An engineered bacillus subtilis strain for producing tagatose, a construction method and application thereof

Abstract

The application discloses an engineered bacillus subtilis strain for producing tagatose, a construction method and application thereof, and belongs to the technical field of bioengineering. The engineered strain takes bacillus subtilis as a host, co-expresses L-arabinose isomerase from Erysipelothrix larvae and beta-galactosidase from escherichia coli, and constructs the bacillus subtilis for co-expressing double enzymes through ribosome binding sites or promoters in series. The double-enzyme bacillus subtilis whole cell catalyzes lactose to produce D-tagatose, and the yield and conversion efficiency of D-tagatose are significantly improved after optimization of the enzymatic reaction. The application adopts single-strain double-enzyme co-expression and one-step whole-cell catalysis, uses low-cost lactose as a substrate, and has the advantages of mild reaction, less by-product, simplified process, no endotoxin safety hazard, and the like. The application solves the problems of low conversion rate, high substrate cost and complicated strain compounding in the prior art, and is suitable for industrialized production of food-grade D-tagatose.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an engineered Bacillus subtilis strain that produces tagatose, its construction method, and its application. Background Technology

[0002] Among numerous artificial sweeteners, D-tagatose (TAG) is highly favored. It is a rare, naturally occurring ketone sugar found in some dairy products, fruits, and cocoa. As an isomer of D-galactose, it is considered a potential sucrose substitute. Its sweetening power is almost equivalent to sucrose and higher than similar ingredients such as mannitol and sorbitol, but its caloric value is lower, only 1.5 kcal per gram, and its energy content is only one-third that of sucrose. D-tagatose has low absorption efficiency in the human body, with only 20%-25% being absorbed by the small intestine, and its metabolic rate is slower, only 50% that of fructose. With these health benefits, it can be used to enhance food flavor, assist in the treatment of obesity, alleviate symptoms associated with type 2 diabetes, and lower high blood sugar. Furthermore, research indicates that it also plays an important role in preventing tooth decay, lowering cholesterol, and enhancing intestinal immunity. Therefore, D-tagatose is an ideal sweetener suitable for dietary products, beverages, and health foods.

[0003] Naturally occurring D-tagatose is extremely rare. To achieve large-scale production, existing technologies typically employ chemical and biocatalytic methods. Chemical production processes require high temperatures and pressures, resulting in high energy consumption. Furthermore, they easily generate byproducts such as sorbitol and mannose, increasing purification costs. Simultaneously, they require large amounts of acids, alkalis, and metal catalysts, polluting the environment and increasing environmental costs. While the yield of this method is 40%-50%, the actual yield is even lower due to the large number of byproducts. In contrast, enzymatic catalysis offers milder reaction conditions, lower energy consumption, fewer byproducts, and simpler purification, resulting in less environmental pollution. Therefore, enzymatic isomerization biotechnology for producing D-tagatose has attracted widespread attention from researchers. In biosynthesis, the single-enzyme reaction primarily uses L-arabinose isomerase (L-AI) to isomerize galactose to tagatose. Currently, L-AI from various sources is being used in tagatose synthesis research. However, due to the high production cost of galactose as a substrate, some studies have attempted to employ a multi-enzyme co-expression strategy to construct a multi-enzyme cascade reaction system, using the cheaper lactose as a substrate to achieve efficient synthesis of D-tagatose. β-galactosidase (β-GAL) can break the glycosidic bonds of lactose, catalyzing its hydrolysis and transglycosylation. β-galactosidases derived from Bacillus tend to catalyze the transglycosylation of lactose to obtain galacto-oligosaccharides. β-galactosidases derived from Escherichia coli have stronger hydrolytic activity and tend to hydrolyze lactose into glucose and galactose, which is then isomerized into tagatose via L-AI. Through in vitro dual-enzyme action, a one-step synthesis from lactose to tagatose can be achieved. This dual-enzyme expression system can not only reduce the cost of substrate raw materials but also reduce fermentation production costs. Some studies have successfully co-expressed arabinose isomerase and β-galactosidase in Escherichia coli. However, since E. coli itself secretes endotoxins, this limits the application of biosynthesis in food industrial production.

