Random integration expression of D-psicose 3-epimerase by bacillus subtilis

Abstract

The invention relates to molecular biology and industrial enzymes, and provides a system for randomly integrating and expressing D-psicose 3-epimerase (DAEase) in a bacillus subtilis chromosome and application of the system. The preparation method comprises the following steps: integrating an expression cassette containing PgsiB / PnprE / PHpaII / PmyE, 7-20bp RBS and SEQ ID NO: 2 by adopting mariner / Himar1 or Tn7; a temperature-sensitive replicon is removed at 42-51 DEG C, and a resistance marker can be deleted. A KpRD / NADH 340nm continuous spectrum is used for screening, and HPLC (High Performance Liquid Chromatography) rechecking is performed. The crude enzyme activity is greater than or equal to 3000U / mL within 68-72 hours by supplementing 3L of materials. Under the conditions that the temperature is 55-70 DEG C, 0.1 mM Co < 2 + > and the substrate is 300 g / L, the D-psicose is obtained by catalyzing D-fructose through whole cells / crude enzyme, the conversion rate is larger than or equal to 25%, stability and compliance are both considered, and the method is suitable for industrialization.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and industrial biotechnology, and relates to a method, expression cassette, and application of D-allulose 3-epimerase for random integration and expression in the chromosome of Bacillus subtilis, a food-grade host. Background Technology

[0002] D-Allulose (also known as D-Psicose) is a rare sugar with a sweetness approximately 70% that of sucrose. It is low in calories, has a good taste, and exhibits some process tolerance, leading to demand for its use in healthy sweeteners and related ingredient systems. Engineered preparation primarily utilizes enzymatic methods. A common approach involves heterologously expressing D-allulose 3-epimerase (DAEase) in microorganisms to epimerize D-fructose into D-allulose. The enzyme preparation can be purified before use or directly catalyzed using a whole-cell model. This pathway is widely adopted in public literature and industry practice. To ensure integrated evaluation across sections, this field typically focuses on effective enzyme activity (U / mL), conversion rate at representative substrate loads and temperatures, genetic and process stability during scale-up, and compliance indicators related to food applications.

[0003] In terms of detection and screening, the KpRD / NADH continuous spectroscopy method monitors absorbance changes in real time at 340 nm, characterizing the reaction rate with ΔA340 / min. This method can be used for plate-based high-throughput screening and can be integrated with subsequent HPLC quantification. In terms of process, the transformations are mostly performed at 55-70℃ and contain trace amounts of metal ions (such as Co). 2+ The assay was conducted under conditions of approximately 0.1 mM, and evaluated in conjunction with a higher substrate loading. The aforementioned detection aperture (KpRD / NADH 340 nm continuous spectrum, characterized by ΔA340 / min and supplemented by HPLC quantification) and representative operating conditions (55-70℃, supplemented with trace amounts of divalent metal ions, e.g., Co) were compared. 2+ Approximately 0.1 mM, combined with a relatively high substrate load, is a commonly used evaluation condition in the field, facilitating horizontal comparison with publicly available technologies.

[0004] In terms of expression strategies, existing approaches mainly include plasmid expression and chromosome integration expression. The former is flexible in construction and quick to start, but the copy number and metabolic burden are significantly affected by culture and passage conditions, and antibiotic resistance markers are not conducive to compliance management for food applications. The latter improves genetic and amplification stability and reduces dependence on resistance markers and replicons by integrating the target sequence into the host chromosome. However, under homologous recombination or fixed-site integration paradigms, it is often affected by the "position effect," and the expression intensity and predictability of single copies are limited, thus limiting the screening efficiency for obtaining highly expressed integrated strains.

