A method for constructing a tagatose-producing engineered strain, and application thereof

Abstract

The application discloses a one-step tagatose engineering bacterium, a construction method and application thereof. An L-arabinose isomerase from Carnobacterium sp. is screened, cloned and expressed in Escherichia coli. Especially, the L-arabinose isomerase is coupled with beta-galactosidase to realize production of D-tagatose from lactose. Under whole-cell biocatalysis conditions, 500 g / L of lactose substrate can be converted into D-tagatose in 3 hours, the yield reaches 95.0 g / L, the average yield is 31.7 g / (L.h), and the conversion rate is 19.0%. The conversion rate is very high, and the D-tagatose has excellent food, health care and medical application potential and economic value.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a one-step engineered strain for producing tagatose, its construction method, and its application. Background Technology

[0002] With the improvement of people's living standards, excessive energy intake has led to a series of chronic diseases, and people are paying more and more attention to their dietary structure, especially sugar intake. The development and research of novel sucrose substitutes has attracted researchers' attention. The development of some functional sweeteners has significant economic and health benefits. Among sucrose substitutes, D-tagatose (TAG) is a low-calorie functional monosaccharide discovered in recent years, belonging to a rare sugar category. D-tagatose's sweetening ability is almost equivalent to sucrose and higher than similar components such as mannitol and sorbitol, but its caloric value is lower at 1.5 kcal / g, with an energy content only one-third that of sucrose. After ingestion, only 20%-25% of D-tagatose is absorbed by the small intestine. The vast majority directly enters the colon and is utilized by the gut microbiota.

[0003] D-tagatose was first discovered in the gum of the tropical plant *Sterculia* (an evergreen tree), and has also been detected in mosses, lichens, and some dairy products, though in very small amounts. There are two main methods for preparing D-tagatose: chemical and biological methods. Due to the high energy consumption, severe chemical pollution, and numerous byproducts of chemical methods, coupled with the increasing consumer demand for natural products in recent years, biological methods have become the mainstream research trend due to their milder reactions, simpler purification, and green, pollution-free nature.

[0004] L-arabinose isomerase (L-AI, EC 5.3.1.4) is an important biocatalyst for the in vitro catalysis of D-galactose to D-tagatose. Enzymes from different microbial sources have different biochemical properties and exhibit substrate heterogeneity. They can catalyze the conversion of arabinose to L-ribulose, and some arabinose isomerases have been reported to have certain catalytic activities on glucose, xylose, and allose (DOI: 10.1016 / j.micres.2013.07.001). It has been reported that high temperatures favor the shift of the isomerization reaction between D-galactose and D-tagatose towards D-tagatose (DOI: 10.1016 / S0378-1097(02)00715-2). Therefore, thermostable L-arabinose isomerases have always been a research focus. However, higher temperatures lead to browning reactions of sugars, and the browning reaction accelerates exponentially with higher temperatures, thereby introducing byproducts and causing pigment deposition. Therefore, screening an L-arabinose isomerase that exhibits high catalytic activity for D-galactose under mesophilic conditions without inducing browning reactions has significant practical implications and industrial application value.

[0005] β-galactosidase (β-GAL) hydrolyzes lactose glycosidic bonds to produce glucose and galactose. Studies have shown that mixing β-GAL and L-AI in a specific ratio catalyzes the production of TAG from lactose, a cheaper substrate than galactose, thus reducing raw material costs. To further optimize the process, reduce energy consumption, and lower costs, Xu Zheng et al. co-expressed L-AI from *Lactobacillus fermentum* and β-galactosidase from *Thermophilus thermophilus* in *Escherichia coli*, using borate to increase the final TAG yield, achieving a maximum yield of 20.2% (DOI:10.1016 / j.bej.2015.12.015). Li Zhiyue et al. cloned the arabinose isomerase gene *araA* and the β-galactosidase gene *lacZ* from the *Escherichia coli* K-12 genome, tandemly linking the two genes with the ribosome binding site sequence SD-AS as a linker, and expressed them in *E. coli*. With the addition of 0.5 mol / L boric acid and 0.1% SDS, the highest yield of tagatose was 83.81 ± 1.38 g / L when the whole cell was transformed with 500 g / L lactose substrate (DOI: 10.13346 / j.cnki.wsxb.20200741). Liu et al. used pET28a as a vector to tandemly express the araA gene from Lactobacillus fermentum CGMCC 2921 and the β-galactosidase gene EclacZ from Escherichia coli. After L-AI mutation modification, 115.21 g / L D-tagatose was obtained after 48 h of transformation with 500 g / L lactose substrate, with a conversion rate of 23.09%, but the yield was low at 2.4 g / (L•h) (DOI:10.13345 / j.cjb.250027).

