Recombinant escherichia coli for producing D-psicose as well as preparation method and application of recombinant escherichia coli

Abstract

The invention discloses recombinant escherichia coli for producing D-psicose as well as a preparation method and application of the recombinant escherichia coli. The recombinant Escherichia coli is obtained by performing multiple modifications on Escherichia coli; the multiple modifications comprise the steps of enhancing the expression of an araE gene, replacing an lpxM gene with a cscB gene and a cscA gene, replacing a glk gene with an Aapp gene, and introducing and expressing a BguiA gene, an HkhkB gene, an Ausp gene, a Snh3e gene and an Rnudt22 gene. According to the recombinant escherichia coli provided by the invention, green organic raw materials such as glycerol, acetic acid or fatty acid which are low in price and easy to obtain can be used as a carbon source for microbial growth, and D-psicose is synthesized by taking sucrose which is low in price as a raw material after the microbial growth reaches a certain biomass. Not only is the production cost reduced, but also the D-psicose is efficiently synthesized, and the method has great economic value and social value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular 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, and pharmaceutical 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, neuroprotective, and immunosuppressive. Because of its special nutritional and biological functions, its preparation method has received increasing 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 traditional production of D-psicose include chemical method and biological method, among which chemical synthesis was mainly used at the beginning. Bilik et al. found that in 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-cost, easily available green organic raw materials. They have a higher energy density than raw materials such as glucose and can 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 oils 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. Sucrose is a low-cost 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 great 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 use sucrose as a raw material to prepare D-psicose.

[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) to (a3):

[0012] Modification (a1): Enhance the expression of 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) to (a3):

[0019] Modification (a1): Enhance the expression of 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 ndK gene;

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

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

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

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

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

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

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

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

[0035] Transformation (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 sp.

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

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

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

[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 sp.

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

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

[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 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 araE-1 / araE-2.

[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 Further, 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. Furthermore, the targeting fragment ndkup-kan-P CPA1 -ndkdown can be a gene fragment (5' to 3' is 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-P CPA1-pyrHdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment pyrHup-kan-P CPA1 -pyrHdown can be a gene fragment (5' to 3' is 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 pyrH-1 / pyrH-2.

[0049] Any of the above-mentioned enhancements of 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 segment udkup-kan-P CPA1 -udkdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment udkup-kan-P CPA1 -udkdown can be a gene fragment (5' to 3' is 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 udk-1 / udk-2.

[0050] Any of the above-mentioned methods for enhancing the expression of the ppa gene is carried out 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 By targeting the 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 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 ppa-1 / ppa-2.

[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 P CPA1By 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 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 of 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 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 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 Further, 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. Furthermore, the targeting fragment sthAup-kan-P CPA1 -sthAdown can be a gene fragment (5' to 3' is 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 PCPA1 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 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 is replaced by 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. Furthermore, the targeting fragment lpxMup-kan-P CPA1 -cscB-cscA-lpxMdown can be a gene fragment (5' to 3' is 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 the 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 is replaced by 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 fragment glkup-kan-P CPA1 -Aapp-glkdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment glkup-kan-P CPA1-Aapp-glkdown can be a gene fragment (5' to 3' is composed of the frt-kan-frt screening marker fragment shown in SEQ ID No.14, the P CPA1 The promoter fragment, Aapp gene and the TrrnB terminator fragment shown in SEQ ID No.15 were used as templates and PCR amplification was performed using primers glk-1 / glk-2.

[0057] Any of the above described poxB gene is replaced by acs gene or the poxB gene is replaced by Escherichia coli constitutive promoter P CPA1 Furthermore, the poxB gene is replaced by the constitutive promoter P CPA1 and acs genes by targeting fragment poxBup-kan-P CPA1 -acs-poxBdown, pCP20 plasmid and pKD46 plasmid. Furthermore, the targeting fragment poxBup-kan-P CPA1 -acs-poxBdown can be a gene fragment (5' to 3' is 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 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 CPA1The 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 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 CPA1 The promoter fragment was used as a template and PCR amplified using primers iclR-1 / iclR-2.

[0062] Any of the above-mentioned BguiA gene, HkhkB gene, Ausp gene, Snh3e gene and Rnudt22 gene is 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'), the HkhkB gene shown in SEQ ID No.4, RBS (5'-AGGAG-3'), the Ausp gene shown in SEQ ID No.6, the TrrnB terminator shown in SEQ ID No.15, the trc promoter shown in SEQ ID No.16, the Snh3e gene shown in SEQ ID No.8, RBS (5'-AGGAG-3'), SEQ ID The Rnudt22 gene composition shown in No. 10) is used as a template and primers BHASR-01F / BHASR-01R are used to perform PCR amplification to obtain it; the pTrc fragment can be obtained by using plasmid pTrc99a as a template and primers pTrc99a-01F / pTrc99a-01R are used to perform PCR amplification to obtain it.