[0005] To balance stability and expression intensity, research and industry have introduced random integration strategies, such as transposon systems, to obtain expression cassette insertions at different locations on the host chromosome. This is combined with rapid screening using 340nm continuous spectroscopy, forming a "construction-screening-scaleup" technical path. Simultaneously, the integration vector should possess removable replicon elements, and the integrated strain must meet the requirements of being free of resistance markers and possessing genetic stability to adapt to food applications. Despite some engineering experience, several challenges remain: the balance between "position effect" and "metabolic burden" in achieving high-level and stable expression in food-grade hosts; the transferability of evaluation criteria between plate screening and fermenter scale-up; and the systemic impact of metal ion management, mass transfer and heat management, and separation and purification on continuous or intermittent production costs. Further optimization is still needed. Summary of the Invention

[0006] Technical issues This case addresses the technical field of engineering systems for obtaining D-allulose 3 epimerase (DAEase) in food-grade hosts for D-allulose preparation. Existing methods suffer from instability due to copy number and metabolic burden in plasmid-replicon-dependent expression, and antibiotic resistance markers hinder compliance management for food applications. Chromosomal integration expression is affected by the "position effect" of the insertion site, resulting in insufficient single-copy expression intensity and predictability, limiting the efficiency of obtaining highly active integrated strains. Furthermore, the lack of a unified and transferable evaluation caliber between plate screening and fermenter scale-up leads to the risk of inconsistencies between small-scale ranking and scale-up performance. In continuous or intermittent production scenarios, it is also necessary to balance genetic stability, process stability, and compliance for food applications. Based on these objective circumstances, it is necessary to achieve stable and scalable high-level expression in food-grade hosts without relying on plasmid replicons and resistance markers, and to establish a consistent evaluation link from screening to scale-up to improve the predictability of candidate strain scale-up and the feasibility of industrial implementation. Technical solution To address the aforementioned technical problems, a plasmid-free, randomly integrated D-allulose 3-epimerase (DAEase) expression method and cassette are provided. Combined with continuous spectrum high-throughput screening and fed-batch scale-up processes, this method yields highly active, genetically stable, and resistance-marker-free recombinant Bacillus subtilis for D-allulose preparation. This technical solution includes at least the following key points: First, the DAEase expression cassette is randomly integrated into the host chromosome using a Mariner / Himar1 or Tn7 transposon system; the integration vector is equipped with a temperature-sensitive replicon so that the replicon can be removed at 42-51℃, thereby obtaining an integrated strain without plasmid replicon.

[0007] Secondly, the expression cassette includes a promoter (selected from PgsiB, PnprE, PHpaII, PamyE, allowing single or dual promoter combinations), an RBS (7-20 bp), a nucleotide sequence encoding DAEase (SEQ ID NO:2 or its ≥90% identity variant), and a transcription terminator (preferably a dual terminator) for achieving stable expression within the host.

[0008] Third, for positive clones, high-throughput screening was carried out in microplates using KpRD / NADH spectroscopy with continuous monitoring at 340 nm. ΔA340 / min was used as the ranking index, combined with HPLC verification, to form an integrated evaluation method of screening and quantification.

[0009] Fourth, site-specific recombination can be selectively performed on the integrated strain to delete the selection marker, thereby obtaining engineered bacteria that do not contain the resistance marker.

[0010] Fifth, in the scale-up stage, 3L fed-batch fermentation was used to achieve stable expression output, with representative batches of fermentation broth showing crude enzyme activity ≥3000U / mL (preferably ≥3900U / mL); in transformation applications, fermentation was carried out at 55-70℃ (preferably 55-65℃) using Co... 2+ D-allulose was prepared by whole-cell or crude enzyme catalysis under conditions of 0.1 mM and 300 g / L D-fructose, with a conversion rate of ≥25% (preferably ≥29%). The above elements can be used in combination and are not limited to a specific order or a single method, to cover reasonable variations required for industrial implementation.

[0011] Beneficial effects Under the synergistic effect of the aforementioned technical features, the relevant effects remain stable in engineering applications and comparative working conditions. The following effects are quantitative representations of the embodiments and comparative records, used for illustration and not for limiting the claims: 1. Consistency between stable high expression and scale-up: Without integration containing plasmid replicons, 3L fed-batch fermentation can repeatedly achieve crude enzyme activity ≥3000 U / mL (preferred batch ≥3900 U / mL), maintaining stable output within a 68-72 h time window, meeting the scale-up threshold requirements from shake flask to fermenter. The above-mentioned specifications and units (U / mL, h) use consistent measurement standards in the examples and figures to facilitate data integration with detection and screening.

