Construction of Efficient Multienzyme Cascade Reactions for D-Tagatose Biosynthesis from D-Fructose

Abstract

D-tagatose is an ideal sucrose substitute with potential applications in food and healthcare. The combined catalysis of polyphosphate kinase (PPK), fructose kinase (FRK), D-tagatose-6-phosphate 3-differential anisomerase (FbaA) and phytase provides a low-cost and convenient pathway for the biosynthesis of D-tagatose from D-fructose; however, there is still a problem of low catalytic efficiency that needs to be solved urgently. Therefore, this study enhanced the biosynthesis of D-tagatose by optimizing the expression levels of PPK, FRK and FbaA in a polycistronic system and knocking out the gene pfka of Escherichia coli. With 30 g/L D-fructose as a substrate, the conversion rate increased to 52%, which was the highest after 24 h. In addition, by constructing a multienzyme self-assembly system with SpyTag and SpyCatcher to improve the whole-cell catalytic ability, the conversion rate was further increased to 75%. Finally, through the fed-batch strategy, the optimal strain Ec-7 produced 68.1 g/L D-tagatose from 100 g/L D-fructose. The multienzyme cascade route reported herein provides an efficient and elegant innovative solution for the generation of D-tagatose.

1. Introduction

D-tagatose is a naturally occurring rare sugar and isomer of D-galactose and D-fructose. It has about one-third the calories of sucrose but is close in sweetness. In recent years, with the increasing demand for low-calorie sweeteners among consumers and the rising prevalence of metabolic diseases such as diabetes and obesity, D-tagatose is considered an ideal sucrose substitute. It can reduce the calories of food and improve the taste without causing tooth decay. In the field of healthcare, its extremely low glycemic index (GI ≈ 3) and prebiotic properties help control sugar and regulate intestinal flora. It has obtained safety certifications such as the FDA. Although excessive intake may cause mild gastrointestinal discomfort, its tolerance is better than that of sugar alcohols, making it an ideal choice for diabetic foods and functional health products [1].

Traditional methods for producing D-tagatose, such as natural extraction and chemical synthesis, are usually limited by high cost, low efficiency and environmental pollution. In recent years, enzymatic biotransformation has emerged as a more viable alternative due to its cost-effectiveness and eco-friendliness [2]. Among the enzymatic approaches studied, simultaneous lactose hydrolysis and D-galactose isomerization by dual enzyme coupling is one of the most commonly explored methods, which uses D-galactose obtained from lactose hydrolysis to produce D-tagatose [3,4,5,6,7]. However, approximately half of the D-galactose cannot be converted due to the thermodynamic equilibrium limitations of the isomerization reaction catalyzed by arabinose isomerase (L-AI) [8]. One way to change the thermodynamic equilibrium is to add boric acid to the reaction system [9]. Since boric acid has a much higher affinity for ketose than aldose, D-tagatose can form complexes with boric acid. This disrupts the reaction equilibrium, driving the reaction toward production and significantly increasing the yield of the final product. However, this strategy has problems such as the toxicity of boric acid and the cumbersome steps of subsequent separation and purification. To overcome the above problems, researchers have investigated alternative substrates and enzymatic pathways. Some researchers have developed tagatose 4-epimerase that could convert D-fructose to D-tagatose in one step, but conversion rates remained low due to similar limitations [10,11]. In addition, some researchers have established a redox pathway by introducing xylose reductase and galactitol dehydrogenase (GDH) in Bacillus subtilis or yeast to reduce the adverse effects of reversible reactions. Still, there were problems with the long fermentation times and the low yields [12,13]. Moreover, a route to produce D-tagatose in one pot via a multienzyme cascade has been proposed, including fructose phosphorylation, fructose-6-phosphate isomerization and tagatose-6-phosphate dephosphorylation [14,15]. This method eliminates the tedious separation and purification steps of reaction intermediates and can handle unstable intermediates and change unfavorable reaction equilibrium. The multienzyme cascade pathway achieved high conversion using D-fructose as a substrate, but the cost was greatly increased because the fructose phosphorylation process required a large amount of ATP [16]. Liu et al. used an in vitro multienzyme cascade route and introduced an ATP regeneration system to effectively reduce costs while achieving a conversion rate of 70%. However, the low product titer and the laborious enzyme purification step were not conducive to industrial-scale production [17]. Although expression of α-glucan phosphorylase in Escherichia coli phosphorylated starch or maltodextrin to glucose-1-phosphate in the absence of ATP, multiple enzyme-mediated reversible reactions as well as non-specific phosphatases resulted in conversions of less than 40% [18,19].