[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 the 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 belongs to 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 protection scope 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 acid can be obtained by extracting from oils and fats. The oils and fats can be obtained by refining from kitchen waste oils and fats. Specifically, the refining method includes the steps of recovery, pretreatment, esterification and distillation.

[0074] The fatty acids can also be obtained from oil processing byproducts, palm fatty acid distillate (PFAD), kitchen waste oil recovery and other sources.

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

[0076] The acetic acid can be produced by a fermentation method or a synthetic method.

[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 Escherichia coli for bioconversion to obtain D-psicose. Further, the method comprises the following steps: inoculating the above-mentioned recombinant Escherichia coli in a glycerol growth medium, culturing at 30°C for 3-4h until the OD 600nm After the pH value is 0.6-0.8, add IPTG at a final concentration of 0.5mM, continue to culture at 30°C for 12h, centrifuge, and collect the bacteria; resuspend the bacteria in the transformation medium, react at 37°C for 24h (keep the pH at 7.0 during the reaction), centrifuge, and collect the supernatant. Wherein, the glycerol growth medium solvent is water, and the solutes and final concentrations are as follows: Na2HPO425 mM, KH2PO425 mM, NH4Cl50 mM, Na2SO45 mM, MgSO42 mM, glycerol 3% (volume percentage concentration), yeast powder 0.5% (mass percentage concentration), FeCl350μM, CaCl220μM, MnCl210μM, ZnSO410μM, CoCl22μM, NiCl22μM, Na2MO42μM, Na2SeO32μM, H3BO3 2μM. 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, 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 in an acetic acid growth medium, culturing at 30°C for 3-4h until the OD 600nmAfter the pH value is 0.6-0.8, add IPTG at a final concentration of 0.5mM, continue to culture at 30°C for 12h, centrifuge, and collect the bacteria; resuspend the bacteria in the transformation medium, react at 37°C for 24h (keep the pH at 7.0 during the reaction), centrifuge, and collect the supernatant. Wherein, the acetic acid growth medium solvent is water, and the solutes and final concentrations are as follows: Na2HPO425mM, KH2PO425mM, NH4Cl50mM, Na2SO45mM, MgSO42mM, sodium acetate 2% (mass percentage concentration), yeast powder 0.5% (mass percentage concentration), FeCl350μM, CaCl220μM, MnCl210μM, ZnSO410μM, CoCl22μM, NiCl22μM, Na2MO42μM, Na2SeO32μM, H3BO3 2μM. 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, 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 Escherichia coli for bioconversion to obtain D-psicose. Further, the method comprises the following steps: inoculating the above-mentioned recombinant Escherichia coli into medium A, inoculating into medium B at a 1% inoculation amount after overnight culture, and culturing at 30°C for 3-4h until OD 600nmAfter the pH value is 0.6-0.8, add IPTG at a final concentration of 0.5mM, continue to culture at 30°C for 12h, centrifuge, and collect the bacteria; resuspend the bacteria in the transformation medium, react at 37°C for 24h (keep the pH at 7.0 during the reaction), centrifuge, and collect the supernatant. Wherein, the A medium solvent is water, and the solutes and final concentrations are as follows: Na2HPO425 mM, KH2PO425 mM, NH4Cl50 mM, Na2SO45 mM, MgSO42 mM, glycerol 0.5% (volume percentage concentration), yeast powder 0.5% (mass percentage concentration), FeCl350μM, CaCl220μM, MnCl210μM, ZnSO410μM, CoCl22μM, NiCl22μM, Na2MO42μM, Na2SeO32μM, H3BO32μM. The B medium is a medium obtained by mixing the A medium with palmitic acid and polyoxyethylene ether Brij58 emulsifier, wherein the mass percentage concentration of palmitic acid is 0.5%, and the mass percentage concentration of polyoxyethylene ether Brij58 emulsifier is 0.2%. 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, sucrose 15% (mass percentage concentration).

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

[0081] The nucleotide sequence of any of the above-mentioned cscB genes is shown in the 3171-4418th position 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 the 5619-7052th position 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 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 above-mentioned glk genes is shown in Gene ID: 946858 in Gene Bank, and the amino acid sequence of the glucose kinase encoded by the gene is shown in reference sequence number NP_416889.1 in NCBI.

[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 glucose 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 glucose isomerase 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 glucose isomerase 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 glucose isomerase encoded by the gene is shown as SEQ ID No.7.

[0090] The nucleotide sequence of any of the above Rnudt22 genes is shown as SEQ ID No.10, and the amino acid sequence of the glucose isomerase 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 ID: 948652 in Gene Bank, and the amino acid sequence of the fatty acid degradation transcription factor encoded by the gene is shown in reference sequence number NP_415705.1 in NCBI.