[0012] 2. Transferability of screening indicators to in-tank indicators: Positive clones were ranked using KpRD / NADH-340nm continuous spectroscopy (with ΔA340 / min as the indicator) and verified by HPLC, forming a unified evaluation chain of "screening-quantification-scale-up". This chain supports subsequent activity and transformation evaluation under in-tank conditions, reducing the uncertainty of candidate strain scale-up and the cost of repeated screening.

[0013] 3. Representative conversion performance: at 55-70℃ (preferably 55-65℃), Co 2+ Under representative conversion conditions of 0.1 mM and approximately 300 g / L of D-fructose, a D-allulose conversion rate of ≥25% (preferably ≥29%) can be achieved using whole-cell or crude enzyme catalysis. Furthermore, a control can be established with the activity level (U / mL) of the fermentation stage, facilitating process window optimization.

[0014] 4. Genetic stability and food application compliance: By removing the temperature-sensitive replicon and (optionally) deleting the resistance marker, the resulting integrative strain does not contain plasmid replicons or antibiotic resistance markers, meeting the compliance expectations for food-grade applications; it maintains activity and phenotypic stability in continuous passage scenarios, facilitating subsequent continuous production.

[0015] 5. Engineering usability in comparative scenarios: Using representative data from plasmid expression, targeted integration, and random integration as a reference, the random integration strain can still achieve transformation performance comparable to or higher than that of the plasmid system in a single-copy background, and significantly improve intergenerational stability (e.g., comparative example: plasmid system crude enzyme activity approximately 376.9 U / mL, transformation rate approximately 28.3%, intergenerational stability ≥70 generations; random integration crude enzyme activity approximately 294.8 U / mL, transformation rate approximately 29.2%, intergenerational stability >200 generations), demonstrating the robustness and reproducibility of engineering implementation. Attached Figure Description

[0016] To facilitate understanding of the embodiments described in the specification, the accompanying drawings are given in schematic form and are not necessarily drawn to scale, and should not be construed as limiting the claims. The elements and symbols shown in the figures are for illustrative purposes only.

[0017] Figure 1 This is a schematic diagram of sequencing and identification of random integration sites.

[0018] Figure 2 a is a schematic diagram of the random integration structure of expression cassettes on the Bacillus subtilis chromosome.

[0019] Figure 2 b is a representative curve of the screening results by KpRD / NADH continuous spectroscopy (340nm).

[0020] Figure 3 OD during 3L fed-batch fermentation 600 A graph showing the change in crude enzyme activity (U / mL) over time.

[0021] Figure 4 Time-conversion curve for whole-cell catalytic preparation of D-allulose.

[0022] Figure 5A comparison of crude enzyme activity (U / mL) for plasmid / random integration strategies (n=3). Detailed Implementation

[0023] Terms and Definitions Unless otherwise stated, the following terms have the following meanings in this specification: D-Allulose 3-epimerase (DAEase): D-allulose 3-epimerase.

[0024] RBS (ribosome binding site): A nucleotide sequence located 7-20 bp upstream of the start codon, used to promote translation initiation.

[0025] ΔA340 / min: The slope of the absorbance change over time at a wavelength of 340 nm, calculated after subtracting the blank wells, and used as a rate index for continuous spectrum screening.

[0026] Crude enzyme activity (U / mL): The amount of enzyme that generates 1 μmol of D allulose per minute under specified substrate, buffer system and temperature is recorded as 1U. Sample activity is expressed as U / mL.

[0027] OD 600 Cell density at a wavelength of 600nm.

[0028] Random integration: refers to the integration of expression cassettes into non-predetermined sites on the host chromosome via a transposon system.

[0029] Cross-boundary PCR: A PCR identification method for amplifying expression cassettes at the boundary with the host genome.

