Recombinant Escherichia coli for producing D-psicose and preparation method and application thereof

Abstract

The present invention discloses recombinant Escherichia coli for producing D-psicose, and its preparation method and application. The recombinant Escherichia coli is obtained by subjecting Escherichia coli to multiple modifications; the multiple modifications include enhancing the expression of the araE gene, replacing the lpxM gene with the cscB gene and the cscA gene, replacing the glk gene with the Aapp gene, and introducing and expressing the BguiA gene, the HkhkB gene, the Ausp gene, the Snh3e gene, and the Rnudt22 gene. The present invention provides recombinant Escherichia coli that can achieve the growth of microorganisms using low-cost, easily available green organic raw materials such as glycerol, acetic acid, or fatty acids as carbon sources. After the microbial growth reaches a certain biomass, D-psicose is synthesized using low-cost sucrose as raw material. Not only does it reduce production costs, but it also efficiently synthesizes D-psicose, which has great economic and social value.

Description

Technical Field

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

[0002] D-allulose is a safe and healthy rare monosaccharide that can be widely used in the food, beverage, pharmaceutical 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 is not only moderately sweet and extremely low in calories, but also has multiple pharmacological effects such as lowering blood sugar response, reducing liver fat production, maintaining weight, anti-inflammatory, neuroprotective and immunosuppressive. Because of its special nutritional and biological functions, its preparation method has attracted more and more attention from researchers. The content of D-allulose in nature is extremely small. It is generally found in wheat, fruit and various other foods. Therefore, it cannot be prepared by natural extraction.

[0003] Japan was the first country in the world to research and develop allulose. In 2012, Matsutani Chemical Industries, a Japanese company, launched a rare sugar syrup containing a certain percentage of D-allulose nationwide, which proved popular with consumers. In 2015, South Korea's CJ, in collaboration with Anderson Pharmaceuticals of the United States, launched the AllSweet brand, primarily selling syrup and powdered sugar containing a certain percentage of D-allulose to North America and other regions. Around the same time, the UK's Tate & Lyle Dolcia Prima low-calorie allulose syrup entered the market. While research on allulose started relatively late in China, Jiangnan University was one of the earliest institutions to undertake this research. Their research encompasses gene discovery and strain development, enzyme immobilization, and the isolation and purification of D-allulose. The published technology shows that the enzymes and enzyme expression performance developed by the Jiangnan University team are comparable to those published by major international companies and are essentially in line with international standards. The team has applied for multiple patents related to this research. However, the mainstream production method using diastereomerase on the market currently 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 fully converted into the product, requiring a complex product separation process and expensive production equipment.

[0004] Traditional methods for producing D-psicose include chemical and biological methods, with chemical synthesis initially the primary method. Bilik et al. discovered that D-fructose can be converted to D-psicose in an acidic aqueous solution under the catalytic action of molybdate ions. In 1997, Donald et al. synthesized D-psicose from 1,2:4,5-di-O-isopropylidene-β-D-fructopyranose through chemical synthesis. D-psicose can also be synthesized by boiling ethanol and triethylamine. Further research has revealed that chemical synthesis involves complex purification steps, the generation of chemical waste, and the production of worthless byproducts. Furthermore, chemically synthesized sweeteners often lack a pure sweetness, resulting in limited application.

[0005] Biosynthesis utilizes abundant natural raw materials and enzymes as catalysts to produce allulose. This not only reduces industrial production costs but also adheres to current green and environmentally friendly production principles. Currently, the predominant method is enzyme-catalyzed biotransformation. For example, identified wild-type isomerases generally have low thermal stability. To meet the needs of industrial applications, targeted discovery, thermostability analysis, and modification of isomerases with diverse properties are being extensively studied. Furthermore, enzyme preservation is crucial during use; extending the shelf life and maintaining enzyme stability are fundamental to commercial application. Currently, the biocatalysts that have been discovered and characterized for the biosynthesis of D-psicose include D-psicose 3-epimerase (DPEase) and D-tagatose 3-epimerase. These enzymes are often used to catalyze the conversion of D-fructose to produce D-psicose.

[0006] Fatty acids are inexpensive, readily available, green organic raw materials. They have a higher energy density than raw materials like glucose and provide more reducing power for biosynthesis, thereby improving the conversion rate of biomanufacturing. They are ideal raw materials for biomanufacturing and can be obtained inexpensively from sources such as crude oil processing products and waste cooking oils. Glycerol and acetic acid, byproducts of chemical production, are widely available and inexpensive. Fermenting strains using glycerol or acetic acid as carbon sources can help address food shortages. Sucrose is a cost-effective alternative. Establishing a route for synthesizing D-psicose using glycerol, acetic acid, and fatty acids as carbon sources for microbial growth and sucrose as a raw material has significant economic and social value. Summary of the Invention

[0007] The technical problem to be solved by the present invention is how to utilize glycerol, acetic acid and fatty acids as carbon sources to supply microbial growth and prepare D-psicose using sucrose as raw material.

[0008] In order to solve the above technical problems, the present invention first provides a recombinant Escherichia coli.

[0009] The recombinant Escherichia coli provided by the present invention is obtained by subjecting Escherichia coli to multiple transformations;

[0010] The plurality of modifications include modification (a) and modification (d);

[0011] Modification (a) includes the following modifications (a1)-(a3):

[0012] Modification (a1): Enhanced expression of the araE gene;

[0013] Transformation (a2): lpxM gene was replaced by cscB gene and cscA gene;

[0014] Transformation (a3): glk gene replaced by Aapp gene;

[0015] Transformation (d): Introduction and expression of BguiA gene, HkhkB gene, Ausp gene, Snh3e gene and Rnudt22 gene.

[0016] In order to solve the above technical problems, the present invention further provides a method for constructing a recombinant Escherichia coli that produces D-psicose.

[0017] The method for constructing a recombinant Escherichia coli producing D-psicose provided by the present invention comprises the following steps: performing multiple transformations on the Escherichia coli; the multiple transformations include transformation (a) and transformation (d);

[0018] Modification (a) includes the following modifications (a1)-(a3):

[0019] Modification (a1): Enhanced expression of the araE gene;

[0020] Transformation (a2): lpxM gene was replaced by cscB gene and cscA gene;

[0021] Transformation (a3): glk gene replaced by Aapp gene;

[0022] Transformation (d): Introduction and expression of BguiA gene, HkhkB gene, Ausp gene, Snh3e gene and Rnudt22 gene.

[0023] In one embodiment of the present invention, the plurality of modifications further comprises modification (b);

[0024] The transformation (b) includes the following transformations (b1) to (b12):

[0025] Transformation (b1): knockout of fadR gene;

[0026] Modification (b2): Enhanced expression of the ndK gene;

[0027] Modification (b3): ​​Enhanced expression of the pyrH gene;

[0028] Modification (b4): Enhanced expression of udk gene;

[0029] Modification (b5): Enhanced expression of the ppa gene;

[0030] Modification (b6): Enhanced expression of the fadD gene;

[0031] Modification (b7): Enhanced expression of fadL gene;

[0032] Modification (b8): Enhanced expression of the sthA gene;

[0033] Modification (b9): Enhanced expression of the atoSC gene cluster;

[0034] Transformation (b10): knockout of the fabF gene;

[0035] Modification (b11): knockout of the fabH gene;

[0036] Modification (b12): Knockout of the iclR gene.

[0037] In another embodiment of the present invention, the multiple modifications further include modification (c); the modification (c): the poxB gene is replaced by the acs gene.

[0038] In a specific embodiment of the present invention, the BguiA gene is derived from Bacillus ( Bacillus sp. ).

[0039] In a specific embodiment of the present invention, the HkhkB gene is derived from human ( Homo sapiens ).

[0040] In a specific embodiment of the present invention, the Ausp gene is derived from Arabidopsis thaliana ( Arabidopsis thaliana ).

[0041] In a specific embodiment of the present invention, the Snh3e gene is derived from Streptomyces ( Streptomyces sp. ).

[0042] In a specific embodiment of the present invention, the Rnudt22 gene is derived from rat ( Rattus norvegicus ).

[0043] In a specific embodiment of the present invention, the Aapp gene is derived from Aspergillus ( Aspergillus sp.).

[0044] In a specific embodiment of the present invention, the cscB gene is derived from Escherichia coli W strain ( Escherichia coli W).

[0045] In a specific embodiment of the present invention, the cscA gene is derived from Escherichia coli W strain ( Escherichia coli W).

[0046] Any of the above-mentioned methods for enhancing the expression of the araE gene is carried out by replacing the araE gene promoter with the Escherichia coli constitutive promoter P CPA1 Furthermore, the araE gene promoter is replaced with the Escherichia coli constitutive promoter P CPA1 Through the targeting fragment araEup-kan-P CPA1 -araEdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment araEup-kan-P CPA1 -araEdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment was used as a template and primers araE-1 / araE-2 were used for PCR amplification.