[0092] The nucleotide sequence of any of the above-mentioned ndK genes is shown in the reference sequence number NP_417013 in NCBI, and the amino acid sequence of the nucleoside diphosphate kinase encoded by the gene is shown in the reference sequence number ECK2514 in NCBI.

[0093] The nucleotide sequence of any of the above-mentioned pyrH genes is shown in the reference sequence number NP_414713 in NCBI, and the amino acid sequence of the uridylate kinase encoded by the gene is shown in the reference sequence number ECK0170 in NCBI.

[0094] The nucleotide sequence of any of the above-mentioned udK genes is shown in the reference sequence number NP_416570 in NCBI, and the amino acid sequence of the uridine kinase encoded by the gene is shown in the reference sequence number ECK2060 in NCBI.

[0095] The nucleotide sequence of any of the above-mentioned ppa genes is shown in the reference sequence number ECK4222 in NCBI, and the amino acid sequence of the inorganic pyrophosphatase encoded by the gene is shown in the reference sequence number ECK2060 in NCBI.

[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 ID: 946820 in Gene Bank, and the amino acid sequence of the long-chain fatty acid transport protein encoded by the gene is shown in reference sequence number NP_416846.2 in NCBI.

[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 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 ID: 946665 in Gene Bank, and the amino acid sequence of β-ketoacyl-ACP synthase II encoded by the gene is shown in reference sequence number NP_415613.1 in NCBI.

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

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

[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 ID: 948572 in Gene Bank, and the amino acid sequence of acetyl-CoA synthetase encoded by the gene is shown in reference sequence number NP_418493.1 in NCBI.

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

[0113] The nucleotide sequence of any of the above-mentioned gene clusters atoSC promoter is shown in positions 2320000-2320042 of the genome sequence of 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 realize the growth of microorganisms using glycerol, acetic acid or fatty acid as carbon sources, and can also use mixed carbon sources containing the above carbon source components for growth. After the microorganisms grow to a certain biomass, sucrose is used as a raw material to synthesize D-psicose. The recombinant Escherichia coli engineered strain can be used for the industrial production of D-psicose. On the one hand, low-priced and easily available green organic raw materials are used as carbon sources for microbial growth, and sucrose is used as a raw material to synthesize D-psicose, thereby reducing production costs. On the other hand, D-psicose is efficiently synthesized, and the yield of D-psicose is increased, which has great economic value and social value.

[0118] Collection Instructions

[0119] Bacteria species: Escherichia coli

[0120] Latin name: Escherichia coli

[0121] Strain ID: AS10.03

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

[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 is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0128] 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.

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

[0130] The E. coli MG1655 (CGSC#: 6300), pKD46 plasmid (CGSC#: 7739), and pCP20 plasmid (CGSC#: 7629) in the following examples are all products of the E. coli Genetic Collection Center of Yale University, USA. pKD46 plasmid: contains the temperature-sensitive replication origin oriR101, which can replicate normally when cultured at 30°C and will be automatically lost when cultured above 37°C; E. coli carrying the PKD46 plasmid is cultured at 30°C, and Gam, Exo, and Beta are efficiently expressed after the addition of arabinose induction, and once the exogenous dsDNA is electrotransferred into the cell, homologous recombination can occur with the genomic target sequence; it carries the 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 a 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 screening marker.

[0131] The Escherichia coli W strain in the following examples is recorded in the document "Colin T Archer, Jihyun F Kim, Haeyoung Jeong, Jin Hwan Park, Claudia E Vickers, Sang Yup Lee, Lars K Nielsen. 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 material from the applicant. The obtained above-mentioned biological material is 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.

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

[0134]

[0135]

[0136]

[0137] Example 1. Construction of recombinant Escherichia coli ASU19

[0138] In this example, strain ASU19 and several intermediate strains thereof were prepared, which can use fatty acids, acetic acid, and glycerol as carbon sources for bacterial growth and convert sucrose into D-psicose. The preparation method is as follows, and the primers used are shown in Table 1.

[0139] (1) Enhancement of the expression of the arabinose transporter (araE) gene by promoter replacement

[0140] Starting from Escherichia coli MG1655, the promoter of the araE gene in the strain (shown in the genomic sequence of reference sequence number NC_000913.3 in NCBI, position 2982183-2982313, submission date: 2022-03-09) was replaced with the constitutive promoter P of Escherichia coli. CPA1 (SEQ ID No.13), and obtain recombinant Escherichia coli ASU01, the specific steps are as follows:

[0141] (1-a) Preparation of the targeting fragment araEup-kan-PCPA1-araEdown

[0142] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the following DNA fragment A: from 5' to 3', it contains the frt-kan-frt screening 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. Among them, araEup contains the 70bp homology arm fragment upstream of the araE gene promoter (the lowercase part in the primer table), and araEdown contains the 70bp homology arm reverse complementary fragment downstream of the araE gene promoter (the lowercase part in the primer table).

