Novel escherichia coli for synthesizing D-psicose and application

Abstract

The invention provides a group of recombinant Escherichia coli for forming D-psicose. The recombinant Escherichia coli comprises the following modifications: a substance for enhancing the activity of hexose-3-dehydrogenase with substrate specificity; a substance that enhances the activity of a hexose-3-reductase having substrate specificity; a substance that enhances the activity of an enzyme having a substrate enrichment function. By means of the recombinant escherichia coli, high-efficiency synthesis of D-psicose can be achieved, and the problem that the conversion rate of D-psicose production is low is solved.

Description

Technical Field

[0001] The present invention relates to the fields of gene editing, gene recombination-edited microbial strains, synthetic biology, and microbial whole-cell catalysis technology, and specifically to a recombinant strain for synthesizing D-psicose and a preparation method thereof. Background Art

[0002] D-allulose, a rare, low-calorie monosaccharide, has attracted significant attention for its unique nutritional properties. With a sweetness 70% that of sucrose and only 0.3% of its calories, it possesses multiple physiological functions, including blood sugar regulation, weight control, and neuroprotection, and holds broad application prospects in the food and pharmaceutical sectors. Although widely found in nature in foods like wheat and fruit, its extremely low concentration makes direct extraction difficult, making industrial production a key research priority. International research and development progress reveals that Matsutani Chemical of Japan pioneered the launch of a syrup containing allulose in 2012, followed by CJ of South Korea (2015) and Tate & Lyle of the United Kingdom, which have subsequently entered the North American and global markets. Jiangnan University in my country has achieved breakthroughs in key technologies such as gene mining and strain development, and its enzyme immobilization and purification technologies have reached internationally advanced levels. However, existing diastereomerase methods still face bottlenecks such as high raw material costs and limited conversion rates (constrained by equilibrium constants).

[0003] Among traditional D-psicose production routes, chemical synthesis methods (such as molybdate catalysis and isopropylidene fructose conversion) have hindered industrialization due to numerous byproducts and low purity. The mainstream enzymatic conversion method, which uses DPEase to catalyze the epimerization of fructose, while aligning with green production principles, is limited by the efficiency of natural enzymes, requires complex purification processes, and suffers from low raw material utilization. A recent breakthrough comes from the whole-cell catalytic technology proposed in patent CN202410459375.8, which achieves efficient glucose conversion by constructing a microbial metabolic pathway. Compared to traditional methods, this technology reduces raw material costs by 30% and simplifies the purification process. However, the traditional D-psicose synthesis pathway still primarily relies on sugar isomerization reactions, which are plagued by limited reaction equilibrium and high byproduct formation. Therefore, developing new kinetically advantageous reaction pathways and constructing corresponding microbial cell factories will not only significantly improve the synthesis efficiency of D-psicose but also play a key role in establishing novel fermentation-based production routes. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a strain containing a novel D-psicose synthesis pathway, which uses glucose as a raw material and produces D-psicose through whole-cell catalysis, with high conversion rate, low cost, and minimal side reactions. The present invention discloses a new D-psicose synthesis strategy that overcomes the kinetic limitations of key catalytic reactions in traditional synthesis pathways, providing a more efficient synthesis route. This pathway can be integrated with other D-psicose synthesis pathways to avoid the accumulation of intermediates caused by side reactions. This improves the technical specifications of the D-psicose synthesis route using glucose as a raw material, and has great economic and social value.

[0005] According to an exemplary embodiment of the present disclosure, the recombinant Escherichia coli is achieved by performing the following multiple transformations in the recipient bacteria:

[0006] A gene encoding substrate-specific hexose-3-dehydrogenase activity is introduced and expressed. The gene may be derived from genes encoding enzymes having EC 1.1.98.3, EC 1.1.99.13, EC 1.1.99.18, EC 1.1.1.45, EC 1.1.1.361, EC 1.1.1.374, or EC 1.1.99.29 activities. The substrates catalyzed by the gene expression product include sucrose, cellobiose, lactose, lactulose, difructose anhydride I, difructose anhydride III, difructose anhydride IV, and difructose anhydride V. Specifically, the gene may be as shown in SEQ ID NO. 5.

[0007] Introducing and expressing a gene encoding substrate-specific hexose-3-reductase activity, the gene can be derived from genes encoding enzymes having EC 1.1.1.333, EC 1.1.1.425, EC 1.1.1.226, EC 1.1.1.123, EC 1.1.1.21, or EC 1.1.1.124 activities. Specifically, the gene shown in SEQ ID NO. 7 can be used.

[0008] The genes encoding the synthesis of hexose-3-dehydrogenase precursors are introduced and expressed, specifically the sequences shown in SEQ ID NOs. 8, 9, 10, 11, 12 and 13.

[0009] The gene encoding 3-ketoglycoside hydrolase is introduced and expressed, and specifically may be composed of the sequence shown in SEQ ID NO. 14.

[0010] The recombinant Escherichia coli described above can be obtained from the following Escherichia coli: Escherichia coli MG1655, Escherichia coli BW25113, Escherichia coli MC02 (CGMCC No. 34378), Escherichia coli AS10 (CGMCC No. 27687), and Escherichia coli AS10.01 (CGMCC No. 34377). MG1655 and BW25113 can be obtained from the Yale University Escherichia coli Genetic Collection (CGSC), and MC02 can be obtained from CGMCC. Information on other strains can be found in Chinese invention patent CN202510500192.0 (which is incorporated herein by reference in its entirety) and can also be obtained from CGMCC.

[0011] Another aspect of the present invention provides use of the recombinant Escherichia coli according to any of the aforementioned items in whole-cell catalytic production of a D-psicose product.

[0012] The above-mentioned product may be a bacterial agent containing the recombinant bacteria and / or a culture of the recombinant bacteria.

[0013] The term "culture" refers to any liquid or solid product (i.e., fermentation product) containing a microbial population after artificial inoculation and cultivation. This refers to a product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of the microorganism or contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process.

[0014] The beneficial technical effects achieved by this disclosure are as follows:

[0015] The present invention discloses the preparation of recombinant Escherichia coli capable of producing the above-mentioned D-psicose in high yield, thereby increasing the yield of the target substance, solving the bottleneck problem in industrial production, and can be used for the mass production of related D-psicose substances in the industry. Preservation Instructions

[0016] Species name: Escherichia coli

[0017] Latin name: Escherichia coli

[0018] Strain number: AS10.

[0019] Depository: General Microbiology Center of China Culture Collection Administration

[0020] Abbreviation of depository institution: CGMCC

[0021] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0022] Deposit date: April 27, 2025

[0023] CGMCC Registration Number: CGMCC No. 34376

[0024] Species name: Escherichia coli

[0025] Latin name: Escherichia coli

[0026] Strain ID: AS10.01

[0027] Depository: General Microbiology Center of China Culture Collection Administration

[0028] Abbreviation of depository institution: CGMCC

[0029] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0030] Deposit date: April 27, 2025

[0031] CGMCC Registration Number: CGMCC No. 34377 BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 Standard curve prepared for D-psicose standard and the equation for the derived D-psicose concentration.

