the NCBI reference sequence number of the amino acid sequence of the isocitrate lyase inhibitor is NP_418442.2 (09-MAR-2022). The nucleotide sequence of the isocitrate lyase inhibitor gene (NCBI Gene ID: 948524) is respectively from position 4222804 to position 4223628 in NC_000913.3 (09-MAR-2022).

[0147] In the present application, the recipient bacteria is Escherichia coli BW25113.

[0148] In this application, the promoter P 113 The nucleotide sequence is SEQ ID No.2.

[0149] In this application, the promoter CPA1 The nucleotide sequence is SEQ ID No.3.

[0150] Furthermore, the active ingredient of the above-mentioned bacterial agent may be the above-mentioned recombinant Escherichia coli or / and the culture of the above-mentioned recombinant Escherichia coli (such as a fermentation product). The active ingredient of the above-mentioned bacterial agent may also contain other biological components or non-biological components, and the other active ingredients of the above-mentioned bacterial agent may be determined by those skilled in the art according to the effect of the bacterial agent.

[0151] The term "culture" refers to a liquid or solid product (all materials in a culture container) that has a microbial colony after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or can contain a certain amount of culture medium, metabolites, or other components produced during the culture process. It can also be a mixture containing a certain amount of culture medium and microbial metabolites without the microbial cells.

[0152] The modification of the recipient bacteria in this application includes 1), 2), 3) and 4):

[0153] 1) Knock out the glucose-6-phosphate dehydrogenase, the G subunit gene of the phosphotransferase system, the galactose repressor gene, and the lipid A biosynthesis myristoyltransferase gene in the biological cells. Weaken the expression level of glucose kinase in the biological cells. Introduce or enhance the glucose transport auxiliary factor Zglf into the biological cells.

[0154] 2) Introduce or enhance the glucose isomerase gene BguiA, the hexoketose kinase gene HkhkB, the UDP-glucose pyrophosphorylase gene Augp, the NDP-hexose-3-isomerase gene Snh3e, the NDP sugar hydrolase gene Rnudt22, the nucleoside diphosphate kinase gene ndK, the uridylate kinase gene pyrH, the uridine kinase gene udK, the inorganic pyrophosphatase gene ppa, the acid phosphatase gene Aapp, the nucleoside diphosphate kinase gene ndK, the uridylate kinase gene pyrH, the uridine kinase gene udK, and the inorganic pyrophosphatase gene ppa into biological cells.

[0155] 3) The gene encoding pyruvate oxidase is replaced by a gene encoding acetyl-CoA synthetase.

[0156] 4) Introduce or enhance the fatty acyl-CoA synthetase gene, long-chain fatty acid transport protein gene, NAD(P) transhydrogenase gene, short-chain fatty acid degradation regulatory gene cluster atoS and atoC genes into biological cells, and knock out the fatty acid degradation transcription factor gene (fatty acid degradation repressor, fadR), β-ketoacyl-ACP synthase II (3-oxoacyl-[acyl carrier protein] synthase 2) gene, β-ketoacyl-ACP synthase III gene (3-oxoacyl-[acyl carrier protein] synthase 3) and isocitrate lyase repressor gene (isocitrate lyase repressor).

[0157] In the methods described in the present application, method 1) prevents the ability of utilizing D-glucose for microbial growth and accumulation of microbial biomass, thereby completely converting D-glucose into D-psicose, thereby improving substrate utilization efficiency. Method 2) provides the microorganism with the ability to synthesize D-psicose from D-glucose. Method 3) provides or enhances the ability of microbial strains to utilize acetic acid to accumulate microbial biomass. Method 4) provides or enhances the ability of microbial strains to utilize oil to accumulate microbial biomass.

[0158] The beneficial technical effects achieved by this application are as follows:

[0159] The recombinant strain provided in the present application can realize the growth of microorganisms using glycerol, acetic acid or fatty acid as carbon source, and can also use mixed carbon sources containing the above carbon source components for growth. After the microorganisms grow to a certain biomass, D-psicose is synthesized using D-glucose as raw material. And then the corresponding technical route for synthesizing D-psicose is established. BRIEF DESCRIPTION OF THE DRAWINGS

[0160] Figure 1 This is the map of pTrc-BHASR.

[0161] Figure 2 Standard curve prepared for D-psicose standard and the equation formula for the derived D-psicose concentration.

[0162] Figure 3 HPLC peak profiles of D-psicose standards of different concentrations. The values ​​of data 1 to data 5 in the upper left corner of the figure correspond to the concentrations (g / L) of the D-psicose standards, respectively.

[0163] Figure 4 Comparison of the ability of different strains to produce D-psicose.

[0164] Figure 5 This is the HPLC peak pattern of D-psicose in the reaction product. DETAILED DESCRIPTION

[0165] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0166] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0167] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0168] Escherichia coli BW25113 (CGSC#:7636), pKD46 plasmid (CGSC#:7739), and pCP20 (CGSC#:7629) were from the Escherichia coli Genetic Collection Center of Yale University CGSC.

[0169] The plasmid pTrc99a in the following examples is a product of Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd., product number ZK1610.

[0170] The D-psicose standard in the following examples is from Shanghai Yuanye Biological Products, product number: S48276.

[0171] Escherichia coli JW1176 was donated by Tao Yong's group at the Institute of Microbiology, Chinese Academy of Sciences, and is disclosed in the document "Xiaoyu Piao, Lei Wang, Baixue Lin, Hao Chen, Weifeng Liu, Yong Tao. Metabolic engineering of Escherichia coli for production of L-aspartate and its derivative β-alanine with high stoichiometric yield. Metabolic Engineering. 2019 54: 244-254." The public can obtain the above-mentioned biological materials from the applicant, and the obtained above-mentioned biological materials are only used for repeating the experiments of this application and cannot be used for other purposes.

[0172] Unless otherwise specified, the NCBI reference sequence number in the following embodiments refers to the NCBI Reference Sequence number on the NCBI website.

[0173] Example 1. Construction of recombinant Escherichia coli AL10 and production of psicose

[0174] 1. In this example, a basic bacterium AL10 was prepared that can use glycerol as a carbon source for bacterial growth and convert D-glucose into D-psicose. The preparation method of the strain is as follows, and the primers used are shown in Table 2.

[0175] (1) Knockout of 6-phosphate glucose dehydrogenase gene (hereinafter referred to as zwf gene)

[0176] Starting from Escherichia coli BW25113, the zwf gene of Escherichia coli BW25113 was knocked out to obtain the recombinant strain AL01 of Escherichia coli BW25113. The specific steps are as follows:

[0177] (1-a) Preparation of the targeting fragment zwfup-kan-zwfdown containing the zwf gene knockout trait

[0178] The target fragment zwfup-kan-zwfdown is from an E. coli strain, which is a W3110 series strain - E. coli JW1176, which contains a kanamycin resistance gene with FRT sites at both ends (denoted as frt-kan-frt screening marker fragment, SEQ ID No.1 (corresponding to GenBank: AY048744.1(32..1334(-)),11-SEP-2001)). Primers zwf-1 / zwf-2 were designed, and the JW1176 strain genome was used as a template to amplify the target fragment zwfup-kan-zwfdown containing 70bp homology arms upstream and downstream of the zwf gene site.

[0179] The sequence of the targeting fragment zwfup-kan-zwfdown is as follows: 5' to 3', in order: zwf1, frt-kan-frt (SEQ ID No. 1), zwf2 (the reverse complementary sequences of zwf1 and zwf2 are marked in the primer table).

[0180] SEQ ID No.1 frt-kan-frt screening marker fragment, 1303 bp, specifically as follows:

[0181]

[0182] (1-b) Preparation of host bacteria containing pKD46 plasmid

[0183] The pKD46 plasmid was transformed into E. coli BW25113 by calcium chloride transformation method, and clones were selected after overnight culture at 30°C on LB plates containing ampicillin to obtain recombinant E. coli BW25113 / pKD46 containing plasmid pKD46. After arabinose induction, BW25113 / pKD46 expressed three recombinant proteins of λ phage, and the host bacteria had the ability of homologous recombination. Then, the competent cells of BW25113 / pKD46 were prepared by washing with 10g / L glycerol aqueous solution.

[0184] (1-c) Homologous recombination

[0185] The target fragment zwfup-kan-zwfdown prepared in (1-a) was electroporated into the BW25113 / pKD46 competent cells prepared in (1-b), and incubated overnight at 37°C on an LB plate containing kanamycin (concentration of 50 μg / ml). The clones were selected to extract genomic DNA, and PCR amplification was performed using zwf-3 / Kan-R as primers to identify the positive clones that amplified the target band of about 1300 bp. The selected positive clones were named AL01-kan. The sequencing analysis results showed that the zwfup-kan-zwfdown fragment was contained in the genome of AL01-kan.