[0143] (1-b) Preparation of host bacteria containing pKD46 plasmid

[0144] The pKD46 plasmid was transformed into E. coli MG1655 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 MG1655 / pKD46 containing the pKD46 plasmid. After arabinose induction, the recombinant E. coli MG1655 / pKD46 expressed three recombinant proteins of λ phage, and the host bacteria had the ability of homologous recombination. Then, MG1655 / pKD46 competent cells were prepared by washing with 10% glycerol.

[0145] (1-c) Homologous recombination

[0146] 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 (at a concentration of 50 μg / ml). The clones were selected to extract genomic DNA and PCR amplified using araE-3 / Kan-R as primers. The amplified target band of about 1700 bp was positive and 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.

[0147] ASU01-kan was cultured at 42°C overnight to eliminate the temperature-sensitive plasmid pKD46.

[0148] (1-d) Elimination of resistance

[0149] The pCP20 plasmid was transformed into ASU01-kan by calcium chloride transformation. The culture was carried out at 30°C overnight on an LB plate containing ampicillin, and the FLP recombinase on the pCP20 plasmid was used to eliminate kanamycin resistance. The culture was carried out at 42°C overnight to eliminate the temperature-sensitive plasmid pCP20. The monoclonal strain was amplified and identified by PCR using araE-2 / araE-3 primers. The amplified target band of about 500 bp was positive, and the recombinant strain ASU01 was obtained.

[0150] The nucleotide sequence of the araE gene is shown in Gene ID: 947341 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the arabinose transporter encoded by the gene is shown in reference sequence number NP_417318.1 in NCBI (submission date: 2022-03-09).

[0151] (2) Integrate the sucrose permease (derived from Escherichia coli W strain, hereinafter referred to as cscB) gene and the sucrose hydrolase (derived from Escherichia coli W strain, hereinafter referred to as cscA) gene into the lipid A biosynthesis myristoyltransferase (hereinafter referred to as lpxM) gene locus on the chromosome of Escherichia coli

[0152] Starting from the recombinant strain ASU01, the lpxM gene in the strain was replaced by the constitutive promoter P CPA1 , cscB gene and cscA gene to obtain recombinant Escherichia coli ASU02, the specific steps are as follows:

[0153] (2-a) Targeting fragment lpxMup-kan-P CPA1 Preparation of -cscB-cscA-lpxMdown

[0154] 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). PCR amplification was performed using lpxM-1 / lpxM-2 as primers and gene synthesized DNA fragment B as template to obtain the target fragment lpxMup-kan-P CPA1-cscB-cscA-lpxMdown. Among them, lpxMup contains the 70 bp homology arm fragment upstream of the lpxM gene (the lowercase part in the primer table), and lpxMdown contains the 70 bp homology arm reverse complementary fragment downstream of the lpxM gene (the lowercase part in the primer table).

[0155] (2-b) The subsequent operations are the same as steps (1-b), (1-c) and (1-d) in (1) above, to obtain a protein expressed as a constitutive promoter P CPA1 The recombinant strain ASU02 in which the lpxM gene was replaced by the cscB-cscA gene was the only difference between them: the primer combination araE-1 / araE-2 / araE-3 was replaced by the primer combination lpxM-1 / lpxM-2 / lpxM-3.

[0156] The nucleotide sequence of the cscB gene is shown in the 3171-4418th position of the genome sequence of Gene ID: X81461.2 (submission date: 2016-7-26) 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 (submission date: 2016-7-26).

[0157] The nucleotide sequence of the cscA gene is shown in the 5619-7052th position of the genome sequence of Gene ID: X81461.2 (submission date: 2016-07-26) 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.

[0158] The nucleotide sequence of the lpxM gene is shown in Gene ID: 945143 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the lipid A biosynthesis myristoyltransferase encoded by the gene is shown in reference sequence number NP_416369.1 in NCBI (submission date: 2022-03-09).

[0159] (3) Replace the glucokinase (glk) gene with the acid phosphatase (from Aspergillus sp., Aapp) gene

[0160] Starting from the recombinant E. coli ASU02, the glk gene in this strain was replaced by the constitutive promoter P CPA1 and Aapp genes to obtain recombinant E. coli ASU03. The specific steps are as follows:

[0161] (3-a) Targeting fragment glkup-kan-P CPA1Preparation of -AAPP-GLKdown

[0162] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the following DNA fragment C: 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), Aapp gene (SEQ ID No. 12) and TrrnB terminator fragment (SEQ ID No. 15) were prepared. PCR amplification was performed using glk-1 / glk-2 as primers and the gene synthesized DNA fragment C as a template to obtain the target fragment glkup-kan-P CPA1 -Aapp-glkdown. Among them, glkup contains the 70 bp homology arm fragment upstream of the glk gene (the lowercase part in the primer table), and glkdown contains the 70 bp homology arm reverse complementary fragment downstream of the glk gene (the lowercase part in the primer table).