Since the reversibility of the isomerase reaction hurts the biotransformation of D-tagatose, the use of multienzyme cascade reactions to eliminate the unfavorable thermodynamic trend is a promising method. An equal amount of ATP is consumed during fructose phosphorylation, which becomes a significant cost burden in industrial production. Therefore, it is particularly necessary to introduce an ATP regeneration system. Compared with other ATP regeneration systems, such asacetyl phosphate/acetate kinase system [20] and the acetate kinase–acetyl phosphate system [21], the use of PPK is an attractive solution to the problem of ATP regeneration because it enables the use of a variety of inexpensive and stable polyphosphates (PolyP) as phosphate donors [22] and is more suitable for industrial scale-up.

In summary, the enzymatic synthesis of D-tagatose from D-fructose faces several key challenges. First, although tagatose-4-epimerase can catalyze the single-step conversion of D-fructose to D-tagatose, its catalytic efficiency is limited by thermodynamic equilibrium and low catalytic activity. Second, multienzyme cascade reactions usually involve multiple enzymes (such as fructose kinase, D-tagatose-6-phosphate 3-differential anisomerase and phosphatases), and it is more complicated to optimize the optimal reaction conditions of each enzyme. In addition, the ATP-dependent phosphorylation step significantly increases the production cost. Finally, phosphatases often exhibit nonspecific dephosphorylation activity on sugar-6-phosphate intermediates (such as D-fructose-6-phosphate and D-tagatose-6-phosphate), resulting in a decrease in yield.

This study aimed to explore the coupling of phosphorylation–dephosphorylation modules with a cost-effective ATP regeneration system to achieve efficient synthesis of D-tagatose. First, we optimized the co-expression of polyphosphate kinase (PPK), fructose kinase (FRK) and D-tagatose-6-phosphate 3-differential anisomerase (FbaA) in Escherichia coli. To reduce the adverse effects of bypass metabolism, we used the CRISPR-Cas9 system to knock out the gene pfka [23]. While tuning enzyme expressions can improve efficiency, it is challenging to alter the natural properties of the enzyme. By constructing multienzyme complexes with well-defined structures such as protein fusion, RNA, DNA or protein scaffolds and scaffold-free self-assembly, it is possible to build substrate channels that promote intermediate transfer between active residues to enhance metabolic flux and mitigate the diffusion of intermediates [24,25,26]. Among them, SpyTag/SpyCatcher covalently links individual proteins through newly formed covalent (isopeptide) bonds and may be an effective method to enhance enzymatic cascade reactions [27,28,29]. Therefore, we used the SpyTag and SpyCatcher covalent self-assembly system to further improve catalytic efficiency. Based on the above findings, we developed an economical and efficient method for synthesizing D-tagatose from D-fructose and provided a reference for the synthesis of rare sugars limited by thermodynamic equilibrium.

2. Materials and Methods

2.1. Materials

Phytase was purchased from Weifang (Shandong, China). Standards of D-tagatose, glucose, fructose, antibiotics (kanamycin and ampicillin) and isopropyl β-D-thiogalactoside (IPTG) were purchased from Aladdin (Shanghai, China). D-fructose was purchased from Sangon Biotech (Shanghai, China). The plasmid extraction and DNA purification kit were purchased from Vazyme (Nanjing, China). The 2X MultiF Seamless Assembly Mix Clone Expression II kit was purchased from ABclonal Technology (Wuhan, China). All other chemicals were purchased from Macklin (Shanghai, China).

2.2. Strains, Plasmids and Primers

Table 1. Strains and plasmids are used in this work.