[0030] No plasmid replicon / no antibiotic resistance marker: refers to an integrated strain that does not contain plasmid origin of replication and resistance gene.

[0031] "Preferred": indicates a more advantageous scope or implementation method compared to "Example or General Conditions", and does not constitute a limitation on the scope of protection of the claims.

[0032] Statistical definition: Unless otherwise stated, experiments are repeated n≥3, and results are expressed as mean or mean ± standard deviation.

[0033] Monitoring and Measurement Methods Continuous spectral screening (KpRD / NADH): Microplates (total system 200 μL) were used, and absorbance was recorded in real time at 340 nm; blank wells were enzyme-free controls, and positive wells were known high-activity samples; sorting was based on ΔA340 / min, and the threshold was determined by the mean ±2SD of positive and negative controls; nonlinear segments were not included in the slope calculation.

[0034] HPLC quantification: See Table 2 for details (column, mobile phase / flow rate, column temperature / detection mode); sample was diluted and filtered through a 0.22 μm filter before injection; D-allulose and D-fructose concentrations were calculated using the external standard method.

[0035] Enzyme activity definition: Reaction system (1 mL): D-fructose 80 g / L, HEPES 20 mM (pH 8.0), Co 2+ 0.1mM; 55℃ for 10min reaction, followed by boiling water for 10min to terminate; 1U definition and calculation caliber are given in Example 6.

[0036] Fermentation process monitoring: Recording OD 600 Parameters such as crude enzyme activity, feeding rate, dissolved oxygen, and temperature; fermentation curves are shown below. Figure 3 .

[0037] Conversion rate calculation: Conversion rate = C(allulose) / [C(allulose) + C(fructose)] × 100%, representative curves are shown below. Figure 4 .

[0038] Statistical processing: Each experiment was repeated n≥3 times; if comparisons were involved, a two-tailed t-test or an equivalent non-parametric test was used, and significance was considered as p<0.05, which does not limit the claims.

[0039] The term "identity" as used in this specification is calculated using BLASTN (v2.13.0 or equivalent, default parameters; word_size=11, match / mismatch=2 / −3, gap_open=5, gap_extend=2) or the Needleman-Wunsch global alignment algorithm (EMBOSS needle, default parameters); when the results of the two methods are inconsistent, the BLASTN result shall prevail.

[0040] Example 1: Design of Expression Box and Construction of Transpose Carrier The expression cassette includes: a promoter (selected from PgsiB, PnprE, PHpaII, and PamyE, which can be a single or dual promoter combination), a ribosome binding site (RBS, 7-20 bp, designed according to translation initiation ΔG hierarchy), a nucleotide sequence encoding DAEase (SEQ ID NO:2 or its ≥90% identity variant, preferably codon-optimized for Bacillus subtilis), and a transcription terminator (preferably a dual terminator to reduce read penetration). A transposon vector containing the above expression cassette and a subsequently removable selection marker is constructed. The vector backbone includes a thermosensitive replicon and a mariner / Himar1 or Tn7 transposon element. The expression cassette is assembled using Gibson or Golden Gate.

[0041] The recombinant fragment and the linkage site were verified to be free of mutations by Sanger sequencing; the resulting transposon vector simultaneously met the process requirements for subsequent random integration and dereplication, and served as the input material for Example 2.

[0042] Example 2: Random Transposition Integration and Carrier Removal The transposon vector obtained in Example 1 was introduced into Bacillus subtilis and cultured at 30-37℃ for 3-24 h to induce random transposition; positive clones were obtained by selection using antibiotic resistance plates. Subsequently, the cells were cultured at 42-51℃ to remove temperature-sensitive replicons, and integrated strains without plasmid replicons were isolated and rescreened. Integration events were confirmed using cross-boundary PCR and Sanger sequencing to confirm the insertion site; the integrated structure of the expression cassette on the genome is described in [reference needed]. Figure 2 a, Typical sequencing results can be found in Figure 1 .