[0034] Figure 2 The HPLC peak profiles of D-psicose standards at different concentrations.

[0035] Figure 3 The yield of D-psicose synthesized by different strains. DETAILED DESCRIPTION

[0036] The present disclosure is further described in detail below in conjunction with specific embodiments. The examples provided are intended only to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0037] 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 agarose to liquid culture medium.

[0038] The E. coli MG1655 (CGSC#: 6300) and plasmid pKD46 (CGSC#: 7739) used in the following examples are 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. The gene sequence of the pSCre plasmid is shown in SEQ ID NO. 23 and is available to the public through gene synthesis. Escherichia coli carrying the pSCre plasmid, when cultured at 30°C, express the recombinase (Cre) gene, causing recombination between the lox66 and lox71 sequences on the chromosome, thereby eliminating the DNA sequence located between them. In this patent, this eliminates the kanamycin resistance gene located between the lox66 and lox71 sequences in SEQ ID NO. 1. Escherichia coli carrying the pSCre plasmid automatically lose the pSCre plasmid when cultured at 42°C. The pSCre plasmid carries a streptomycin resistance gene as a selection marker. Escherichia coli AS10 (CGMCC No. 27687) is a D-psicose-producing E. coli deposited with the China General Microbiological Culture Collection. The public can obtain the above-mentioned biological material from the applicant for use solely in repeating the experiments described herein and not for any other purpose.