Name	Description	Source
Strains
E. coli BL21(DE3)	The expressing host for recombinant protein	Lab stock
E. coli JM109	General cloning host	Lab stock
Ec-pfkb	E. coli BL21 with plasmid pET28a-pfkb	This work
Ec-fbaA	E. coli BL21 with plasmid pET28a-fbaA	This work
Ec-ppk	E. coli BL21 with plasmid pET28a-ppk	This work
Ec-1	E. coli BL21 with plasmid pETDuet-ppk-fbaA-pfkb	This work
Ec-2	E. coli BL21 with plasmid pETDuet-ppk-pfkb-fbaA	This work
Ec-3	E. coli BL21 with plasmid pETDuet-pfkb-fbaA-ppk	This work
Ec-4	E. coli BL21 with plasmid pETDuet-pfkb-ppk-fbaA	This work
Ec-5	E. coli BL21 with plasmid pETDuet-fbaA-pfkb-ppk	This work
Ec-6	E. coli BL21 with plasmid pETDuet-fbaA-ppk-pfkb	This work
Ec-2-Δp	E. coli BL21 with plasmid pETDuet-ppk-pfkb-fbaA, Δpfka	This work
Ec-7	E. coli BL21 with plasmid pETDuet-ppk-SpyTag-pfkb-SpyCatcher-fbaA, Δpfka	This work
Plasmids
pET28a	Kanr, lacIq, PT7, expression vector	Lab stock
pETDuet	Ampr, lacIq, PT7, expression vector	Lab stock
pET28a-pfkb	pET28a containing pfkb	This work
pET28a-fbaA	pET28a containing fbaA	This work
pET28a-ppk	pET28a containing ppk	This work
pETDuet-ppk-fbaA-pfkb	pETDuet containing ppk-fbaA-pfkb	This work
pETDuet-ppk-pfkb-fbaA	pETDuet containing ppk-pfkb-fbaA	This work
pETDuet-pfkb-fbaA-ppk	pETDuet containing pfkb-fbaA-ppk	This work
pETDuet-pfkb-ppk-fbaA	pETDuet containing pfkb-ppk-fbaA	This work
pETDuet-fbaA-pfkb-ppk	pETDuet containing fbaA-pfkb-ppk	This work
pETDuet-fbaA-ppk-pfkb	pETDuet containing fbaA-ppk-pfkb	This work
pETDuet-ppk-SpyTag-pfkb-SpyCatcher-fbaA	pETDuet containing ppk-SpyTag-pfkb-SpyCatcher-fbaA	This work

lists the strains and plasmids used in this work. The specific fructose kinase (FRK) gene, pfkb (WP_011067996.1), from BifiDobacterium longum [30] was derived from previous reports and codon-optimized, synthesized by Azenta (Suzhou, China). The ppk gene from Cytophaga hutchinsonii and SpyTag/SpyCatcher genes were stored in our laboratory [31,32]. The FbaA gene fbaA was obtained from the E. coli k12 genome and subjected to recombinant plasmid construction. For example, the genome of E. coli k12 was used as a template, and fbaA was amplified via PCR by fbaA-F and fbaA-R. Then, the pET28a plasmids skeleton was linearized via the reverse PCR by 28a-F and 28a-R at multiple cloning sites. The gene fragment was connected with the linearized vector by homologous recombination. The resulting plasmid, pET28a-fbaA, was chemically transformed into E. coli BL21.

To construct co-expression plasmid, take pETDuet-ppk-pfkb-fba as an example, the ppk was cloned into the MCS1 of pETDuet to generate plasmid pETDuet-ppk. pfkb-T7 fbaA cluster was inserted into MCS2 of pETDuet-ppk to generate plasmid pETDuet-ppk-pfkb-fbaA. To construct co-expression plasmid pETDuet-ppk-SpyTag-pfkb-SpyCatcher-fbaA, the SpyTag with fbaA and SpyCatcher with ppk and pfkb were first fused to form the SpyTag-ppk and pfkb-SpyCatcher-fbaA, respectively, using overlapping PCR. The two fusion gene fragments were then inserted into the plasmid MCS1 and MCS2 of pETDuet to generate plasmid pETDuet-ppk-SpyTag-pfkb-SpyCatcher-fbaA.

Table S1 listed all PCR primers for the plasmids constructed in this work (see Supplementary Materials).