[0186] AL01-kan was cultured at 42°C overnight to eliminate the temperature-sensitive plasmid pKD46.

[0187] (1-d) Elimination of resistance

[0188] The pCP20 plasmid was transformed into AL01-kan by calcium chloride transformation. Cultured overnight at 30°C on an LB plate containing ampicillin, and the FLP recombinase on the pCP20 plasmid eliminated kanamycin resistance. Cultured overnight at 42°C to eliminate the temperature-sensitive plasmid pCP20. The monoclonal strain was amplified and identified by PCR using the zwf-2 / zwf-3 primers. The amplified target band of about 200 bp was positive, and the recombinant strain AL01 was obtained.

[0189] Compared with Escherichia coli BW25113, the 6-phosphate glucose dehydrogenase gene of the recombinant bacteria AL01 was knocked out.

[0190] Among them, the NCBI reference sequence number of the amino acid sequence of 6-phosphate glucose dehydrogenase is AAC74922.1 (17-NOV-2022), and the nucleotide sequence of 6-phosphate glucose dehydrogenase gene (NCBI Gene ID: 946370) is the 1934839 to 1936314 positions in the reference sequence number NC_000913.3 (09-MAR-2022). The NCBI reference sequence number of the amino acid sequence of 6-phosphate glucose dehydrogenase corresponding to Gene ID: 946370 is NP_416366.1 (09-MAR-2022).

[0191] (2) Weakening the expression of the glucokinase gene (hereinafter referred to as the glk gene) by promoter replacement

[0192] Starting from the recombinant strain AL01, the promoter of the glk gene in the strain was replaced with the Escherichia coli constitutive weak promoter P 113 (SEQ ID No.2), and obtain recombinant Escherichia coli AL02, the specific steps are as follows:

[0193] (2-a) Preparation of targeting fragment glkup-kan-P113-glkdown

[0194] Gene synthesis (GenScript) DNA fragment frt-kan-frt-P 113 , DNA fragment frt-kan-frt-P 113 It is a double-stranded DNA molecule, which consists of the following segments from upstream to downstream: frt-kan-frt screening marker fragment (SEQ ID No.1) and P 113 Promoter fragment (nucleotide sequence is SEQ ID No. 2).

[0195] Using glk-1 / glk-2 as primers and DNA fragment frt-kan-frt-P 113 PCR amplification was performed as a template to obtain the target fragment glkup-kan-P 113 -glkdown. Targeting fragment glkup-kan-P 113 The sequence of -glkdown is as follows: 5' to 3': glk1, frt-kan-frt (SEQ ID No. 1), P 113 Promoter (SEQ ID No. 2), glk2 (the reverse complementary sequences of glk1 and glk2 are shown in the primer table).

[0196] (2-b) The subsequent operations refer to steps b, c, and d in (1) (steps 1-b to 1-d) to obtain a constitutive promoter P 113 Recombinant bacteria AL02 with glk promoter replaced.

[0197] The targeting fragment introduced in step c is glkup-kan-P 113 -glkdown, using primer identification step glk-3 / Kan-R to identify a 1300bp positive clone. In step d, using primers glk-2 / glk-3 to identify the 400bp test bacteria as positive clone AL02.

[0198] Compared with AL01, the promoter of the glucokinase gene in AL02 was replaced by the weak promoter P 113 . Weak promoter P 113 The introduction site is the glucokinase gene promoter of the recipient bacteria.

[0199] P 113 The promoter sequence is SEQ ID No.2, 170 bp, and the nucleotide sequence is as follows:

[0200]

[0201] The NCBI reference sequence number of the amino acid sequence of glucokinase is NP_416889.1 (09-MAR-2022), and the nucleotide sequence of the glucokinase gene (NCBI Gene ID: 946858) is positions 2508461 to 2509426 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0202] The promoter sequence of the glucokinase gene is from position 2509427 to 2509522 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0203] (3) Enhancement of the expression of the nucleoside diphosphate kinase gene (hereinafter referred to as ndK gene) by promoter replacement

[0204] Starting from the recombinant strain AL02, the promoter of the ndK gene in the strain was replaced with the strong constitutive promoter P of Escherichia coli. CPA1 (SEQ ID No.3), and obtain recombinant Escherichia coli AL03, the specific steps are as follows:

[0205] (3-a) Preparation of targeting fragment ndKup-kan-PCPA1-ndKdown

[0206] Gene synthesis (GenScript) DNA fragment frt-kan-frt-P CPA1 , DNA fragment frt-kan-frt-P CPA1 It is a double-stranded DNA molecule, which consists of the following segments from upstream to downstream (5' to 3' in sequence): frt-kan-frt screening marker fragment (SEQ ID No. 1) and P CPA1 Promoter fragment (SEQ ID No. 3).

[0207] Using ndk-1 / ndk-2 as primers and DNA fragment frt-kan-frt-P CPA1 PCR amplification was performed as a template to obtain the target fragment ndK up-kan-P CPA1 -ndKdown. Targeting fragment ndKup-kan-P CPA1 -ndKdown sequence 5' to 3' is: ndk1, frt-kan-frt (SEQ ID No.1), P CPA1 Promoter fragment (SEQ ID No.3), ndk2 (see the primer table for the reverse complementary sequences of ndk1 and ndk2).

[0208] (3-b) The subsequent operations refer to steps b, c, and d in (1) (steps 1-b to 1-d) to obtain a constitutive promoter P CPA1Recombinant bacteria AL03 with the ndK gene promoter replaced.

[0209] The targeting fragment introduced in step c is the targeting fragment ndKup-kan-P CPA1 -ndKdown, using primers ndk-3 / Kan-R to identify 1300bp as a positive clone. In step d, using primers ndk-2 / ndk-3 to identify 400bp as a positive clone.

[0210] Compared with AL02, the promoter of the nucleoside diphosphate kinase gene in AL03 was replaced by the constitutive promoter P CPA1 . Constitutive promoter P CPA1 The introduction site is the nucleoside diphosphate kinase gene promoter of the recipient bacteria.

[0211] P CPA1 The nucleotide sequence of the promoter fragment is SEQ ID No. 3, 178 bp, and the sequence is as follows:

[0212]

[0213] The NCBI reference sequence number of the amino acid sequence of nucleoside diphosphate kinase is NP_417013.1 (09-MAR-2022), and the nucleotide sequence of the nucleoside diphosphate kinase gene (NCBI GeneID: 945611) is positions 2644433 to 2644864 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0214] The promoter sequence of the nucleoside diphosphate kinase gene is from position 2644865 to 2644993 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0215] (4) Replace the phosphotransferase system G subunit gene (hereinafter referred to as ptsG gene) with the acid phosphatase gene (hereinafter referred to as Aapp gene)

[0216] Starting from the recombinant strain AL03, the ptsG gene in the strain was replaced by the constitutive promoter P CPA1 and Aapp genes to obtain recombinant Escherichia coli AL04. The specific steps are as follows:

[0217] (4-a) Preparation of targeting fragment ptsGup-kan-P CPA1 -Aapp-ptsGdown

[0218] Gene synthesis (commissioned by GenScript) DNA fragment frt-kan-frt-P CPA1 -Aapp-TrrnB, DNA fragment frt-kan-frt-PCPA1 -Aapp-TrrnB is a double-stranded DNA molecule, which is composed of the following segments from upstream to downstream: frt-kan-frt screening marker fragment (SEQ ID No. 1), P CPA1 Promoter fragment (SEQ ID No. 3), Aapp gene (SEQ ID No. 5) and TrrnB terminator fragment (SEQ ID No. 4).

[0219] Using ptsG-1 / ptsG-2 as primers and DNA fragment frt-kan-frt-P CPA1 -Aapp-TrrnB was used as a template for PCR amplification to obtain the target fragment ptsGup-kan-P CPA1 -Aapp-ptsGdown. Targeting fragment ptsGup-kan-P CPA1 -Aapp-ptsGdown sequence 5' to 3' is: ptsG1, frt-kan-frt (SEQ ID No.1), P CPA1 (SEQ ID No.3), Aapp gene (SEQ ID No.5), TrrnB terminator fragment (SEQ ID No.4), ptsG2 (the reverse complementary sequences of ptsG1 and ptsG2 are shown in the primer table).