[0004] Bacillus subtilis, as a model bacterium of Gram-positive bacteria, occupies an important position in the field of biological research. From a genetic perspective, the genetic background of Bacillus subtilis is clear, and its whole genome has been sequenced, enabling researchers to gain a deep understanding of its genetic composition and function, thus laying a solid foundation for subsequent molecular-level research. In terms of cultivation, Bacillus subtilis is characterized by a short culture cycle and rapid growth rate, and its fermentation technology is mature and easily applied industrially. Furthermore, it is relatively easy to manipulate at molecular levels, facilitating traceless gene manipulation and genetic engineering. Patent CN112852702A (publication date: 2021.05.28) expresses β-galactosidase and arabinose isomerase derived from Escherichia coli in Bacillus subtilis, respectively. When the two recombinant strains were combined to catalyze 500 g / L of lactose, the highest yield of tagatose was 96.76 g / L, less than 20% conversion rate. Patent CN117660562A (publication date: 2024.03.08) describes the expression of arabinose isomerase in Bacillus subtilis to catalyze the conversion of D-galactose to tagatose, but the substrate price is higher than that of lactose. Zhang Xian et al. constructed an integrated recombinant using the plasmid of the arabinose isomerase gene araA from Bacillus subtilis 168 D2, achieving a maximum D-tagatose yield of 96.8 g / L from 500 g / L lactose (Zhang, Xian, et al. "Production of d-Tagatose by Whole-Cell Conversion of Recombinant." Biology10.12(2021).).

[0005] Current research on Bacillus subtilis mainly employs two methods: one is to express two enzymes separately and then use the cells in a specific ratio; the other is to use galactose as a substrate for catalysis. However, the yield of D-tagatose is relatively low when using gene integration or plasmid co-expression of two genes. Therefore, constructing a lactose-based dual-enzyme co-expression system in Bacillus subtilis is of great significance for food production and industrial applications. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention constructs a tandem co-expression system of L-arabinose isomerase and β-galactosidase in Bacillus subtilis, optimizes enzyme catalytic conditions and methods, improves the enzyme's catalytic conversion ability, and shortens the reaction time of the entire process using a single-step method, thereby saving energy consumption.

[0007] On one hand, the present invention provides a Bacillus subtilis engineered strain of tagatose, which uses Bacillus subtilis as the starting strain and co-expresses L-arabinose isomerase and β-galactosidase in tandem to obtain a Bacillus subtilis engineered strain with dual enzyme co-expression.

[0008] Specifically, the L-arabinose isomerase is derived from the strain *Erysipelothrix larvae*, whose amino acid sequence is shown in SEQ ID NO.1, and the β-galactosidase is derived from *Escherichia coli*, whose amino acid sequence is shown in SEQ ID NO.3.

[0009] Specifically, the tandem linking involves linking the gene encoding L-arabinose isomerase and the gene encoding β-galactosidase in tandem via a linker or promoter. The nucleotide sequence of the gene encoding L-arabinose isomerase is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene encoding β-galactosidase is shown in SEQ ID NO. 4.

[0010] Specifically, the linker is the SD-AS linker, whose nucleotide sequence is shown in SEQ ID NO.5, and the promoter is Pveg and / or PftsZ, whose nucleotide sequences are shown in SEQ ID NO.6 and SEQ ID NO.7. Preferably, if promoter tandem is used, the promoter sequence also has a terminator sequence to terminate the transcription of the E1AI sequence, and the nucleotide sequence is shown in the sequence listing SEQ ID NO.8.

[0011] Specifically, the originating bacterium is Bacillus subtilis SCK6, or Bacillus subtilis 168 and its derivatives, wherein the Bacillus subtilis derivatives are selected from Bacillus subtilis DB series or Bacillus subtilis WB series.

[0012] On one hand, the present invention provides a method for constructing the engineered Bacillus subtilis strain, wherein the gene encoding L-arabinose isomerase and the gene encoding β-galactosidase are tandemly linked by a linker or promoter, and the tandem fragments are connected to a vector using seamless cloning to construct a co-expression plasmid, which is then transformed into Bacillus subtilis and screened to obtain the engineered Bacillus subtilis strain; preferably, the nucleotide sequence of the gene encoding L-arabinose isomerase is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding β-galactosidase is shown in SEQ ID NO.4; preferably, the linker is an SD-AS linker, the nucleotide sequence of which is shown in SEQ ID NO.5, and the promoter is Pveg and / or PftsZ, the nucleotide sequences of which are shown in SEQ ID NO.6 and SEQ ID NO.7; preferably, if promoter tandem is used, the promoter sequence also contains a terminator sequence to terminate the transcription of the ElAI sequence, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO.8.