2.3. Gene Editing by CRISPR-Cas9

The gene pfka was deleted using the CRISPR-Cas9 system, following the standardized protocols of the gene editing method [33]. The plasmids and primers for gene manipulation were listed in Supplementary Tables S2 and S3. At first, the primers (gRNA-pfka-F and gRNA-pfka-R) were annealed to form dsDNA. Then, the pGRB-pfka plasmid was constructed through homologous recombination of the dsDNA and the linearized vector. The donor DNA was obtained by amplifying and fusing the two corresponding arms of the desired gene. After that, the donor DNA and pGRB sequences were transfected into E. coli containing pREDCas9 via electrotransformation and then transformed cells were cultured in 800 μL of LB medium. After cultivating for 2 h at 30 °C, the cells were plated on LB agar plates, and then the positive single colony was verified by colony PCR. To eliminate the targeting pGRB, positive recombinants were cultured in an LB medium containing 2 g/L L-arabinose. Finally, the bacterial fluid was further cultured at 42 °C to lose the pREDCas9 plasmid.

2.4. Expression and Purification of Enzymes

The recombinant strain was activated by plate, and the individual colony was inoculated in 10 mL LB medium with appropriate antibiotics and cultured at 37 °C for 8–10 h. For protein expression assays, 1% of the inoculated volume was transferred to 100 mL LB medium and cultured at 37 °C for 2 h. Then, 1 mmol/L IPTG was added and the culture was incubated at 200 r/min and 20 °C. After 24 h, the induced cells were centrifuged at 4 °C and 8000× g for 10 min and resuspended in 10 mL of PBS buffer. After two repetitions of resuspension, the cells were broken by an ultrasonic breaker for 10 min (300 W, 2 s pulses, 3 s pauses). The cell-free extracts were obtained by centrifugation at 12,000× g for 10 min to remove cellular debris. The His tag was inserted into the N-terminus of the enzyme, and the crude enzyme was purified by Ni-NTA affinity chromatography [34]. Specifically, the supernatant was applied to a Ni-NTA resin that had been pre-equilibrated with a 50 mM PBS buffer (pH 7.4) containing 500 mM NaCl. The elution of the target protein was carried out using a 50 mM PBS buffer (pH 7.4), supplemented with 500 mM NaCl and 250 mM imidazole. The expression of target enzymes was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

2.5. Whole-Cell Biotransformation and Determination of Catalytic Activity

At the end of fermentation, the recombinant strain was collected by centrifugation at 8000× g for 10 min. The induction culture was collected and the cell catalyst with an OD₆₀₀ of 50 was suspended in 50 mmol/L PBS buffer (pH 7.5) containing 10 mmol/L Mg²⁺, 10 mmol/L ATP, 35 mmol/L sodium hexametaphosphate (polyP6) and 30 g/L D-fructose. The whole-cell biotransformation was carried out at 200 r/min and 40 °C for 24 h and then boiled for 15 min to terminate the reaction. Then, 1 U/L phytase was added and dephosphorylation was carried out for 5 h at 40 °C.

Whole-cell catalytic activity was determined based on the yield of tagatose 6-phosphate. One unit of whole-cell activity was determined as the number of cells needed to produce 1 μmol of tagatose 6-phosphate from D-fructose per minute.

Phytase catalytic activity was determined based on the production of D-tagatose. One unit of activity was determined as the amount of enzyme required to produce 1 μmol of D-tagatose from tagatose 6-phosphate per minute.

2.6. Optimization of Whole-Cell Reaction Conditions

To optimize the biotransformation conditions, key factors such as temperature, pH, Mg²⁺ concentration, ATP to fructose ratio, cellular catalyst addition and polyP6 concentration were optimized. Whole-cell biotransformation was performed at 25–55 °C to confirm the optimal temperature. In order to assess the optimal pH, the activity in the pH range of 5.0–9.0 was determined by using PBS buffer (pH 5.0–7.0) and Tris-HCl buffer (pH 7.0–9.0). Concentrations of 2–20 mmol/L Mg²⁺ were also evaluated for optimal additions. The optimal ratio of ATP to fructose was assessed from 1:200 to 1:5. Later, the optimum cellular catalyst addition was determined by performing experiments using different cell concentrations (OD₆₀₀ values of 20, 30, 40, 50, 60 and 70). To evaluate the effect polyP6, concentrations of 15–55 mmol/L polyP6 were added to the reaction system. The maximum whole-cell biocatalytic activity was used as the standard (100%).

2.7. Analytical Approaches

The biomass was measured as the absorbance value at a wavelength of 600 nm (OD₆₀₀) by a UV–VIS spectrophotometer (Aoesh, Shanghai, China). The samples were centrifuged and filtered through a 0.22 μm filter membrane. HPLC was used to Detect the compounds in the reaction solution. HPLC used a Carbomix-Ca-NP chromatographic column and a refractive index detector, eluted with ultrapure water at a flow rate of 0.4 mL/min at 80 °C. All experiments were conducted in triplicate. The statistical procedures were analyzed using Statistical Packages for the Social Sciences (SPSS).