[0220] (4-b) The subsequent operations refer to steps b, c, and d in (1) (steps 1-b to 1-d), and the ptsG gene is replaced with a constitutive promoter P CPA1 The recombinant bacteria AL04 of the Aapp gene that initiates transcription and is terminated by the TrrnB terminator. Among them, the target fragment ptsGup-kan-P is introduced in step c. CPA1 -Aapp-ptsGdown, the primer pairs for identifying positive clones were replaced with ptsG-3 / Kan-R, the primer pairs for identifying positive clones in step d were ptsG-2 / ptsG-3, and the size of the positive clone fragment was about 1200 bp.

[0221] Compared with recombinant E. coli AL03, recombinant E. coli AL04 is a recombinant bacterium obtained by modifying E. coli AL03 as follows: the G subunit gene of the phosphosugar transferase system (ptsG gene) of E. coli AL03 is knocked out, and an acid phosphatase gene (Aapp gene) expression cassette is introduced into the site of the G subunit gene of the phosphosugar transferase system (ptsG gene) of E. coli AL03. The acid phosphatase gene expression cassette is composed of P CPA1 The protein is composed of a promoter fragment (SEQ ID No. 3), an Aapp gene (SEQ ID No. 5) and a TrrnB terminator fragment (SEQ ID No. 4).

[0222] The NCBI reference sequence number of the amino acid sequence of the phosphotransferase system G subunit is NP_415619.1 (09-MAR-2022), and the nucleotide sequence of the phosphotransferase system G subunit gene (NCBI Gene ID: 945651) is positions 1157869 to 1159302 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0223] SEQ ID No.4TrrnB terminator sequence is as follows:

[0224]

[0225] The amino acid sequence of the acid phosphatase is SEQ ID No.6, and the nucleotide sequence of the acid phosphatase gene is SEQ ID No.5.

[0226] The coding sequence of SEQ ID No.5 acid phosphatase is as follows:

[0227]

[0228] The amino acid sequence of SEQ ID No.6 acid phosphatase is as follows:

[0229]

[0230] (5) The galactose repressor gene (hereinafter referred to as galR gene) was replaced with a glucose transport cofactor gene (derived from Zymomonas mobilis, hereinafter referred to as Zglf gene)

[0231] Starting from the recombinant strain AL04, the galR gene in the strain was replaced by the constitutive promoter P CPA1 and Zglf genes to obtain recombinant Escherichia coli AL05. The specific steps are as follows:

[0232] (5-a) Preparation of targeting fragment galRup-kan-P CPA1 -Zglf-galRdown

[0233] Gene synthesis (commissioned by GenScript) DNA fragment frt-kan-frt-P CPA1 -Zglf-TrrnB, DNA fragment frt-kan-frt-P CPA1 -Zglf-TrrnB is a double-stranded DNA molecule, which is composed of the following segments from upstream to downstream: frt-kan-frt screening marker fragment (nucleotide sequence is SEQ ID No.1), P CPA1Promoter fragment (nucleotide sequence is SEQ ID No. 3), Zglf gene (nucleotide sequence is 368681 to 370102 (+) in reference sequence No. NC_006526.2) and TrrnB terminator fragment (nucleotide sequence is SEQ ID No. 4).

[0234] Using galR-1 / galR-2 as primers and DNA fragment frt-kan-frt-P CPA1 -Zglf-TrrnB was used as a template for PCR amplification to obtain the target fragment galRup-kan-P CPA1 -Zglf-galRdown. Targeting fragment galRup-kan-P CPA1 -Zglf-galRdown is composed of the following segments from upstream to downstream: galR1, frt-kan-frt screening marker fragment (nucleotide sequence is SEQ ID No.1), P CPA1 Promoter fragment (nucleotide sequence is SEQ ID No.3), Zglf gene (nucleotide sequence is 368681 to 370102 (+) positions in reference sequence number NC_006526.2), TrrnB terminator fragment (nucleotide sequence is SEQ ID No.4) and galR2 (the reverse complementary sequences of galR1 and galR2 are shown in the primer table).

[0235] (5-b) The subsequent operations refer to steps b, c, and d in (1) (steps 1-b to 1-d) to obtain the galR gene replaced by the constitutive promoter P CPA1 and galR gene recombinant bacteria AL05. Wherein, the targeting fragment introduced in step c is galRup-kan-P CPA1 -Zglf-galRdown, the primer pairs for identifying positive clones were replaced with galR-3 / Kan-R, the primer pairs for identifying positive clones in step d were galR-2 / galR-3, and the size of the positive clone fragment was about 2000 bp.

[0236] Compared with recombinant E. coli AL04, recombinant E. coli AL05 is a recombinant bacterium obtained by modifying E. coli AL04 as follows: the galactose repressor gene (galR gene for short) of E. coli AL04 is knocked out, and a glucose transport cofactor gene (Zglf gene for short) expression cassette is introduced into the galactose repressor gene site of E. coli AL04. The glucose transport cofactor gene expression cassette is composed of P CPA1The protein comprises a promoter fragment (SEQ ID No. 3), a Zglf gene (the nucleotide sequence is positions 368681 to 370102 (+) in reference sequence No. NC_006526.2) and a TrrnB terminator fragment (SEQ ID No. 4).

[0237] The NCBI reference sequence number of the amino acid sequence of the galactose repressor factor is NP_417314.1 (09-MAR-2022), and the nucleotide sequence of the galactose repressor factor gene (NCBI Gene ID: 947314) is positions 2976599 to 2977630 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0238] The NCBI reference sequence number of the amino acid sequence of the glucose transport cofactor (derived from Zymomonas mobilis) is AAA27691.1 (BCT 26-APR-1993), and the nucleotide sequence of the glucose transport cofactor gene is positions 368681 to 370102 (+) in the NCBI reference sequence number NC_006526.2.

[0239] (6) Starting from strain AL05, the expression of uridylate kinase gene (hereinafter referred to as pyrH gene), uridine kinase gene (hereinafter referred to as udK gene), and inorganic pyrophosphatase gene (hereinafter referred to as ppa gene) were enhanced by promoter replacement, and the lipid A biosynthesis myristoyltransferase gene (lpxM gene) was knocked out to obtain recombinant strains AL06, AL07, AL08, and AL09. The specific operation steps for constructing recombinant strains AL06, AL07, and AL08 are as described in Example 1 (3). The difference is that:

[0240] Starting from strain AL05, the expression of pyrH gene was enhanced by promoter replacement to obtain recombinant strain AL06. Using pyrH-1 / pyrH-2 as primers and DNA fragment frt-kan-frt-P CPA1 PCR amplification was performed as a template to obtain the target fragment pyrHup-kan-P CPA1 -pyrHdown. Targeting fragment pyrHup-kan-P CPA1 -pyrHdown sequence 5' to 3' is: pyrH1, frt-kan-frt (SEQ ID No.1), P CPA1 Promoter fragment (SEQ ID No. 3), pyrH2 (the reverse complementary sequences of pyrH1 and pyrH2 are shown in the primer table). Positive clones were identified using primer pairs pyrH-3 / Kan-R and pyrH-2 / pyrH-3.

[0241] Starting from strain AL06, the expression of udk gene was enhanced by promoter replacement to obtain recombinant strain AL07. CPA1 PCR amplification was performed as a template to obtain the target fragment udkup-kan-P CPA1 -udkdown. Target shooting segment udkup-kan-P CPA1 -udkdown sequence 5' to 3' is: udk1, frt-kan-frt (SEQ ID No.1), P CPA1 Promoter fragment (SEQ ID No. 3), udk2 (the reverse complementary sequences of udk1 and udk2 are shown in the primer table). Positive clones were identified using primer pairs udk-3 / Kan-R and udk-2 / udk-3.

[0242] Starting from strain AL07, the expression of ppa gene was enhanced by promoter replacement to obtain recombinant strain AL08. CPA1 PCR amplification was performed as a template to obtain the target fragment ppaup-kan-P CPA1 -ppadown. Targeting fragment ppaup-kan-P CPA1 -ppadown sequence 5' to 3' is: ppa1, frt-kan-frt (SEQ ID No.1), P CPA1 Promoter fragment (SEQ ID No. 3), ppa2 (the reverse complementary sequences of ppa1 and ppa2 are shown in the primer table). Positive clones were identified using primer pairs ppa-3 / Kan-R and ppa-2 / ppa-3.