[0006] Many L-AI catalytic reactions require high temperatures to maintain high conversion rates. In the construction of multi-enzyme cascade catalysis, most require the addition of boric acid to increase TAG yield; however, boric acid is not a food additive. The Food Safety Law explicitly prohibits the production and sale of food made from non-food raw materials or the addition of chemical substances other than food additives. Boric acid, as a non-food additive, is strictly prohibited from being added to food. Therefore, it is necessary to screen for L-AIs with strong medium-temperature catalytic ability to produce D-tagagose from D-galactose, which can achieve high TAG yields when used in combination with β-GAL without the addition of non-food raw materials. Summary of the Invention

[0007] This invention screened and identified an L-arabinose isomerase that can efficiently catalyze the conversion of D-galactose to D-tagatose under mesophilic conditions and used it for the fermentation production of D-tagatose. Furthermore, the selected L-arabinose isomerase and β-galactosidase sequences were expressed in tandem in *E. coli*, using lactose as a substrate for catalytic conversion to D-tagatose. By optimizing whole-cell catalytic conditions, the catalytic conversion capacity of the whole cell was improved. Therefore, the genetically engineered bacteria co-expressing the enzyme can be used in the preparation of D-tagatose.

[0008] Specifically, this invention screened 122 sequences and identified L-AI derived from Carnobacterium sp. CP1, named CaAI. Its amino acid sequence is shown in SEQ ID NO.1. Its NCBI accession number is WP_058919357.1, containing 473 amino acids and a molecular weight of 53.3 kDa. It exhibits catalytic activity in the formation of D-tagatose from D-galactose. The nucleotide sequence encoding the polypeptide is shown in SEQ ID NO.2 of the sequence listing. This invention also provides methods for high-throughput screening of strains and optimization of CaAI enzyme reaction conditions.

[0009] Then, high-throughput screening was performed on recombinant strains BL21(DE3)-pET-28a-araA in 96-well polystyrene microplates containing 1 mL of LB medium. The strains were induced in a high-throughput shaker. The supernatant was discarded after centrifugation at low temperature. The bacteria were resuspended in the reaction solution to 400 μL and incubated overnight at 55°C for 20 h. 2 μL of the reaction solution was transferred to 96-well polystyrene microplates, and strains with high activity were screened using the cysteine-bazol-sulfuric acid (CCSA) assay.

[0010] The optimal reaction conditions for CaAI enzyme were investigated. After inducing bacterial expression, 4 mL of the solution was centrifuged and the supernatant was discarded. Different pH solutions and metal ion cofactors were prepared using a reaction solution containing 5 g / L galactose. The solutions were resuspended to 400 μL, and 1 g / L lysozyme was added to disrupt the bacterial suspension. The reaction was carried out at different temperatures (30-70℃) for 0.5 h-1 h. TAG yield was detected using the CCSA method. The optimal reaction temperature, pH, metal ion concentration, and reaction time were screened.

[0011] CaAI exhibits high activity, and its enzymatic properties indicate favorable conditions for industrial application. A vector was constructed by tandemly combining the gene sequences of L-arabinose isomerase and β-galactosidase from *E. coli* using two strategies, and then further transformed into *E. coli* for expression. This invention relates to *E. coli*, a β-galactosidase source, named *EcGAL*. Its amino acid sequence is shown in SEQ ID NO. 3. Its NCBI accession number is WP_000443089.1, containing 1024 amino acids with a molecular weight of 116.5 kDa. It exhibits catalytic activity in the production of D-galactose and glucose from lactose. The nucleotide sequence encoding the polypeptide is shown in SEQ ID NO. 4. Strains with high transformation rates were selected for subsequent experiments.

[0012] The optimal reaction conditions were optimized. After the above-mentioned bacteria were cultured and expressed, the bacterial cells were collected, resuspended to 250 OD, and sonicated. The crude enzyme solution was then added to the reaction system, which included solutions containing 10 g / L of the substrate lactose. Different pH solutions and different metal ion cofactors were prepared, and the solutions were resuspended to 400 μL. The reaction was carried out at different temperatures (30-70℃) for 1 h, and samples were taken. The TAG yield was detected by CCSA (sulfuric acid-cysteine ​​carbazole) method. The optimal reaction temperature, pH, metal ion concentration, and reaction time were screened.