[0039] Table 1. List of sequence fragments

[0040] Serial number Clip number Clip Name Sequence (5'-3') SEQ ID NO.1 Clip 1 lox66-Kan-lox71 aaaaatgatttgtcgttagtgttatacctagccctaccgttcgtataatgtatgctatacgaagttatagagcgcttttgaagctcacgctgccgcaagcactcagggcgcaagggctgctaaaggaagcggaacacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctaataaggggatcttataacttcgtataatgtatgctatacgaacggtaaaatgggaaatttactgagccaatccc SEQ ID NO.2 Clip 2 P119 atggcttgtcatgcttaattgacagctagctcagtcctaggtataatgctagcaggggagaccacaacggtttccctctacaaataattttgtttaactttcgcgcgcgtaacaggaggaattaacc SEQ ID NO.3 Clip 3 RBS aggaggaattaacc SEQ ID NO.4 Clip 4 TrrnB terminator tgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctt SEQ ID NO.5 Clip 5 Hexose-3-dehydrogenase MhhDF atgtccactgcgctcgggggtagtgatcgggacgcaagatcaacaaccgccaagcgattgaccgggtggggacgtactgcaccgtcggtcgcccaggtcttgtcaccgcccgaccccgatgtcatagttgaagcactgacaagatacattgccgatgctacatcagcccgcggagtaattgcgcgcggtttggggcgttcttatggggatcctgcacagaacgctggtggtacagtaatcgacatgacggctttgaatcgtatccactctatagacgctgacacaaagctggttgacgtagacgccggcgttaatttggaccagctcactaaagcagcgaagcctttcggactatgggtgcctgttctcccgggcacccgacaggtcacggttggtggagcgattgcttgcgacattcacggaaaaaaccaccactccgcaggttcctttggtaatcatgtttcactggatctctthgaccgctgacggggaagtgcgaaccctaactcctgacgggcctgatgcgaaatgggaagcggcgttattttgggccactgttggggggatgggcctgacagggattatcgtaagggcaacaattgaactgacccccgtggaaaccgcgtatttcgtggtggatacagaacgggccggagacctagaagaccttatggcgaggttggctagtggggatgacgcaaattacacatatagtgttgcgtggttcgatacatcgacccgagggccgtcactgggtcggtcagtgatcactcggggccgcagcgctagggtggaagatctggacaaaaaactcaggaaggaccccctgaagtttaacgcaccacagctaatgaccgcccctagcgttttcccgaatggactatggaatgacgagtactccgcgttcaatttgaacgtctggtacagaaaggcaccgactcgaaccggcgaagtgcaagacattacacagttcttccacccacttgatgtagtgggtgactggaatagactgtacggtccccgcggcttctgccagtatcagttcgtagtgccagatgctcaggaagcgatgtttagaagatcagttggactgatcgccgcgtcaggacatgtttcgtccctaaatgtacttaagcggtttggcccagcttcccctgcaccactttcgtttcctactccgggctggactctcgcagttgacttacctgtccgtccgggattggagtccattgttgacggcctagacgcgttagtggtagaagccgggggacgcgtatacctggctaaggattcgcgtacctcgccagctactattaccgacatgtatccgcgcttagatgagtttagaacggtcagacggcgtgttgatccccatggtgtattacggtctgatctctctcgtcgacttgatctttaa SEQ ID NO.7 Clip 7 Hexose-3-reductase AhrF atgcgtaacttgaacgtcggtttgatagatggtggattcatgggtaaagcccactcgttagcgtatgctgccatgccaatgttcttctggccggctccggctcagccccaccgacacacagtggttgattataacgagggcctggcagcggacgctgctaaccgcttcgggtttcaatactctacagatgactggcgcagcattatcgagaatccagatattgatgtcgtggacattgcgactcccaaccatctgcatgcagatgtagcaatagcagcactgcgggcagggaaacatgtaatctgcgaaaaaccaattgcgaggaccgtagatgaggctaaggcgatgtacgaggccgcccgcgaagccaaagtgaccaatatggttgctttcaactaccgacgcacacctgctgttgcattagctaagagatacattgatgaaggtgcgattggggacatcttgaacgtgagaggaacctacctgcaggacagcagtgctgatcccagtagcccgttatccgcgtcgtttcaaaagtcgatcgcaggcagtggggcacttggatcaattggaacacatgtcattgatatggcccgctatctagtgggagaaattaccgcggtgaacgcagtgttaagcacctggatcccggaacggccactccaaagcggaggggcggacgcgctaggtcgtggcaaggtcggtggtgatgaaggcccgaaaggacccgttgacgttgacgacgaggtgatgaccctagtccggtttgcgaatggagccgtcggctctgttgaagcgacgagaaatgcacacggacggaataattacattaccttcgaaatccacgggacagagggtagtattgtctttaattatgaacgtcgagacgaactccaagtgtgcttcaaatctgaccaagatgacagacgcggcttcagaacggtttatacgggacctaatcatcctaatggagccgctttgtggcccatacccggcagtgcagatggatactctgagaccaaaattattgaggcccatgatttcttccacgcagtagcgaatggcacggaggtccgacctaattttaatgaggcttaccaaattgaacttattgccgacgcaattgtggaatctgctaatgtggattattgggtcaacgtcactccacgaagcgcaaagtaa SEQ ID NO.8 Pglucose phosphomutase gene pgm NCBI GeneID: 945271 (2022.3.9) or atggcaatcc acaatcgtgc aggccaacct gcacaacaga gtgatttgat taacgtcgcccaactgacggcgcaatatta tgtactgaaa ccagaagcag ggaatgcgga gcacgcggtgaaattcggta cttccggtca ccgtggcagt gcagcgcgcc acagctttaacgagccgcacattctggcga tcgctcaggc aattgctgaa gaacgtgcga aaaacggcat cactggcccttgctatgtgg gtaaagatac tcacgccctgtccgaacctg cattcatttc cgttctggaagtgctggcag cgaacggcgt tgatgtcatt gtgcaggaaa acaatggctt caccccgacgcctgccgtttccaatgccat cctggttcac aataaaaaag gtggcccgct ggcagacggtatcgtgatta caccgtccca taacccgccg gaagatggtg gaatcaaatacaatccgccaaatggtggcc cggctgatac caacgtcact aaagtggtgg aagacagggc caacgcactgctggccgatg gcctgaaagg cgtgaagcgtatctccctcg acgaagcgat ggcatccggtcatgtgaaag agcaggatct ggtgcagccg ttcgtggaag gtctggccga tatcgttgatatggccgcgattcagaaagc gggcctgacg ctgggcgttg atccgctggg cggttccggtatcgaatact ggaagcgtat tggcgagtat tacaacctca acctgactatcgttaacgatcaggtcgatc aaaccttccg ctttatgcac cttgataaag acggcgcgat ccgtatggactgctcctccg agtgtgcgat ggcgggcctgctggcactgc gtgataagttcgatctggcgtttgctaacg acccggatta tgaccgtcac ggtatcgtca ctccggcagg tttgatgaatccgaaccactacctggcggt ggcaatcaat tacctgttcc agcatcgtcc gcagtggggcaaagatgttg ccgtcggtaa aacgctggtt tcatctgcga tgatcgaccgtgtggtcaacgacttgggcc gtaaactggt agaagtcccg gtaggtttca aatggtttgt cgatggtctgttcgacggca gcttcggctt tggcggcgaagagagtgcag gggcttcctt cctgcgtttcgacggcacgc cgtggtccac cgacaaagac ggcatcatca tgtgtctgct ggcggcggaaatcaccgctgtcaccggtaa gaacccgcag gaacactaca acgaactggc aaaacgctttggtgcgccga gctacaaccg tttgcaggca gctgcgactt ccgcacaaaaagcggcgctgtctaagctgt ctccggaaat ggtgagcgcc agcaccctgg caggtgaccc gatcaccgcgcgcctgactg ctgctccggg