[0013] Specifically, the skeletal carrier is PUC980, the Bacillus subtilis is Bacillus subtilis SCK6, or Bacillus subtilis 168 and its derivatives, and the Bacillus subtilis derivatives are selected from Bacillus subtilis DB series or Bacillus subtilis WB series.

[0014] On the one hand, the present invention provides the application of the engineered Bacillus subtilis strain in the preparation of D-tagatose.

[0015] On the other hand, the present invention provides a method for preparing D-tagatose, wherein the engineered Bacillus subtilis bacteria are treated, and the treatment solution is added to a reaction system containing 0.5 mM-1 mM metal cofactor as a substrate. The pH of the reaction system is 6.0-7.0, the reaction temperature is controlled at 50-60°C, and the reaction time is within 24 hours. Preferably, the metal cofactor is selected from one or any of the following: Mg 2+ Mn 2+ and Co 2+ .

[0016] Specifically, the engineered Bacillus subtilis bacteria are treated using any one of the following methods: whole cell, ultrasonic disruption, repeated freeze-thaw cycles, or 1% surfactant. Preferably, the surfactant is Tween-20, Tween-80, or Triton X-100. The pH of the reaction system is 7.0, the reaction temperature is controlled at 55°C, and the reaction time is 24 hours.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The Bacillus subtilis dual-enzyme tandem co-expression system constructed in the present invention has significant technical advantages in D-tagatose production. (1) Using food-grade safe Bacillus subtilis as the host, it is free of endotoxins and non-pathogenic, meeting the requirements of food industry production and completely avoiding the safety hazards of Escherichia coli expression system; (2) Using single strain tandem co-expression of L-arabinose isomerase and β-galactosidase, no strain matching is required, simplifying the process and reducing operation and fermentation costs; (3) Using low-cost lactose as the substrate to replace high-priced galactose, significantly reducing raw material costs; (4) The dual enzyme expression tandemly via SD-AS linker is balanced and has high catalytic activity. The optimal reaction conditions are 55℃, pH 7.0, and 1mMCo. 2+ The half-life at 55℃ is about 12h, and the thermal stability is excellent; (5) The conversion of lactose to tagatose can be completed in one step of whole cell catalysis without cell disruption. The process is simple, energy consumption is low, and there are few by-products. Under a lactose substrate of 500g / L, the yield of D-tagatose reached 123.5g / L in 24h reaction time, with a conversion rate of 24.7%, which is significantly higher than the existing technology and is suitable for industrial-scale high-efficiency production of food-grade D-tagatose. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the SDS-PAGE detection results of protein expression in various recombinant bacteria, where M is the marker, T is the whole-cell lysate, and S is the supernatant of the lysate cells.

[0019] Figure 2 This is a schematic diagram showing the conversion rate of lactose to tagatose catalyzed by recombinant bacteria with different connection methods.

[0020] Figure 3 This is a schematic diagram illustrating the effect of temperature on the activity of L-AI-SDAS-β-GAL enzyme.

[0021] Figure 4 This is a schematic diagram illustrating the effect of pH on the activity of L-AI-SDAS-β-GAL enzyme.

[0022] Figure 5 This is a schematic diagram of the half-life of L-AI-SDAS-β-GAL at 55℃.

[0023] Figure 6 This is a schematic diagram illustrating the impact of different bacterial cell treatments on TAG conversion rate. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Example 1: Construction of a co-expression vector for L-arabinose isomerase and β-galactosidase

[0028] The L-arabinose isomerase used in this invention is derived from the strain *Erysipelothrix larvae*, named ElAI, with its amino acid sequence shown in SEQ ID NO.1 and NCBI accession number WP_067634338.1. It contains 474 amino acids and has a molecular weight of 53.5 kDa. It exhibits catalytic activity in the formation of D-galactose from D-tagatose. The nucleotide sequence encoding the polypeptide, after codon optimization, is shown in SEQ ID NO.2. This invention also relates to a β-galactosidase derived from *Escherichia coli*, named *EcGAL*, with its amino acid sequence shown in SEQ ID NO.3 and NCBI accession number WP_000443089.1. It contains 1024 amino acids and has a molecular weight of 116.5 kDa. It exhibits catalytic activity in the formation of D-galactose and glucose from lactose. The nucleotide sequence encoding the polypeptide is shown in SEQ ID NO.4.