[0013] The catalytic effects of whole cells and crushed crude enzyme solution as catalysts on high-concentration substrates were also compared. Under optimal reaction conditions, using 500 g / L lactose as the substrate, whole cells and crude enzyme solution were added for conversion, and the difference in conversion rates between whole cells and crushed crude enzyme solution was detected. The catalytic effect of whole cells on lactose was also tested. Under optimal reaction conditions, with a high concentration of lactose (500 g / L) and a certain amount of bacteria added, the reaction was allowed to reach equilibrium, and the maximum conversion rate of lactose to tagatose by the whole cells of the bacteria was detected.

[0014] Therefore, the present invention provides the following technical solution.

[0015] The present invention first provides a one-step engineered strain for producing tagatose, which is obtained by coupled expression of L-arabinose isomerase and β-galactosidase in Escherichia coli, wherein the L-arabinose isomerase is derived from Carnobacterium sp. CP1 and the β-galactosidase is derived from Escherichia coli.

[0016] Preferably, the amino acid sequence of the L-arabinose isomerase is shown in SEQ ID NO.1, and the amino acid sequence of the β-galactosidase is shown in SEQ ID NO.3.

[0017] Specifically, the originating bacteria are strains of Escherichia coli B strain series, Origami series, SHuffle series, and Rosetta series.

[0018] The present invention also provides a method for constructing the engineered strain that produces tagatose in one step, which includes the following steps: introducing the encoding gene of L-arabinose isomerase and the encoding gene of β-galactosidase into Escherichia coli.

[0019] Specifically, the genes encoding L-arabinose isomerase and β-galactosidase are introduced into Escherichia coli via expression vectors or integrated into Escherichia coli genes for expression through genetic engineering.

[0020] More preferably, the L-arabinose isomerase encoding gene and the β-galactosidase encoding gene are expressed in tandem by being constructed on the same expression vector.

[0021] Tandem methods include using promoter tandem, non-coding ribosome binding site (RBS) tandem, and various short peptide nucleotide sequences to link the nucleotide sequences of two proteins.

[0022] In a specific implementation, the arabinose isomerase gene CaaraA and the β-galactosidase gene EclacZ were inserted into two T7 promoter sequences using the pETDuet-1 vector. CaaraA was inserted after the first promoter, and EclacZ was inserted after the second. The pETDuet-CaaraA-EclacZ plasmid was then transformed into E. coli BL21(DE3). The recombinant strain constructed was BL21(DE3)-pETDuet-CaaraA-EclacZ, named E1.

[0023] The CaaraA and EclacZ gene sequences were tandemly linked using the SD-AS (GAAGGAGATATACC) linker, and the entire tandem fragment was constructed into the pET-28a vector. The constructed plasmid pET-28a-CaaraA-SDAS-EclacZ was transformed into E. coli BL21(DE3) competent cells, and positive transformants were selected for protein co-expression. The constructed recombinant strain was BL21(DE3)-pET-28a-CaaraA-SDAS-EclacZ, named E2.

[0024] This invention also provides a method for preparing tagatose, which uses the crude enzyme solution or whole cells of the engineered bacteria that produce tagatose in one step as a catalyst, and lactose as a substrate to form a reaction system, and produces tagatose through a catalytic reaction.

[0025] Preferably, the reaction system also contains the metal ion Co. 2+ ;

[0026] The method for preparing the crude enzyme solution is as follows: after centrifuging the fermented strain, add buffer solution to the bacterial sludge, sonicate and break it up, then centrifuge and take the supernatant to prepare the crude enzyme solution.

[0027] The whole cell preparation method is as follows: after centrifuging the fermented strain, buffer solution is added to the bacterial sludge.

[0028] Specifically, the reaction conditions are as follows: per 1 mL of reaction system, the concentration is 20-80 g / L lactose and 1-4 mM Co. 2+ Crude enzyme solution 1-4 mg / mL, reaction 20-60 min;

[0029] Or bacterial cell concentration OD 600 =200 whole-cell and substrate concentrations of 200-500 g / L lactose, pH 6-8, 0.5-4 mM Co 2+ React at 50-65℃ for 3-24 hours.

