According to the International Diabetes Federation (IDF), approximately 366 million people were living with diabetes worldwide in 2011. By 2021, this number had increased to 537 million, and it is projected to reach 643 million by 2030 and 783 million by 2045. Consequently, addressing the growing prevalence of diabetes has become a critical global challenge.
Figure 1 Distribution of Diabetes Patients (Figure Source IDF official website)
Figure 2 Ranking of the number of people with diabetes in different countries (figure source IDF official website)
Peripheral insulin resistance and gradual pancreatic β-cell failure are the primary causes of type II diabetes. As a result, there is an urgent need to identify a molecule that not only maintains blood glucose levels within a normal range but also slows the progression of diabetes by restoring β-cell function, reducing cardiovascular risk factors, and aiding weight management. D-tagatose, a functional sugar, emerges as a promising therapeutic molecule for type II diabetes. It is approximately 90% as sweet as sucrose but contains only one-third the calories, making it an ideal sucrose substitute. Tagatose has a wide range of applications in both the pharmaceutical and food industries.
Fructose, as an isomer of tagatose, is inexpensive and stable, making it an ideal substrate for tagatose production through the action of tagatose 4-epimerase. However, reports of naturally occurring tagatose 4-epimerases that catalyze the synthesis of tagatose from fructose are scarce. Currently, the production of tagatose from fructose is hindered by limited enzyme sources and low conversion rates. For instance, the conversion rate of tagatose from D-fructose using tagatose 4-epimerase from Thermotogae bacterium was only 14% when using 200 g/L of D-fructose, which falls short of industrial production standards and presents significant opportunities for improvement. Therefore, our goal is to identify new tagatose 4-epimerases and enhance their catalytic activity.
Identify four proteins of unknown function with potential tagatose 4-epimerase activity through enzyme mining.
Improve the soluble expression of the enzymes by optimizing molecular chaperones, pro-fusion labeling, fermentation conditions, and growth media.
Employ targeted mutagenesis to further increase the activity of tagatose 4-epimerase. Characterize the enzymatic properties and kinetic parameters of the most effective mutant strain.
Develop a green fluorescent biosensor based on tagatose specificity to facilitate high-throughput screening of tagatose isomerase directed evolution.
The discovery of new tagatose 4-epimerases is crucial for the industrial production of tagatose. The sequence of the hypothetical protein UxaE derived from T. petrophila was used as a template for BLASTp searches in NCBI's non-redundant protein sequence database. A phylogenetic tree was constructed based on the search results, and the sequences were structurally modeled using AlphaFold2. Binding free energy calculations of receptor-ligand complexes were performed using CHARMM by Discovery Studio. Combining docking free energy, phylogenetic branching, and source information, four candidate tagatose isomerase sequences were selected and named AJC7, TET, MBC, and HDM. The wild-type enzyme activities were initially assessed using high-performance liquid chromatography (HPLC), with AJC7 exhibiting the highest activity.
E. coli is a commonly used host for protein expression; however, the expression of exogenous genes, particularly those from extremophilic microorganisms, often results in the formation of inclusion bodies. This complicates the study of their enzymatic properties and subsequent applications. Inclusion body formation is primarily due to incorrect protein folding. To address this issue, we utilized E. coli BL21 (DE3) as the host and wild-type AJC7 as the base strain. We implemented fusion tags, molecular chaperones, and fermentation optimization strategies to resolve the inclusion body problem associated with tagatose 4-epimerase expression, providing an effective approach for the efficient soluble expression of AJC7.
Figure 3 Inclusion body formation schematic
Fusion tags are well-characterized proteins or peptides that can be attached to a target protein to enhance its solubility and facilitate expression. Certain fusion tags promote soluble expression by binding to and preventing the aggregation of folding intermediates. The DNA sequence of the target protein is linked to the fusion tag sequence via a linker, resulting in the formation of a recombinant plasmid, as illustrated in Figure 4.
Figure 4 Mapping of fusion marker plasmid constructs
Based on this approach, the following fusion tags were selected for integration into target proteins to address the issue of inclusion body formation:
1.Thioredoxin A (TrxA)[3]: This tag reduces disulfide bonding by displacing thiodisulfides. Its key features include thermal stability, robust folding properties, and exceptional solubilization efficiency.
2.Transcription Anti-Termination Factor A (NusA) [4]: NusA is a highly soluble protein that binds to and separates aggregation-prone folding intermediates, thereby preventing their self-association and aggregation.
3.Maltose-Binding Protein (MBP)[5]: MBP is known for its broad solubility profile and high pro-solubilization efficiency, making it an effective choice for enhancing protein expression.
Molecular chaperones are a class of proteins that, despite lacking sequence similarity, share a common function: they assist in the proper assembly of polypeptide structures within the cell. Once assembly is complete, chaperones detach and do not become part of the final protein complexes. Co-expression with molecular chaperones has been shown to effectively reduce the misfolding of polypeptide chains and enhance the soluble expression of proteins.