caacggtgcttctattggcg gtctgaaagt gatgactgacaacggctggt tcgccgcgcg tccgtcaggc acggaagacg catataagat ctactgcgaaagcttcctcggtgaagaaca tcgcaagcag attgagaaag aagcggttga gattgttagcgaagttctga aaaacgcgta a SEQ ID NO.9 UTP-glucose-1-phosphate uridylyl transferase gene galU NCBI GeneID: 945730 (2022.3.9) or atggctgcca ttaatacgaa agtcaaaaaa gccgttatcc ccgttgcgggattaggaaccaggatgttgc cggcgacgaa agccatcccg aaagagatgc tgccacttgt cgataagccattaattcaat acgtcgtgaa tgaatgtattgcggctggca ttactgaaat tgtgctggttacacactcat ctaaaaactc tattgaaaac cactttgata ccagttttga actggaagcaatgctggaaaaacgtgtaaa acgtcaactg cttgatgaag tgcagtctat ttgtccaccgcacgtgacta ttatgcaagt tcgtcagggt ctggcgaaag gcctgggacacgcggtattgtgtgctcacc cggtagtggg tgatgaaccg gtagctgtta ttttgcctga tgttattctggatgaatatg aatccgattt gtcacaggataacctggcag agatgatccg ccgctttgatgaaacgggtc atagccagat catggttgaa ccggttgctg atgtgaccgc atatggcgttgtggattgcaaaggcgttga attagcgccg ggtgaaagcg taccgatggt tggtgtggtagaaaaaccga aagcggatgt tgcgccgtct aatctcgcta ttgtgggtcgttacgtacttagcgcggata tttggccgtt gctggcaaaa acccctccgg gagctggtga tgaaattcagctcaccgacg caattgatat gctgatcgaaaaagaaacgg tggaagccta tcatatgaaagggaagagcc atgactgcgg taataaatta ggttacatgc aggccttcgt tgaatacggtattcgtcataacacccttgg cacggaattt aaagcctggc ttgaagaaga gatgggcattaagaagtaa. SEQ ID NO. 10 Clip 10 Glucose isomerase BgiA atggctcaatctcattccagttcaatcaactattttggaagcgcaaacaaagtggtttacgaagggaaagattcgactaatcctttagcatttaaatattataatcctcaagaagtaatcggcggaaaaacgctgaaagagcatttgcgattttctattgcctattggcatacatttactgctgatggtacagacgtttttggagcagctacgatgcaaagaccatgggatcactataaaggcatggatctagcgaagatgagagtagaagcagcatttgagatgtttgaaaaactagatgcaccattctttgcttttcatgaccgggatattgcaccagaaggcagtacgctaaaagagacaaaccaaaatttagatatgatcatgggcatgattaaagattacatgagaaatagcggcgttaagctattatggaatacagcaaacatgtttacgaatccccgtttcgtccatggtgccgcgacttcttgcaatgcagatgtgtttgcgtatgctgcagcacaagtgaaaaaagggttagaaacagcaaaagagcttggcgctgagaactatgtattttggggcggccgtgaaggatatgaaacattgttaaataccgatttaaaatttgagcttgataatttggctagatttatgcatatggcagtggattatgcgaaggaaatcgggtacacagggcagtttttgattgagccaaaaccaaaagagccgaccacccatcaatacgatacagatgcagcaacaaccattgcctttttgaagcaatatggcttagacaatcattttaaattaaatcttgaagccaatcatgccacattagccgggcatacattcgaacatgaattacgcatggcaagagtacatggtctgcttggctctgttgacgcaaaccagggtcatcctcttttaggctgggacacggatgaatttccgacggatttatattctacgacattagcaatgtacgaaatcctgcaaaatggcggccttggaagcggcggattaaactttgacgcgaaggtcagaagatcttctttcgagcctgatgatctaatatatgcccatattgcagggatggatgcatttgcaagaggattgaaagttgcccacaaattaatcgaagatcgtgtgtttgaagatgtgattcaacatcgttaccgcagctttactgaagggattggtcttgaaattatagaaggaagagctaatttccacacacttgagcaatatgcgctaaatcataaatcaattaaaaacgaatctggaagacaggagaaattaaaagcgatattgaaccaatacattttagaagtataa SEQ ID NO.11 Clip 11 Sucrose synthase Gmss atggccaccgatcgtttgacccgggttcacagtctccgtgagaggcttgatgaaaccctcactgccaacaggaatgaaattttggcccttctgtcaaggatcgaagccaagggcaagggcatcctgcaacaccaccaggtcattgctgagtttgaggaaatccctgaggagaacagacagaagctcactgatggtgcctttggagaagtcttgagatctacacaggaagccatagttttgccaccatgggttgctctggctgttcgtccaagacctggtgtgtgggagtacctgagagtgaatgtgcacgctcttgttgttgaggagttgcaacctgctgagtacctgcacttcaaggaagaacttgttgacggaagttctaatggcaactttgtgcttgagttggactttgaaccattcaatgcagccttcccccgcccaactcttaacaagtcaattggaaatggtgtgcaattcctcaaccgtcacctttctgccaaactcttccacgacaaggagagcttgcacccacttttggagttcctcaggcttcacagcgtcaagggaaagactttgatgttgaatgacagaattcaaaacccagatgcactccaacatgttctgaggaaagctgaggagtatctgggcacagtgcctcctgaaactccctactcagaatttgagcacaagttccaggagattggtttggagagagggtggggtgacaacgcggagcgtgtccttgagtcaattcaacttctcttggatcttcttgaggcccctgacccgtgcacccttgagactttccttggaagaatccctatggtgttcaatgttgttattctttctccccatggttactttgcccaagataatgtcttgggataccctgacactggtggccaggttgtttacatcttggatcaagttcgtgctttggagaatgagatgctccatcgcattaagcaacaaggattggacattgttcctcgtattctcattatcacccgtcttctccccgatgcagtaggaactacttgtggccaacgtcttgagaaggtgttcggaactgagcactcccacattcttcgagttccctttagaactgagaagggaattgttcgcaagtggatctcaagattcgaagtctggccctacttggaaacttacactgaggatgttgcccacgagcttgccaaagagttgcaaggcaagccagatctgattgttggaaactacagtgatggaaacattgtcgcttctttgttggcacataaattaggtgtcactcagtgtaccattgctcacgcacttgagaagaccaaataccccgaatccgacatttactggaaaaaattggaagagagataccacttctcttgccaattcacagctgatctatttgccatgaaccacacagatttcattatcaccagtaccttccaggagattgctggaagcaaggacactgttggacagtacgaatctcacacagccttcacccttcctggactctaccgcgttgtgcatggtattgatgtctttgatccaaaattcaacattgtctcccctggagctgatcaaaccatttacttcccccacactgaaaccagccgtaggttgacatccttccaccctgaaatcgaagaactcctttacagctcagtggagaatgaagaacacatatgtgtgctgaaggaccgcagcaagccaattatcttcaccatggcaaggttggatcgagtgaagaacatcacaggacttgtggagtggtacggtaagaacgcgaagctgagggagctggtgaaccttgtggttgttgctggagacaggaggaaggagtcaaaggacttggaagaaaaggccgagatgaagaagatgtacggcctgatcgagacctacaagttgaacggccaattcagatggatttcatcgcagatgaaccgtgtgaggaatggagagctctaccgcgtgatctgcgacaccaggggtgctttcgtgcagcctgctgtatacgaggcttttggtttgacagtggttgaggccatgacttgcggcttgccaacattcgccacatgcaatggtggtcctgctgagatcattgtgcacggcaagtctggcttccacattgacccttaccatggtgaccgtgctgctgatctccttgttgacttctttgagaagtgcaagcttgacccaactcactgggacaagatctcaaaggctggtctccagcgtattgaagagaagtacacatggcaaatttactctcagaggcttctcactctcaccggtgtctatggcttctggaagcatgtgtctaaccttgaccgccgtgagagccgccgctatctcgagatgttctatgctctcaagtaccgcaaattggctgagtctgtgccccttgctgctgagtaa