[0030] The L-arabinose isomerase screened in this invention is derived from Carnobacterium sp. CP1, and its optimized gene sequence was overexpressed in E. coli. D-galactose was used as a substrate to produce D-tagatose. Optimal reaction conditions were screened under mild conditions, with an optimal temperature of 60°C and an optimal pH of 7.0. Co metal ions were used. 2+ Maximum activity can be achieved at a concentration of 1 mM. The reaction conditions for producing TAG using the described isomerase are favorable for industrial production, and its high conversion rate offers significant advantages in industrial applications.

[0031] In particular, this invention also co-expresses two enzymes in *E. coli*: L-AI from *Carnobacterium sp. CP1* and β-galactosidase from *E. coli*. A multi-gene plasmid of L-AI and β-GAL for single-step synthesis of D-tagatose was constructed. In whole-cell biocatalysis, using inexpensive lactose as a raw material, a high-concentration substrate (500 g / L) was catalyzed for 3 h to produce 19.0% D-tagatose, with a yield as high as 31.7 g / (L·h). Attached Figure Description

[0032] Figure 1 The graph shows the transformation rate results of high-throughput screening strains.

[0033] Figure 2 The graph shows the effect of temperature on CaAI enzyme activity.

[0034] Figure 3 The graph shows the effect of pH on CaAI enzyme activity.

[0035] Figure 4 The graph shows the effect of metal ions on the thermal stability of CaAl.

[0036] Figure 5 Displaying a diagram of the co-expression vector construction.

[0037] Figure 6 The SDS-PAGE images of protein expression in E1 and E2 bacteria are shown. M represents the marker, T represents the whole-cell lysate, and S represents the supernatant from the lysate cells.

[0038] Figure 7 The graph shows the comparison of the conversion rates of crude enzyme solutions E1 and E2. Detailed Implementation

[0039] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation of the present invention.

[0040] Example 1: Screening for L-arabinose isomerases that catalyze the conversion of D-galactose to D-tagatose and constructing recombinant plasmids

[0041] To obtain homologous protein sequences related to the function of the target enzyme, the inventors used the reported amino acid sequence of *Acidothermus cellulolyticus* ATCC 43068 (GenBank accession number: ACZ67491) as a probe. Using bioinformatics platforms such as NCBI, UniProt, and AlphaFold, sequence alignment and structural similarity analysis were conducted to screen for 122 candidate sequences with sequence / structural similarity ranging from 40% to 80%. Subsequently, Tianjin Zhonghe Gene Technology Co., Ltd. was commissioned to optimize the codons of all candidate gene sequences to adapt them to the *E. coli* expression system. Finally, seamless cloning technology was used to construct the optimized gene fragment between the NcoI and XhoI restriction sites of the expression vector pET-28a.

[0042] Example 2: High-throughput screening of recombinant strain BL21(DE3)-pET28a-araA

[0043] The constructed recombinant plasmid pET28a-araA was introduced into the expression host *Escherichia coli* BL21(DE3) via chemical transformation. The transformation product was plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C inverted position. Positive transformants were screened. Single colonies were picked from the plates and inoculated into 96-well polystyrene deep-well plates containing 1 mL of LB liquid medium (containing 50 μg / mL kanamycin). The plates were placed in a high-throughput shaker and incubated at 37°C and 800 r / min until OD (dose elapsed). 600The concentration was increased to 0.6-0.8. Subsequently, the culture temperature was slowly reduced to 16℃ over 1 hour, and isopropyl-β-D-thiogalactoside was added to bring the final concentration to 0.5 mM. Expression was then induced for another 18 hours at 16℃ and 800 r / min.

[0044] Configure the activity detection reaction system: 5 g / L galactose, 0.6 mM MnCl2, 0.8 mM CoCl2, 20 mM PB (pH 7.0) buffer, and 1 g / L lysozyme (add fresh before use).

[0045] The induced bacterial culture was centrifuged at 10,000 r / min for 10 minutes at 4°C, and the supernatant was carefully discarded, retaining the bacterial precipitate. 400 μL of the pre-prepared reaction mixture was precisely added to each well of a 96-well plate containing the bacterial precipitate to completely resuspend the bacteria in the reaction system. The resuspended reaction system was incubated at 35°C for 15 minutes. Subsequently, the 96-well plate was transferred to a high-throughput shaker and reacted continuously at 55°C and 800 rpm for 16 hours.

[0046] Active bacterial strains were screened using a cysteine-carbazole sulfate colorimetric method. The reaction system consisted of 2 μL of reaction solution, 5 μL of 15 g / L cysteine ​​hydrochloride, 150 μL of 70% (w / w) concentrated sulfuric acid, and 5 μL of 0.12% (w / v) carbazole ethanol. The colorimetric reaction solution was placed in 96-well polystyrene shallow-well plates and reacted at 60°C for 10 min. The reaction was terminated by incubation at 4°C for 10 min. After cooling, the color turned reddish-purple. The values ​​were detected at 560 nm using a microplate reader.