[0243] Starting from the recombinant bacteria AL08, the lpxM gene of AL08 was knocked out to obtain the mutant AL09 of AL08. The specific steps refer to b, c, and d (steps 1-b to 1-d) in (1) of reference Example 1. The difference is that the targeting fragment is replaced by lpxMup-kan-lpxMdown. The preparation process is: using lpxM-1 / lpxM-2 as primers and the JW1176 strain genome as a template, amplify the targeting fragment lpxMup-kan-lxpMdown containing the upstream and downstream homologous arms of the lpxM gene site. The sequence of the targeting fragment lpxMup-kan-lpxMdown is as follows: 5' to 3' is: lpxM1, frt-kan-frt (SEQ ID No.1), lpxM2 (the reverse complementary sequences of lpxM1 and lpxM2 are marked in the primer table). The primer pairs lpxM-3 / Kan-R and lpxM-2 / lpxM-3 are used to identify positive clones.

[0244] Compared with AL05, AL06 is a gene in which the promoter of the uridylate kinase gene in the recipient bacteria is replaced by the constitutive promoter P CPA1 . Constitutive promoter P CPA1 The introduction site is the uridylate kinase gene promoter of the recipient bacteria AL05.

[0245] Compared with AL06, the promoter of the uridine kinase gene in AL07 was replaced by the constitutive promoter P CPA1 . Constitutive promoter P CPA1 The introduction site is the uridine kinase gene promoter of the recipient bacteria AL06.

[0246] Compared with AL07, the promoter of inorganic pyrophosphatase gene in AL08 was replaced by constitutive promoter P CPA1 . Constitutive promoter P CPA1 The introduction site is the inorganic pyrophosphatase gene promoter of the recipient bacteria AL07.

[0247] Compared with AL08, the lipid A biosynthesis myristoyltransferase gene (lpxM gene) of AL09 was knocked out.

[0248] The NCBI reference sequence number of the amino acid sequence of uridylate kinase is NP_414713.1 (09-MAR-2022), and the nucleotide sequence of the uridylate kinase gene (NCBI GeneID: 944989) is positions 191855 to 192580 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0249] The NCBI reference sequence number of the amino acid sequence of uridine kinase is NP_416570.2 (09-MAR-2022), and the nucleotide sequence of the uridine kinase gene (NCBI GeneID: 946597) is positions 2142307 to 2142948 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0250] The NCBI reference sequence number of the amino acid sequence of inorganic pyrophosphatase is NP_418647.1 (09-MAR-2022), and the nucleotide sequence of the inorganic pyrophosphatase gene (NCBI GeneID: 948748) is positions 4449122 to 4449652 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0251] The promoter sequence of the uridylate kinase gene is from position 191752 to 191854 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0252] The promoter sequence of the uridine kinase gene is from position 2142949 to 2143088 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0253] The promoter sequence of the inorganic pyrophosphatase gene is from position 4449653 to 4449806 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0254] The NCBI reference sequence number of the amino acid sequence of lipid A biosynthesis myristoyltransferase is NP_416369.1 (09-MAR-2022), and the nucleotide sequence of the lipid A biosynthesis myristoyltransferase gene (NCBI Gene ID: 945143) is from position 1939222 to position 1940193 in NC_000913.3 (09-MAR-2022).

[0255] (7) Introduce the glucose isomerase gene (BguiA gene), the hexoketol kinase gene (HkhkB gene), the UDP-glucose pyrophosphorylase gene (Augp gene), the NDP-hexose-3-isomerase gene (Snh3e gene), and the NDP sugar hydrolase gene (Rnudt22 gene) into Escherichia coli.

[0256] Starting from the recombinant bacteria AL09, the expression plasmid pTrc-BHASR containing the BguiA gene, the HkhkB gene, the Augp gene, the Snh3e gene, and the Rnudt22 gene was introduced into AL09 to obtain AL10. The specific steps are as follows:

[0257] (7-a) Preparation of pTrc-BHASR plasmid

[0258] The recombinant expression vector pTrc-BHASR containing BguiA gene, HkhkB gene, Augp gene, Snh3e gene, Rnudt22 gene and related expression elements was constructed. The map of the recombinant expression vector is shown in FIG. Figure 1 , whose nucleotide sequence is SEQ ID NO. 7. Among them, SEQ ID NO. 7 has trc promoter at positions 193-266, BguiA gene at positions 267-1604, HkhkB gene at positions 1620-2516, Augp gene at positions 2532-3941, rrnB terminator at positions 3942-4028, trc promoter at positions 4049-4122, Snh3e gene at positions 4123-4743, Rnudt22 gene at positions 4759-5646, and rrnB terminator at positions 5647-5733.

[0259] The sequence of SEQ ID NO.7 is as follows:

[0260]

[0261]

[0262]

[0263] E. coli DH5α competent cells (Beijing Quanshijin Biotechnology Co., Ltd., product catalog CD201) were transformed by CaCl2 method. They were evenly spread on LB plates containing ampicillin and cultured at 37°C overnight. Clones were selected, and the clones that could amplify the target fragment were identified and sequenced using primers RV-M / pTrcHis-R. Positive clones were selected to extract plasmids, and the obtained positive plasmids were named pTrc-BHASR.

[0264] (7-b) Plasmid transformation

[0265] The pTrc-BHASR plasmid was transformed into the recombinant bacteria AL09 by the calcium chloride transformation method, and clones were selected after overnight culture at 37°C on LB plates containing ampicillin, and positive clones were identified with primers RV-M / pTrcHis-R and frozen at -80°C. The strain was named Escherichia coli AL10.

[0266] Compared with AL09, AL10 contains expression cassettes for glucose isomerase gene (BguiA gene), ketokinase gene (HkhkB gene), UDP-glucose pyrophosphorylase gene (Augp gene), NDP-hexose-3-isomerase gene (Snh3e gene), and NDP sugar hydrolase gene (Rnudt22 gene).

[0267] The amino acid sequence of the glucose isomerase is SEQ ID No. 8, and the nucleotide sequence of the glucose isomerase gene is SEQ ID No. 7, positions 267-1604.

[0268] The amino acid sequence of the hexolose kinase is SEQ ID No. 9, and the nucleotide sequence of the hexolose kinase gene is SEQ ID No. 7, positions 1620-2516.

[0269] The amino acid sequence of the UDP-glucose pyrophosphorylase is SEQ ID No. 10, and the nucleotide sequence of the UDP-glucose pyrophosphorylase gene is SEQ ID No. 7, positions 2532-3941.

[0270] The amino acid sequence of the NDP-hexose-3-epimerase is SEQ ID No. 11, and the nucleotide sequence of the NDP-hexose-3-epimerase gene is SEQ ID No. 7, positions 4123-4743.

[0271] The amino acid sequence of the NDP glycohydrolase is SEQ ID No. 12, and the nucleotide sequence of the NDP glycohydrolase gene is SEQ ID No. 7, positions 4759-5646.

[0272] The amino acid sequence of SEQ ID No.8 is as follows:

[0273]

[0274] The amino acid sequence of SEQ ID No.9 is as follows:

[0275]

[0276] The amino acid sequence of SEQ ID No.10 is as follows:

[0277]

[0278] SEQ ID No.11,206AA, the amino acid sequence is as follows:

[0279]

[0280] SEQ ID No.12 295AA, the amino acid sequence is as follows:

[0281]

[0282] 2. Cultivation of recombinant bacteria AL10 using glycerol as carbon source and preparation of D-psicose using glucose as substrate

[0283] 1. Glycerol Growth Medium Composition

[0284] Growth medium components and final concentrations are as follows:

[0285] Na2HPO4: 25mM, KH2PO4: 25mM, NH4Cl: 50mM, Na2SO4: 5mM, MgSO4: 2mM, glycerol: 3g / 100mL, yeast powder: 0.5g / 100mL, trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3, and the rest is water.

[0286] 2. Transformation medium components

[0287] Na2HPO4: 25mM, KH2PO4: 25mM, MgCl2: 50mM, D-glucose: 15g / 100mL, and the rest is water.

[0288] 3. Bacterial Cultivation and Enzyme Induction

[0289] The wild-type strain BW25113, recombinant strain AL09 and engineered strain AL10 cultured overnight were inoculated at 1% in a shake flask containing 200 mL of glycerol growth medium (wherein the AL10 growth medium contained ampicillin at a final concentration of 100 mg / L) and cultured at 30°C for 3-4 h until the OD 600nm When the concentration of IPTG reached 0.6-0.8, add IPTG to a final concentration of 0.5 mM, continue culturing at 30°C for 12 h, and collect the bacteria by centrifugation at 8000 rpm for 10 min.