An isopeptide bond is a stable covalent bond that forms spontaneously between the side chains of lysine (Lys) and asparagine/aspartic acid (Asn/Asp) residues. The SpyTag/SpyCatcher system consists of two short peptide sequences, where the Catcher specifically recognizes and binds to the Tag peptide, thereby promoting the formation of isopeptide bonds between the side chains. Subsequently, the amino acid sequence of PPK-SpyTag-FRK-SpyCatcher-FbaA was analyzed by liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) [35]. Briefly, this multienzyme complex was detected by polyacrylamide gel electrophoresis. After Coomassie Brilliant Blue staining, the target protein band was cut out from the gel, broken into small fragments, treated with ammonium bicarbonate and acetonitrile to remove the stain and then digested with trypsin for 20 h. The resulting digestion products were analyzed by LC-MS/MS system to confirm the presence of the multienzyme complex.

3. Results and Discussion

3.1. Construction of a Multienzyme Cascade Pathway for Producing D-Tagatose from D-Fructose

Efficient ATP regeneration is critical to the overall reaction rate. PPK is mainly divided into two major families, PPK1 and PPK2. Although both enzyme families can catalyze the synthesis of PolyP, PPK1 preferentially synthesizes PolyP from nucleoside triphosphates, while PPK2 preferentially consumes polyP to phosphorylate nucleoside monophosphates or diphosphates [36]. Therefore, PPK2 was selected for ATP regeneration in the pathway. The specific reaction process was shown in Figure 1A: in the presence of ATP, FRK catalyzed the conversion of fructose to fructose 6-phosphate, FbaA catalyzed the conversion of fructose 6-phosphate to tagatose 6-phosphate, PPK was introduced for ATP regeneration and phytase catalyzed dephosphorylation of tagatose 6-phosphate to produce D-tagatose. Although the isomerization was a reversible reaction, FbaA established an equilibrium ratio of 1:9 between fructose 6-phosphate (F6P) and tagatose 6-phosphate (T6P) [37], which was significantly higher than that of tagatose 4-epimerase and L-arabinose isomerase. The fructose phosphorylation reaction catalyzed by FRK and the dephosphorylation of tagatose 6-phosphate catalyzed by phytase are both irreversible and complete reactions. Therefore, the maximum theoretical conversion rate of D-tagatose in this pathway should reach 90%. We introduced these three enzymes into Escherichia coli for overexpression. The expression of PPK, FbaA and FRK was verified by SDS-PAGE, which were consistent with the theoretical molecular weights of approximately 35.4, 39.1 and 33.4 kDa, respectively (Figure 1B).

Multienzyme cascade reactions can generally be classified as either sequential or concurrent reactions [38]. Since no phosphatase is specific to tagatose 6-phosphate in nature, we chose to add phytase at the end of the reaction process to dephosphorylate tagatose 6-phosphate to reduce interference with intermediates. In this work, we first added the crude enzyme of the above three enzymes and substrates (fructose, ATP and polyP6) into a pot and reacted at 40 °C for 12 h, and then added phytase and continued the reaction for 3 h. The results showed that 4 g/L D-tagatose was produced from 9 g/L D-fructose, proving the feasibility of this multienzyme cascade reaction. In the biosynthesis of D-tagatose, the synergistic effects of PPK (polyphosphate kinase), FRK (fructokinase), FbaA (fructose-1,6-bisphosphate aldolase) and phytase improve efficiency by optimizing energy utilization and reaction pathways. In the first step, PPK uses cheap polyphosphate to efficiently regenerate ATP, providing continuous energy support for the phosphorylation of D-fructose catalyzed by FRK to fructose-6-phosphate (F6P), greatly reducing the cost of external ATP supplementation. At the same time, the efficient fructokinase irreversibly converts fructose into F6P, laying the foundation for subsequent isomerization. In the second step, non-specific phosphatase (phytase) hydrolyzes the phosphate bonds of tagatose-6-phosphate (T6P) and also hydrolyzes other high-energy phosphate compounds. By optimizing the reaction conditions (time series control), undesirable dephosphorylation can be suppressed, and the product tagatose is finally released.