[0047] Standard curve preparation: Tag sugar solutions with concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1, 2, and 3 g / L were prepared, and 2 μL of each solution was used for reaction to prepare standard curves. A good linear relationship was observed between TAG concentration and A560 concentration, with the standard curve equation Y = 0.9328X + 0.0703 and R² = 0.9993.

[0048] Fourteen strains with a tagatose conversion rate of 20%-50% were selected, ten strains with a conversion rate of 8%-20%, and the remaining strains with a conversion rate of less than 8%. Figure 1 Among them, CaAI derived from Carnobacterium sp. CP1 has a high conversion rate for galactose, with a conversion rate of 50% to tagatose (its nucleotide sequence is shown in SEQ ID No. 2, and the encoded amino acid sequence is shown in SEQ ID No. 1).

[0049] CaAI (SEQ ID No.1):MLQTNAKEFWFVVGSQNLYGEETLNQVKEHAAQIVEGLNKSGVLSYPLVFKDLVTTSDEIKHVMKEVNYQDNVAGVITWMHTFSPAKMWIAGTKLLQKPLLHLATQFNEKIPWD TIDMDFMNLNQSAHGDREYGFINARLKNNKIVVGYWGNKSVQKDISLWMDAAIGFIESQNIKVARFGDNMRHVAVTEGDKVEAAIQFGWTVDYGIGDLVAEMDKVTNEEIQSTYEECQ CLYEFEQGDNDPAYYEEHVKEQIKIEIALRRFLEAGGYTAFTTNFEDLHGMKQLPGMAVQRLNAEGYGFAGEGDWKTAALDRLMKIMAKNKQTGFMEDYTYDLTEGSEMILQSHMLEVDP TLASNKPKVIVHPLGIGDKEDPARLVFDGAAGSGVVVSMLDLGTHYRLLINAVEAEIPTQSAPNLPVARVLWKPKPNFKEGVTKWIQSGGGHHTVVSLVLTVEQIQDWAKLVNLETVVI。

[0050]

[0051] Example 3: Optimization of CaAI enzymatic reaction conditions

[0052] 1. To determine the effect of temperature on the catalytic activity of recombinant CaAI enzyme (L-arabinose isomerase), 4 mL of the induced bacterial culture was centrifuged and the supernatant was discarded. The bacterial cells were resuspended in 400 μL of 20 mM phosphate buffer (pH 7.0) containing 5 g / L galactose, 0.6 mM MnCl2, and 0.8 mM CoCl2, and then sonicated to obtain crude enzyme solution. Equal volumes of the reaction solution were incubated at 30℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 1 h, respectively. The product yield was determined by CCSA method, and the conversion rate was calculated. The enzyme activity corresponding to the highest conversion rate was defined as 100%, and the relative enzyme activity at each temperature was calculated and compared accordingly.

[0053] The results are as follows Figure 2 As shown, the enzyme's catalytic activity is affected by temperature. Within the temperature range of 30℃ to 60℃, the reaction rate increases with rising temperature, and the relative enzyme activity continuously rises, reaching a peak at 60℃. At 65℃, the enzyme activity remains essentially unchanged. When the temperature rises to 70℃, the enzyme activity drops sharply, retaining only 40% of its peak activity. Based on these results, the optimal reaction temperature for this recombinant CaAI enzyme is determined to be 60℃.

[0054] 2. To determine the effect of pH on the catalytic activity of recombinant CaAI enzyme, a series of reaction buffers with pH values ​​ranging from 4.0 to 9.0 were prepared at the determined optimum temperature (60℃) 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). Equal amounts of substrate and metal ions were added to each buffer to construct reaction systems with different pH values. These were added to bacterial cultures, sonicated, and reacted at 60℃ for 1 hour. Enzyme activity was then measured. The results are as follows: Figure 3 As shown in the figure. Experimental results show that the recombinant CaAI enzyme exhibits maximum catalytic activity at pH 7.0. This indicates that its optimal reaction pH is 7.0, under which it has the highest catalytic efficiency.