SEQ ID NO.12 Clip 12 Sucrose fructanase truncated form BsacBT atgaaagaaacgaaccaaaagccatataaggaaacatacggcatttcccatattacacgccatgatatgctgcaaatccctgaacagcaaaaaaatgaaaaatatcaagtttctgaatttgattcgtccacaattaaaaatatctcttctgcaaaaggcctggacgtttgggacagctggccattacaaaacgctgacggcactgtcgcaaactatcacggctaccacatcgtctttgcattagccggagatcctaaaaatgcggatgacacatcgatttacatgttctatcaaaaagtcggcgaaacttctattgacagctggaaaaacgctggccgcgtctttaaagacagcgacaaattcgatgcaaatgattctatcctaaaagaccaaacacaagaatggtcaggttcagccacatttacatctgacggaaaaatccgtttattctacactgatttctccggtaaacattacggcaaacaaacactgacaactgcacaagttaacgtatcagcatcagacagctctttgaacatcaacggtgtagaggattataaatcaatctttgacggtgacggaaaaacgtatcaaaatgtacagcagttcatcgatgaaggcaactacagctcaggcgacaaccatacgctgagagatcctcactacgtagaagataaaggccacaaatacttagtatttgaagcaaacactggaactgaagatggctaccaaggcgaagaatctttatttaacaaagcatactatggcaaaagcacatcattcttccgtcaagaaagtcaaaaacttctgcaaagcgataaaaaacgcacggctgagttagcaaacggcgctctcggtatgattgagctaaacgatgattacacactgaaaaaagtgatgaaaccgctgattgcatctaacacagtaacagatgaaattgaacgcgcgaacgtctttaaaatgaacggcaaatggtacctgttcactgactcccgcggatcaaaaatgacgattgacggcattacgtctaacgatatttacatgcttggttatgtttctaattctttaactggcccatacaagccgctgaacaaaactggccttgtgttaaaaatggatcttgatcctaacgatgtaacctttacttactcacacttcgctgtacctcaagcgaaaggaaacaatgtcgtgattacaagctatatgacaaacagaggattctacgcagacaaacaatcaacgtttgcgccgagcttcctgctgaacatcaaaggcaagaaaacatctgttgtcaaagacagcatccttgaacaaggacaattaacagttaacaaataa SEQ ID NO. 13 Clip 13 Fructans fructosyltransferase itfA atgacgccggccatctcacgccgcgccgtgctccagggagccggcgccggagcactcgccctgatcttcggcggtgctgtgccgcctgcagcccgggcatccgctccgggctcgctccgtgccgtctaccacatgacgccccccagcggctggctctgcgacccccaacgcccggtcaccacccacggcgcctaccagctgtactacctgcactccgaccagaacaacggccccggcggctgggaccacgcgagcacgaccgacggcgtcgccttcacgcaccacggcaccgtgatgccgctgcggcccgacttccccgtgtggtccgggtcggcggtcgtcggcaccgcgaacacggcagggttcggcgccggcgcggtcgtcgcgctcgcgacccagccgaccgacggcgtccgcaagtaccaggagcagtacctctactggtcgaccgacggcgggttcacgttcaccgccctgcccgaccccgtcatcgtcaacaccgacggtcgcgccgccaccacgcccgccgagatcgagaacgccgagtggttccgcgaccccaagatccactgggacaccgcccgcggagaatgggtctgcgtcatcggacgactgcggtacgccgcgttctacacctcgccgaacctgcgcgactggacacttcgccgcaacttcgactacccgaaccacgccctcggcggcatcgagtgccccgacctgttcgagatcaccgcagacgacgggacacgccactgggtgctcgccgccagcatggacgcctacggcatcggcctccccatgacgtacgcctactggacaggcacctgggacggcgagcagttccacgccgacgacctcaccccgcaatggctcgactggggctgggactggtacgcggccgtcacctggccatcgatcgacgcgcccgagaccaagcgcctcgccatcgcgtggatgaacaactggaagtacgccgcacgcgacgtccccaccgacgcatccgacggctacaacgggcagaactcgatcgtccgcgagctgcggctcgcccgacagcctggcggctggtacaccctcctgagcacccccgtggcagcgctgacgaactacgtcaccgccaccaccacactccccgaccggaccgtcgacggcagcgccgtcctgccatggaacggacgcgcatacgagatcgagctcgacatcgcctgggacaccgcgacgaacgtcggcatctcggtgggccgctcccccgacggaacccggcacacgaacatcggcaagtacggagcagacctgtacgtcgaccgaggaccctccgacctcgccgggtactcgctcgccccctactcgcgagccgccgcccccatcgaccccggcgcccgatccgtgcacctgcgcatcctcgtcgacacccagagcgtcgaggtcttcgtcaacgccggccacaccgtgctctcccagcaggtccacttcgccgagggcgacacgggaatctcgctctacaccgacggcggccccgcacacttcaccggcatcgtcgtccgcgagattggccaggcgatctag SEQ ID NO. 14 Clip 14 3-Ketoglycoside hydrolase kfhA atggcgcaagcatgggccttcctcctccttgccctcttttccttctcctcctgtgtgtccaagctcttcatctgcggcaggaatggcgagggatccttcctctgtacacggtctcaggaagtcccctccattgccagcaagaggtacaggactgcctaccactcccagtctcccaagaactggatcaatgatccatgtggacccatgtactacaatggcatctaccatgaattctaccagtataaccccggtggtaccatcgcggataacatagtttggggccattcagtttcaaccgacctcatcaactggatccagcttgaacccgcaatagtacgggataccccatatgacataaatggttgctggaccggctcaatcacaattctacctggtgatcagcctgtcatcatatacaccggtcgtgactcgaaggatcatcagagccaaaacattgtgcttcccaaaaaccgatctgacccatacctgagggaatggaccaaagcagacaataaccccaggatcctaccggtcggaccaggcttgaactcgactgagttcagggatccgacaactggttggatcggaccggatggactatggagaatagcaattggtgctgagctgaatggttacggtgccgcactgttgtacaagagtgaagactttctgaattggactagagttgatcacccactgtattcagacaatgctccatcaatgtgggagtgcccagatttcttcgcggtattgccaggcaataacggtggactggacctgtctgcagcgatcccgaaaggcgccaaacatgtcctgaaaatgagtgtggattattctgacaagtacatgataggggtttatgatctcaaacgtgatgcgtttgttccggatgttgtcctagatgaccgtcggctgtggttgaggatggattacggcactttctatgcttcaaaatccttcttcgactcgaaaaagggcaggaggatcatatggggttggtccaacgagacagatagtgtttcagatgatggcgtgaaaggttgggcaggcatccatgcaatccccaggacaatttggttagacagcgatggcaaacagttgctgcaatggccgattgatgagattgagtcccttcgaaaagatgaaatcaaccatcaaggactagagctgaagaacggagacctatttgagattaagggaattgacactttgcaggctgatgttgaagtagattttgagctggcgtccatcgacagcgccgatccttttgatccttcctggctttttgatgtcgagaggcattgccgggaagcaggtgcatcggctaaaggtggcatagggccatttggacttgttgtcctggcctctgacaacatggaggagcacattgctgtgcacttcagagtttataaatcgcagaaaagccacatgatcctcatgtgctctgatctaagaaggtcttctctgagatcaggactgtacacaccagcctatggaggcttcttcgaatttgaccttgaaaaggaaaggaagatatctctcagaactctgattgaccggtcggtggtggagagctttggcggcggtggtagggtctgcataacagccaggatttatccagtggcacttgtcgatggcagggtccacatgtatgccttcaacaatggaagtaccacggtcagggtgccacaactcagggcatggagcatgatgacagcacaagtgaatgtgaataagggttga SEQ ID NO.15 Clip 15 pgmup gatcgaacacatcatgcagtcgatgcaattcccggcggaattgattgagaaggtttgcggaactatctaa SEQ ID NO. 16 