3.2. Optimizing D-Tagatose Production by Transcriptional Fine-Tuning

D-fructose and ATP can diffuse freely within cells, so the reaction can be carried out directly using intact whole cells as catalysts without cell disruption. Whole-cell biotransformation provides an excellent basis for efficient and sustainable catalysis, and the intracellular environment can also enhance enzyme stability while simplifying the operational process. In order to further improve the mass transfer efficiency, we chose to co-express multiple enzymes, which is an efficient way to couple multiple reactions [39]. When a polycistronic system places multiple genes in the same plasmid for expression, the gene order will affect the expression level of the enzyme [40]. Therefore, to adjust the expression level of PPK, FRK and FbaA in the co-expression strain to the maximum activity, the order of the three genes was optimized and six plasmids with different gene orders were constructed (Figure 2A). As shown in Figure 2B, the engineered strain Ec-2 has the highest D-tagatose concentration among all six strains, reaching 12.3 g/L, so this strain was selected for the next experiment. This phenomenon may be related to the characteristics of prokaryotic transcription–translation coupling. Because genes close to the promoter are preferentially expressed, the expression of distal genes may be reduced due to transcriptional attenuation or mRNA degradation. The gene arrangement of Ec-2 optimizes the expression ratio of each enzyme, thereby achieving the best efficiency of the multienzyme cascade reaction. Although the yield of D-tagatose has been improved after gene order optimization, it was not significant. We speculated that the low production of D-tagatose may be due to the existence of bypass pathways and diffusion of intermediates, which ultimately limited the conversion of D-fructose to D-tagatose.

3.3. Block of the Alternative Pathway of Fructose-6-Phosphate to Enhance D-Tagatose Production

Although the cells in the resuspension buffer have stopped growing, some enzymes still have catalytic activity under certain conditions, leading to side reactions and intermediate degradation. In the glycolysis pathway, fructose-6-phosphate kinase (PfkA) can convert fructose-6-phosphate into fructose-1,6-bisphosphate, thereby competitively inhibiting FbaA. Therefore, we blocked the glycolysis pathway by knocking out the pfkA gene in Escherichia coli, resulting in a large accumulation of fructose-6-phosphate (F6P), thereby providing more precursors for the synthesis of D-tagatose. The resulting engineered strain was named Ec-2-Δp and the effects of pfka gene deletion on cell growth and whole-cell catalytic efficiency were further studied. In E. coli, F6P can still be slowly metabolized through the Entner–Doudoroff pathway of pentose phosphate or phosphofructokinase II (Pfkb) [41]. As shown in Figure 3A, after the pfka gene was deleted, the growth rate of Ec-2-Δp was significantly reduced and the final OD₆₀₀ after 24 h was 16% lower than Ec-2. In terms of catalytic activity, Ec-2-Δp produced 15.6 g/L D-tagatose after 24 h using 30 g/L fructose, which was 26% higher than Ec-2. The results showed that blocking the bypass pathway of the intermediate effectively increased the production of D-tagatose.

3.4. Effects of Biotransformation Conditions on Whole-Cell Catalytic Activity

In multienzyme cascade reactions, each enzyme typically has different optimal conditions, such as pH, temperature and substrate concentration. When performing these reactions in one pot, a set of reaction conditions must be determined that balances the requirements of all the enzymes involved to maximize overall efficiency. Reaction conditions such as temperature, pH, Mg²⁺ concentration, ATP to fructose ratio, cellular catalyst addition and polyP6 concentration were optimized in order to improve the whole-cell catalytic activity in Figure 4.