[0055] 3. Metal ions are key cofactors of monosaccharide isomerases, typically binding to the enzyme's active site and playing a crucial role in maintaining protein spatial stability and catalytic processes. To investigate the effect of metal ions on CaAI enzyme activity, Mn was added to the reaction system at a final concentration of 1 mM. 2+ Co 2+ Ni 2+ Mg 2+ Ca2+ Zn 2+ Cu 2+ Fe 2+ Ions, and combined metal ions 1 mM Mn 2+ and 1 mM Co 2+ 0.5 mM Mn 2+ and 0.5 mM Co 2+ A blank control without any metal ions was used, and the measured enzyme activity was set to 100%. Samples were reacted at 60℃ and pH 7.0 with different metal ions. The results are shown in Table 1.

[0056] Table 1. Effects of metal ions on CaAl activity

[0057] ;

[0058] As shown in the table above, the metal ion Mg 2+ Mn 2+ Co2+ and Mg have an enhancing effect on enzyme activity. 2+ It can increase activity by 9%. Adding the metal ion Mn 2+ and Co 2+ The subsequent enzyme activity was 1.8 and 1.9 times that of the blank control. Ni 2+ Ca 2+ Zn 2+ Fe 2+ It has an inhibitory effect on enzyme activity. Especially Cu. 2+ The addition of [a specific ingredient] had the strongest inhibitory effect on enzyme activity, resulting in an 81% loss of enzyme activity. 2+ and Co 2+ When both are present, enzyme activity does not have separate Co 2+ High presence. CaAI at 1 mM Co 2+ Maximum activity is achieved in the presence of [a substance].

[0059] Example 4: Study on Factors Affecting the Thermal Stability of CaAl

[0060] The thermostability of the recombinant CaAI enzyme was determined by centrifuging 4 mL of the induced bacterial culture and discarding the supernatant. The cells were resuspended in 300 μL of 20 mM phosphate buffer (pH 7.0) and sonicated to obtain the crude enzyme solution. The crude CaAI enzyme solution was heat-treated by incubating at 45℃, 50℃, 55℃, 60℃, and 65℃ for 1 h. Galactose was added to the treated enzyme solution to a final concentration of 5 g / L, and 1 mM Co was added to a final concentration of... 2+Metal ions, 20 mM phosphate buffer (pH 7.0), final volume 400 μL. React at 60 °C for 1 h. The blank control is the sample reacted directly without heat treatment, and the enzyme activity measured in it is set as 100% of the reference baseline.

[0061] After induction, 4 mL of bacterial culture was centrifuged and the supernatant was discarded. Then, 300 μL of 20 mM phosphate buffer (pH 7.0) containing 1 mM Co was added. 2+ The bacterial cells were resuspended in buffer solution and sonicated to obtain crude enzyme solution. The sonicated crude enzyme solution was heat-treated at different temperatures of 45℃, 50℃, 55℃, 60℃, and 65℃ for 1 h, and then 400 μL of galactose and 20 mM phosphate buffer (pH 7.0) were added for enzyme reaction. The reaction was carried out at 60℃ for 1 h, and the remaining conversion rate was measured.

[0062] Test results as follows Figure 4 As shown, the enzyme maintained good stability after heat treatment at 45℃, with little difference in activity compared to the untreated enzyme. Enzyme activity began to decrease above 45℃, with a slow decrease at 45-55℃, a significant decrease at 60℃, and near-complete inactivation at 65℃.

[0063] Heat treatment of the crude enzyme solution in a buffer solution with added metal ions significantly improved the enzyme's thermostability. Even after heat treatment at 65°C for 1 h, the enzyme activity remained unaffected and unchanged compared to the blank. This indicates that the thermostability of this L-arabinose isomerase is metal-dependent, and the addition of metal ions can improve the enzyme's thermostability.

[0064] Based on the comprehensive test results, this invention yielded a reaction condition of 60℃ and pH 7.0, with a metal ion concentration of 1 mM Co. 2+ This enzyme, CaAI, is an L-arabinose isomerase that efficiently catalyzes the production of TAG from galactose. It exhibits good heat resistance; even with the prior addition of metal ions, its activity remains essentially unchanged after heating at 65°C for 1 hour. It can be used as a preferred enzyme in conjunction with β-galactosidase to produce TAG from lactose.