[0047] Any of the above-mentioned enhancements to ndk gene expression are achieved by replacing the ndk gene promoter with the Escherichia coli constitutive promoter P CPA1 Furthermore, the ndk gene promoter is replaced with the Escherichia coli constitutive promoter P CPA1 By targeting the fragment ndkup-kan-P CPA1 -ndkdown, pCP20 plasmid and pKD46 plasmid. CPA1 -ndkdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment was used as a template and PCR amplified using primers ndk-1 / ndk-2.

[0048] Any of the above-mentioned methods for enhancing the expression of the pyrH gene is carried out by replacing the pyrH gene promoter with the Escherichia coli constitutive promoter P CPA1 Furthermore, the pyrH gene promoter is replaced with the Escherichia coli constitutive promoter P CPA1 By targeting the fragment pyrHup-kan-PCPA1 -pyrHdown, pCP20 plasmid and pKD46 plasmid. Further, the targeting fragment pyrHup-kan-P CPA1 -pyrHdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment was used as a template and primers pyrH-1 / pyrH-2 were used for PCR amplification.

[0049] Any of the above-mentioned enhancements to udk gene expression are achieved by replacing the udk gene promoter with the Escherichia coli constitutive promoter P CPA1 Furthermore, the udk gene promoter is replaced with the Escherichia coli constitutive promoter P CPA1 Through the target shooting segment udkup-kan-P CPA1 -udkdown, pCP20 plasmid and pKD46 plasmid. Further, the targeting fragment udkup-kan-P CPA1 -udkdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment was used as a template and primers udk-1 / udk-2 were used for PCR amplification.

[0050] Any of the above-mentioned enhancements to ppa gene expression are achieved by replacing the ppa gene promoter with the Escherichia coli constitutive promoter P CPA1 Furthermore, the ppa gene promoter is replaced with the Escherichia coli constitutive promoter P CPA1 Through the targeting fragment ppaup-kan-P CPA1 -ppadown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment ppaup-kan-P CPA1 -ppadown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment was used as a template and primers ppa-1 / ppa-2 were used for PCR amplification.

[0051] Any of the above-mentioned enhancements of fadD gene expression are achieved by replacing the fadD gene promoter with the Escherichia coli constitutive promoter P CPA1 Furthermore, the fadD gene promoter is replaced with the Escherichia coli constitutive promoter PCPA1 By targeting the fragment fadDup-kan-P CPA1 -fadDdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment fadDup-kan-P CPA1 -fadDdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment was used as a template and primers fadD-1 / fadD-2 were used for PCR amplification.

[0052] Any of the above-mentioned enhancements to fadL gene expression are achieved by replacing the fadL gene promoter with the Escherichia coli constitutive promoter P CPA1 Furthermore, the fadL gene promoter is replaced with the Escherichia coli constitutive promoter P CPA1 By targeting the fragment fadLup-kan-P CPA1 -fadLdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment fadLup-kan-P CPA1 -fadLdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment was used as a template and primers fadL-1 / fadL-2 were used for PCR amplification.

[0053] Any of the above-mentioned enhancements of sthA gene expression is achieved by replacing the sthA gene promoter with the Escherichia coli constitutive promoter P CPA1 Furthermore, the sthA gene promoter is replaced with the Escherichia coli constitutive promoter P CPA1 By targeting the fragment sthAup-kan-P CPA1 -sthAdown, pCP20 plasmid and pKD46 plasmid. Further, the targeting fragment sthAup-kan-P CPA1 -sthAdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment was used as a template and primers sthA-1 / sthA-2 were used for PCR amplification.

[0054] Any of the above-mentioned enhancements to the expression of the atoSC gene cluster are achieved by replacing the atoSC gene cluster promoter with the Escherichia coli constitutive promoter P CPA1 Furthermore, the atoSC gene cluster promoter is replaced with the Escherichia coli constitutive promoter P CPA1 By targeting the fragment atoSCup-kan-P CPA1 -atoSCdown, pCP20 plasmid and pKD46 plasmid. Further, the targeting fragment atoSCup-kan-P CPA1 -atoSCdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No. 14, the P CPA1 The promoter fragment was used as a template and primers atoSC-1 / atoSC-2 were used for PCR amplification.

[0055] Any of the above lpxM genes are replaced by cscB gene and cscA gene, and the lpxM gene is replaced by Escherichia coli constitutive promoter P CPA1 , cscB gene and cscA gene. Further, the lpxM gene is replaced by the Escherichia coli constitutive promoter P CPA1 , cscB gene and cscA gene by targeting fragment lpxMup-kan-P CPA1 -cscB-cscA-lpxMdown, pCP20 plasmid and pKD46 plasmid. CPA1 -cscB-cscA-lpxMdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment, cscB gene, RBS (5'-AGGAG-3'), cscA gene and TrrnB terminator fragment shown in SEQ ID No. 15 were used as templates and PCR amplified using primers lpxM-1 / lpxM-2.

[0056] Any of the above glk genes is replaced by Aapp gene or glk gene is replaced by Escherichia coli constitutive promoter P CPA1 Furthermore, the glk gene is replaced by the Escherichia coli constitutive promoter P CPA1 and Aapp genes by targeting the glkup-kan-P CPA1-Aapp-glkdown, pCP20 plasmid and pKD46 plasmid. Further, the targeting fragment glkup-kan-P CPA1 -Aapp-glkdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment, Aapp gene and TrrnB terminator fragment shown in SEQ ID No. 15 were used as templates and PCR amplified using primers glk-1 / glk-2.

[0057] Any of the above-mentioned poxB genes are replaced by acs genes or the poxB gene is replaced by the Escherichia coli constitutive promoter P CPA1 Furthermore, the poxB gene is replaced by the Escherichia coli constitutive promoter P CPA1 and acs genes by targeting the fragment poxBup-kan-P CPA1 -acs-poxBdown, pCP20 plasmid and pKD46 plasmid. CPA1 -acs-poxBdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment, acs gene and TrrnB terminator fragment shown in SEQ ID No. 15 were used as templates and PCR amplified using primers poxB-1 / poxB-2.

[0058] The knockout of the fadR gene described above is achieved by using the targeting fragment fadRup-kan-fadRdown, the pCP20 plasmid, and the pKD46 plasmid. Further, the targeting fragment fadRup-kan-fadRdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No. 14, the P shown in SEQ ID No. 13). CPA1 The promoter fragment was used as a template and primers fadR-1 / fadR-2 were used for PCR amplification.

[0059] The knockout of the fabF gene described above is achieved by using the targeting fragment fabFup-kan-fabFdown, the pCP20 plasmid, and the pKD46 plasmid. Further, the targeting fragment fabFup-kan-fabFdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No. 14, the P shown in SEQ ID No. 13) CPA1 The promoter fragment was used as a template and primers fabF-1 / fabF-2 were used for PCR amplification.

[0060] The knockout of the fabH gene described above is achieved by using the targeting fragment fabHup-kan-fabHdown, the pCP20 plasmid, and the pKD46 plasmid. Further, the targeting fragment fabHup-kan-fabHdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No. 14, the P shown in SEQ ID No. 13) CPA1 The promoter fragment was used as a template and primers fabH-1 / fabH-2 were used for PCR amplification.

[0061] Any of the above-mentioned knockout of the iclR gene is achieved by using the targeting fragment iclRup-kan-iclRdown, the pCP20 plasmid and the pKD46 plasmid. Further, the targeting fragment iclRup-kan-iclRdown can be a gene fragment (5' to 3' is sequentially composed of the frt-kan-frt screening marker fragment shown in SEQ ID No. 14, the P shown in SEQ ID No. 13) CPA1 The promoter fragment was used as a template and primers iclR-1 / iclR-2 were used for PCR amplification.

[0062] Any of the above-mentioned BguiA genes, HkhkB genes, Ausp genes, Snh3e genes and Rnudt22 genes are introduced through the recombinant expression vector pTrc99a-BHASR. The recombinant expression vector pTrc99a-BHASR can be obtained by connecting the BHASR fragment with the pTrc fragment by the Gibson assembly method; the BHASR fragment can be a gene fragment (5' to 3' is sequentially composed of the BguiA gene shown in SEQ ID No. 2, RBS (5'-AGGAG-3'), HkhkB gene shown in SEQ ID No. 4, RBS (5'-AGGAG-3'), Ausp gene shown in SEQ ID No. 6, TrrnB terminator shown in SEQ ID No. 15, trc promoter shown in SEQ ID No. 16, Snh3e gene shown in SEQ ID No. 8, RBS (5'-AGGAG-3'), SEQ ID The Rnudt22 gene shown in No. 10) was used as a template and primers BHASR-01F / BHASR-01R were used to perform PCR amplification to obtain the fragment; the pTrc fragment was obtained by using plasmid pTrc99a as a template and primers pTrc99a-01F / pTrc99a-01R were used to perform PCR amplification to obtain the fragment.

[0063] Any of the above-mentioned Escherichia coli is an Escherichia coli containing the araE gene, the lpxM gene, the glk gene, the fadR gene, the ndK gene, the pyrH gene, the udk gene, the ppa gene, the fadD gene, the fadL gene, the sthA gene, the atoSC gene cluster, the fabF gene, the fabH gene, the iclR gene, and the poxB gene.