[0043] All positive clones obtained from screening showed specific amplification bands crossing the expression cassette-genome junction; the sequencing reads were consistent with the expected adapter / flanking sequences, confirming successful integration. After removing thermosensitive replicons at 42-51℃, no plasmid replicon-related fragments were detected in the rescreened samples, which were determined to be integrated strains without plasmid replicons. The corresponding sequencing chromatograms for the above confirmation are shown below. Figure 1 See integrated structure Figure 2 a.

[0044] Example 3: PCR identification of integration site Site-specific recombination systems were used to delete selection markers; left and right boundary-crossing primers were designed to cross the left / right wing-expression cassette-genome junction for PCR identification, and the junction sequence was confirmed by Sanger sequencing (see [link to article]). Figure 1 , Figure 2 a).

[0045] PCR system (25 μL): 2.5 μL 10× buffer, Mg 2 ⁺ 1.5mM, dNTP 200μM, each primer 0.4μM, high-fidelity DNA polymerase 1U, template genome 50-100ng, ddH2O to bring the total to 25μL.

[0046] Cyclic program: 95℃ for 3 min; 30-35 cycles (95℃ for 10 s, 60℃ for 20 s [optimization range 58-62℃], 72℃ for 30 s / kb); 72℃ for 5 min.

[0047] Table 1. Cross-boundary primers and their uses (examples) Note: LF / LR indicates left-side out-of-bounds, RF / RR indicates right-side out-of-bounds; annealing temperature is set according to Tm−3~5℃ for each primer; if "N" is included, it indicates a linker / random primer and is marked in the description.

[0048] Table 1 lists the cross-boundary PCR primers and their uses (examples), including name, sequence (5′→3′), and purpose; LF / LR indicates left-side cross-boundary, and RF / RR indicates right-side cross-boundary; annealing temperature is set according to Tm−3~5℃ for each primer. If a primer contains "N", it indicates an adapter / random site, which is noted in the table footnote. After amplification and sequencing, the obtained reads are consistent with the inserted flanking sequences, confirming the integration structure with... Figure 1 , Figure 2 a matches.

[0049] Example 4 High-throughput activity screening KpRD / NADH continuous spectroscopy was used to monitor absorbance changes in real time at 340 nm, and DAEase activity was evaluated using ΔA340 / min as the rate index. The total volume of the plate reaction system was 200 μL, and the buffer was 20 mM HEPES (pH 8.0) containing Co. 2+ 0.1 mM of D-fructose substrate (in the same proportions as the assay system in Example 6) was used; blank and positive controls were set up, and the slope of the linear segment was calculated after subtracting the blank control. The screening threshold was set at the mean ± 2 SD of the positive and negative controls, and the experiment was repeated n≥3 times.

[0050] Candidate clones were obtained by sorting according to ΔA340 / min and verified by HPLC; the selected strains were used for subsequent shake flask and fermenter scale-up evaluation, achieving [the desired results]. Figure 3 / Figure 4 Consistency of data scope. Representative continuous spectral curves or sorting results can be found in [reference needed]. Figure 2 b.

[0051] Example 5: Shake flask and 3L fed-batch fermentation Seeds were prepared using a conventional two-stage propagation method; a 0.9L initial culture solution was added to a 3L fermenter, with an initial pH of 7.0, dissolved oxygen set at 100%, and the temperature maintained at 37℃; the fed-batch culture medium consisted of 150g / L glycerol and 300g / L peptone, added in stages according to dissolved oxygen or substrate consumption. Process monitoring included OD (oxidative stress). 600 Crude enzyme activity (U / mL), feeding rate, dissolved oxygen and temperature were measured at 2-4 h intervals.

[0052] Representative batches showed crude enzyme activity ≥3000 U / mL at 68-72h, while preferred batches could reach ≥3900 U / mL; Figure 3 Give OD 600 Changes in crude enzyme activity over time.