[0163] (3-b) The subsequent operations are the same as steps (1-b), (1-c) and (1-d) in (1) above, to obtain a constitutive promoter P CPA1 and recombinant E. coli ASU03 in which the glk gene is replaced by the Aapp gene. 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.

[0164] The nucleotide sequence of the glk gene is shown in Gene ID: 946858 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of glucose kinase encoded by the gene is shown in reference sequence number NP_416889.1 in NCBI (submission date: 2022-03-09).

[0165] 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.

[0166] (4) Introducing the glucose isomerase gene (derived from Bacillus sp., hereinafter referred to as BguiA), the hexokinokinase gene (derived from Homo sapiens, hereinafter referred to as HkhkB), the UDP-sugar pyrophosphorylase gene (derived from Arabidopsis thaliana, hereinafter referred to as Ausp), the NDP-hexose-3-epimerase gene (derived from Streptomyces sp., hereinafter referred to as Snh3e), and the NDP sugar hydrolase gene (derived from rat (Rattus norvegicus, hereinafter referred to as Rnudt22) into Escherichia coli

[0167] Starting from the recombinant E. coli ASU03, the expression plasmid pTrc99a-BHASR containing the BguiA gene, the HkhkB gene, the Ausp gene, the Snh3e gene, and the Rnudt22 gene was introduced into the recombinant E. coli ASU03 to obtain the recombinant E. coli ASU04. The specific steps are as follows:

[0168] (4-a) Preparation of pTrc99a-BHASR plasmid

[0169] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the following DNA fragment D: from 5' to 3', it contains 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).

[0170] The synthesized DNA fragment D was used as a template, BHASR-01F and BHASR-01R were used as primers, and high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog number AP221) was used for PCR amplification to obtain the BHASR fragment containing the above 5 genes, and the target fragment was recovered by agarose gel electrophoresis. Then, primers pTrc99a-01F and pTrc99a-01R were used, and plasmid pTrc99a (Beijing Zhuangmeng International Biogene Technology Co., Ltd., product number ZK1610) was used as a template to PCR amplify the pTrc fragment and recover the vector large 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., product catalog CD201) by calcium chloride transformation method. It was evenly spread on LB plates containing ampicillin and cultured at 37°C overnight. The clones were selected, and the clones that could amplify the target fragment were identified and sequenced using primers RV-M / pTrcHis-R. The positive clones were selected to extract the plasmids, and the obtained positive plasmids were named pTrc99a-BHASR.

[0171] (4-b) Plasmid transformation

[0172] The pTrc99a-BHASR plasmid was transformed into recombinant Escherichia coli ASU03 by the calcium chloride transformation method. After overnight culture at 37°C on an LB plate containing ampicillin, clones were selected and positive clones were identified using primers RV-M / pTrcHis-R. The positive clones were named ASU04 and stored at -80°C.

[0173] The nucleotide sequence of the BguiA gene is shown as SEQ ID No.2, and the amino acid sequence of the glucose isomerase encoded by the gene is shown as SEQ ID No.1.

[0174] The nucleotide sequence of the HkhkB gene is shown in SEQ ID No.4, and the amino acid sequence of the glucose isomerase encoded by the gene is shown in SEQ ID No.3.

[0175] The nucleotide sequence of the Ausp gene is shown as SEQ ID No.6, and the amino acid sequence of the glucose isomerase encoded by the gene is shown as SEQ ID No.5.

[0176] The nucleotide sequence of the Snh3e gene is shown as SEQ ID No.8, and the amino acid sequence of the glucose isomerase encoded by the gene is shown as SEQ ID No.7.

[0177] The nucleotide sequence of the Rnudt22 gene is shown as SEQ ID No.10, and the amino acid sequence of the glucose isomerase encoded by the gene is shown as SEQ ID No.9.

[0178] (5) Knockout of fatty acid degradation transcription factor (fadR) gene

[0179] Starting from the recombinant E. coli AS03, the fadR gene in the strain was knocked out to obtain the recombinant E. coli ASU05. The specific steps are as follows:

[0180] (5-a) Preparation of targeting fragment fadRfup-kan-fadRdown

[0181] Using fadR-1 / fadR-2 primers and the DNA fragment A described in step (1) above as a template, PCR amplification was performed to obtain a targeting fragment fadRup-kan-fadRdown containing 70 bp homology arms upstream and downstream of the fadR gene site.

[0182] (5-b) The subsequent operations refer to steps (1-b), (1-c) and (1-d) in (1) above to obtain recombinant Escherichia coli ASU05 with the fadR gene knocked out. The only difference is that the primer combination araE-1 / araE-2 / araE-3 is replaced by the primer combination fadR-1 / fadR-2 / fadR-3. At the same time, during the operation, PCR amplification and identification are performed using fadR-3 / Kan-R as primers, and the amplified target band of about 1400 bp is positive. PCR amplification and identification are performed on monoclonal strains using fadR-2 / fadR-3 as primers, and the amplified target band of about 200 bp is positive.