[0064] In a specific embodiment of the present invention, the Escherichia coli is Escherichia coli MG1655 strain.

[0065] In a specific embodiment of the present invention, the deposit number of the recombinant Escherichia coli may be CGMCC No.32627.

[0066] The sequences of any of the above primers are shown in Table 1 in the Examples below.

[0067] The recombinant Escherichia coli prepared according to the above method also falls within the protection scope of the present invention.

[0068] The use of the above-mentioned recombinant Escherichia coli or the recombinant Escherichia coli prepared according to the above-mentioned method in the preparation of D-psicose also falls within the scope of protection of the present invention.

[0069] In order to solve the above technical problems, the present invention finally provides a method for preparing D-psicose.

[0070] The preparation method of D-psicose provided by the present invention comprises the following steps: using sucrose as a raw material and adopting the above-mentioned recombinant Escherichia coli to carry out biotransformation to obtain D-psicose.

[0071] The method further comprises the step of culturing the recombinant Escherichia coli using at least one of glycerol, acetic acid or fatty acid as a carbon source.

[0072] The fatty acids include saturated fatty acids such as palmitic acid and myristic acid and unsaturated fatty acids such as oleic acid, as well as glycerol fatty acid esters containing the above fatty acid components and raw materials containing the above components.

[0073] The fatty acids can be extracted from oils and fats. The oils and fats can be extracted from waste cooking oils and fats. Specifically, the extraction method includes the steps of recovery, pretreatment, esterification, and distillation.

[0074] The fatty acids can also be obtained from oil processing by-products, palm fatty acid distillate (PFAD), recycling of waste cooking oil, and other sources.

[0075] The glycerol can be produced by fermentation or synthesis (eg, from carbon dioxide or petrochemical raw materials).

[0076] The acetic acid can be produced by fermentation or synthesis.

[0077] In a specific embodiment of the present invention, the method comprises the following steps: using sucrose as a raw material and glycerol as a carbon source to culture the above-mentioned recombinant E. coli for bioconversion to obtain D-psicose. Further, the method comprises the following steps: inoculating the above-mentioned recombinant E. coli into a glycerol growth medium and culturing at 30°C for 3-4 hours until the OD 600nmAfter the pH value reaches 0.6-0.8, IPTG is added to a final concentration of 0.5 mM, and the cells are cultured at 30°C for 12 hours. The cells are then centrifuged and harvested. The cells are then resuspended in transformation medium and reacted at 37°C for 24 hours (maintaining the pH at 7.0 during the reaction). The supernatant is then collected by centrifugation. The glycerol growth medium contains water as the solvent, and the solutes and final concentrations are as follows: Na2HPO4 25 mM, KH2PO4 25 mM, NH4Cl 50 mM, Na2SO4 5 mM, MgSO4 2 mM, glycerol 3% (volume percentage concentration), yeast extract 0.5% (mass percentage concentration), FeCl3 50 μM, CaCl2 20 μM, MnCl2 10 μM, ZnSO4 10 μM, CoCl2 2 μM, NiCl2 2 μM, Na2MO4 2 μM, Na2SeO 32 μM, and H3BO 32 μM. The transformation medium solvent is water, and the solutes and their final concentrations are as follows: Na2HPO4 25 mM, KH2PO4 25 mM, MgCl2 5 mM, and sucrose 15% (mass percentage concentration).

[0078] In another specific embodiment of the present invention, the method comprises the following steps: using sucrose as a raw material and acetic acid as a carbon source to cultivate the above-mentioned recombinant Escherichia coli for bioconversion to obtain D-psicose. Further, the method comprises the following steps: inoculating the above-mentioned recombinant Escherichia coli into acetic acid growth medium, culturing at 30°C for 3-4 hours until the OD 600nm After the pH value reaches 0.6-0.8, IPTG is added to a final concentration of 0.5 mM, and the cells are cultured at 30°C for 12 hours. The cells are then centrifuged and collected. The cells are then resuspended in transformation medium and reacted at 37°C for 24 hours (maintaining the pH at 7.0 during the reaction). The cells are then centrifuged and the supernatant is collected. The acetic acid growth medium contains water as the solvent, and the solutes and final concentrations are as follows: 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4, 2% sodium acetate (mass percentage concentration), 0.5% yeast extract (mass percentage concentration), 50 μM FeCl3, 20 μM CaCl2, 10 μM MnCl2, 10 μM ZnSO4, 2 μM CoCl2, 2 μM NiCl2, 42 μM Na2MO4, 32 μM Na2SeO, and 32 μM H3BO. The transformation medium solvent is water, and the solutes and their final concentrations are as follows: Na2HPO4 100 mM, NaH2PO4 100 mM, MgCl2 5 mM, and sucrose 15% (mass percentage concentration).

[0079] In another specific embodiment of the present invention, the method comprises the following steps: using sucrose as a raw material and fatty acids as a carbon source to culture the above-mentioned recombinant E. coli for bioconversion to obtain D-psicose. Furthermore, the method comprises the following steps: inoculating the above-mentioned recombinant E. coli into medium A, culturing overnight and then inoculating 1% of the inoculum into medium B, culturing at 30°C for 3-4 hours until the OD reaches 600nm After the pH reaches 0.6-0.8, IPTG is added to a final concentration of 0.5 mM. The cells are cultured at 30°C for 12 hours, centrifuged, and harvested. The cells are resuspended in transformation medium and reacted at 37°C for 24 hours (maintaining the pH at 7.0 during the reaction). The supernatant is then collected by centrifugation. The solvent in medium A is water, and the solutes and final concentrations are as follows: Na2HPO4 25 mM, KH2PO4 25 mM, NH4Cl 50 mM, Na2SO4 5 mM, MgSO4 2 mM, glycerol 0.5% (volume percentage concentration), yeast extract 0.5% (mass percentage concentration), FeCl3 50 μM, CaCl2 20 μM, MnCl2 10 μM, ZnSO4 10 μM, CoCl2 2 μM, NiCl2 2 μM, Na2MO4 2 μM, Na2SeO 32 μM, and H3BO 32 μM. Medium B is prepared by mixing Medium A with palmitic acid and a polyoxyethylene ether Brij 58 emulsifier, wherein the mass percentage concentration of palmitic acid is 0.5% and the mass percentage concentration of polyoxyethylene ether Brij 58 emulsifier is 0.2%. The transformation medium solvent is water, and the solutes and their final concentrations are as follows: 100 mM Na2HPO4, 100 mM NaH2PO4, 5 mM MgCl2, and 15% sucrose (mass percentage concentration).

[0080] The nucleotide sequence of any of the above-mentioned araE genes is shown in Gene Bank as Gene ID: 947341, and the amino acid sequence of the arabinose transporter encoded by the gene is shown in NCBI as reference sequence number NP_417318.1.

[0081] The nucleotide sequence of any of the above-mentioned cscB genes is shown in positions 3171-4418 of the genome sequence of Gene ID: X81461.2 in Gene Bank, and the amino acid sequence of the sucrose permease encoded by the gene is shown in the reference sequence number CAA57217.1 in NCBI.

[0082] The nucleotide sequence of any of the above-mentioned cscA genes is shown in positions 5619-7052 of the genome sequence with Gene ID: X81461.2 in Gene Bank, and the amino acid sequence of the sucrose permease encoded by the gene is shown in the reference sequence number CAA57219.1 in NCBI.

[0083] The nucleotide sequence of any of the above-mentioned lpxM genes is shown in Gene Bank as Gene ID: 945143, and the amino acid sequence of the lipid A biosynthesis myristoyltransferase encoded by the gene is shown in NCBI as reference sequence number NP_416369.1.

[0084] The nucleotide sequence of any of the glk genes mentioned above is shown in Gene Bank as Gene ID: 946858, and the amino acid sequence of the glucokinase encoded by the gene is shown in NCBI as reference sequence number NP_416889.1.

[0085] The nucleotide sequence of any of the above-mentioned Aapp genes is shown as SEQ ID No. 12, and the amino acid sequence of the acid phosphatase encoded by the gene is shown as SEQ ID No. 11.

[0086] The nucleotide sequence of any of the above-mentioned BguiA genes is shown as SEQ ID No. 2, and the amino acid sequence of the xylose isomerase encoded by the gene is shown as SEQ ID No. 1.

[0087] The nucleotide sequence of any of the above-mentioned HkhkB genes is shown as SEQ ID No. 4, and the amino acid sequence of the hexokinokinase encoded by the gene is shown as SEQ ID No. 3.

[0088] The nucleotide sequence of any of the above-mentioned Ausp genes is shown as SEQ ID No. 6, and the amino acid sequence of the UDP-sugar pyrophosphorylase encoded by the gene is shown as SEQ ID No. 5.

[0089] The nucleotide sequence of any of the above-mentioned Snh3e genes is shown as SEQ ID No. 8, and the amino acid sequence of the NDP-hexose-3-epimerase encoded by the gene is shown as SEQ ID No. 7.