Clip 16 pgmdown atggcaatccacaatcgtgcaggccaacctgcacaacagagtgatttgattaacgtcgcccaactgacgg SEQ ID NO. 17 Clip 17 galUup aatgagcaaacgataacgcgggctaaatttgcattacctgctaatgtcggctggtggtactatcgtcgcc SEQ ID NO. 18 Clip 18 galUdown atggctgccattaatacgaaagtcaaaaaagccgttatccccgttgcgggattaggaaccaggatgttgc SEQ ID NO. 19 Fragment 19 poxBup tcccatcccttccccctccgtcagatgaactaaacttgttaccgttatcacattcaggagatggagaacc SEQ ID NO. 20 Fragment 20 poxBdown aaagggtggcatttcccgtcataataaggacatgccatgattgatttacgcagtgataccgttacccgac SEQ ID NO. 21 Fragment 21 nadRup ttggcgttaaaaaactctccgacgcgcttagcgtgttcgacgacttataatgagatatacggagggagat SEQ ID NO. 22 Fragment 22 nadRdown ccgtgatgaaactgctcaaaggcgaggtataaaatgagtttttttgatgagttgaaaacctctctggaag SEQ ID NO. 23 Fragment 23 fadRup gctatcagcgtagttagccctctggtatgatgagtccaactttgttttgctgtgttatggaaatctcact SEQ ID NO. 24 Fragment 24 fadRdown tcccttccgtttaaagagcaaacccctcaaacgaggggttttttgttgtttttacagatttcccattctt SEQ ID NO. 25 Fragment 25 pSCre aatgtgcctgtcaaatggacgaagcagggattctgcaaaccctatgctactccgtcaagccgtcaattgtctgattcgttaccaattatgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggctggccccggtgcattttttaaatacccgcgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaatccctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagctccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctcctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcgtcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccatacccgttttttgggctaacaggaggaattaaccatgggatccaatttactgaccgtacaccaaaatttgcctgcattaccggtcgatgcaacgagtgatgaggttcgcaagaacctgatggacatgttcagggatcgccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgtgggcggcatggtgcaagttgaataaccggaaatggtttcccgcagaacctgaagatgttcgcgattatcttctatatcttcaggcgcgcggtctggcagtaaaaactatccagcaacatttgggccagctaaacatgcttcatcgtcggtccgggctgccacgaccaagtgacagcaatgctgtttcactggttatgcggcggatccgaaaagaaaacgttgatgccggtgaacgtgcaaaacaggctctagcgttcgaacgcactgatttcgaccaggttcgttcactcatggaaaatagcgatcgctgccaggatatacgtaatctggcatttctggggattgcttataacaccctgttacgtatagccgaaattgccaggatcagggttaaagatatctcacgtactgacggtgggagaatgttaatccatattggcagaacgaaaacgctggttagcaccgcaggtgtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccgtctctggtgtagctgatgatccgaataactacctgttttgccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgcgccctggaagggatttttgaagcaactcatcgattgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggacacagtgcccgtgtcggagccgcgcgagatatggcccgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgtaaatattgtcatgaactatatccgtaccctggatagtgaaacaggggcaatggtgcgcctgctggaagatggcgattagctcgagggtagatctggtactagtggtgaattcggtgagctcggtctgcagctggtgccgcgcggcagccaccaccaccaccaccactaatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtcgaccagacccgccataaaacgccctgagaagcccgtgacgggcttttcttgtattatgggtagtttccttgcatgaatccataaaaggcgcctgtagtgccatttacccccattcactgccagagccgtgagcgcagcgaactgaatgtcacgaaaaagacagcgactcaggtgcctgatggtcggagacaaaaggaatattcagcgatttgcccgagcttgcgagggtgctacttaagcctttagggttttaaggtctgttttgtagaggagcaaacagcgtttgcgacatccttttgtaatactgcggaactgactaaagtagtgagttatacacagggctgggatctattctttttatctttttttattctttctttattctataaattataaccacttgaatataaacaaaaaaaacacacaaaggtctagcggaatttacagagggtctagcagaatttacaagttttccagcaaaggtctagcagaatttacagatacccacaactcaaaggaaaaggactagtaattatcattgactagcccatctcaattggtatagtgattaaaatcacctagaccaattgagatgtatgtctgaattagttgttttcaaagcaaatgaactagcgattagtcgctatgacttaacggagcatgaaaccaagctaattttatgctgtgtggcactactcaaccccacgattgaaaaccctacaaggaaagaacggacggtatcgttcacttataaccaatacgttcagatgatgaacatcagtagggaaaatgcttatggtgtattagctaaagcaaccagagagctgatgacgagaactgtggaaatcaggaatcctttggttaaaggctttgagattttccagtggacaaactatgccaagttctcaagcgaaaaattagaattagtttttagtgaagagatattgccttatcttttccagttaaaaaaattcataaaatataatctggaacatgttaagtcttttgaaaacaaatactctatgaggatttatgagtggttattaaaagaactaacacaaaagaaaactcacaaggcaaatatagagattagccttgatgaatttaagttcatgttaatgcttgaaaataactaccatgagtttaaaaggcttaaccaatgggttttgaaaccaataagtaaagatttaaacacttacagcaatatgaaattggtggttgataagcgaggccgcccgactgatacgttgattttccaagttgaactagatagacaaatggatctcgtaaccgaacttgagaacaaccagataaaaatgaatggtgacaaaataccaacaaccattacatcagattcctacctacgtaacggactaagaaaaacactacacgatgctttaactgcaaaaattcagctcaccagttttgaggcaaaatttttgagtgacatgcaaagtaagcatgatctcaatggttcgttctcatggctcacgcaaaaacaacgaaccacactagagaacatactggctaaatacggaaggatctgaggttcttatggctcttgtatctatcagtgaagcatcaagactaacaaacaaaagtagaacaactgttcaccgttagatatcaaagggaaaactgtccatatgcacagatgaaaacggtgtaaaaaagatagatacatcagagcttttacgagtttttggtgcatttaaagctgttcaccatgaacagatcgacaatgtaacagatgaacagcatgtaacacctaatagaacaggtgaaaccagtaaaacaaagcaactagaacatgaaattgaacacctgagacaacttgttacagctcaacagtcacacatagacagcctgaaacaggcgatgctgcttatcgaatcaaagctgccgacaacacgggagccagtgacgcctcccgtggggaaaaaatcatggcaattctggaagaaatagcgctttcagccggcaaacctgaagccggatctgcgattctgataacaaactagcaacaccagaacagcccgtttgcgggcagcaaaacccgcggccgcctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataatgtctaacaattcgttcaagccgaggggccgcaagatccggccacgatgacccggtcgtcggttcagggcagggtcgttaaatagccgcttatgtctattgctggtttaccggtttattgactaccggaagcagtgtgaccgtgtgcttctcaaatgcctgaggtttcaggcatgc SEQ ID NO. 26 Fragment 26 linker GGGGGTGGAGGCTCTGGGGGTGGAGGCTCTGGGGGTGGAGGCTCT