[0183] The nucleotide sequence of the fadR gene is shown in Gene ID: 948652 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the fatty acid degradation transcription factor encoded by the gene is shown in reference sequence number NP_415705.1 in NCBI (submission date: 2022-03-09).

[0184] (6) Construction of recombinant E. coli ASU06, ASU07, ASU08, ASU09, ASU10, ASU11, ASU12, and ASU13

[0185] 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, acyl-CoA synthase (hereinafter referred to as fadD) gene, long-chain fatty acid transporter (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 2644433-2644864th position of the genome sequence of the reference sequence number NC_000913.3 in NCBI, submission date: 2022-03-09), the pyrH gene promoter (the 191752-191854th position of the genome sequence of the reference sequence number NC_000913.3 in NCBI, submission date: 2022-03-09), the udK gene promoter (the 2142949-2143088th position of the genome 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 4449653-4449806th position of the genome sequence of the reference sequence number NC_000913.3 in NCBI, submission date: 2022-03-09), fadD gene promoter (shown in the 1889747-1889867th position of the genome sequence of the reference sequence number NC_000913.3 in NCBI, submission date: 2022-03-09), fadL gene promoter (shown in the 1889747-1889867th position of the genome sequence of the reference sequence number NC_000913.3 in NCBI, 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 reference sequence number NC_000913.3 in NCBI, submission date: 2022-03-09), and the atoSC gene cluster promoter (the 2320000-2320042 positions of the genome sequence of the reference sequence number NC_000913.3 in NCBI, 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.

[0186] The specific operation steps refer to step (1) in Example 1. The only difference is that the primer araE-1 / araE-2 / araE-3 combination is replaced by the primer ndk-1 / ndk-2 / ndk-3 combination, the primer pyrH-1 / pyrH-2 / pyrH-3 combination, the primer udk-1 / udk-2 / udk3 combination, the primer ppa-1 / ppa-2 / ppa-3 combination, the primer fadD-1 / fadD-2 / fadD-3 combination, the primer fadL-1 / fadL-2 / fadL-3 combination, the primer sthA-1 / sthA-2 / sthA-3 combination, and the primer atoSC-1 / atoSC-2 / atoSC-3 combination.

[0187] The nucleotide sequence of the ndK gene is shown in Gene ID: 945611 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the nucleoside diphosphate kinase encoded by the gene is shown in reference sequence number NP_417013.1 in NCBI (submission date: 2022-03-09).

[0188] The nucleotide sequence of the pyrH gene is shown in Gene ID: 944989 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the uridylate kinase encoded by the gene is shown in reference sequence number NP_417013.1 in NCBI (submission date: 2022-03-09).

[0189] The nucleotide sequence of the udK gene is shown in Gene ID: 946597 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the uridine kinase encoded by the gene is shown in reference sequence number NP_417013.1 in NCBI (submission date: 2022-03-09).

[0190] The nucleotide sequence of the ppa gene is shown in Gene ID: 948748 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the inorganic pyrophosphatase encoded by the gene is shown in reference sequence number NP_417013.1 in NCBI (submission date: 2022-03-09).

[0191] The nucleotide sequence of the fadD gene is shown in Gene ID: 946327 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the acyl-CoA synthase encoded by the gene is shown in reference sequence number NP_416319.1 in NCBI (submission date: 2022-03-09).

[0192] The nucleotide sequence of the fadL gene is shown in Gene ID: 946820 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the long-chain fatty acid transport protein encoded by the gene is shown in reference sequence number NP_416846.2 in NCBI (submission date: 2022-03-09).

[0193] The nucleotide sequence of the sthA gene is shown in Gene ID: 948461 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the NAD(P) transhydrogenase encoded by the gene is shown in the reference sequence number NP_418397.2 (2022-03-09) in NCBI.

[0194] The atoSC gene cluster includes the atoS gene and the atoC gene. The nucleotide sequences of the atoS gene and the atoC gene are shown in Gene ID: 949011 (submission date: 2023-04-14) and Gene ID: 947444 (submission date: 2023-04-14) in Gene Bank, respectively. The amino acid sequences of the atos protein and atoC protein encoded by the atoS gene and the atoC gene are shown in the reference sequence numbers NP_416723.1 (submission date: 2022-03-09) and NP_416724.1 (submission date: 2022-03-09) in NCBI, respectively.

[0195] (7) Construction of recombinant E. coli ASU14, ASU15, and ASU16

[0196] Starting from the recombinant Escherichia coli ASU13, the β-ketoacyl-ACP synthase II (hereinafter referred to as fabF) gene, β-ketoacyl-ACP synthase III (hereinafter referred to as fabH) gene and isocitrate lyase inhibitor (hereinafter referred to as iclR) gene were knocked out in sequence to obtain recombinant Escherichia coli ASU14, ASU15 and ASU16.