[0029] The ElAI gene underwent codon optimization by Tianjin Zhonghe Gene Technology Co., Ltd. to adapt it to the Bacillus subtilis expression system. Using a seamless cloning method, it was constructed between the NcoI and XhoI restriction sites in plasmid pET-28a, forming plasmid pET-28a-ElAI. Homologous recombination was used to tandemly link the ElAI and EcGAL gene sequences via the SD-AS linker (as shown in SEQ ID NO. 5, GAGGAGGTTATTTCA) or by inserting a promoter. The inserted promoters were Pveg and PftsZ, and their nucleotide sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 7. A trpA terminator sequence was designed before the promoter sequence to terminate ElAI gene transcription; its nucleotide sequence is shown in SEQ ID NO. 8.

[0030] Specifically, primers LF and LR were designed to amplify the ElAI gene using plasmid pET-28a-ElAI as a template. Primers GF and GR were designed to amplify the β-galactosidase EcGAL gene sequence using the *E. coli* BL21(DE3) genome as a template. Primers VF1 and VR2 were designed to amplify the PUC980 fragment using plasmid PUC980 as a template. The two target gene sequences were ligated into plasmid PUC980 using a seamless cloning method. The two target gene sequences were tandemly linked by an SD-AS linker (as shown in SEQ ID NO. 5, GAGGAGGTTATTTCA), and the SD-AS sequence was designed into the primers for synthesis. The constructed plasmid was named PUC980-L-AI-SDAS-β-GAL, where PUC980 is the commercial plasmid name, L-AI is the name of the first linked enzyme arabinose isomerase sequence, SDAS is the name of the linker sequence connecting the two genes, and β-GAL is the name of the second linked enzyme β-galactosidase sequence.

[0031] Primers VEG-F and VEG-R were designed to amplify the Pveg promoter sequence using the Bacillus subtilis SCK6 genome as a template. Primers VF2 and VR2 were designed to amplify the plasmid PUC980-L-AI-SDAS-β-GAL sequence. The two fragments were ligated to obtain the plasmid PUC980-L-AI-Pveg-β-GAL. The terminator sequence was designed and incorporated into primers for synthesis.

[0032] Primers ftsZ-F and ftsZ-R were designed to amplify the PftsZ promoter sequence using the Bacillus subtilis SCK6 genome as a template. This sequence was then ligated to the plasmid PUC980-L-AI-SDAS-β-GAL amplified from VF2 and VR2 sequences, resulting in the plasmid PUC980-L-AI-PftsZ-β-GAL. The primer sequences are shown in Table 1.

[0033] Table 1. Primer sequence information

[0034] ;

[0035] Example 2: Expression of recombinant co-expression strain SCK6-PUC980-L-AI-β-GALs

[0036] The recombinant co-expression plasmids PUC980-L-AI-SDAS-β-GAL, PUC980-L-AI-Pveg-β-GAL, and PUC980-L-AI-PftsZ-β-GAL constructed in Example 1 were transformed into the expression strain Bacillus subtilis SCK6. The strains were cultured overnight at 37°C on LB agar plates containing 50 μg / mL kanamycin, and positive transformants were screened. Single clones of bacteria were selected and placed in 100 mL Erlenmeyer flasks containing 50 mL of LB broth (50 μg / mL kanamycin). The flasks were then incubated at 37 °C and 220 rpm for 24 h on a shaker to obtain strains B1 (a recombinant strain containing the co-expression plasmid PUC980-L-AI-SDAS-β-GAL), B2 (a recombinant strain containing PUC980-L-AI-Pveg-β-GAL), and B3 (a recombinant strain containing PUC980-L-AI-PftsZ-β-GAL). The enzymes expressed by bacteria B1, B2, and B3 were named L-AI-SDAS-β-GAL, L-AI-Pveg-β-GAL, and L-AI-PftsZ-β-GAL, respectively. L-AI-SDAS-β-GAL represents the names of the two enzyme products co-expressed by SCK6-PUC980-L-AI-SDAS-β-GAL(B1) bacteria: arabinose isomerase and β-galactosidase. L-AI-Pveg-β-GAL and L-AI-PftsZ-β-GAL are the names of the two enzyme products expressed by B2 and B3, respectively. The three bacterial strains (B1, B2, and B3) were ultrasonically disrupted, and their expression was detected by SDS-PAGE. The results are as follows: Figure 1 Both L-AI and β-GAL protein bands were expressed normally. In B1, both proteins were expressed well. B2 expressed L-AI at a level similar to that of B1, but its β-GAL expression was the worst among the three strains. B3 expressed more β-GAL than B1 and B2, and its L-AI expression was the lowest compared to the other two strains.