[0053] Example 6 Enzyme activity assay Reaction system (1 mL): D-fructose 80 g / L, HEPES 20 mM (pH 8.0), Co 2+ 0.1 mM; add 200 μL of enzyme solution, react at 55℃ for 10 min, then stop with boiling water for 10 min; quantify D-allulose by HPLC. 1 U is defined as 1 μmol of D-allulose generated per minute; sample activity (U / mL) is converted according to the generation rate measured by HPLC. Table 2 gives recommended HPLC conditions (examples).

[0054] Table 2 Recommended HPLC conditions for quantification (examples, not the only limit). The sample activity (U / mL) calculated under the above criteria was used for the evaluation of the fermentation process in Example 5 and the strategy comparison statistics in Example 8; the methodological conditions and statistical criteria were kept consistent to facilitate cross-stage comparison.

[0055] Example 7 Preparation of D-allulose Whole-cell or crude enzyme catalysis was employed: 2% (v / v) fermentation broth was added to 10 mL of a 300 g / L D-fructose solution, and incubated at 60 °C. 2+ The reaction was carried out at 0.1 mM and 200 rpm; samples were taken at set time intervals, and the substrate and product concentrations were determined by HPLC after the reaction was terminated with boiling water. The conversion rate was calculated using the formula: Conversion Rate = C(allulose) / [C(allulose) + C(fructose)] × 100%. To evaluate the process window, the temperature range of 55-70℃ (preferably 55-65℃) was investigated.

[0056] Representative transformation curves are shown below. Figure 4 Stable epimerization can be achieved under the above representative operating conditions, with a conversion rate reaching the specified range, and preferably ≥25% (preferably ≥29%). This approach is consistent with the activity calculation method in Example 6 and the fermentation output in Example 5.

[0057] Example 8: Control and Unexpected Effects Two control strategies were set up: plasmid expression and random integration expression. Under uniform detection and statistical standards (enzyme activity in U / mL, conversion rate calculated according to Example 7; repetition n≥3), crude enzyme activity, process scale-up stability, and multi-generational passaging stability were compared; if necessary, a two-tailed t-test was used to assess the significance of differences (p<0.05). This statistical data is for illustrative purposes only and does not constitute a limitation of the claims.

[0058] Under the same evaluation conditions, the randomly integrated strains achieved transformation performance comparable to or higher than the plasmid system in a single-copy background, and significantly improved intergenerational stability. Representative data are shown in Table 3; corresponding comparison figures are shown in […]. Figure 5.

[0059] Table 3. Summary of Comparative Data (Example) Industrial applicability The Bacillus subtilis integration expression system described in this application, which does not contain plasmid replicons or antibiotic resistance markers, can be implemented continuously or intermittently using conventional fermentation equipment. The resulting fermentation broth or whole cells / crude enzyme can be used to reduce process complexity under representative operating conditions and to epimerize D-fructose to D-allulose. The relevant evaluation and scale-up parameters are consistent with the detection and process specifications described in the examples, making it suitable for industrial implementation.

[0060] Industrial Implementation and Compliance To ensure the stability and compliance of continuous or intermittent production, it is recommended to establish the following control and recording mechanisms: (1) Plasmid-free replicons: Thermosensitive replicons were removed; whole genome PCR and sequencing were used for confirmation; batch records and electronic archives were generated.

[0061] (2) No antibiotic resistance marker: the screening marker was deleted by site-specific recombination; confirmed by cross-boundary PCR and sequencing.

[0062] (3) Genetic stability: After ≥50 generations of continuous passage, crude enzyme activity and phenotype were tested at fixed time points, and the consistency met the preset control limits.

[0063] (4) Metal ion management: Control Co according to process formula 2+ Dosage; establish waste liquid and residue assessment and recycling programs.

[0064] (5) Production consistency: Set control limits for pH, dissolved oxygen, feeding, and temperature, and record and handle deviations; see the corresponding fermentation curves. Figure 3 .

[0065] (6) Microbial safety: Confirm that the strain is non-pathogenic; establish an environmental release monitoring and emergency response plan.

[0066] The aforementioned records and determinations do not limit the claims, but serve to support the reproducibility of industrial implementation.