For our study, we selected the Chaperone Plasmid Set, which includes the molecular chaperones pG-KJE8, pGro7, pKJE7, pG-Tf2, and pTf16. These chaperone plasmids were co-expressed alongside the pET-28a(+)-AJC7 plasmid in E. coli to establish a dual plasmid co-expression system. The synergistic effects of the chaperones facilitated improved protein folding and increased the recovery of soluble proteins.
Various fermentation temperatures, inducer concentrations, and media types can significantly impact protein solubility during expression. Therefore, the optimization of fermentation conditions for AJC7 was approached from three key aspects.
Induction temperature plays a crucial role in determining the expression level, folding quality, and growth status of the target protein. Low-temperature induction can reduce the likelihood of misfolding and inclusion body formation in E. coli. Although low temperatures may slow cell growth, they generally minimize stress on the cells, which can be beneficial for prolonged target protein expression. Conversely, high-temperature induction often accelerates cell growth and protein expression but may increase the risk of inclusion body formation due to rapid expression rates. To balance these factors, optimal temperatures for bacterial growth and enzyme synthesis were evaluated by inducing at 16°C, 20°C, 30°C, and 37°C, while keeping other factors, such as bacterial density at induction and IPTG concentration, constant.
The concentration of isopropylthiogalactoside (IPTG) directly influences gene expression levels. Typically, lower IPTG concentrations may only partially induce target gene expression, while higher concentrations often lead to greater expression levels. However, excessively high IPTG concentrations can negatively affect cell health and growth. Elevated IPTG levels may also result in overexpression of target proteins, increasing the likelihood of inclusion body formation, which can compromise protein folding and solubility. Therefore, final IPTG concentrations of 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM were tested in shake flasks to determine the optimal concentration for maximizing gene expression.
The formation of inclusion bodies is believed to be influenced by the culture conditions of the host bacteria, including media composition. In a self-inducing medium, the host bacterium utilizes glucose as a carbon source, allowing for sustained growth until the logarithmic phase. After this phase, the bacteria switch to lactose to induce the expression of target genes via the lac operon system. This approach not only mitigates the cytotoxic effects on target proteins but also reduces the risk of contamination by adding the inducer during the initial preparation of the medium. Additionally, this method enhances the expression of soluble proteins.
To optimize this process, we designed 16 sets of orthogonal experiments with various combinations of carbon sources. The results were analyzed using polar analysis to determine the optimal combination for maximizing soluble protein expression.
Among the four enzymes screened through gene mining—AJC7, MBC, TET, and HDM—the wild-type AJC7 exhibited the highest activity; however, its catalytic efficiency has not yet reached the desired level. Targeted mutagenesis has been widely reported as an effective approach to enhance enzyme activity. This study aimed to improve AJC7’s ability to catalyze the conversion of fructose to tagatose by identifying effective mutation sites.
Based on an analysis of AJC7’s structural features and reaction mechanism, five potential mutation sites were identified for iterative mutagenesis, drawing from relevant literature. The resulting mutant plasmids were introduced into E. coli BL21 (DE3) cells for induced expression. After induction, the cells were lysed using ultrasonication, and the protein was extracted and purified. The purified enzyme was then reacted with fructose, and the products were analyzed by high-performance liquid chromatography (HPLC). The results were compared to those obtained from wild-type AJC7 to identify the mutant strain with the highest enzyme activity. Additionally, the enzymatic properties and kinetic parameters of the most effective mutant were determined.
The formation of inclusion bodies is believed to be influenced by the culture conditions of the host bacteria, including media composition. In a self-inducing medium, the host bacterium utilizes glucose as a carbon source, allowing for sustained growth until the logarithmic phase. After this phase, the bacteria switch to lactose to induce the expression of target genes via the lac operon system. This approach not only mitigates the cytotoxic effects on target proteins but also reduces the risk of contamination by adding the inducer during the initial preparation of the medium. Additionally, this method enhances the expression of soluble proteins.
Rational design can yield well-characterized mutant enzymes, but it is often constrained by the current knowledge of the enzyme, which limits further performance enhancements. Directed evolution offers a way to mutate enzymes without such constraints; however, it requires high-throughput screening methods to identify optimal mutants. Therefore, developing effective screening techniques is essential to accelerate directed evolution and obtain high-performance enzymes targeting tagatose.To facilitate this, we constructed a recombinant plasmid containing a tagatose-specific promoter, a reporter protein, and a fluorescent protein. This design enables the plasmid to specifically recognize tagatose and express green fluorescence. E. coli Nissle 1917 (EcN) has been shown to utilize tagatose as a carbon source and contains a cluster of genes involved in tagatose utilization, including tagatose transporter proteins and tagatose-responsive transcription factors.Leveraging this characteristic, we assembled the EcN Tagose system using tagatose-responsive transcription factors and promoters, along with green fluorescent proteins, to create tagatose-responsive biosensors. These constructs were transferred into EcN strains. Different concentrations of tagatose solution were introduced to induce fluorescence expression under varying temperatures. Control and parallel experiments were conducted to validate the biological feasibility of the biosensor. The goal was to assist in the directed evolution of tagatose 4-epimerase by facilitating efficient screening of mutants.
Figure 5 Green fluorescent biosensor based on tagatose specificity
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