[0037] Example 3: Comparison of conversion rates of SCK6-PUC980-L-AI-β-GALs

[0038] Preparation of crude enzyme solution: Culture bacteria B1, B2, and B3 in 100 mL of water, centrifuge at 3,000 g for 20 min at 4°C to remove the supernatant. Dissolve the bacterial sludge in 20 mM PB buffer (pH 7.0) to dilute the bacterial solution to approximately 250 OD600. After ultrasonic disruption of the bacterial solution, centrifuge at 12,000 g for 30 min at 4°C and collect the supernatant to prepare the crude enzyme solution. Take 200 μL of the crude enzyme solution and add 100 g / L lactose, 0.5 mM MnCl2, 0.5 mM CoCl2, and 20 mM PB (pH 7.0) buffer, for a total reaction volume of 1 mL. React at 55°C for 24 h. The amount of reaction product in the sample is determined by high-performance liquid chromatography (HPLC). The detection conditions are: Waters Sugar Pak I column, column temperature 80°C, differential detector, injection volume 10 μL, ultrapure water as the mobile phase, and flow rate 0.5 mL / min. After testing Figure 2 As shown, the conversion rate of tagatose in the crude enzyme solution of B1 was 24.1%, B2 was 22.2%, and B3 was 21.2%. Among them, the enzyme L-AI-SDAS-β-GAL expressed by the recombinant strain B1, which consists of two genes linked in tandem with the SD-AS linker, had the highest conversion rate. Subsequent experiments will use strain B1 for further research.

[0039] Example 4: Optimization of enzyme L-AI-SDAS-β-GAL reaction conditions

[0040] First, the effect of temperature on L-AI-SDAS-β-GAL activity was investigated. After expression of the recombinant co-expression strain, the bacterial cells were collected by centrifugation and resuspended in 20 mM PB buffer (pH 7.0) to OD. 600 The concentration was 250, and the mixture was ultrasonically disrupted. After centrifugation, the supernatant was collected.

[0041] 10 g / L lactose was dissolved in 20 mM PB buffer (pH 7.0), and metal ions MnCl2 and CoCl2 were added to a final concentration of 0.5 mM. 30 μL of crude enzyme solution was added to the reaction mixture, bringing the total reaction volume to 500 μL. The reaction was carried out at 30℃, 40℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 0.5 h, respectively. The conversion rate of tagatose was determined by HPLC to identify the optimal reaction temperature, with the highest conversion rate representing 100% relative enzyme activity. The test results are as follows: Figure 3 As shown, the activity of enzyme-catalyzed reactions is affected by temperature. As temperature increases, the rate of enzyme-catalyzed reactions accelerates. Based on the trend of enzyme activity with temperature, enzyme activity gradually increases with increasing temperature from 30-55℃, but decreases significantly above 55℃. Therefore, the optimal reaction temperature for L-AI-SDAS-β-GAL is 55℃.

[0042] To investigate the effect of pH on L-AI-SDAS-β-GAL, buffer solutions with different pH values ​​(pH 4.0-5.0) were prepared using 20 mM sodium acetate buffer (pH 4.0-5.0), 20 mM sodium phosphate buffer (pH 6.0-7.0), and 20 mM Tris-HCl buffer (pH 8.0-9.0). Metal ions MnCl2 and CoCl2 were added to a final concentration of 0.5 mM, along with 10 g / L lactose substrate and 30 μL of crude enzyme solution. 500 μL of reaction solutions at different pH values ​​were prepared and reacted at 55℃ for 0.5 h to observe enzyme activity. The maximum relative enzyme activity was set to 100%. Figure 4 As shown, L-AI-SDAS-β-GAL exhibits maximum activity at pH 7.0.