The effect of temperature (25–55 °C) and pH (5.0–9.0) were investigated in this study. As shown in Figure 3A, the catalytic reaction exhibited an optimum temperature at 40 °C, maintaining more than 70% of the maximum activity between 40 and 45 °C. However, a decrease in catalytic activity was observed as the temperature of the reaction increased, which could be attributed to the high temperature reducing the enzyme activity. Recombinant cells were more active at neutral and weakly alkaline pH than at acidic pH. At least 80% of the maximum activity was observed at pH 7.0–8.0 with an optimum pH of 7.5. 50 mmol/L Tris-HCl buffer and PBS buffer at pH 7.0–8.0 had little effect on the activity. The FRK and PPK usually require the addition of Mg²⁺ [42,43], so the effects of Mg²⁺ on the biocatalytic activity of the whole-cell reaction were primarily investigated. Activity increased by 77% after a 2 mmol/L Mg²⁺ increase. Subsequently, the Mg²⁺ concentration was optimized to 10 mmol/L. Excess Mg²⁺ may be chelated to form a precipitate in the reaction system, leading to a decrease in cellular activity. To promote fructose phosphorylation, the effect of different ATP concentrations on catalytic activity was also investigated. The highest biocatalytic activity was obtained at an ATP:fructose ratio of 1:15. However, the activity instead decreased rapidly with increasing ATP concentration, which could be attributed to the inhibitory effect of excess ATP. For higher D-tagatose production capacity, the cellular catalyst addition was optimized in the reaction system. Whole-cell activity increased with an increase in cellular catalyst addition and reached a maximum under OD₆₀₀ of 50. As the cellular catalyst addition continued to increase, the yield decreased slightly, which may be due to the slow circulation of the medium and the reduced fluidity of the catalytic system. Sodium Hexametaphosphate (polyP6) is the phosphate donor for ATP regeneration by PPK2, so the amount of polyP6 was optimized. The increase in polyP6 stimulated the relative activity concentration, but it gradually weakened when polyP6 exceeded 35 mmol/L, which may be due to excessive chelation of Mg²⁺ by polyP6 to form precipitates [44]. After optimizing the reaction conditions, Ec-2-Δp finally produced 15.6 g/L D-tagatose with the biocatalytic activity of 1.20 U/OD₆₀₀. The conversion rate was only 52%, with almost half of the D-fructose remaining. We suspected that this may be due to the insufficient overall driving force of the multienzyme and free diffusion of intermediates.

3.5. Improving Whole-Cell Catalytic Efficiency by Constructing Multienzyme Complex

Cascade reactions have great potential, but when multiple enzymes coexist in cells, it is difficult for each enzyme to efficiently bind to its specific substrate, resulting in limited reaction efficiency. The SpyTag-SpyCatcher system precisely couples multiple enzymes through covalent self-assembly to form a tightly cascaded enzyme complex, thereby increasing substrate flux, reducing intermediate diffusion and improving overall catalytic efficiency. Although this multienzyme cascade strategy can theoretically produce up to 0.9 mol of D-tagatose per 1 mol of D-fructose, the synthesis of D-tagatose is not ideal due to the lack of ability of the downstream pathway to process excess intermediates, low diffusion efficiency and the accumulation of intermediate metabolites. Consequently, it is essential to modify and optimize the in vivo enzyme cascade pathway to enhance conversion efficiency. We wished to improve catalytic efficiency by constructing multienzyme complexes for spatial clustering of biosynthetic pathway enzymes in recombinant E. coli using the self-assembling peptide tags SpyTag and SpyCatcher to increase the efficiency and yield of D-tagatose production (Figure 5B). Specifically, the three-armed star-shaped protein was made by constructing the plasmid pETDuet-ppk-SpyTag-pfkb-SpyCatcher-fbaA ligating the end-functionalized protein SpyTag-PPK and the internally functionalized protein FRK-SpyCatcher-FbaA. The plasmid was transformed into the engineered E coli-∆pfkA, which was named Ec-7, and the recombinant protein was validated using SDS-PAGE (Figure 5C). The actual molecular weight of the multienzyme complex formed by SpyTag and SpyCatcher technology far exceeded the theoretical molecular weight, which had not been reported in previous studies and was likely due to protein aggregation. The proteins extracted from the labeled band were further subjected to an LC-MS/MS analysis to ensure the formation of the self-assembling protein (see Supplementary Materials).

Under the same reaction conditions as above, the D-tagatose yield of Ec-7 reached 22.6 g/L with a conversion rate of 75%, and the residual D-fructose was further reduced to 6.4 g/L. The catalytic level of the multienzymes complex increased to 1.74 U/OD_600, which was 45% higher than Ec-2-Δp (Figure 4D). The multienzymes complex in the self-assembly system has higher catalytic efficiency and product yield, which is mainly achieved by aggregating pathway enzymes together, effectively shortening the substrate diffusion distance, enhancing the synergistic effect of the cascade reaction, improving substrate utilization efficiency and catalytic rate and thus improving the conversion rate of D-tagatose [45].