[0290] 4. Whole-cell catalysis of D-psicose

[0291] The collected bacteria (wild-type strain BW25113, recombinant strain AL09 and engineered strain AL10) were resuspended in a shake flask containing 10 mL of transformation medium and reacted at 220 r / min and 37 ° C. The start of the reaction was recorded as 0 h. The reaction process was regularly monitored using pH test paper, and 0.1 M NaOH and HCl solutions were used to adjust the pH to 7.0. The supernatant was centrifuged and the D-psicose content was detected at 4h, 8h, 12h, 16h, 20h and 24h of the reaction. Three replicates were set for each reaction time.

[0292] The content of D-psicose was detected by HPLC using the external standard method. HPLC used 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 ° C; the detector was a differential refractive index detector; the temperature was 40 ° C. In the HPLC analysis, D-psicose (Shanghai Yuanye Biological Products (Cat. No.: S48276)) was used as the standard to qualitatively analyze D-psicose according to the retention time of the standard and the standard curve method (external standard method) was used for quantitative analysis. Figure 2 The standard curve prepared for the D-psicose standard and the equation for the derived D-psicose concentration are as follows: Y=1254.11X-1629.77, r2=0.9999, r=0.9999. In the formula, Y is the characteristic peak area, and X is the D-psicose concentration of the sample on the machine (in g / L). Among them: the yield is defined as the concentration of D-psicose produced in the reaction solution; the conversion rate is defined as the ratio of the mass of D-psicose produced in the reaction solution to the mass of D-glucose raw material consumed. Figure 3The HPLC peak diagram of D-psicose standard with different concentrations (the numbers in the upper left corner are different standard concentrations (g / L)). The content of D-glucose was detected by HPLC using the external standard method. The HPLC detection method and conditions are the same as the above-mentioned D-psicose detection method, wherein D-glucose (Beijing Mairida product (item number: M043597)) is used as the standard to perform qualitative analysis based on the retention time of the standard and the standard curve method (external standard method) is used for quantitative analysis of D-glucose.

[0293] The results of the test are as follows Figure 4 As shown in the results, after 24 hours of reaction, the production of D-psicose of wild-type strain BW25113 and recombinant strain AL09 was 0, and the average production of D-psicose of engineered strain AL10 was 103.6±5.4 g / L. D-glucose can be converted into D-psicose using engineered strain AL10, and the average conversion rate after 24 hours of reaction is 69.1±3.6%. In addition, the purity of D-psicose in the reaction solution is high, and no other sugars are obviously detected ( Figure 5 ), which is conducive to simplifying the downstream separation and purification process.

[0294] Example 2: Construction of recombinant Escherichia coli AL12 and production of psicose

[0295] 1. In this example, a basic bacterium AL12 was prepared that can use acetic acid as a carbon source for bacterial growth and convert D-glucose into D-psicose. The preparation method of the strain is as follows, and the primers used are shown in Table 2.

[0296] (1) Replace the pyruvate oxidase gene (hereinafter referred to as poxB gene) with the acetyl-CoA synthetase gene (hereinafter referred to as acs gene)

[0297] Starting from the recombinant strain AL09, the poxB gene of Escherichia coli AL09 was replaced with P CPA1 The acs gene expressed by the promoter was activated to obtain the mutant AL11 of Escherichia coli AL09. The specific operation steps are as described in (4) of Example 1. The specific steps are as follows:

[0298] (1-a) Preparation of targeting fragment poxBup-kan-P119-acs-poxBdown

[0299] Gene synthesis (commissioned by GenScript) DNA fragment frt-kan-frt-P CPA1 -acs-TrrnB, DNA fragment frt-kan-frt-P CPA1 -acs-TrrnB is a double-stranded DNA molecule, which is composed of the following segments from upstream to downstream: frt-kan-frt screening marker fragment (SEQ ID No.1), PCPA1 Promoter fragment (SEQ ID No. 3), acs gene (the nucleotide sequence of acs gene is 4285413 to 4287371 in reference sequence number NC_000913.3 (09-MAR-2022)) and TrrnB terminator fragment (SEQ ID No. 4).

[0300] Using poxB-1 / poxB-2 as primers, the gene synthesized DNA fragment frt-kan-frt-P CPA1 -acs-TrrnB was used as a template for PCR amplification to obtain the targeting fragment poxBup-kan-P CPA1 -acs-poxBdown. Targeting fragment poxBup-kan-P CPA1 The sequence of -acs-poxBdown from 5' to 3' end is poxB1, frt-kan-frt (SEQ ID No.1), P CPA1 Promoter fragment (SEQ ID No. 3), acs gene, poxB2 (see the primer table for the reverse complementary sequences of poxB1 and poxB2).

[0301] (1-b) The subsequent operations refer to steps b, c, and d in Example 1 (1) (steps 1-b to 1-d) to obtain the poxB gene replaced with the constitutive promoter P CPA1 and acs gene recombinant bacteria AL11. The targeting fragment introduced in step c is poxBup-kan-P CPA1 -acs-poxBdown, the primer pair for identifying positive clones was replaced with poxB-3 / Kan-R, and the primer pair for identifying positive clones in step d was poxB-2 / poxB-3, respectively.

[0302] Compared with recombinant E. coli AL09, recombinant E. coli AL11 is a recombinant bacterium obtained by modifying E. coli AL09 as follows: the pyruvate oxidase gene (poxB gene) of E. coli AL09 is knocked out, and an acetyl-CoA synthetase gene (acs gene) expression cassette is introduced into the pyruvate oxidase gene site of E. coli AL09. The acetyl-CoA synthetase gene expression cassette is composed of P CPA1 The promoter fragment (SEQ ID No. 3), the acs gene (the nucleotide sequence of the acs gene is the 4285413 to 4287371 positions in the reference sequence number NC_000913.3 (09-MAR-2022)) and the TrrnB terminator fragment (SEQ ID No. 4).

[0303] The NCBI reference sequence number of the amino acid sequence of pyruvate oxidase is NP_415392.1 (09-MAR-2022), and the nucleotide sequence of the pyruvate oxidase gene (NCBI Gene ID: 946132) is positions 909331 to 911049 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0304] The NCBI reference sequence number of acetyl-CoA synthetase is NP_418493.1 (09-MAR-2022), and the nucleotide sequence of the acetyl-CoA synthetase gene (NCBI Gene ID 948572) is positions 4285413 to 4287371 in the reference sequence number NC_000913.3 (09-MAR-2022).

[0305] (2) Starting from the recombinant bacterium AL11, the plasmid pTrc-BHASR was introduced into AL11 to obtain AL12. The specific steps are as described in (7) of Example 1.

[0306] Compared with AL11, AL12 contains expression cassettes for glucose isomerase gene (BguiA gene), hexoketokinase gene (HkhkB gene), UDP-glucose pyrophosphorylase gene (Augp gene), NDP-hexose-3-isomerase gene (Snh3e gene), and NDP sugar hydrolase gene (Rnudt22 gene).

[0307] 2. Cultivation of recombinant strain AL10 using acetic acid as carbon source and preparation of D-psicose using glucose as substrate

[0308] 1. Acetic acid growth medium composition

[0309] Na2HPO4: 25mM, KH2PO4: 25mM, NH4Cl: 50mM, Na2SO4: 5mM, MgSO4: 2mM, sodium acetate: 2g / 100mL, yeast powder: 0.5g / 100mL, trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3, and the rest is water.

[0310] 2. Transformation medium components

[0311] Na2HPO4: 100mM, NaH2PO4: 100mM, MgCl2: 5mM, D-glucose: 15g / 100mL, and the rest is water.

[0312] 3. Bacterial Cultivation and Enzyme Induction

[0313] The overnight cultured recombinant bacteria AL11 and engineered strain AL12 were inoculated into a shake flask containing 200 mL of acetic acid growth medium (AL12 growth medium contained ampicillin at a final concentration of 100 mg / L) and cultured at 30°C for 3-4 h until the OD 600nm When the concentration of IPTG reached 0.6-0.8, add IPTG to a final concentration of 0.5 mM, continue culturing at 30°C for 12 h, and collect the cells by centrifugation at 8000 rpm for 10 min.

[0314] 4. Whole-cell catalysis of D-psicose

[0315] The collected bacteria (recombinant bacteria AL11 and engineered strain AL12) were resuspended in a shake flask containing 10 mL of transformation medium and reacted at 220 r / min and 37°C. The start of the reaction was recorded as 0 h. The reaction process was regularly monitored using pH test paper, and 0.1 M NaOH and HCl solutions were used to adjust the pH to 7.0. The supernatant was centrifuged and taken at 4 h, 8 h, 12 h, 16 h, 20 h and 24 h of the reaction, and the content of D-psicose was detected by HPLC according to the method of Example 1. Three replicates were set for each reaction time.