[0043] To investigate the effect of metal ions on the activity of L-AI-SDAS-β-GAL enzyme, Mn was added to the reaction system at a final concentration of 1 mM. 2+ Co 2+ Ni 2+ Mg 2+ Ca 2+ Zn 2+ Cu 2+ Ions and combined metal ions 1mM Mn 2+ and 1mM Co 2+ 0.5mM Mn 2+ and 0.5mM Co 2+ A blank control was used without any metal ions. The samples were reacted at 55℃ and pH 7.0 with different metal ions. The enzyme activity was set to a maximum relative enzyme activity of 100%, and the results are shown in Table 2.

[0044] Table 2. Effects of metal ions on the activity of L-AI-SDAS-β-GAL

[0045] ;

[0046] Mg 2+ Mn 2+ and Co 2+ It has an enhancing effect on enzyme activity, especially Mn. 2+ and Co 2+ The activity was 2.0 and 2.3 times that of the blank. Ni 2+ Ca 2+ Zn 2+ and Cu 2+ It inhibits enzyme activity, among which Cu 2+ The inhibitory effect was strongest, resulting in a 67% loss of enzyme activity. Co 2+When present alone, the enzyme activity reaches its maximum, being 2.3 times the original enzyme activity. A metal ion cofactor of 1 mMCo is selected. 2+ .

[0047] The optimal reaction conditions for the enzyme catalysis described above were: temperature 55℃, pH 7.0, and metal ion concentration of 1 mM Co. 2+ It can achieve maximum activity.

[0048] Example 5: Detection of the half-life of L-AI-SDAS-β-GAL

[0049] The half-life of L-AI-SDAS-β-GAL at the optimal reaction temperature of 55℃ was determined. 250 OD bacteria were lysed in 20 mMPB buffer (pH 7.0), and CoCl2 metal ions were added to a final concentration of 1 mM. The mixture was incubated at 55℃ for 1 h, 2 h, 3 h, 4 h, and 5 h. After incubation, 50 μL of crude enzyme solution was added to the reaction solution, along with 10 g / L lactose, for a total reaction volume of 0.5 mL. The enzyme reaction solution was incubated at 55℃ for 0.5 h, and the reaction was terminated by heating. The conversion rate of L-AI-SDAS-β-GAL was assessed by HPLC detection of tagatose production, with the conversion rate of the unincubated crude enzyme solution representing a relative conversion of 100%. The half-life of L-AI-SDAS-β-GAL at 55℃ is as follows: Figure 5 As shown, the tandemly coupled dual enzymes exhibit good thermostability, remaining relatively stable at 55℃, with a half-life of t0. 1 / 2 Around 12 hours.

[0050] Example 6: Effects of different bacterial treatments on dual-enzyme catalysis

[0051] SCK6-PUC980-L-AI-SDAS-β-GAL cells were collected and resuspended in 20 mM PB (pH 7.0) buffer to 200 OD. The cells were then added to the reaction system using whole-cell disruption, sonication, repeated freeze-thaw disruption, and disruption with 1% surfactants (Tween-20, Tween-80, Triton X-100). 400 μL of L-AI-SDAS-β-GAL cells from different treatments were added to a 500 μL reaction solution containing 400 g / L lactose, 1 mM CoCl2, and 20 mM PB (pH 7.0) buffer. The reaction was carried out at 55 °C for 1 h, 5 h, and 24 h, and samples were taken. The amount of reaction product in the samples was determined by HPLC. The yield of D-tagatose (TAG) was as follows: Figure 6As shown, when L-AI-SDAS-β-GAL was added to the reaction system in whole-cell form, the conversion rate of TAG in the whole-cell reaction system was slightly lower than that of other treatments at 1 hour, but the advantage became obvious after 5 hours, outperforming ultrasound and freeze-thaw. The surfactants showed inconsistent results after 5 hours, with Trixon-100 showing better performance; at 24 hours, the conversion rate of TAG in the whole-cell reaction system reached the same level as that of the crude enzyme solution after ultrasound disruption, with a conversion rate of 24.7% at 24 hours. Therefore, the tandem expression strain can be directly added to the reaction system in whole-cell form to produce tagatose in a one-step process using lactose as a substrate, simplifying the production process and reducing production costs.