[0197] The specific operation steps refer to step (5) in Example 1. The only difference is that the primer combination of fadR-1 / fadR-2 / fadR-3 is replaced by the primer combination of fabF-1 / fabF-2 / fabF-3, the primer combination of fabH-1 / fabH-2 / fabH-3, and the primer combination of iclR-1 / iclR-2 / iclR-3.

[0198] The nucleotide sequence of the fabF gene is shown in Gene ID: 946665 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of β-ketoacyl-ACP synthase II encoded by the gene is shown in reference sequence number NP_415613.1 in NCBI (submission date: 2022-03-09).

[0199] The nucleotide sequence of the fabH gene is shown in Gene ID: 946003 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of β-ketoacyl-ACP synthase III encoded by the gene is shown in reference sequence number NP_415609.1 in NCBI (submission date: 2022-03-09).

[0200] The nucleotide sequence of the iclR gene is shown in Gene ID: 948524 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the isocitrate lyase inhibitor encoded by the gene is shown in reference sequence number NP_418442.2 in NCBI (submission date: 2022-03-09).

[0201] (8) Replace the pyruvate oxidase (poxB) gene with the acetyl-CoA synthetase (acs) gene

[0202] Starting from the recombinant Escherichia coli ASU16, the poxB gene in this strain was replaced with P CPA1 promoter and acs gene to obtain recombinant Escherichia coli ASU17.

[0203] 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.

[0204] The nucleotide sequence of the poxB gene is shown in Gene ID: 946132 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the pyruvate oxidase encoded by the gene is shown in reference sequence number NP_415392.1 in NCBI (submission date: 2022-03-09).

[0205] The nucleotide sequence of the acs gene is shown in Gene ID: 948572 in Gene Bank (submission date: 2023-04-14), and the amino acid sequence of the acetyl-CoA synthetase encoded by the gene is shown in reference sequence number NP_418493.1 in NCBI (submission date: 2022-03-09).

[0206] (9) Construction of recombinant E. coli ASU18 and ASU19

[0207] The 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 in step (4) above.

[0208] The recombinant bacterium ASU19 was deposited in the General Microbiology Center of China Microorganism Culture Collection (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.

[0209] 2. Cultivation of recombinant E. coli ASU04 using glycerol as a carbon source and preparation of D-psicose using sucrose as a raw material

[0210] 1. Glycerol Growth Medium Composition

[0211] The components and final concentrations of glycerol growth medium are as follows (the solvent is water): Na2HPO4 25mM, KH2PO4 25mM, NH4Cl 50mM, Na2SO4 5mM, MgSO4 2mM, glycerol 3% (volume percentage concentration), yeast powder 0.5% (mass percentage concentration), FeCl3 50μM, CaCl2 20μM, MnCl2 10μM, ZnSO4 10μM, CoCl2 2μM, NiCl2 2μM, Na2MO4 2μM, Na2SeO3 2μM, H3BO3 2μM.

[0212] 2. Transformation medium composition

[0213] 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, sucrose 15% (mass percentage concentration).

[0214] 3. Bacterial culture and enzyme induction

[0215] The control strain ASU03 and the engineered strain ASU04 cultured overnight were 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 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, centrifuge at 8000 rpm for 10 min, and collect the bacteria.

[0216] 4. Whole-cell catalysis of D-psicose

[0217] The collected bacteria were resuspended in a beaker containing 10 mL of transformation medium. The reaction process was monitored regularly using pH test paper, and 0.1 M NaOH and HCl solutions were used to adjust the pH to 7.0. After reacting at 37°C for 24 hours, the supernatant was centrifuged and filtered. The content of D-psicose was detected by HPLC. 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. The D-psicose standard was a product of Shanghai Yuanye Biotechnology (Cat. No.: S48276).

[0218] The results showed that the D-psicose yield of strain ASU03 was 0, and the average D-psicose yield of strain ASU04 was 83 g / L. Therefore, strain ASU04 can convert sucrose into D-psicose with a conversion rate of 55%. In addition, the purity of D-psicose in the reaction solution was high, and no other sugars were significantly detected, which is conducive to simplifying the downstream separation and purification process.

[0219] 3. Cultivation of recombinant E. coli ASU19 using acetic acid as a carbon source and preparation of D-psicose using sucrose as a raw material

[0220] 1. Acetic acid growth medium composition

[0221] The components and final concentrations of acetic acid growth medium are as follows (the solvent is water): Na2HPO4 25mM, KH2PO4 25mM, NH4Cl 50mM, Na2SO4 5mM, MgSO4 2mM, sodium acetate 2% (mass percentage concentration), yeast powder 0.5% (mass percentage concentration), FeCl3 50μM, CaCl2 20μM, MnCl2 10μM, ZnSO4 10μM, CoCl2 2μM, NiCl2 2μM, Na2MO4 2μM, Na2SeO3 2μM, H3BO3 2μM.