[0090] The nucleotide sequence of any of the above-mentioned Rnudt22 genes is shown as SEQ ID No. 10, and the amino acid sequence of the NDP glycohydrolase encoded by the gene is shown as SEQ ID No. 9.

[0091] The nucleotide sequence of any of the above-mentioned fadR genes is shown in Gene Bank as Gene ID: 948652, and the amino acid sequence of the fatty acid degradation transcription factor encoded by the gene is shown in NCBI as reference sequence number NP_415705.1.

[0092] The nucleotide sequence of any of the above-mentioned ndK genes is shown in Gene Bank as Gene ID: 945611, and the amino acid sequence of the nucleoside diphosphate kinase encoded by the gene is shown in NCBI as reference sequence number NP_417013.1.

[0093] The nucleotide sequence of any of the above-mentioned pyrH genes is shown in Gene Bank as Gene ID: 944989, and the amino acid sequence of the uridylate kinase encoded by the gene is shown in NCBI as reference sequence number NP_414713.1.

[0094] The nucleotide sequence of any of the above-mentioned udK genes is shown in Gene Bank as Gene ID: 946597, and the amino acid sequence of uridine kinase encoded by the gene is shown in NCBI as reference sequence number NP_416570.2.

[0095] The nucleotide sequence of any of the above-mentioned ppa genes is shown in Gene Bank as Gene ID: 948748, and the amino acid sequence of the inorganic pyrophosphatase encoded by the gene is shown in NCBI as reference sequence number NP_418647.1.

[0096] The nucleotide sequence of any of the above-mentioned fadD genes is shown in Gene Bank as Gene ID: 946327, and the amino acid sequence of the acyl-CoA synthase encoded by the gene is shown in NCBI as reference sequence number NP_416319.1.

[0097] The nucleotide sequence of any of the above-mentioned fadL genes is shown in Gene Bank as Gene ID: 946820, and the amino acid sequence of the long-chain fatty acid transport protein encoded by the gene is shown in NCBI as reference sequence number NP_416846.2.

[0098] The nucleotide sequence of any of the above-mentioned sthA genes is shown in Gene Bank as Gene ID: 948461, and the amino acid sequence of the NAD(P) transhydrogenase encoded by the gene is shown in NCBI as reference sequence number NP_418397.2.

[0099] Any of the above-mentioned atoSC gene clusters contains the atoS gene and the atoC gene, the nucleotide sequences of the atoS gene and the atoC gene are shown in Gene ID: 949011 and Gene ID: 947444 in Gene Bank, respectively, and the amino acid sequences of the atoS protein and the atoC protein encoded by the atoS gene and the atoC gene are shown in the reference sequence numbers NP_416723.1 and NP_416724.1 in NCBI, respectively.

[0100] The nucleotide sequence of any of the above-mentioned fabF genes is shown in Gene Bank as Gene ID: 946665, and the amino acid sequence of β-ketoacyl-ACP synthase II encoded by the gene is shown in NCBI reference sequence number NP_415613.1.

[0101] The nucleotide sequence of any of the above-mentioned fabH genes is shown in Gene Bank as Gene ID: 946003, and the amino acid sequence of β-ketoacyl-ACP synthase III encoded by the gene is shown in NCBI reference sequence number NP_415609.1.

[0102] The nucleotide sequence of any of the above-mentioned iclR genes is shown in Gene Bank as Gene ID: 948524, and the amino acid sequence of the isocitrate lyase inhibitor encoded by the gene is shown in NCBI as reference sequence number NP_418442.2.

[0103] The nucleotide sequence of any of the above-mentioned poxB genes is shown in Gene Bank as Gene ID: 946132, and the amino acid sequence of the pyruvate oxidase encoded by the gene is shown in NCBI as reference sequence number NP_415392.1.

[0104] The nucleotide sequence of any of the above-mentioned acs genes is shown in Gene Bank as Gene ID: 948572, and the amino acid sequence of the acetyl-CoA synthetase encoded by the gene is shown in NCBI as reference sequence number NP_418493.1.

[0105] The nucleotide sequence of any of the above-mentioned araE gene promoters is shown in positions 2982183-2982313 of the genome sequence with reference sequence number NC_000913.3 in NCBI.

[0106] The nucleotide sequence of any of the above-mentioned ndK gene promoters is shown in positions 2644433-2644864 of the genome sequence with reference sequence number NC_000913.3 in NCBI.

[0107] The nucleotide sequence of any of the above-mentioned pyrH gene promoters is shown in positions 191752-191854 of the genome sequence with reference sequence number NC_000913.3 in NCBI.

[0108] The nucleotide sequence of any of the above-mentioned udK gene promoters is shown in positions 2142949-2143088 of the genome sequence of reference sequence number NC_000913.3 in NCBI.

[0109] The nucleotide sequence of any of the above-mentioned ppa gene promoters is shown in positions 4449653-4449806 of the genome sequence with reference sequence number NC_000913.3 in NCBI.

[0110] The nucleotide sequence of any of the above-mentioned fadD gene promoters is shown in positions 1889747-1889867 of the genome sequence with reference sequence number NC_000913.3 in NCBI.

[0111] The nucleotide sequence of any of the above-mentioned fadL gene promoters is shown in positions 2461145-2461305 of the genome sequence with reference sequence number NC_000913.3 in NCBI.

[0112] The nucleotide sequence of any of the above-mentioned sthA gene promoters is shown in positions 4160791-4160862 of the genome sequence with reference sequence number NC_000913.3 in NCBI.

[0113] The nucleotide sequence of any of the above-mentioned gene cluster atoSC promoters is shown at positions 2320000-2320042 of the genome sequence with reference sequence number NC_000913.3 in NCBI.

[0114] Any of the above P CPA1 The nucleotide sequence of the promoter is shown in SEQ ID No.13.

[0115] The nucleotide sequence of any of the above-mentioned TrrnB terminators is shown in SEQ ID No.15.

[0116] The nucleotide sequence of any of the above-mentioned trc promoters is shown in SEQ ID No.16.

[0117] The recombinant Escherichia coli engineered strain provided by the present invention can achieve microbial growth using glycerol, acetic acid, or fatty acids as carbon sources, and can also utilize mixed carbon sources containing these carbon source components. After the microorganisms reach a certain biomass, D-psicose can be synthesized using sucrose as a raw material. This recombinant Escherichia coli engineered strain can be used for the industrial production of D-psicose. On the one hand, it uses inexpensive, readily available green organic raw materials as carbon sources for microbial growth and synthesizes D-psicose using sucrose as a raw material, reducing production costs. On the other hand, it efficiently synthesizes D-psicose and increases D-psicose yield, thus having great economic and social value.

[0118] Deposit description

[0119] Bacteria species: Escherichia coli

[0120] Latin name:Escherichia coli

[0121] Strain ID: AS10.03

[0122] Depository: General Microbiology Center of China Culture Collection Administration

[0123] Abbreviation of depository institution: CGMCC

[0124] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0125] Deposit date: November 14, 2024

[0126] CGMCC registration number: CGMCC No.32627 DETAILED DESCRIPTION

[0127] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0128] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0129] The liquid LB medium (pH 7.0) in the following examples contained 1 g / 100 mL NaCl, 1 g / 100 mL tryptone, 0.5 g / 100 mL yeast extract, and the balance was water. Solid culture medium is obtained by adding agar to liquid culture medium.

[0130] The E. coli MG1655 (CGSC#: 6300), pKD46 plasmid (CGSC#: 7739), and pCP20 plasmid (CGSC#: 7629) used in the following examples are all products of the Yale E. coli Genetic Collection (CGSC). The pKD46 plasmid contains the temperature-sensitive replication origin, oriR101, which replicates normally at 30°C but is lost at temperatures above 37°C. E. coli carrying the pKD46 plasmid are cultured at 30°C and, upon induction with arabinose, efficiently express Gam, Exo, and Beta. Once exogenous dsDNA is electroporated into the cells, it undergoes homologous recombination with the genomic target sequence. The pKD46 plasmid also carries an ampicillin resistance gene as a selection marker. pCP20 plasmid: Escherichia coli carrying the pCP20 plasmid can express the flippase recombinase (FLP) gene when cultured at 30°C, eliminating one FRT site and the kanamycin resistance gene; Escherichia coli carrying the pCP20 plasmid will automatically lose the pCP20 plasmid when cultured at 42°C; it carries the ampicillin resistance gene as a selection marker.

[0131] The Escherichia coli W strain in the following examples is described in the literature "Colin T Archer, Jihyun F Kim, Haeyoung Jeong, Jin Hwan Park, Claudia E Vickers, Sang Yup Lee, Lars KNielsen. The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genomics. 2011:12:9.” The public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0132] The primer sequences involved in the following examples are shown in Table 1 below.

[0133] Table 1. Primer names and primer nucleotide sequences in the examples

[0134]

[0135] Example 1. Construction of recombinant Escherichia coli ASU19

[0136] In this example, strain ASU19 and several intermediate strains were prepared that can utilize fatty acids, acetic acid, and glycerol as carbon sources for bacterial growth and can convert sucrose into D-psicose. The preparation method is as follows, and the primers used are shown in Table 1.