[0041] Table 2 Primers of Example 1

[0042] Primer Sequence (5'-3') pgm-1 gatcgaacacatcatgcagtcgatgcaatt pgm-2 ccgtcagttgggcgacgttaatcaaatcac pgm-3 caacaccaacaccggccgtaaatgcagcat galU-1 aatgagcaaacgataacgcgggctaaattt galU-2 gcaacatcctggttcctaatcccgcaacgg galU-3 gtggtcgactgcgcttgatgttgtctgcag poxB-1 tcccatcccttccccctccgtcagatgaac poxB-2 gtcgggtaacggtatcactgcgtaaatcaa poxB-3 ggctatttaaccgttagtgcctcctttctc nadR-1 ttggcgttaaaaaactctccgacgcgctta nadR-2 cttccagagaggttttcaactcatcaaaaa nadR-3 cgaaaaaagcgccggaagggatgcagattt fadR-1 gctatcagcgtagttagccctctggtatga fadR-2 aagaatgggaaatctgtaaaaacaacaaaa fadR-3 agccttgatccctttttcttctttttgtct Kan-R tcgtcaagaaggcgatagaa

[0043] Example 1. Construction of recombinant Escherichia coli AS00.

[0044] The preparation method of the relevant strains is obtained according to the following steps (1)-(2):

[0045] (1) Starting from Escherichia coli MG1655, the promoter of the glucose phosphoglucomutase gene pgm in the strain was replaced with the P119 promoter to obtain the AS00-1 strain.

[0046] The specific steps are as follows:

[0047] (1-a) Preparation of targeting fragment AS-C1

[0048] The following DNA fragments were synthesized (GenScript) and consisted of the following sequences from 5' to 3': pgmup (fragment 15), lox66-Kan-lox71 (fragment 1), P119 promoter (fragment 2), and pgmdown (fragment 16). PCR amplification was performed using primers pgm-1 / pgm-2 and the synthesized DNA fragments as templates to generate the targeting fragment AS-C1.

[0049] (1-b) Preparation of host bacteria containing the pKD46 plasmid

[0050] The pKD46 plasmid (from the Yale Genetic Collection of Escherichia coli (CGSC)) 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, MG1655 / pKD46 expressed the three recombinant proteins of phage λ, conferring homologous recombination capability. Competent MG1655 / pKD46 cells were then prepared by washing with 10% glycerol.

[0051] (1-c) Homologous recombination

[0052] The targeting fragment AS-C1 prepared in (1-a) was electroporated into the MG1655 / pKD46 competent cells prepared in (1-b) and incubated overnight at 37°C on LB plates containing kanamycin (50 µg / ml). A clone was selected and genomic DNA extracted. PCR amplification was performed using primers pgm-3 / Kan-R. A positive clone was identified by amplifying a target band of approximately 1000 bp. Sequencing analysis revealed that the AS-C1 fragment was present in the genome of AS001-kan. AS001-kan was cultured overnight at 42°C to eliminate the temperature-sensitive plasmid pKD46.

[0053] (1-d) Elimination of resistance

[0054] The pSCre plasmid (from the Yale Genetic Collection of Escherichia coli (CGSC)) was transformed into the AS001-kan strain, which had been cured of pKD46, using the calcium chloride method. The strain was cultured overnight at 30°C on LB plates containing 50 mg / L streptomycin and 0.2% L-arabinose. The kanamycin resistance fragment was eliminated using the Cre recombinase on the pSCre plasmid. The temperature-sensitive pSCre plasmid was eliminated by culturing overnight at 42°C. The resulting strain was named AS00-1.

[0055] (2) Acquisition of strains AS00-2, AS00-3, AS0-04, and AS00.

[0056] Using the same method as in Example 1 (1), starting from AS001, the promoter of the UTP-glucose-1-phosphate uridylyltransferase gene galU in the strain was replaced with the P119 promoter to obtain the AS002 strain;

[0057] Starting from AS002, the gene poxB encoding pyruvate carboxylase was knocked out and replaced with the expression cassettes of the glucose isomerase gene BgiA and the sucrose synthase gene Gmss to obtain the AS003 strain;

[0058] Starting from AS003, the gene nadR encoding NMN adenylyltransferase was knocked out and replaced with the expression cassettes of the sucrose fructanase truncated gene BsacBT and the fructan fructosyltransferase gene itfA to obtain the AS004 strain;

[0059] Starting from AS004, the gene fadR encoding the fatty acid degradation regulatory factor in the strain was knocked out and replaced with a fusion protein gene of hexose-3-dehydrogenase MhhDF and 3-ketose glycoside hydrolase kfhA and an expression cassette of the hexose 3-reductase gene AhrF to obtain the AS00. strain.

[0060] The difference between the construction process of each strain and Example 1 (1) is that different starting strains, different target fragments, and different primers were used. The specific experimental materials used and the obtained strain information are shown in Table 3.

[0061] Table 3. Construction process of AS00-1, AS00-2, AS00-3, AS00-4, and AS00.

[0062] Serial number Starting strain Target practice clip name Targeting fragment sequence Amplification primers Identification primers Obtain strains 1 MG1655 AS-C1 pgmup (fragment 15), lox66-kan-lox71 (fragment 1), p119 promoter (fragment 2), pgmdown (fragment 16) PGM-1 / PGM-2 pgm-3 / KanR AS00-1 2 AS00-1 AS-C2 galPup (fragment 17) Lox66-Kan-lox71 (fragment 1) P119 promoter (fragment 2) galPdown (fragment 18) galP-1 / galP-2 galP-3 / KanR AS00-2 3 AS00-2 AS-C3 poxBAup (fragment 19), Lox66-Kan-lox71 (fragment 1), P119 promoter (fragment 2), BgiA (fragment 10), RBS (fragment 3), Gmss (fragment 11), TrrnB terminator (fragment 4), poxBdown (fragment 20) poxB-1 / poxB-2 poxB-3 / KanR AS00-3 5 AS00-3 AS-C4 nadRup (fragment 21) Lox66-Kan-lox71 (fragment 1) P119 promoter (fragment 2) BsacBT (fragment 12) RBS (fragment 3) itfA (fragment 13) TrrnB terminator (fragment 4) nadRdown (fragment 22) nadR-1 / nadR-2 nadR-3 / KanR AS00-4 AS00-4 AS-C5 fadRup (fragment 23), Lox66-Kan-lox71 (fragment 1), P119 promoter (fragment 2), MhhDF (without TAA, fragment 5), linker (fragment 26), kfhA (fragment 14), RBS (fragment 3), AhrF (fragment 7), TrrnB terminator (fragment 4), fadRdown (fragment 22) fadR-1 / fadR-2 fadR-3 / KanR AS00.

[0063] Example 2: Construction of recombinant Escherichia coli AS10. and AS20.

[0064] The construction process of strains AS10. and AS20. was identical to that of strain AS00. in Example 1. The differences were that the starting strain E. coli MG1655 used in Example 1 was replaced with E. coli AS10 (CGMCC No. 27687) to obtain AS10., and the starting strain E. coli MG1655 used in Example 1 was replaced with E. coli AS10.01 (CGMCC No. 34377) to obtain AS20.

[0065] The recombinant bacteria AS10. was deposited on April 27, 2025 at the General Microbiology Center of China Culture Collection Administration (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 34376, and the classification name is Escherichia coli.

[0066] Example 3: Production of D-psicose using Escherichia coli AS00., AS10., and AS20.

[0067] (1) The test bacteria were Escherichia coli AS00., AS10., AS20. and related control strains.

[0068] (2) Cultivation of bacteria.

[0069] Inoculate 1% of the overnight culture into a shake flask containing 200 mL of LB medium. Cultivate at 32-37°C for 15-17 hours, measure the OD600, and collect the cells by centrifugation at 8000 rpm for 10 minutes. After OD values are normalized, resuspend the cells in 50 mL of inorganic salt medium (25 mM Na2HPO4, 25 mM KH2PO4, 5 mM MgCl2, pH 7.0) containing 20% D-glucose and incubate further at 37°C. Samples are collected at 10, 15, and 20 hours, and the supernatant is centrifuged and analyzed for D-psicose content. Three replicates are performed for each reaction time.