[0052] Example 7: Application of L-AI-SDAS-β-GAL whole-cell catalysis for the production of tagatose from high-concentration substrates

[0053] In a 10 mL reaction system, the bacterial cell OD 600 Diluted to 200, with a substrate of lactose at 500 g / L and a metal ion concentration of 1 mMCo. 2+ The bacterial culture was dissolved in 20 mM PB (pH 7.0) buffer, and the reaction temperature was controlled at 55℃ for 24 h. The yield of D-tagatose was detected by HPLC. The results showed that the yield of D-tagatose in the bacterial culture was 123.5 g / L, the yield was 5.1 g / (L·h), and the conversion rate was 24.7%.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A Bacillus subtilis engineered strain that produces tagatose in one step, characterized in that, Using Bacillus subtilis as the starting strain, L-arabinose isomerase and β-galactosidase were co-expressed in tandem to obtain engineered Bacillus subtilis strains with dual enzyme co-expression.

2. The engineered Bacillus subtilis strain according to claim 1, characterized in that, The L-arabinose isomerase is derived from the strain Erysipelothrix rhusiopathiae, and its amino acid sequence is shown in SEQ ID NO.

1. The β-galactosidase is derived from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO.

3.

3. The engineered Bacillus subtilis strain according to claim 1, characterized in that, The tandem linking is the tandem linking of the gene encoding L-arabinose isomerase and the gene encoding β-galactosidase via a linker or promoter. The nucleotide sequence of the gene encoding L-arabinose isomerase is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding β-galactosidase is shown in SEQ ID NO.

4.

4. The engineered Bacillus subtilis strain according to claim 3, characterized in that, The linker is the SD-AS linker, and its nucleotide sequence is shown in SEQ ID NO.

5. The promoter is Pveg and / or PftsZ, and its nucleotide sequence is shown in SEQ ID NO.6 and SEQ ID NO.

7. If promoter tandem is used, the promoter sequence also has a terminator sequence to terminate the transcription of the ElAI sequence, and its nucleotide sequence is shown in the sequence listing SEQ ID NO.

8.

5. The engineered Bacillus subtilis strain according to claim 1, characterized in that, The originating bacteria are Bacillus subtilis SCK6, or Bacillus subtilis 168 and its derivatives, wherein the Bacillus subtilis derivatives are selected from Bacillus subtilis DB series bacteria or Bacillus subtilis WB series bacteria.

6. The method for constructing the engineered Bacillus subtilis strain according to any one of claims 1-5, characterized in that, The gene encoding L-arabinose isomerase and the gene encoding β-galactosidase are tandemly linked via a linker or promoter. A co-expression plasmid is constructed by seamless cloning and linking the tandem fragment to a vector. This plasmid is then transformed into *Bacillus subtilis*, and engineered *Bacillus subtilis* strains are obtained through screening. The nucleotide sequence of the gene encoding L-arabinose isomerase is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding β-galactosidase is shown in SEQ ID NO.

4. The linker is the SD-AS linker, and its nucleotide sequence is shown in SEQ ID NO.

5. The promoter is Pveg and / or PftsZ, and its nucleotide sequences are shown in SEQ ID NO.6 and SEQ ID NO.

7. If tandem promoters are used, the promoter sequence also includes a terminator sequence to terminate the transcription of the ElAI sequence; the nucleotide sequence of this terminator is shown in SEQ ID NO.

8.

7. The construction method according to claim 6, characterized in that, The skeletal carrier is PUC980, and the Bacillus subtilis is Bacillus subtilis SCK6, or Bacillus subtilis 168 and its derivatives. The Bacillus subtilis derivatives are selected from Bacillus subtilis DB series or Bacillus subtilis WB series.

8. The use of the engineered Bacillus subtilis strain according to any one of claims 1-5 in the preparation of D-tagatose.

9. A method for preparing D-tagatose, characterized in that, The engineered Bacillus subtilis strain according to any one of claims 1-5 is treated, and the treatment solution is added to a reaction system containing 0.5 mM-1 mM metal cofactor as a substrate. The pH of the reaction system is 6.0-7.0, the reaction temperature is controlled at 50-60℃, and the reaction time is within 24 h. The metal cofactor is selected from one or any of the following: Mg 2+ Mn 2+ and Co 2+ .

10. The method according to claim 9, characterized in that, The engineered Bacillus subtilis bacteria were treated using any one of the following methods: whole cell, ultrasonic disruption, repeated freeze-thaw cycles, or 1% surfactant. The surfactant was Tween-20, Tween-80, or Triton X-100. The pH of the reaction system was 7.0, the reaction temperature was controlled at 55°C, and the reaction time was 24 hours.

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