[0137] (1) Enhanced expression of the arabinose transporter (araE) gene by promoter replacement

[0138] Starting from Escherichia coli MG1655, the araE gene promoter in the strain (shown in the genomic sequence of the reference sequence number NC_000913.3 in NCBI, position 2982183-2982313, submission date: 2022-03-09) was replaced with the Escherichia coli constitutive promoter P CPA1 (SEQ ID No. 13), and recombinant Escherichia coli ASU01 was obtained by the following steps:

[0139] (1-a) Preparation of the targeting fragment araEup-kan-PCPA1-araEdown

[0140] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the following DNA fragment A: from 5' to 3', it contains the frt-kan-frt selection marker fragment (SEQ ID No. 14) and P CPA1 Promoter fragment (SEQ ID No. 13). PCR amplification was performed using araE-1 / araE-2 as primers and gene-synthesized DNA fragment A as template to obtain the target fragment araEup-kan-P CPA1 -araEdown. araEup contains a 70 bp homology arm fragment upstream of the araE gene promoter (lowercase portion in the primer table), and araEdown contains a 70 bp reverse complementary homology arm fragment downstream of the araE gene promoter (lowercase portion in the primer table).

[0141] (1-b) Preparation of host bacteria containing the pKD46 plasmid

[0142] The pKD46 plasmid was transformed into E. coli MG1655 using the calcium chloride method. After overnight culture at 30°C on LB plates containing ampicillin, colonies were selected to obtain recombinant E. coli MG1655 / pKD46 containing the pKD46 plasmid. After induction with arabinose, the recombinant E. coli MG1655 / pKD46 expressed the three recombinant proteins of phage lambda, conferring homologous recombination capability. Competent MG1655 / pKD46 cells were then prepared by washing with 10% glycerol.

[0143] (1-c) Homologous recombination

[0144] The targeting fragment araEup-kan-P prepared in step (1-a) CPA1 -araEdown was electroporated into the MG1655 / pKD46 competent cells prepared in step (1-b) and incubated overnight at 30°C on an LB plate containing kanamycin (50µg / ml). A clone was selected to extract genomic DNA and amplified using araE-3 / Kan-R primers. A positive clone was identified by amplifying a target band of approximately 1700bp. The selected positive clone was named ASU01-kan. Sequencing analysis results showed that the genome of ASU01-kan contained the target fragment araEup-kan-P CPA1 -araEdown's sequence.

[0145] ASU01-kan was cultured at 42°C overnight to eliminate the temperature-sensitive plasmid pKD46.

[0146] (1-d) Elimination of resistance

[0147] The pCP20 plasmid was transformed into ASU01-kan using the calcium chloride method. Cultured overnight at 30°C on LB plates containing ampicillin, the FLP recombinase on the pCP20 plasmid eliminated kanamycin resistance. Cultured overnight at 42°C eliminated the temperature-sensitive plasmid pCP20. Single clones were identified by PCR amplification using the araE-2 / araE-3 primers. A positive result indicated the presence of a target band approximately 500 bp, indicating recombinant strain ASU01.

[0148] The nucleotide sequence of the araE gene is shown in Gene Bank as Gene ID: 947341 (submission date: 2023-04-14), and the amino acid sequence of the arabinose transporter encoded by this gene is shown in NCBI reference sequence number NP_417318.1 (submission date: 2022-03-09).

[0149] (2) Sucrose permease (derived from Escherichia coli W strain ( Escherichia coli W), hereinafter referred to as cscB) gene and sucrose hydrolase (derived from Escherichia coli W strain ( Escherichia coli The cscA gene was integrated into the myristoyltransferase (lpxM) gene locus for lipid A biosynthesis in the Escherichia coli chromosome.

[0150] Starting from the recombinant strain ASU01, the lpxM gene in the strain was replaced with the constitutive promoter P CPA1 , cscB gene and cscA gene to obtain recombinant Escherichia coli ASU02. The specific steps are as follows:

[0151] (2-a) Targeting fragment lpxMup-kan-P CPA1Preparation of cscB-cscA-lpxMdown

[0152] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the following DNA fragment B: from 5' to 3', it contains the frt-kan-frt screening marker fragment (SEQ ID No. 14), P CPA1 The promoter fragment (SEQ ID No. 13), cscB gene, RBS (5'-AGGAG-3'), cscA gene and TrrnB terminator fragment (SEQ ID No. 15) were amplified by PCR using lpxM-1 / lpxM-2 as primers and gene-synthesized DNA fragment B as template to obtain the targeting fragment lpxMup-kan-P CPA1 -cscB-cscA-lpxMdown. lpxMup contains the 70 bp homology arm fragment upstream of the lpxM gene (lowercase part in the primer table), and lpxMdown contains the reverse complementary fragment of the 70 bp homology arm downstream of the lpxM gene (lowercase part in the primer table).

[0153] (2-b) The subsequent operations are the same as steps (1-b), (1-c) and (1-d) in (1) above, and the constitutive promoter P is obtained. CPA1 The recombinant strain ASU02, in which the lpxM gene was replaced by the cscB-cscA gene, differed only in that the primer combination araE-1 / araE-2 / araE-3 was replaced by the primer combination lpxM-1 / lpxM-2 / lpxM-3.

[0154] The nucleotide sequence of the cscB gene is shown in the genomic sequence of Gene ID: X81461.2 (submission date: 2016-7-26) at positions 3171-4418 in Gene Bank, and the amino acid sequence of the sucrose permease encoded by this gene is shown in the reference sequence number CAA57217.1 in NCBI (submission date: 2016-7-26).

[0155] The nucleotide sequence of the cscA gene is shown in the genomic sequence of Gene ID: X81461.2 (submission date: 2016-07-26) at positions 5619-7052 in Gene Bank, and the amino acid sequence of the sucrose permease encoded by the gene is shown in the reference sequence number CAA57219.1 (2016-07-26) in NCBI.

[0156] The nucleotide sequence of the lpxM gene is shown in Gene Bank as Gene ID: 945143 (submission date: 2023-04-14), and the amino acid sequence of the lipid A biosynthesis myristoyltransferase encoded by the gene is shown in NCBI reference sequence number NP_416369.1 (submission date: 2022-03-09).

[0157] (3) Replace the glucokinase (glk) gene with an acid phosphatase (derived from Aspergillus Aspergillus sp. ), hereinafter referred to as Aapp) gene

[0158] Starting from the recombinant Escherichia coli ASU02, the glk gene in the strain was replaced with the constitutive promoter P CPA1 and Aapp genes to obtain recombinant E. coli ASU03. The specific steps are as follows:

[0159] (3-a) Targeting fragment glkup-kan-P CPA1 Preparation of -Aapp-glkdown

[0160] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the following DNA fragment C: from 5' to 3', it contains the frt-kan-frt selection marker fragment (SEQ ID No. 14), P CPA1 The promoter fragment (SEQ ID No. 13), Aapp gene (SEQ ID No. 12) and TrrnB terminator fragment (SEQ ID No. 15) were amplified by PCR using glk-1 / glk-2 as primers and the gene-synthesized DNA fragment C as template to obtain the targeting fragment glkup-kan-P CPA1 -Aapp-glkdown. glkup contains the 70 bp homology arm fragment upstream of the glk gene (lowercase part in the primer table), and glkdown contains the reverse complementary fragment of the 70 bp homology arm downstream of the glk gene (lowercase part in the primer table).

[0161] (3-b) The subsequent operations are the same as steps (1-b), (1-c) and (1-d) in (1) above, and the constitutive promoter P is obtained. CPA1 The recombinant E. coli ASU03 in which the glk gene is replaced by the Aapp gene is derived. The only difference is that the primer combination araE-1 / araE-2 / araE-3 is replaced by the primer combination glk-1 / glk-2 / glk-3.

[0162] The nucleotide sequence of the glk gene is shown in Gene Bank as Gene ID: 946858 (submission date: 2023-04-14), and the amino acid sequence of the glucokinase encoded by the gene is shown in NCBI reference sequence number NP_416889.1 (submission date: 2022-03-09).

[0163] The nucleotide sequence of the Aapp gene is shown as SEQ ID No. 12, and the amino acid sequence of the acid phosphatase encoded by the gene is shown as SEQ ID No. 11.

[0164] (4) Xylose isomerase (derived from Bacillus Bacillus sp. ), hereinafter referred to as BguiA) gene, hexokine kinase (derived from human ( Homo sapiens ), hereinafter referred to as HkhkB) gene, UDP-sugar pyrophosphorylase (from Arabidopsis thaliana ( Arabidopsis thaliana ), hereinafter referred to as Ausp) gene, NDP-hexose-3-epimerase (from Streptomyces ( Streptomyces sp. ), hereinafter referred to as Snh3e) gene, NDP glycohydrolase (derived from rat ( Rattus norvegicus ), hereafter referred to as Rnudt22) gene was introduced into Escherichia coli

[0165] Starting from recombinant E. coli ASU03, the expression plasmid pTrc99a-BHASR containing the BguiA gene, HkhkB gene, Ausp gene, Snh3e gene, and Rnudt22 gene was introduced into the recombinant E. coli ASU03 to obtain recombinant E. coli ASU04. The specific steps are as follows:

[0166] (4-a) Preparation of pTrc99a-BHASR plasmid

[0167] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the following DNA fragment D: from 5' to 3', it contains the BguiA gene (SEQ ID No. 2), RBS (5'-AGGAG-3'), HkhkB gene (SEQ ID No. 4), RBS (5'-AGGAG-3'), Ausp gene (SEQ ID No. 6), TrrnB terminator (SEQ ID No. 15), trc promoter (SEQ ID No. 16), Snh3e gene (SEQ ID No. 8), RBS (5'-AGGAG-3'), and Rnudt22 gene (SEQ ID No. 10).