3.6. Improving D-Tagatose Production Using the Fed-Batch Strategy

To further enhance the application potential of the multienzyme cascade, we increased the initial concentration of D-fructose to 100 g/L and maintained the reaction under optimal conditions. Since high concentrations of polyP6 will form precipitation with Mg²⁺, we chose to add equal amounts of sodium hexametaphosphate in batches at the beginning, after 6 h and after 12 h. However, the conversion rate of D-tagatose was found to be 46.2% when the fructose concentration was increased to 100 g/L, which still needs further improvement. We speculated that this was due to the change in pH conditions during the reaction or the inactivation of the enzyme [46]. In this study, pH control and enzyme stability were investigated during the reaction to further improve the yield of D-tagatose.

To verify the first possibility, we measured the pH of the reaction system during the reaction, and the results showed that the pH gradually decreased to 6.5 in the later stage. Although the acidic substances produced after the hydrolysis of polyP6 changed the pH, this did not significantly reduce the enzyme activity. Subsequently, whole-cell activity was measured at different time intervals at 40 °C, and the catalytic activity dropped significantly to 30% of the maximum activity after 12 h (Figure 6A), indicating that the low conversion rate was due to the instability of the enzyme.

To solve the problem of whole-cell catalyst deactivation, a fed-batch strategy was adopted, and 15 g/L of wet cells were added at 6 h and 12 h. By adopting the feeding strategy, sodium hexametaphosphate is effectively prevented from combining with magnesium ions to produce precipitation, thereby avoiding the reduction in enzyme activity. At the same time, fresh enzymes are added regularly to compensate for the decrease in catalytic efficiency caused by enzyme inactivation, thereby maintaining high catalytic efficiency and increasing the final yield of D-tagatose. Finally, the concentration of D-tagatose in the reaction system increased from 46.2 g/L at 24 h to 68.1 g/L, and the conversion rate was 68%.

Finally, we used an initial 100 g/L of D-fructose, 40 mmol/L ATP and a strain of Ec-7 with an OD₆₀₀ of 50 for one-pot production of D-tagatose. During the reaction, we optimized the reaction process and improved the overall conversion rate by adding sodium hexametaphosphate and fresh cells in batches. After 24 h of reaction, phytase was added for dephosphorylation. The final yield of D-tagatose reached 68.1 g/L, with a conversion rate of 68%.

4. Conclusions

In this work, a multienzymatic cascade reaction with Escherichia coli-based was successfully constructed for the biosynthesis of D-tagatose from D-fructose. The expression of the enzymes was optimized by transcriptional fine-tuning, and Ec-2 was obtained to produce 12.3 g/L D-tagatose. The gene pfka was knocked out to reduce bypass metabolic, and Ec-2-Δp could further produce 15.6 g/L of D-tagatose, although their growth was affected. Eventually, the catalytic level of the multienzyme system E. coli-7 was further enhanced by a multienzyme self-assembly system with SpyTag and SpyCatcher. Under optimized conversion conditions, D-tagatose titers and conversions of 22.6 g/L and 75% were achieved with 30 g/L D-fructose. The highest yield of 68.1 g/L D-tagatose was obtained using a fed-batch strategy under the optimized reaction conditions. Compared with traditional chemical methods and other enzymatic methods, this study not only simplified the operation process by using whole-cell catalysis, avoiding the steps of cell disruption and enzyme purification but also reduced costs by introducing an ATP regeneration system, showing higher economic benefits. Compared with the traditional arabinose isomerase method using lactose and galactose as substrates, this study selected cheaper and more readily available fructose as the reaction substrate, and the reaction conditions were milder, further improving the reaction efficiency and reducing energy consumption, which makes this method have great potential and advantages in industrial applications. However, the current production of D-tagatose is mainly limited by low enzyme activity and poor stability, as well as the lack of highly specific tagatose-6-phosphate phosphatases. To achieve the large-scale industrial application of D-tagatose, on the one hand, key enzymes can be modified through protein engineering to improve their catalytic efficiency and reduce substrate competition. On the other hand, enzyme immobilization technology can be used to improve the stability of the enzyme, making it tolerant to pH and temperature changes while facilitating recovery and reuse, thereby reducing production costs and improving conversion efficiency. Moreover, cheaper dextrin starch can be used as a substrate to further reduce costs. This study provides new ideas and insights into the production of rare sugars.