[0065] Example 5: Construction of a co-expression vector for L-arabinose isomerase and β-galactosidase

[0066] Primers L1F and L1R were used to amplify the CaaraA gene sequence using pET28a-CaaraA as a template, and primers V1F and V1R were used to amplify the vector gene sequence using pETDuet-1 as a template. The two fragments were seamlessly cloned and ligated to construct the vector pETDuet-CaaraA. Primers G1F and G1R were used to amplify the EclacZ gene (its nucleotide sequence is shown in SEQ ID No. 4, and the encoded amino acid sequence is shown in SEQ ID NO. 0. 3) using the BL21(DE3) genome as a template, and primers V2F and V2R were used to amplify the vector sequence using pETDuet-CaaraA as a template. The two fragments were ligated to construct the plasmid pETDuet-CaaraA-EclacZ. Figure 5

[0067]

[0068] Primers L2F and L2R were designed to amplify the CaaraA gene sequence, and primers G2F and G2R were designed to amplify the EclacZ sequence. Primer G2F contains the forward sequence of SD-AS (GAAGGAGATATACC, SEQ ID NO.5), and primer L2R contains the reverse sequence of SD-AS. The SD-AS sequence was introduced between the CaaraA and EclacZ sequences using primer design. Primers V3F and V3R amplified the pET-28a sequence. The three target gene fragments were then ligated using a seamless cloning method to construct the plasmid pET-28a-CaaraA-SDAS-EclacZ. Figure 5 After being transformed into BL21(DE3), the bacterium BL21(DE3)-pET-28a-CaaraA-SDAS-EclacZ was obtained and named bacterium E2, which overexpressed the proteins CaAI and EcGAL.

[0069] Table 2. Primer sequence list

[0070] ;

[0071] Example 6: Expression and Enzyme Activity Detection of Recombinant Strains

[0072] Strains E1 and E2 constructed in Example 5 were cultured in LB medium at 37°C and 200 r / min, with 100 μg / mL ampicillin and 50 μg / mL kanamycin added, respectively. The cultures were cultured until OD... 600 The expression level was increased to 0.6-0.8, the temperature was lowered to 16℃, and 0.5 mM IPTG was added for induction for 18 h. The two bacterial strains, E1 and E2, were then sonicated and their expression levels were detected by SDS-PAGE. Figure 6 Both construction methods can correctly express CaAI and EcGAL. Comparing the expression levels, EcGAL expression is better in E1 than in E2, while CaAI expression is better in E2 than in E1. The amount of AI required for the catalytic lactose-to-tagatose reaction is higher than that of GAL; therefore, strain E2 is superior to strain E1 in terms of expression level.

[0073] Example 7: Enzyme activity detection and transformation rate comparison of recombinant strains

[0074] The enzyme activity of crude enzyme solutions E1 and E2 at 60℃ was detected. The crude enzyme solution was prepared according to Example 4. 100 mL of the induced bacterial culture was centrifuged and the supernatant was discarded. 1 mL of 20 mM phosphate buffer (pH 7.0) containing 1 mM Co was added. 2+ Bacterial cells resuspended in buffer solution, bacterial OD 600Approximately 250. The bacteria were ultrasonically disrupted to obtain a crude enzyme solution. The concentration of the crude enzyme solution after ultrasonic disruption was determined by the BCA method to be 50 mg / mL. Enzyme reaction conditions: 0.5 mL reaction system, lactose concentration 20 g / L, final crude enzyme solution concentration 1 mg / mL, buffer solution 20 mM phosphate (pH 7.0), 1 mM Co 2+ The reaction was carried out at 60℃ for 30 min. The amount of reaction product in the sample was determined by high performance liquid chromatography (HPLC); the detection conditions were: Waters Sugar Pak I column, column temperature 80℃, differential detector, injection volume 10 μL, ultrapure water as mobile phase, flow rate 0.5 mL / min. The amount of enzyme required to catalyze the conversion of 1 µmol of substrate per minute is defined as one enzyme activity unit, denoted by U. The enzyme activities of crude enzyme solutions E1 and E2 were 0.25 U / mg and 0.34 U / mg, respectively. The enzyme activity of E2 was higher than that of E1. In Example 6, the expression level of CaAI in E2 was higher than that in E1, and E2 was more suitable for enzyme-catalyzed reactions than E1.