[0168] Using the synthesized DNA fragment D as a template, BHASR-01F and BHASR-01R as primers, and high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog number AP221), PCR amplification was performed to obtain a BHASR fragment containing the above five genes. The target fragment was recovered by agarose gel electrophoresis. Then, using primers pTrc99a-01F and pTrc99a-01R, plasmid pTrc99a (Beijing Zhuangmeng International Biogene Technology Co., Ltd., product number ZK1610) as a template, PCR amplification of the pTrc fragment was performed to recover the large vector fragment pTrc. The above BHASR fragment was ligated with the pTrc fragment using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. methods. 2009; 6 (5): 343-345) to obtain the recombinant expression vector pTrc99a-BHASR. The recombinant expression vector pTrc99a-BHASR was transformed into Escherichia coli DH5α competent cells (Beijing Quanshijin Biotechnology Co., Ltd., catalog number CD201) using the calcium chloride method. The cells were evenly spread on LB plates containing ampicillin and cultured overnight at 37°C. Clones were selected and identified using primers RV-M / pTrcHis-R to amplify the target fragment. These clones were sequenced and the plasmids were extracted from positive clones. The resulting plasmids were named pTrc99a-BHASR.

[0169] (4-b) Plasmid transformation

[0170] The pTrc99a-BHASR plasmid was transformed into recombinant Escherichia coli ASU03 by the calcium chloride transformation method. After overnight culture at 37°C on LB plates containing ampicillin, clones were selected and positive clones were identified using primers RV-M / pTrcHis-R. The positive clone was named ASU04 and stored at -80°C.

[0171] The nucleotide sequence of the BguiA gene is shown as SEQ ID No. 2, and the amino acid sequence of the xylose isomerase encoded by the gene is shown as SEQ ID No. 1.

[0172] The nucleotide sequence of the HkhkB gene is shown in SEQ ID No. 4, and the amino acid sequence of the hexokinokinase encoded by the gene is shown in SEQ ID No. 3.

[0173] The nucleotide sequence of the Ausp gene is shown as SEQ ID No. 6, and the amino acid sequence of the UDP-sugar pyrophosphorylase encoded by the gene is shown as SEQ ID No. 5.

[0174] The nucleotide sequence of the Snh3e gene is shown in SEQ ID No. 8, and the amino acid sequence of the NDP-hexose-3-epimerase encoded by the gene is shown in SEQ ID No. 7.

[0175] The nucleotide sequence of the Rnudt22 gene is shown in SEQ ID No. 10, and the amino acid sequence of the NDP glycohydrolase encoded by the gene is shown in SEQ ID No. 9.

[0176] (5) Knockout of the fatty acid degradation transcription factor (fadR) gene

[0177] Starting from recombinant E. coli AS03, the fadR gene in the strain was knocked out to obtain recombinant E. coli ASU05. The specific steps are as follows:

[0178] (5-a) Preparation of the targeting fragment fadRfup-kan-fadRdown

[0179] PCR amplification was performed using fadR-1 / fadR-2 primers and the DNA fragment A described in step (1) above as a template to obtain the targeting fragment fadRup-kan-fadRdown containing 70 bp homology arms upstream and downstream of the fadR gene site.

[0180] (5-b) Subsequent operations were performed in accordance with steps (1-b), (1-c), and (1-d) of (1) above to obtain recombinant E. coli ASU05 with the fadR gene knocked out. The only difference was that the primer combination araE-1 / araE-2 / araE-3 was replaced with the primer combination fadR-1 / fadR-2 / fadR-3. During the operation, PCR amplification was performed using fadR-3 / Kan-R as primers, and the amplification of a target band of approximately 1400 bp was considered positive. PCR amplification of the monoclonal strain using fadR-2 / fadR-3 as primers was performed, and the amplification of a target band of approximately 200 bp was considered positive.

[0181] The nucleotide sequence of the fadR gene is shown in Gene Bank as Gene ID: 948652 (submission date: 2023-04-14), and the amino acid sequence of the fatty acid degradation transcription factor encoded by this gene is shown in NCBI reference sequence number NP_415705.1 (submission date: 2022-03-09).

[0182] (6) Construction of recombinant E. coli ASU06, ASU07, ASU08, ASU09, ASU10, ASU11, ASU12, and ASU13

[0183] Starting from the recombinant Escherichia coli ASU05, the expression of nucleoside diphosphate kinase (hereinafter referred to as ndK) gene, uridylate kinase (hereinafter referred to as pyrH) gene, uridine kinase (hereinafter referred to as udK) gene, inorganic pyrophosphatase (hereinafter referred to as ppa) gene, fatty acyl-CoA synthase (hereinafter referred to as fadD) gene, long-chain fatty acid transport protein (hereinafter referred to as fadL) gene, NAD(P) transhydrogenase (hereinafter referred to as sthA) gene, and short-chain fatty acid degradation regulatory gene cluster atoSC were successively enhanced by promoter replacement. The ndK gene promoter ( The 2644433-2644864 positions of the genomic sequence of the reference sequence number NC_000913.3 in NCBI, submission date: 2022-03-09), the pyrH gene promoter (the 191752-191854 positions of the genomic sequence of the reference sequence number NC_000913.3 in NCBI, submission date: 2022-03-09), the udK gene promoter (the 2142949-2143088 positions of the genomic sequence of the reference sequence number NC_000913.3 in NCBI, submission date: 2022-03-09), Submission date: 2022-03-09), ppa gene promoter (shown in the genomic sequence of the reference sequence number NC_000913.3 in NCBI at positions 4449653-4449806, submission date: 2022-03-09), fadD gene promoter (shown in the genomic sequence of the reference sequence number NC_000913.3 in NCBI at positions 1889747-1889867, submission date: 2022-03-09), fadL gene promoter (shown in the genomic sequence of the reference sequence number NC_000913.3 in NCBI at positions 1889747-1889867, submission date: 2022-03-09), The 2461145-2461305 positions of the genome sequence, submission date: 2022-03-09), the sthA gene promoter (the 4160791-4160862 positions of the genome sequence of the NCBI reference sequence number NC_000913.3, submission date: 2022-03-09), and the gene cluster atoSC promoter (the 2320000-2320042 positions of the genome sequence of the NCBI reference sequence number NC_000913.3, submission date: 2022-03-09) were replaced by P CPA1 Promoter, and obtained recombinant Escherichia coli ASU06, ASU07, ASU08, ASU09, ASU10, ASU11, ASU12, and ASU13.

[0184] The specific operation steps refer to step (1) in Example 1. The only difference is that the primer combination araE-1 / araE-2 / araE-3 is replaced by the primer combination ndk-1 / ndk-2 / ndk-3, the primer combination pyrH-1 / pyrH-2 / pyrH-3, the primer combination udk-1 / udk-2 / udk3, the primer combination ppa-1 / ppa-2 / ppa-3, the primer combination fadD-1 / fadD-2 / fadD-3, the primer combination fadL-1 / fadL-2 / fadL-3, the primer combination sthA-1 / sthA-2 / sthA-3, and the primer combination atoSC-1 / atoSC-2 / atoSC-3.

[0185] The nucleotide sequence of the ndK gene is shown in Gene Bank as Gene ID: 945611 (submission date: 2023-04-14), and the amino acid sequence of the nucleoside diphosphate kinase encoded by the gene is shown in NCBI reference sequence number NP_417013.1 (submission date: 2022-03-09).

[0186] The nucleotide sequence of the pyrH gene is shown in Gene Bank as Gene ID: 944989 (submission date: 2023-04-14), and the amino acid sequence of the uridine kinase encoded by this gene is shown in NCBI reference sequence number NP_414713.1 (submission date: 2022-03-09).

[0187] The nucleotide sequence of the udK gene is shown in Gene Bank as Gene ID: 946597 (submission date: 2023-04-14), and the amino acid sequence of the uridine kinase encoded by this gene is shown in NCBI reference sequence number NP_416570.2 (submission date: 2022-03-09).

[0188] The nucleotide sequence of the ppa gene is shown in Gene Bank as Gene ID: 948748 (submission date: 2023-04-14), and the amino acid sequence of the inorganic pyrophosphatase encoded by the gene is shown in NCBI reference sequence number NP_418647.1 (submission date: 2022-03-09).