[0222] 2. Transformation medium composition

[0223] 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, sucrose 15% (mass percentage concentration).

[0224] 3. Bacterial culture and enzyme induction

[0225] 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 concentration of IPTG reached 0.6-0.8, add IPTG to a final concentration of 0.5 mM and continue culturing at 30°C for 12 h. Centrifuge at 8000 rpm for 10 min to collect the cells.

[0226] 4. Whole-cell catalysis of D-psicose

[0227] The collected bacteria were resuspended in a beaker containing 10 mL of transformation medium, and the pH was maintained at 7.0 during the reaction. After reacting at 37°C for 24 hours, the supernatant was centrifuged and filtered. The content of D-psicose was detected by HPLC according to the method in step 2.

[0228] The results showed that the D-psicose yield of strain ASU17 was 0, and the average D-psicose yield of strain ASU19 was 101.7 g / L, with a conversion rate of 67.8%. In addition, the purity of D-psicose in the reaction solution was high, and no other sugars were significantly detected, which was conducive to simplifying the downstream separation and purification process.

[0229] 4. Cultivation of recombinant E. coli ASU18 using fatty acids as carbon source and preparation of D-psicose using sucrose as raw material

[0230] 1. Fatty acid growth medium composition

[0231] Culture medium A: a sterile culture medium composed of solutes and solvents, the solvent is water, and the solutes and their concentrations are: Na2HPO4 25mM, KH2PO4 25mM, NH4Cl 50mM, Na2SO4 5mM, MgSO4 2mM, glycerol 0.5% (volume percentage concentration), yeast powder 0.5% (mass percentage concentration), FeCl3 50μM, CaCl2 20μM, MnCl210μM, ZnSO4 10μM, CoCl2 2μM, NiCl2 2μM, Na2MO4 2μM, Na2SeO3 2μM, H3BO3 2μM.

[0232] Medium B: A sterile medium obtained by adding palmitic acid and polyoxyethylene ether Brij58 emulsifier (Merck, Inc., Product Number: 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%.

[0233] 2. Transformation medium composition

[0234] 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, sucrose 15% (mass percentage concentration).

[0235] 3. Bacterial culture and enzyme induction

[0236] The control strain ASU16 and the engineered strain ASU18 cultured overnight in medium A were inoculated at 1% in a shake flask containing 200 mL medium B (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 (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. Centrifuge at 8000 rpm for 10 minutes to collect the cells.

[0237] 4. Whole-cell catalysis of D-psicose.

[0238] The collected bacteria were resuspended in a beaker containing 10 mL of transformation medium, and the pH was maintained at 7.0 during the reaction. After reacting at 37°C for 24 hours, the supernatant was centrifuged and filtered. The content of D-psicose was detected by HPLC according to the method in step 2.

[0239] The results showed that the D-psicose yield of the control strain ASU16 was 0, while the average D-psicose yield of the strain ASU18 was 110.4 g / L, with a conversion rate of 73.6%. In addition, the purity of D-psicose in the reaction solution was high, and no other sugars were significantly detected, which was conducive to simplifying the downstream separation and purification process.

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) to (a3): Modification (a1): Enhance the expression of araE gene; Transformation (a2): lpxM gene was replaced by cscB gene and cscA gene; Transformation (a3): glk gene replaced by Aapp gene; Transformation (d): Introduction and expression of BguiA gene, HkhkB gene, Ausp gene, Snh3e gene and Rnudt22 gene.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The plurality of modifications also includes modification (b); The transformation (b) includes the following transformations (b1) to (b12): Transformation (b1): knockout of fadR gene; Modification (b2): Enhanced expression of ndK gene; Modification (b3): ​​Enhanced expression of the pyrH gene; Modification (b4): Enhance the expression of udk gene; Modification (b5): Enhance the expression of the ppa gene; Modification (b6): Enhanced expression of fadD gene; Modification (b7): Enhanced expression of fadL gene; Modification (b8): Enhanced expression of sthA gene; Modification (b9): Enhanced expression of the atoSC gene cluster; Transformation (b10): knockout of the fabF gene; Transformation (b11): knockout of the fabH gene; Modification (b12): Knockout of the iclR gene.

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 poxB gene is replaced by the acs gene.

4. The recombinant Escherichia coli according to any one of claims 1 to 3, characterized in that: The deposit number of the recombinant bacteria 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 following steps: performing the transformation according to any one of claims 1 to 3.

7. The recombinant Escherichia coli prepared according to the method of claim 6.

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

9. A method for preparing D-psicose, comprising the following steps: using sucrose as a raw material, and using the recombinant Escherichia coli according to any one of claims 1 to 4 or the recombinant Escherichia coli according to claim 7 to perform biotransformation to obtain D-psicose.

10. The method according to claim 9, 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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