[0316] The results of the test are as follows Figure 4 As shown in the figure, the results show that after 24 hours of reaction, the yield of D-psicose of the recombinant strain AL11 is 0, and the average yield of D-psicose of the engineered strain AL12 is 94.2±4.7g / L. Therefore, D-glucose can be converted into D-psicose using the engineered strain AL12, with a conversion rate of 62.8±3.1%. In addition, the purity of D-psicose in the reaction solution is high, and no other sugars are obviously detected, which is conducive to simplifying the downstream separation and purification process.

[0317] Example 3. Construction of recombinant Escherichia coli AL22 and production of psicose

[0318] 1. This example prepared a basic bacterium AL22 that can use fatty acids as a carbon source for bacterial growth and convert D-glucose into D-psicose. The preparation method of the strain is as follows, and the primers used are shown in Table 2.

[0319] (1) Knockout of fatty acid degradation transcription factor gene (hereinafter referred to as fadR gene)

[0320] Starting from the recombinant bacteria AL11, the fadR gene of AL11 was knocked out to obtain the mutant AL13 of AL11. The specific steps refer to b, c, and d (steps 1-b to 1-d) in (1) of reference Example 1. The difference is that the targeting fragment is replaced by fadRup-kan-fadRdown. The preparation process is: using fadR-1 / fadR-2 as primers and the JW1176 strain genome as a template, amplify the targeting fragment fadRup-kan-fadRdown containing the upstream and downstream homologous arms of the fadR gene site. The sequence of the targeting fragment fadRup-kan-fadRdown is as follows: 5' to 3' is: fadR1, frt-kan-frt (SEQ ID No.1), fadR2 (the reverse complementary sequences of fadR1 and fadR2 are marked in the primer table). The primer pairs fadR-3 / Kan-R and fadR-2 / fadR-3 are used to identify positive clones.

[0321] Compared with the recombinant bacteria AL11, the fatty acid degradation transcription factor gene (fadR gene for short) of the recombinant bacteria AL13 was knocked out.

[0322] The NCBI reference sequence number of the amino acid sequence of the fatty acid degradation transcription factor is NP_415705.1 (09-MAR-2022), and the nucleotide sequence of the fatty acid degradation transcription factor gene (NCBI Gene ID: 948652) is positions 1234938 to 1235657 in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0323] (2) Enhancement of the expression of the acyl-CoA synthase gene (hereinafter referred to as fadD gene) by promoter replacement

[0324] Starting from the recombinant strain AL13, the promoter of the fadD gene in the strain was replaced with the constitutive promoter P of Escherichia coli. CPA1 , and obtain recombinant E. coli AL14. The specific steps are as described in Example 1 (3). The difference is that the targeting fragment is fadDup-kan-P CPA1 -fadDdown, replace primers ndk-1 / ndk-2 / ndk-3 with primers fadD-1 / fadD-2 / fadD-3 respectively. The specific steps are as follows:

[0325] (2-a) Preparation of targeting fragment fadDup-kan-P CPA1 -fadDdown

[0326] Using fadD-1 / fadD-2 as primers, the gene synthesized DNA fragment frt-kan-frt-P CPA1PCR amplification was performed as a template to obtain the target fragment fadDup-kan-P CPA1 -fadDdown. Targeting segment fadDup-kan-P CPA1 -fadDdown sequence 5' to 3' is: fadD1, frt-kan-frt (SEQ ID No.1), P CPA1 Promoter fragment (SEQ ID No. 3), fadD2 (see the primer table for the reverse complementary sequences of fadD1 and fadD2).

[0327] (2-b) The subsequent operations refer to steps b, c, and d in (1) (steps 1-b to 1-d) to obtain a constitutive promoter P CPA1 The recombinant strain AL14 with the fadD promoter replaced. The targeting fragment introduced in step c is fadDup-kan-P CPA1 -fadDdown, positive clones were identified as fadD-3 / Kan-R, and 1400bp positive clones were identified. In step d, positive clones were identified as fadD-2 / fadD-3, and 300bp positive clones were identified.

[0328] Compared with AL13, the promoter of the acyl-CoA synthase gene in AL14 was replaced by the constitutive promoter P CPA1 . Constitutive promoter P CPA1 The introduction site is the promoter of the acyl-CoA synthase gene of the recipient bacteria AL13.

[0329] The NCBI reference sequence number of the amino acid sequence of acyl-CoA synthase is NP_416319.1 (09-MAR-2022), and the nucleotide sequence of the acyl-CoA synthase gene (NCBI Gene ID: 946327) is shown at positions 1888061 to 1889746 in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0330] The promoter sequence of the acyl-CoA synthase gene is shown at positions 1889747 to 1889867 in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0331] (3) Using AL14 as the starting strain, the expression of the long-chain fatty acid transporter gene (hereinafter referred to as fadL gene), NAD(P) transhydrogenase gene (hereinafter referred to as sthA gene), and short-chain fatty acid degradation regulatory gene cluster genes (atoS gene and atoC gene) were enhanced by promoter replacement in turn to obtain recombinant bacteria AL15, AL16, and AL17.

[0332] The specific operation steps are the same as those in Example 1 (3). The difference is that:

[0333] Starting from strain AL14, the expression of fadL gene was enhanced by promoter replacement to obtain recombinant strain AL15. CPA1 PCR amplification was performed as a template to obtain the target fragment fadLup-kan-P CPA1 -fadLdown. Targeting fragment fadLup-kan-P CPA1 -fadLdown sequence 5' to 3' is: fadL1, frt-kan-frt (SEQ ID No.1), P CPA1 Promoter fragment (SEQ ID No. 3), fadL2 (the reverse complementary sequences of fadL1 and fadL2 are shown in the primer table). Positive clones were identified using primer pairs fadL-3 / Kan-R and fadL-2 / fadL-3.

[0334] Compared with AL14, the promoter of the long-chain fatty acid transporter gene in AL15 was replaced by the constitutive promoter P CPA1 . Constitutive promoter P CPA1 The introduction site is the promoter of the long-chain fatty acid transporter gene of the recipient bacteria AL14.

[0335] Starting from strain AL15, the expression of sthA gene was enhanced by promoter replacement to obtain recombinant strain AL16. sthA-1 / sthA-2 were used as primers and the DNA fragment frt-kan-frt-P CPA1 PCR amplification was performed as a template to obtain the target fragment sthAup-kan-P CPA1 -sthAdown. Targeting fragment sthAup-kan-P CPA1 -sthAdown sequence 5' to 3' is: sthA1, frt-kan-frt (SEQ ID No.1), P CPA1 Promoter fragment (SEQ ID No. 3), sthA2 (the reverse complementary sequences of sthA1 and sthA2 are shown in the primer table). Positive clones were identified using primer pairs sthA-3 / Kan-R and sthA-2 / sthA-3.

[0336] Compared with AL15, the promoter of the NAD(P) transhydrogenase gene in AL16 was replaced by the constitutive promoter P CPA1 . Constitutive promoter P CPA1 The introduction site is the NAD(P) transhydrogenase gene promoter of the recipient bacteria AL15.

[0337] Starting from strain AL16, the expression of atoS and atoC genes was enhanced by promoter replacement to obtain recombinant strain AL17. CPA1 PCR amplification was performed as a template to obtain the target fragment atoSCup-kan-P CPA1 -atoSCdown. Targeting fragment atoSCup-kan-P CPA1 -atoSCdown sequence 5' to 3' is: atoSC1, frt-kan-frt (SEQ ID No.1), P CPA1 Promoter fragment (SEQ ID No. 3), atoSC2 (the reverse complementary sequences of atoSC1 and atoSC2 are shown in the primer table). Positive clones were identified using primer pairs atoSC-3 / Kan-R and atoSC-2 / atoSC-3.

[0338] Compared with AL16, the promoter of the short-chain fatty acid degradation regulatory gene cluster in AL17 was replaced by the constitutive promoter P CPA1 . Constitutive promoter P CPA1 The introduction site is the promoter of the short-chain fatty acid degradation regulatory gene cluster gene of the recipient bacteria AL16.