[0189] The nucleotide sequence of the fadD gene is shown in Gene Bank as Gene ID: 946327 (submission date: 2023-04-14), and the amino acid sequence of the acyl-CoA synthase encoded by this gene is shown in NCBI reference sequence number NP_416319.1 (submission date: 2022-03-09).

[0190] The nucleotide sequence of the fadL gene is shown in Gene Bank as Gene ID: 946820 (submission date: 2023-04-14), and the amino acid sequence of the long-chain fatty acid transport protein encoded by this gene is shown in NCBI reference sequence number NP_416846.2 (submission date: 2022-03-09).

[0191] The nucleotide sequence of the sthA gene is shown in Gene Bank as Gene ID: 948461 (submission date: 2023-04-14), and the amino acid sequence of the NAD(P) transhydrogenase encoded by this gene is shown in NCBI reference sequence number NP_418397.2 (2022-03-09).

[0192] The atoSC gene cluster contains the atoS gene and the atoC gene. The nucleotide sequences of the atoS gene and the atoC gene are shown in Gene Bank as Gene ID: 949011 (submission date: 2023-04-14) and Gene ID: 947444 (submission date: 2023-04-14), respectively. The amino acid sequences of the atoS protein and the atoC protein encoded by the atoS gene and the atoC gene are shown in NCBI as reference sequence numbers NP_416723.1 (submission date: 2022-03-09) and NP_416724.1 (submission date: 2022-03-09), respectively.

[0193] (7) Construction of recombinant E. coli ASU14, ASU15, and ASU16

[0194] Starting from the recombinant Escherichia coli ASU13, the β-ketoacyl-ACP synthase II (fabF) gene, β-ketoacyl-ACP synthase III (fabH) gene and isocitrate lyase inhibitor (iclR) gene were knocked out in sequence to obtain recombinant Escherichia coli ASU14, ASU15 and ASU16.

[0195] The specific operation steps refer to step (5) in Example 1. The only difference is that the primer combination fadR-1 / fadR-2 / fadR-3 is replaced by the primer combination fabF-1 / fabF-2 / fabF-3, the primer combination fabH-1 / fabH-2 / fabH-3, and the primer combination iclR-1 / iclR-2 / iclR-3.

[0196] The nucleotide sequence of the fabF gene is shown in Gene Bank as Gene ID: 946665 (submission date: 2023-04-14), and the amino acid sequence of β-ketoacyl-ACP synthase II encoded by the gene is shown in NCBI reference sequence number NP_415613.1 (submission date: 2022-03-09).

[0197] The nucleotide sequence of the fabH gene is shown in Gene Bank as Gene ID: 946003 (submission date: 2023-04-14), and the amino acid sequence of β-ketoacyl-ACP synthase III encoded by the gene is shown in NCBI reference sequence number NP_415609.1 (submission date: 2022-03-09).

[0198] The nucleotide sequence of the iclR gene is shown in Gene Bank as Gene ID: 948524 (submission date: 2023-04-14), and the amino acid sequence of the isocitrate lyase inhibitor encoded by the gene is shown in NCBI reference sequence number NP_418442.2 (submission date: 2022-03-09).

[0199] (8) Replace the pyruvate oxidase (poxB) gene with the acetyl-CoA synthetase (acs) gene

[0200] Starting from the recombinant Escherichia coli ASU16, the poxB gene in the strain was replaced with P CPA1 promoter and acs gene to obtain recombinant Escherichia coli ASU17.

[0201] The specific operation steps refer to step (2) in Example 1. The only difference is that the amplification template DNA fragment B is replaced by DNA fragment E, and DNA fragment E contains frt-kan-frt screening marker fragment (SEQ ID No. 14), P CPA1 Promoter fragment (SEQ ID No. 13), acs gene, TrrnB terminator fragment (SEQ ID No. 15); the primer combination lpxM-1 / lpxM-2 / lpxM-3 was replaced with the primer combination poxB-1 / poxB-2 / poxB-3.

[0202] The nucleotide sequence of the poxB gene is shown in Gene Bank as Gene ID: 946132 (submission date: 2023-04-14), and the amino acid sequence of the pyruvate oxidase encoded by this gene is shown in NCBI reference sequence number NP_415392.1 (submission date: 2022-03-09).

[0203] The nucleotide sequence of the acs gene is shown in Gene Bank as Gene ID: 948572 (submission date: 2023-04-14), and the amino acid sequence of the acetyl-CoA synthetase encoded by this gene is shown in NCBI reference sequence number NP_418493.1 (submission date: 2022-03-09).

[0204] (9) Construction of recombinant E. coli ASU18 and ASU19

[0205] Recombinant E. coli ASU16 and ASU17 were used as starting bacteria to transform the plasmid pTrc99a-BHASR prepared in step (4) above to obtain recombinant E. coli ASU18 and ASU19, respectively. The specific steps are as described in step (4) above.

[0206] The recombinant bacterium ASU19 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on November 14, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 32627, and it is named Escherichia coli AS10.03.

[0207] 2. Cultivation of recombinant E. coli ASU04 using glycerol as a carbon source and preparation of D-psicose using sucrose as a raw material

[0208] 1. Glycerol growth medium composition

[0209] The components and final concentrations of glycerol growth medium are as follows (the solvent is water): Na2HPO4 25 mM, KH2PO4 25 mM, NH4Cl 50 mM, Na2SO4 5 mM, MgSO4 2 mM, glycerol 3% (volume percentage concentration), yeast powder 0.5% (mass percentage concentration), FeCl3 50 μM, CaCl2 20 μM, MnCl2 10 μM, ZnSO4 10 μM, CoCl2 2 μM, NiCl2 2 μM, Na2MO4 2 μM, Na2SeO 32 μM, H3BO 32 μM.

[0210] 2. Transformation medium components

[0211] The components and final concentrations of the transformation medium are as follows (the solvent is water): Na2HPO4 25 mM, KH2PO4 25 mM, MgCl2 5 mM, and sucrose 15% (mass percentage concentration).

[0212] 3. Bacterial culture and enzyme induction

[0213] The control strain ASU03 and the engineered strain ASU04 were cultured overnight and inoculated into a shake flask containing 200 mL of glycerol growth medium (containing ampicillin at a final concentration of 100 mg / L) at a 1% inoculum size. The culture was 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, centrifuge at 8000 rpm for 10 min, and collect the bacteria.

[0214] 4. Whole-cell catalysis of D-psicose

[0215] The collected cells were resuspended in a beaker containing 10 mL of transformation medium. The reaction progress was monitored periodically using pH test paper, and the pH was adjusted to 7.0 using 0.1 M NaOH and HCl solutions. After incubation at 37°C for 24 hours, the supernatant was centrifuged and filtered. The D-psicose content was determined by HPLC. The HPLC analysis used a Hi-Plex Ca column (300 mm × 7.7 mm, 8 μm) with ultrapure water as the mobile phase at a flow rate of 0.5 mL / min, a column temperature of 78°C, and a differential refractive index detector at 40°C. The D-psicose standard was obtained from Shanghai Yuanye Biotechnology (Cat. No. S48276).

[0216] The results showed that strain ASU03 produced zero D-psicose, while strain ASU04 produced an average D-psicose yield of 83 g / L. Therefore, the engineered strain ASU04 was capable of converting sucrose into D-psicose with a conversion rate of 55%. Furthermore, the D-psicose in the reaction solution was of high purity, with no significant detection of other sugars, facilitating the simplification of downstream separation and purification processes.

[0217] 3. Cultivation of recombinant Escherichia coli ASU19 using acetic acid as a carbon source and preparation of D-psicose using sucrose as a raw material

[0218] 1. Acetic acid growth medium composition

[0219] The components and final concentrations of acetic acid growth medium are as follows (the solvent is water): Na2HPO4 25 mM, KH2PO4 25 mM, NH4Cl 50 mM, Na2SO4 5 mM, MgSO4 2 mM, sodium acetate 2% (mass percentage concentration), yeast powder 0.5% (mass percentage concentration), FeCl3 50 μM, CaCl2 20 μM, MnCl2 10 μM, ZnSO4 10 μM, CoCl2 2 μM, NiCl2 2 μM, Na2MO4 2 μM, Na2SeO 32 μM, H3BO 32 μM.

[0220] 2. Transformation medium components

[0221] The components and final concentrations of the transformation medium are as follows (the solvent is water): Na2HPO4 100 mM, NaH2PO4 100 mM, MgCl2 5 mM, and sucrose 15% (mass percentage concentration).

[0222] 3. Bacterial culture and enzyme induction

[0223] The control strain AS17 and the engineered strain ASU19 cultured overnight were inoculated into a shake flask containing 200 mL of acetic acid growth medium (containing ampicillin at a final concentration of 100 mg / L) and cultured at 30°C for 3-4 h until the OD 600nm When the pH value is 0.6-0.8, add IPTG to a final concentration of 0.5 mM and continue to culture at 30°C for 12 h. Centrifuge at 8000 rpm for 10 min to collect the cells.

[0224] 4. Whole-cell catalysis of D-psicose

[0225] Resuspend the collected cells in a beaker containing 10 mL of transformation medium. Maintain a pH of 7.0 during the reaction. Incubate at 37°C for 24 hours, centrifuge, and filter the supernatant. Analyze the D-psicose content by HPLC using the same method as in step 2.