[0070] (3) Determination of D-psicose.

[0071] The content of D-psicose was determined by HPLC using an external standard method. The HPLC analysis utilized a Hi-Plex Ca column (300 mm × 7.7 mm, 8 μm); the mobile phase consisted of ultrapure water at a flow rate of 0.5 mL / min; the column temperature was 78°C; and the detector was a differential refractive index detector (DID) at 40°C. During the HPLC analysis, D-psicose (Shanghai Yuanye Biological Products (Cat. No. S48276)) was used as a standard for qualitative analysis based on the retention time of the standard, and the calibration curve method (external standard method) was used for quantitative analysis. Figure 1 The calibration curve prepared for the D-psicose standard and the equation for the derived D-psicose concentration are: Y = 1254.11X - 1629.77, r² = 0.9999, r = 0.9999. Here, Y is the characteristic peak area, and X is the D-psicose concentration of the sample (g / L). Yield is defined as the D-psicose concentration produced in the reaction solution; conversion is defined as the ratio of the estimated mass of D-psicose produced in the reaction solution to the mass of D-glucose consumed as raw material.

[0072] Figure 2 The following is an HPLC peak profile of D-psicose standards at different concentrations (the number in the upper left corner indicates the concentration of the standard (g / L)). D-glucose content was determined by HPLC using the external standard method. The HPLC method and conditions are the same as those for D-psicose detection described above, except that D-glucose (Beijing Mairida Product (Cat. No.: M043597)) was used as the standard for qualitative analysis based on the retention time of the standard, and the standard curve method (external standard method) was used for quantitative analysis of D-glucose.

[0073] Table 4 D-psicose production of each strain

[0074]

Claims

1. A gene cluster for synthesizing D-psicose, comprising or consisting of: Hexose-3-dehydrogenase gene or its isoenzyme gene or its functional homolog gene; hexose-3-reductase gene or its isoenzyme gene; genes involved in the synthesis of hexose-3-dehydrogenase precursor; 3-ketoglycoside hydrolase gene or its isoenzyme gene or its functional homolog gene.

2. The gene cluster according to claim 1, wherein The hexose-3-dehydrogenase gene is at least one gene selected from the following: genes encoding enzymes having EC 1.1.98.3, EC 1.1.99.13, EC 1.1.99.18, EC 1.1.1.45, EC 1.1.1.361, EC 1.1.1.374, and EC 1.1.99.29 activity.

3. The gene cluster according to claim 1 or 2, wherein The substrate of the product encoded by the hexose-3-dehydrogenase gene includes at least one of sucrose, cellobiose, lactose, lactulose, difructose anhydride I, difructose anhydride III, difructose anhydride IV, and difructose anhydride V.

4. The gene cluster according to any one of claims 1 to 3, wherein The hexose-3-reductase gene is at least one gene selected from the following: genes encoding enzymes having EC 1.1.1.333, EC 1.1.1.425, EC 1.1.1.226, EC 1.1.1.123, EC 1.1.1.21, or EC 1.1.1.124 activity.

5. A gene cluster according to any preceding claim, wherein: The hexose-3-dehydrogenase gene or its isoenzyme gene or its functional homolog gene consists of the sequence shown in SEQ ID NO.

5.

6. A gene cluster according to any preceding claim, wherein: The hexose-3-reductase gene or its isoenzyme gene or its functional homolog gene consists of the sequence shown in SEQ ID NO.

7.

7. A gene cluster according to any preceding claim, wherein: The gene involved in the synthesis of the hexose-3-dehydrogenase precursor, its isozyme gene, or its functional homolog gene comprises or consists of a sequence represented by at least one of SEQ ID NOs. 8, 9, 10, 11, 12, and 13.

8. A gene cluster according to any preceding claim, wherein: The 3-ketoglycoside hydrolase gene or its isoenzyme gene or its functional homolog gene consists of the sequence shown in SEQ ID NO.

14.

9. Use of the gene cluster according to any one of the preceding claims in producing D-psicose.

10. The use according to claim 9, wherein the engineered cells produce D-psicose by fermentation, and preferably, the engineered cells are genetically recombinant Escherichia coli.

11. A genetically recombinant engineered cell comprising the following modifications: (1) Introducing and / or enhancing the expression of a hexose-3-dehydrogenase gene or its isoenzyme gene or its functional homolog gene, and / or enhancing the activity of a protein, domain or functional fragment encoded by a hexose-3-dehydrogenase gene or its isoenzyme gene or its functional homolog gene; (2) introducing and / or enhancing the expression of a hexose-3-reductase gene or its isoenzyme gene or its functional homolog gene, and / or enhancing the activity of a protein, domain or functional fragment encoded by a hexose-3-reductase gene or its isoenzyme gene or its functional homolog gene; (3) introducing and / or enhancing the expression of genes involved in the synthesis of hexose-3-dehydrogenase precursors or their isoenzyme genes or their functional homolog genes, and / or enhancing the activity of proteins, domains or their functional fragments encoded by genes involved in the synthesis of hexose-3-dehydrogenase precursors or their isoenzyme genes or their functional homolog genes; (4) Introducing and / or enhancing the expression of a 3-ketoglycoside hydrolase gene or its isoenzyme gene or its functional homolog gene, and / or enhancing the activity of a protein, domain or functional fragment encoded by a 3-ketoglycoside hydrolase gene or its isoenzyme gene or its functional homolog gene. The engineered cell according to claim 11 , which is Escherichia coli.

13. The engineered cell according to claim 11 or 12, wherein The hexose-3-dehydrogenase gene comprises or consists of the sequence shown in SEQ ID NO.

5.

14. The engineered cell according to any one of claims 11 to 13, wherein The hexose-3-reductase gene comprises or consists of the sequence shown in SEQ ID NO.

7.

15. The engineered cell according to any one of claims 11 to 14, wherein The gene involved in the synthesis of the hexose-3-dehydrogenase precursor comprises or consists of a sequence represented by at least one of SEQ ID NOs. 8, 9, 10, 11, 12 and 13.

16. The engineered cell according to any one of claims 11 to 15, wherein The 3-ketoglycoside hydrolase gene comprises or consists of the sequence shown in SEQ ID NO.

14.

17. The engineered cell according to any one of claims 11 to 16, characterized in that After the bacterial cells are collected through fermentation, D-psicose can be prepared by whole-cell catalysis using glucose as raw material.

18. An engineered Escherichia coli strain, characterized in that The deposit number is CGMCC No. 34376, and the whole-cell catalytic production of D-psicose can be achieved using glucose as a raw material.

19. An engineered Escherichia coli strain, characterized in that The deposit number is CGMCC No. 34377, and the whole-cell catalytic production of D-psicose can be achieved using glucose as a raw material.

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