[0339] The NCBI reference sequence number of the amino acid sequence of the long-chain fatty acid transport protein is NP_416846.2 (09-MAR-2022), and the nucleotide sequence of the long-chain fatty acid transport protein gene (NCBI Gene ID: 946820) is 2461306 to 2462646 in the NCBI reference sequence number NC_000913.3 (09-MAR-2022). The promoter sequence of the long-chain fatty acid transport protein gene is 2461145 to 2461305 in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0340] The NCBI reference sequence number of the amino acid sequence of NAD(P) transhydrogenase is NP_418397.2 (09-MAR-2022), and the nucleotide sequence of the NAD(P) transhydrogenase gene (NCBI Gene ID: 948461) is the 4159390 to 4160790 positions in the NCBI reference sequence number NC_000913.3 (09-MAR-2022). The promoter sequence of the NAD(P) transhydrogenase gene is the 4160791 to 4160862 positions in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0341] The nucleotide sequences of the short-chain fatty acid degradation regulatory gene cluster genes atoS and atoC (NCBI Gene ID: 949011 and 947444) are 2320043 to 2321869 and 2321866 to 2323251 in NC_000913.3 (09-MAR-2022), respectively. The NCBI reference sequence numbers of the amino acid sequences of the proteins encoded by the short-chain fatty acid degradation regulatory gene cluster atoS and atoC are NP_416723.1 (09-MAR-2022) and NP_416724.1 (09-MAR-2022), respectively. The promoter sequences of the short-chain fatty acid degradation regulatory gene cluster genes (atoS and atoC) are 2320000 to 2320042 in the NCBI reference sequence number NC_000913.3 (09-MAR-2022).

[0342] (4) Using AL17 as the starting strain, the β-ketoacyl-ACP synthase II gene (fabF gene), β-ketoacyl-ACP synthase III gene (fabH gene) and isocitrate lyase inhibitor gene (iclR gene) were knocked out in sequence to obtain recombinant bacteria AL18, AL20 and AL21.

[0343] Starting from strain AL17, the fabF gene was knocked out to obtain recombinant strain AL18. Using fabF-1 / fabF-2 as primers and the JW1176 strain genome as a template, the targeting fragment fabFup-kan-fabFdown containing the upstream and downstream homologous arms of the fabF gene site was amplified. The sequence of the targeting fragment fabFup-kan-fabFdown from 5' to 3' is: fabF1, frt-kan-frt (SEQ ID No.1), fabF2 (the reverse complementary sequences of fabF1 and fabF2 are shown in the primer table). The primer pairs fabF-3 / Kan-R and fabF-2 / fabF-3 were used to identify positive clones.

[0344] Starting from strain AL18, the fabH gene was knocked out to obtain recombinant strain AL20. Using fabH-1 / fabH-2 as primers and the JW1176 strain genome as a template, the targeting fragment fabHup-kan-fabHdown containing the upstream and downstream homologous arms of the fabH gene site was amplified. The sequence of the targeting fragment fabHup-kan-fabHdown from 5' to 3' is: fabH1, frt-kan-frt (SEQ ID No.1), fabH2 (the reverse complementary sequences of fabH1 and fabH2 are shown in the primer table). The primer pairs fabH-3 / Kan-R and fabH-2 / fabH-3 were used to identify positive clones.

[0345] Starting from strain AL20, the iclR gene was knocked out to obtain recombinant strain AL21. Using iclR-1 / iclR-2 as primers and the JW1176 strain genome as a template, the targeting fragment iclRup-kan-iclRdown containing the upstream and downstream homologous arms of the iclR gene site was amplified. The sequence of the targeting fragment iclRup-kan-iclRdown from 5' to 3' is: iclR1, frt-kan-frt (SEQ ID No.1), iclR2 (the reverse complementary sequences of iclR1 and iclR2 are shown in the primer table). The primer pairs iclR-3 / Kan-R and iclR-2 / iclR-3 were used to identify positive clones.

[0346] Among them, the NCBI reference sequence number of the amino acid sequence of β-ketoacyl-ACP synthase II is NP_415613.1 (09-MAR-2022), and the nucleotide sequence of the β-ketoacyl-ACP synthase II gene (NCBI Gene ID: 946665) is positions 1151939 to 1153180 in NC_000913.3 (09-MAR-2022).

[0347] The NCBI reference sequence number of the amino acid sequence of β-ketoacyl-ACP synthase III is NP_415609.1 (09-MAR-2022), and the nucleotide sequence of the β-ketoacyl-ACP synthase III gene (NCBI Gene ID: 946003) is positions 1148759 to 1149712 in NC_000913.3 (09-MAR-2022).

[0348] The NCBI reference sequence number of the amino acid sequence of the isocitrate lyase inhibitor is NP_418442.2 (09-MAR-2022), and the nucleotide sequence of the isocitrate lyase inhibitor gene (NCBI Gene ID: 948524) is positions 4222804 to 4223628 in NC_000913.3 (09-MAR-2022).

[0349] (7) Starting from the recombinant bacterium AL21, the plasmid pTrc-BHASR was introduced into AL21 to obtain AL22. The specific steps are as described in (7) of Example 1.

[0350] Compared with AL21, AL22 contains expression cassettes for glucose isomerase gene (BguiA gene), ketokinase gene (HkhkB gene), UDP-glucose pyrophosphorylase gene (Augp gene), NDP-hexose-3-isomerase gene (Snh3e gene), and NDP sugar hydrolase gene (Rnudt22 gene).

[0351] 2. Cultivation of recombinant bacteria AL22 using fatty acids as carbon source and preparation of D-psicose using glucose as substrate

[0352] 1. Fatty Acid Growth Medium Composition

[0353] Culture medium A: Culture medium A is a sterile culture medium composed of solutes and solvents, the solvent is water, and the solutes and their concentrations are: 25mM Na2HPO4, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 0.5g / 100mL glycerol, 0.5g / 100mL yeast powder, 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μMCoCl2, 2μM NiCl2, 2μM Na2MO4, 2μM Na2SeO3 and 2μM H3BO3, and the rest is water.

[0354] Medium B: Medium B is a sterile medium obtained by adding palmitic acid and polyoxyethylene ether Brij58 emulsifier to medium A, wherein the concentration of palmitic acid is 0.5 g / 100 mL, and the concentration of polyoxyethylene ether Brij58 emulsifier is 0.2 g / 100 mL.

[0355] 2. Transformation medium components

[0356] Na2HPO4: 100mM, NaH2PO4: 100mM, MgCl2: 5mM, D-glucose: 15g / 100mL, and the rest is water.

[0357] 3. Cultivation of bacteria and induction of enzymes.

[0358] The wild-type recombinant strain AL21 and engineered strain AL22 cultured overnight in medium A were inoculated at 1% in a shake flask containing 200 ml medium B (AL22 growth medium contained ampicillin at a final concentration of 100 mg / L) and cultured at 30°C for 3-4 h until OD 600nm When the pH value is 0.6-0.8 (determined after extracting the oil components with n-hexane), add IPTG at a final concentration of 0.5 mM and continue culturing at 30°C for 12 hours. Collect the cells.

[0359] 4. Whole-cell catalysis of D-psicose.

[0360] The collected bacteria (recombinant bacteria AL21 and engineered strain AL22) were resuspended in a shake flask containing 10 mL of transformation medium and reacted at 220 r / min and 37°C. The start of the reaction was recorded as 0 h. The reaction process was regularly detected using pH test paper, and 0.1 M NaOH and HCl solutions were used to adjust the pH to 7.0. The supernatant was centrifuged and taken at 4 h, 8 h, 12 h, 16 h, 20 h and 24 h of the reaction, and the content of D-psicose was detected by HPLC according to the method of Example 1. Three replicates were set for each reaction time.

[0361] The results of the test are as follows Figure 4 As shown, the results show that after 24 hours of reaction, the yield of D-psicose of the recombinant strain AL21 is 0, and the average yield of D-psicose of the engineered strain AL22 is 109.5±6.6g / L. The engineered strain AL22 can convert D-glucose into D-psicose with a conversion rate of 73.0±4.4%. In addition, the purity of D-psicose in the reaction solution is high, and no other sugars are obviously detected, which is conducive to simplifying the downstream separation and purification process.

[0362] AL22, AL12, and AL10 were grown using different carbon sources, and then the whole cells catalyzed the synthesis of D-psicose. The results showed that the above three strains can effectively synthesize psicose, and the conversion rate exceeded 55% (Table 1).