[0067] Sequence List Description This application relates to nucleotide and amino acid sequences, and the sequence listing is submitted separately and exclusively in WIPO ST.26 (XML) format. Any excerpts or formatting in the specification for ease of reading shall be superseded by the ST.26 (XML) version. SEQ ID NO:1 (amino acid) and SEQ ID NO:2 (nucleotide) are presented in their entirety in the ST.26 file.

Claims

1. A method of constructing a recombinant Bacillus subtilis host cell, characterized in that, The method comprises: (1) providing a transposon vector, the vector comprising an expression cassette for expressing D-Allulose 3-epimerase (DAEase) in the chromosome of Bacillus subtilis and a screening marker, and comprising a temperature-sensitive replicon and a transposition system; (2) introducing the transposon vector into Bacillus subtilis, inducing random transposition, and allowing the expression cassette to be randomly integrated into the chromosome; (3) culturing at 42-51℃ to remove the temperature-sensitive replicon, and obtaining an integrated strain free of plasmid replication; (4) optionally, deleting the screening marker by site-specific recombination, and obtaining an integrated strain free of antibiotic resistance marker.

2. The method of claim 1, wherein, The screening comprises: obtaining positive clones by the screening marker, and performing microplate activity screening by a coupled enzyme method for continuously monitoring the change in coenzyme absorption at 340 nm, the coupled enzyme being an NAD(H) / NADP(H)-dependent dehydrogenase; preferably, the KpRD / NADH method is used, and ΔA340 / min is used as the ranking index, and HPLC is used for rechecking.

3. The method of claim 1 or 2, wherein, The expression cassette comprises: (a) a promoter selected from PgsiB, PnprE, PHpaII, PamyE, and any combination thereof; (b) a ribosome binding site (RBS) having a length of 7-20 bp; (c) a nucleotide sequence encoding DAEase, being SEQ ID NO: 2 or a variant thereof having ≥90% nucleotide identity; (d) a transcription terminator, preferably a double terminator.

4. The method according to any one of claims 1 to 3, wherein, The random transposition is mediated by a mariner / Himar1 or Tn7 system; and the induction condition is 30-37℃ for 3-24h.

5. The method according to any one of claims 1 to 4, wherein, The screening marker is a resistance screening marker; and the site-specific recombination is used for deleting the screening marker.

6. A DAEase expression cassette, comprising, The method comprises: at least one of PgsiB, PnprE, PHpaII, or PamyE; an RBS having a length of 7-20 bp; SEQ ID NO: 2 or a variant thereof having ≥90% nucleotide identity; and a transcription terminator; wherein the expression cassette is suitable for being integrated into the chromosome of Bacillus subtilis by random transposition.

7. A transposon vector for integrating expression of DAEase on Bacillus subtilis chromosome, characterized in that, The vector comprises the expression cassette of claim 6, a mariner / Himar1 or Tn7 transposition element, and a temperature-sensitive replicon.

8. A recombinant Bacillus subtilis host cell, characterized in that, The chromosome of the host cell is integrated with the expression cassette of claim 6, and is free of plasmid replication; and optionally, free of antibiotic resistance marker.

9. A method of preparing DAEase, characterized by, The method comprises culturing the host cell of claim 8 to obtain a fermentation broth, cell lysate, whole-cell catalyst, or crude enzyme containing DAEase; under fed-batch fermentation conditions, the crude enzyme activity of the fermentation broth is ≥3000 U / mL, preferably ≥3900 U / mL, and the culturing time is about 68-72h.

10. A method for preparing D-allulose, characterized by, contacting a reaction solution comprising D-fructose with the fermentation broth, cell lysate, crude enzyme of claim 9 or whole cell catalyst of claim 8 under epimerization conditions to obtain D-psicose; wherein the reaction solution comprises about 300 g / L D-fructose, the reaction is carried out at 55-70 °C and comprises 0.1 mM Co 2+ , the conversion of the resulting D-psicose is > 25%, preferably > 29%.

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