[0075] The conversion rates of two crude enzyme solutions catalyzed for different time periods were compared. The reaction system consisted of 200 g / L lactose substrate, 20 mM PB (pH 7.0), 1 mM CoCl2 buffer, and a crude enzyme concentration of 10 mg / mL. The reaction was carried out at 60℃ for 1–24 h. TAG was determined by HPLC. The results after detection were as follows: Figure 7 As shown, the enzyme reaction rate was relatively fast from 1 to 3 hours, with conversion rates of E1 and E2 reaching 15.5% and 16.6% after 3 hours, respectively. The conversion rate slowed down from 3 to 7 hours, increasing by only about 3% from 7 to 24 hours. After 24 hours, the conversion rates of E1 and E2 were 21.2% and 22.35%, respectively. There were differences in lactose conversion between E1 and E2; the enzymes CaAI and EcGAL, expressed by two genes linked in tandem with the SD-AS linker, showed higher conversion rates than the other linker. E2 exhibited better enzyme activity and conversion efficiency than E1.

[0076] Example 8: Application of E2 whole-cell catalysis for the production of tagatose from high-concentration substrates

[0077] In the whole-cell catalytic reaction, both the bacterial cell concentration and the substrate concentration were simultaneously increased. In a 10 mL reaction system, the bacterial cell concentration E2 OD was used. 600 =200, lactose substrate concentration 500 g / L, 20 mM PB (pH 7.0), 1 mM Co 2+The reaction was carried out at 60℃ for 3-24 h. The D-tagatose yields at 3 h and 24 h were 95.0 g / L and 110.5 g / L, respectively, with yields of 31.7 g / (L·h) and 4.6 g / (L·h), and conversion rates of 19.0% and 22.1%, respectively. In whole-cell catalysis, the expression of CaAI and EcGAL in the same cell enhances the metabolic pathway effect. By bringing the two enzymes closer together, the product of EcGAL can be directly captured and catalyzed by the neighboring CaAI, improving catalytic efficiency. Enzymes are more stable inside the cell, and the intracellular environment can resist the influence of some adverse factors outside the cell, such as temperature changes. Therefore, the tandem co-expression strain E2 can simplify the process, accelerate conversion efficiency, and reduce production costs.

Claims

1. A one-step engineered bacterium for producing tagatose, characterized in that, It is obtained by coupling expression of L-arabinose isomerase and β-galactosidase in Escherichia coli, wherein the L-arabinose isomerase is derived from Carnobacterium sp. and the β-galactosidase is derived from Escherichia coli.

2. The engineered strain for producing tagatose in one step as described in claim 1, characterized in that, The amino acid sequence of the L-arabinose isomerase is shown in SEQ ID NO.1, and the amino acid sequence of the β-galactosidase is shown in SEQ ID NO.

3.

3. The engineered strain for producing tagatose in one step as described in claim 1, characterized in that, Its originating bacteria are strains of Escherichia coli Bstrain series, Origami series, SHuffle series, and Rosetta series.

4. A method for constructing engineered bacteria that produce tagatose in one step as described in any one of claims 1 to 3, characterized in that, The procedure includes the following steps: introducing the genes encoding L-arabinose isomerase and β-galactosidase into Escherichia coli.

5. The construction method as described in claim 4, characterized in that, The L-arabinose isomerase encoding gene and the β-galactosidase encoding gene are introduced into Escherichia coli via expression vectors or integrated into Escherichia coli genes for expression through genetic engineering.

6. The construction method as described in claim 5, characterized in that, The L-arabinose isomerase encoding gene and the β-galactosidase encoding gene are expressed in tandem by being constructed on the same expression vector.

7. The use of the engineered bacteria that produce tagatose in one step as described in any one of claims 1 to 3 in the preparation of tagatose.

8. A method for preparing tagatose, characterized in that, Using crude enzyme solution or whole cells of the engineered bacteria that produce tagatose in one step as described in any one of claims 1 to 3 as a catalyst, and lactose as a substrate to form a reaction system, tagatose is produced through a catalytic reaction.

9. The method as described in claim 8, characterized in that, The reaction system also contains the metal ion Co. 2+ ; The method for preparing the crude enzyme solution is as follows: after centrifuging the fermented strain, add buffer solution to the bacterial sludge, sonicate and break it up, then centrifuge and take the supernatant to prepare the crude enzyme solution. The whole cell preparation method is as follows: after centrifuging the fermented strain, buffer solution is added to the bacterial sludge.

10. The method as described in claim 9, characterized in that, Reaction conditions: 1 mL of reaction system contains 20-80 g / L lactose and 1-4 mM Co 2+ Crude enzyme solution 1-4 mg / mL, reaction 20-60 min; Or bacterial cell concentration OD 600 =200 whole-cell and substrate concentrations of 200-500 g / L lactose, pH 6-8, 0.5-4 mM Co 2 + React at 50-65℃ for 3-24 hours.

Images