[0226] The results showed that strain ASU17 produced zero D-psicose, while strain ASU19 produced an average D-psicose yield of 101.7 g / L, with a conversion rate of 67.8%. Furthermore, the D-psicose in the reaction solution was of high purity, with no significant detection of other sugars, facilitating the simplification of downstream separation and purification processes.

[0227] 4. Cultivation of recombinant E. coli ASU18 using fatty acids as a carbon source and preparation of D-psicose using sucrose as a raw material

[0228] 1. Fatty acid growth medium composition

[0229] Culture medium A: A sterile culture medium composed of solutes and solvents, the solvent is water, and the solutes and their concentrations are: Na2HPO4 25 mM, KH2PO4 25 mM, NH4Cl 50 mM, Na2SO4 5 mM, MgSO4 2 mM, glycerol 0.5% (volume percentage concentration), yeast powder 0.5% (mass percentage concentration), FeCl3 50 μM, CaCl2 20 μM, MnCl2 10 μM, ZnSO4 10 μM, CoCl2 2 μM, NiCl2 2 μM, Na2MO4 2 μM, Na2SeO 32 μM, H3BO 32 μM.

[0230] Medium B: A sterile medium obtained by adding palmitic acid and polyoxyethylene ether Brij58 emulsifier (Merck, Cat. No. P5884-100G) to medium A, wherein the mass percentage concentration of palmitic acid is 0.5% and the mass percentage concentration of polyoxyethylene ether Brij58 emulsifier is 0.2%.

[0231] 2. Transformation medium components

[0232] The components and final concentrations of the transformation medium are as follows (the solvent is water): Na2HPO4 100 mM, NaH2PO4 100 mM, MgCl2 5 mM, and sucrose 15% (mass percentage concentration).

[0233] 3. Bacterial culture and enzyme induction

[0234] The control strain ASU16 and the engineered strain ASU18 cultured overnight in medium A were inoculated into a shake flask containing 200 mL medium B (containing ampicillin at a final concentration of 100 mg / L) at a 1% inoculum size and cultured at 30°C for 3-4 h until the OD 600nm When the pH value is 0.6-0.8 (determined after extracting the oil components with n-hexane), add IPTG to a final concentration of 0.5 mM and continue culturing at 30°C for 12 hours. Centrifuge at 8000 rpm for 10 minutes and collect the cells.

[0235] 4. Whole-cell catalysis of D-psicose.

[0236] Resuspend the collected cells in a beaker containing 10 mL of transformation medium. Maintain a pH of 7.0 during the reaction. Incubate at 37°C for 24 hours, centrifuge, and filter the supernatant. Analyze the D-psicose content by HPLC using the same method as in step 2.

[0237] The results showed that the control strain ASU16 produced zero D-psicose, while strain ASU18 produced an average D-psicose yield of 110.4 g / L, with a conversion rate of 73.6%. Furthermore, the D-psicose in the reaction solution was of high purity, with no significant detection of other sugars, facilitating the simplification of downstream separation and purification processes.

Claims

1. Recombinant E. coli is obtained by performing multiple modifications on E. coli; The plurality of modifications include modification (a) and modification (d); Modification (a) includes the following modifications (a1)-(a3): Modification (a1): Enhanced expression of the arabinose transporter araE gene; Transformation (a2): The lipid A biosynthesis myristoyltransferase lpxM gene was replaced with the sucrose permease cscB gene and the sucrose permease cscA gene; Transformation (a3): Glucokinase glk gene was replaced with acid phosphatase Aapp gene; Transformation (d): introducing and expressing the xylose isomerase BguiA gene, the hexoketol kinase HkhkB gene, the UDP-sugar pyrophosphorylase Ausp gene, the NDP-hexose-3-epimerase Snh3e gene, and the NDP sugar hydrolase Rnudt22 gene; The amino acid sequence of the arabinose transporter araE is shown in the NCBI reference sequence number NP_417318.1; The amino acid sequence of the sucrose permease cscB is shown in the NCBI reference sequence number CAA57217.1; The amino acid sequence of the sucrose permease cscA is shown in the NCBI reference sequence number CAA57219.1; The amino acid sequence of the lipid A biosynthesis myristoyltransferase lpxM is shown in the NCBI reference sequence number NP_416369.1; The amino acid sequence of the glucokinase glk is shown in the NCBI reference sequence number NP_416889.1; The amino acid sequence of the acid phosphatase Aapp is shown in SEQ ID No.11; The amino acid sequence of the xylose isomerase BguiA is shown in SEQ ID No. 1; The amino acid sequence of the hexokinases HkhkB is shown in SEQ ID No. 3; The amino acid sequence of the UDP-sugar pyrophosphorylase Ausp is shown in SEQ ID No. 5; The amino acid sequence of the NDP-hexose-3-epimerase Snh3e is shown in SEQ ID No. 7; The amino acid sequence of the NDP glycohydrolase Rnudt22 is shown in SEQ ID No.

9.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The plurality of transformations also includes transformation (b); The transformation (b) includes the following transformations (b1) to (b12): Modification (b1): Knockout of the fatty acid degradation transcription factor fadR gene; Modification (b2): Enhanced expression of the nucleoside diphosphate kinase ndK gene; Modification (b3): ​​Enhanced expression of the uridine kinase pyrH gene; Modification (b4): Enhanced expression of uridine kinase udk gene; Modification (b5): Enhanced expression of the inorganic pyrophosphatase ppa gene; Modification (b6): Enhanced expression of the acyl-CoA synthase fadD gene; Modification (b7): Enhanced expression of the long-chain fatty acid transporter fadL gene; Modification (b8): Enhanced expression of the NAD(P) transhydrogenase sthA gene; Modification (b9): Enhanced expression of the short-chain fatty acid degradation regulatory gene cluster atoSC; Modification (b10): Knockout of the β-ketoacyl-ACP synthase II fabF gene; Modification (b11): Knockout of the β-ketoacyl-ACP synthase III fabH gene; Modification (b12): Knockout of the isocitrate lyase inhibitor iclR gene; The amino acid sequence of the fatty acid degradation transcription factor fadR is shown in the reference sequence number NP_415705.1 in NCBI; The amino acid sequence of the nucleoside diphosphate kinase ndK is shown in the NCBI reference sequence number NP_417013.1; The amino acid sequence of the uridylate kinase pyrH is shown in the NCBI reference sequence number NP_414713.1; The amino acid sequence of the uridine kinase udK is shown in the reference sequence number NP_416570.2 in NCBI; The amino acid sequence of the inorganic pyrophosphatase ppa is shown in the reference sequence number NP_418647.1 in NCBI; The amino acid sequence of the acyl-CoA synthase fadD is shown in the NCBI reference sequence number NP_416319.1; The amino acid sequence of the long-chain fatty acid transport protein fadL is shown in the reference sequence number NP_416846.2 in NCBI; The amino acid sequence of the NAD(P) transhydrogenase sthA is shown in the NCBI reference sequence number NP_418397.2; The short-chain fatty acid degradation regulatory gene cluster atoSC comprises the atoS gene and the atoC gene, and the amino acid sequences of the atoS protein and the atoC protein encoded by the atoS gene and the atoC gene are shown in the reference sequence numbers NP_416723.1 and NP_416724.1 in NCBI, respectively; The amino acid sequence of the β-ketoacyl-ACP synthase II fabF is shown in the NCBI reference sequence number NP_415613.1; The amino acid sequence of the β-ketoacyl-ACP synthase III fabH is shown in the NCBI reference sequence number NP_415609.1; The amino acid sequence of the isocitrate lyase inhibitor iclR is shown in the reference sequence number NP_418442.2 in NCBI.

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that: The multiple modifications also include modification (c); the modification (c): the pyruvate oxidase poxB gene is replaced with the acetyl-CoA synthetase acs gene; The amino acid sequence of the pyruvate oxidase poxB is shown in the NCBI reference sequence number NP_415392.1; The amino acid sequence of the acetyl-CoA synthetase is shown in the reference sequence number NP_418493.1 in NCBI.

4. Recombinant Escherichia coli, characterized in that: The deposit number of the recombinant Escherichia coli is CGMCC No. 32627.

5. Use of the recombinant Escherichia coli according to any one of claims 1 to 4 in the preparation of D-psicose.

6. A method for constructing a recombinant Escherichia coli that produces D-psicose, comprising the steps of: performing the transformation according to any one of claims 1 to 3.

7. Use of the recombinant Escherichia coli prepared according to the method of claim 6 in the preparation of D-psicose.

8. A method for preparing D-psicose, comprising the steps of: using sucrose as a raw material and performing biotransformation using the recombinant Escherichia coli according to any one of claims 1 to 4 or the recombinant Escherichia coli prepared according to the method of claim 6 to obtain D-psicose.

9. The method according to claim 8, characterized in that: The method further comprises the step of culturing the recombinant Escherichia coli using at least one of glycerol, acetic acid or fatty acid as a carbon source.

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