[0363] Table 1 Comparison of the yield of D-psicose prepared by AL22, AL12 and AL10

[0364] strain Carbon source Allulose yield (g / L) Conversion rate AL10 glycerin 103.6±5.4 79.8±2.4％ AL12 Acetic acid 94.2±4.7 84.2±3.3％ AL22 Palmitic acid 109.5±6.6 90.7±1.6％

[0365] Table 2 Primer names and primer nucleotide sequences in the examples

[0366]

[0367]

[0368]

[0369] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims

1. A recombinant Escherichia coli having the following characteristics: A1), substances containing glucose isomerase gene and / or regulating the expression of glucose isomerase gene; A2), substances containing hexokinakinase gene and / or regulating the expression of hexokinakinase gene; A3), a substance containing a UDP-glucose pyrophosphorylase gene and / or regulating the expression of a UDP-glucose pyrophosphorylase gene; A4), a substance containing an NDP-hexose-3-epimerase gene and / or regulating the expression of an NDP-hexose-3-epimerase gene; A5), substances containing NDP glycohydrolase gene and / or regulating the expression of NDP glycohydrolase gene; A6), containing substances that reduce the expression of glucokinase gene and / or reduce the activity or content of glucokinase; A7), containing substances that enhance the expression of nucleoside diphosphate kinase genes and / or enhance the activity or content of nucleoside diphosphate kinases; A8), containing substances that enhance the expression of uridylate kinase gene and / or enhance the activity or content of uridylate kinase gene; A9), containing substances that enhance uridine kinase gene expression and / or enhance uridine kinase activity or content; A10), containing substances that enhance the expression of inorganic pyrophosphatase genes and / or enhance the activity or content of inorganic pyrophosphatase; A11), substances containing acid phosphatase gene and / or regulating the expression of acid phosphatase gene; A12), substances containing glucose transport cofactor genes and / or regulating the expression of glucose transport cofactor genes; A13), does not contain the G subunit gene of the phosphotransferase system; A14), does not contain the galactose repressor gene; A15), does not contain 6-phosphate glucose dehydrogenase gene; A16) does not contain the lipid A biosynthesis myristoyltransferase gene.

2. The recombinant Escherichia coli according to claim 1, characterized in that The recombinant Escherichia coli also has the following characteristics: A17), substances containing acetyl-CoA synthetase gene and / or regulating the expression of acetyl-CoA synthetase gene; A18) does not contain the pyruvate oxidase gene.

3. The recombinant Escherichia coli according to claim 2, characterized in that The recombinant Escherichia coli also has the following characteristics: A19), containing substances that enhance the expression of acyl-CoA synthase genes and / or enhance the activity or content of acyl-CoA synthase; A20), containing substances that enhance the gene expression of long-chain fatty acid transport proteins and / or enhance the activity or content of long-chain fatty acid transport proteins; A21), containing substances that enhance NAD(P) transhydrogenase gene expression and / or enhance NAD(P) transhydrogenase activity or content; A22), substances containing genes that enhance the expression of the short-chain fatty acid degradation regulatory gene cluster atoS and atoC and / or enhance the activity or content of the short-chain fatty acid degradation regulatory gene cluster atoS and atoC; A23), does not contain fatty acid degradation transcription factor genes; A24), does not contain β-ketoacyl-ACP synthase II gene; A25), does not contain β-ketoacyl-ACP synthase III gene; A26) does not contain the isocitrate lyase inhibitor gene.

4. A method for preparing the recombinant Escherichia coli according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: M1), introducing glucose isomerase gene and / or substance regulating glucose isomerase gene expression into recipient bacteria; M2), introducing the hexokinokinase gene and / or a substance regulating the expression of the hexokinokinase gene into the recipient bacteria; M3), introducing UDP-glucose pyrophosphorylase gene and / or substance regulating UDP-glucose pyrophosphorylase gene expression into the recipient bacteria; M4), introducing NDP-hexose-3-epimerase gene and / or a substance regulating the expression of NDP-hexose-3-epimerase gene into the recipient bacteria; M5), introducing NDP glycohydrolase gene and / or substance regulating the expression of NDP glycohydrolase gene into the recipient bacteria; M6), introducing a substance that reduces the expression of the glucokinase gene and / or reduces the activity or content of glucokinase into a recipient bacterium, wherein the recipient bacterium contains a glucokinase gene; M7), introducing a substance that enhances the expression of a nucleoside diphosphate kinase gene and / or enhances the activity or content of a nucleoside diphosphate kinase into a recipient bacterium, wherein the recipient bacterium contains a nucleoside diphosphate kinase gene; M8), introducing a substance that enhances uridylate kinase gene expression and / or enhances uridylate kinase activity or content into a recipient bacterium, wherein the recipient bacterium contains a uridylate kinase gene; M9), introducing a substance that enhances uridine kinase gene expression and / or enhances uridine kinase activity or content into a recipient bacterium, wherein the recipient bacterium contains a uridine kinase gene; M10), introducing a substance that enhances the inorganic pyrophosphatase gene and / or enhances the activity or content of inorganic pyrophosphatase into a recipient bacterium, wherein the recipient bacterium contains an inorganic pyrophosphatase gene; M11), introducing acid phosphatase gene and / or substance regulating the expression of acid phosphatase gene into the recipient bacteria; M12), introducing glucose transport cofactor genes and / or substances regulating the expression of glucose transport cofactor genes into the recipient bacteria; M13), knocking out the G subunit gene of the phosphosugar transferase system in the recipient bacteria, wherein the recipient bacteria contains the G subunit gene of the phosphosugar transferase system; M14), knocking out the galactose repressor gene in the recipient bacteria, wherein the recipient bacteria contains the galactose repressor gene; M15), knocking out the 6-phosphate glucose dehydrogenase gene in the recipient bacteria, wherein the recipient bacteria contains the 6-phosphate glucose dehydrogenase gene; M16), knocking out the lipid A biosynthesis myristoyltransferase gene in the recipient bacteria, wherein the recipient bacteria contains the lipid A biosynthesis myristoyltransferase gene.

5. The method according to claim 4, characterized in that The method further comprises the steps of: M17), introducing acetyl-CoA synthetase gene and / or substance regulating acetyl-CoA synthetase gene expression into the recipient bacteria; M18), knocking out the pyruvate oxidase gene in the recipient bacteria, wherein the recipient bacteria contains the pyruvate oxidase gene.

6. The method according to claim 5, characterized in that: The method further comprises the steps of: M19), introducing a substance that enhances the expression of acyl-CoA synthase gene and / or enhances the activity or content of acyl-CoA synthase into a recipient bacterium, wherein the recipient bacterium contains an acyl-CoA synthase gene; M20), introducing a substance that enhances the expression of a long-chain fatty acid transport protein gene and / or enhances the activity or content of a long-chain fatty acid transport protein into a recipient bacterium, wherein the recipient bacterium contains a long-chain fatty acid transport protein gene; M21), introducing into the recipient bacteria a substance that enhances the expression of the NAD(P) transhydrogenase gene and / or enhances the activity or content of the NAD(P) transhydrogenase, wherein the recipient bacteria contains the NAD(P) transhydrogenase gene; M22), introducing substances that enhance the short-chain fatty acid degradation regulatory gene cluster atoS and atoC genes and / or enhance the activity or content of the short-chain fatty acid degradation regulatory gene cluster atoS and atoC into the recipient bacteria, wherein the recipient bacteria contain the short-chain fatty acid degradation regulatory gene cluster atoS and atoC genes; M23), knocking out the fatty acid degradation transcription factor gene in the recipient bacteria, wherein the recipient bacteria contains the fatty acid degradation transcription factor gene; M24), knocking out the β-ketoacyl-ACP synthase II gene in the recipient bacteria, wherein the recipient bacteria contains the β-ketoacyl-ACP synthase II gene; M25), knocking out the β-ketoacyl-ACP synthase III gene in the recipient bacteria, wherein the recipient bacteria contains the β-ketoacyl-ACP synthase III gene; M26), knocking out the isocitrate lyase inhibitor gene in the recipient bacteria, wherein the recipient bacteria contains the isocitrate lyase inhibitor gene.

7. The recombinant Escherichia coli prepared by the method according to any one of claims 4 to 6.

8. Use of the recombinant Escherichia coli according to any one of claims 1 to 3, the method according to any one of claims 4 to 6, and / or the recombinant Escherichia coli according to claim 7 in C1) or C2), C1) Application in the production of D-psicose; C2) Application in the preparation of products for producing D-psicose.

9. A composition, characterized in that: The composition is D1) or D2), D1), containing the recombinant Escherichia coli according to any one of claims 1 to 3; D2), a culture containing the recombinant Escherichia coli according to any one of claims 1 to 3.

10. A method for preparing D-psicose, characterized in that: The method comprises using the recombinant Escherichia coli according to any one of claims 1 to 3, the recombinant Escherichia coli according to claim 7 and / or the composition according to claim 9 to prepare D-psicose using D-glucose as a substrate.

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