China is currently facing severe public health challenges. The Report on Chinese Residents' Nutrition and Chronic Disease Status released by the National Health Commission reveals that the number of overweight and obese individuals nationwide has exceeded 600 million, accounting for over 42% of the total population. Behind this statistic lies a significant risk of disease: the prevalence of diabetes among obese individuals is 4.3 times higher than that among those with normal weight, and the risk of cardiovascular diseases increases by 2.8 times. This directly contributes to chronic disease medical expenses making up more than 70% of total health expenditures. Against this backdrop, the widespread adoption of sugar substitutes is seen as a key solution—driven by policy initiatives, the domestic market has experienced explosive growth, with natural sweetener consumption surging from 5,500 tons in 2018 to 41,000 tons in 2020, an increase of 173%.

However, sugar substitutes are not a panacea. Recent studies indicate that excessive intake of artificial sweeteners may lead to gut dysbiosis and decreased insulin sensitivity, while natural alternatives like erythritol have been linked to an increased risk of thrombosis. This health paradox has spurred the development of a new generation of solutions: sweet-tasting proteins.

Sweet-tasting proteins, such as thaumatin, have become a focal point due to their revolutionary advantages: they can be up to 3,000 times sweeter than sucrose, contain zero calories, and do not cause blood sugar spikes. In animal studies, they have even shown potential to suppress appetite and improve metabolic syndrome. Nevertheless, industrial production faces three major bottlenecks: First, inefficient extraction. Producing one kilogram of pure thaumatin requires 10 tons of raw material from the African katemfe fruit, resulting in high costs. Second, barriers in biosynthesis technology. Although genetically engineered strains can express sweet-tasting proteins, the misfolding rate exceeds 60%, and the fermentation process is vulnerable to microbial contamination. Third, stability issues. Sweet-tasting proteins are prone to inactivation in acidic environments or during high-temperature sterilization, with a half-life less than 30% of that of traditional sweeteners. These bottlenecks limit the current global production of sweet-tasting proteins to only 200 tons per year, meeting less than 0.1% of market demand.

The core drivers propelling the sugar substitute industry span four dimensions:

• Health Needs: With 140 million diabetic patients in China, the WHO emphasizes that reducing added sugar intake to 25 grams per day can lower obesity-related disease risks by 26%. This compels consumers to turn to sugar substitutes, though safety concerns over traditional artificial sweeteners are accelerating the shift toward natural and protein-based alternatives.
• Policy and Regulation: Globally, 58 countries have implemented "sugar taxes". China’s Healthy China 2030 Initiative mandates a 30% sugar reduction by food enterprises, and the newly revised Standards for the Use of Food Additives (2024) includes novel sugar substitutes in the approved list, providing a regulatory pathway for technological innovation.
• Market Expansion: The global sugar substitute market is projected to grow at an annual compound rate of 11.2%, reaching $24.6 billion by 2025. China’s market growth is even more explosive, with the low-sugar food industry expected to exceed ¥800 billion by 2030.
• Technological Breakthroughs: Synthetic biology is tackling the industrial challenges of sweet-tasting proteins. Companies like Weiyuan Synthesis plan to build thousand-ton-scale smart fermentation facilities, aiming to reduce costs below $300 per kilogram.

Despite representing a healthier sweetening solution, the industrialization of sweet-tasting proteins remains constrained by complex production processes and high costs. Current breakthroughs rely on interdisciplinary innovations: gene editing to optimize expression efficiency, continuous-flow bioreactors to enhance production capacity, and food colloid technology to improve stability. If an 80% reduction in technical costs can be achieved within the next five years, sweet-tasting proteins could replace 30% of the artificial sweetener market, becoming a strategic weapon in combating the obesity crisis.

This experiment is a systematic, multi-stage molecular biology project. Its core objective is to construct recombinant plasmids containing the target genes PDI and Thaumatin isoforms A/B/C/D, transform them into different E. coli host strains (DH5α, BL21(DE3), Origami 2(DE3)) for expression, and subsequently purify the expressed recombinant protein Thaumatin followed by preliminary enzymatic activity assay for β-1,3-glucanase. The entire process has a clear logic, with stages closely linked, covering the core techniques of molecular cloning.

Stage 1:

Basic Preparation and Primary Vector Construction: The starting point involves preparing basic materials and acquiring target genes. LB liquid and solid media were repeatedly prepared and autoclaved to ensure sufficient supply for subsequent bacterial culture, transformation, and plating. Reagents required for PCR, agarose gel electrophoresis, and SDS-PAGE were also prepared in advance.

PCR Amplification and Vector Preparation: The PDI gene and the four Thaumatin gene fragments (Thaum-A/B/C/D) were amplified separately using PCR. A versatile pre-mixed PCR Master Mix was used with a standard three-step cycling program. Concurrently, the original empty vector pRSFDuet (Kanamycin resistant) was cultured in a shaking incubator for activation, preparing it for plasmid extraction. High-purity plasmid DNA was extracted from the cultured pRSFDuet bacteria using a spin-column based kit. The extracted pRSFDuet plasmid was linearized via double digestion.

Vector Linearization Verification and Purification: Successful double digestion was verified by 1% agarose gel electrophoresis. The target linearized vector fragment was quickly and precisely excised under UV light and purified using a gel extraction kit.

Homologous Recombination Ligation (Primary Vector): The purified linearized pRSFDuet vector and the amplified PDI gene fragment were joined via seamless cloning using a 2× recombinase at 50°C for 15 minutes, forming the recombinant plasmid pRSFDuet-PDI.

Heat-Shock Transformation and Screening (Primary Vector): The recombinant pRSFDuet-PDI product was transformed into chemically competent E. coli DH5α cells. The transformed cells were spread onto pre-prepared LB solid plates containing Kanamycin antibiotic (100 μg/ml) and incubated at 37°C overnight.

Colony PCR Screening (Primary Vector): Single colonies were picked from the Kan+ plates and used directly as templates for colony PCR. Specific primers for the PDI gene were used. PCR products were analyzed by agarose gel electrophoresis. Positive clones were expected to show a band of the predicted size for PDI. Colonies identified as positive for pRSFDuet-PDI were subjected to small-scale expansion culture in a shaking incubator to provide bacterial culture for the next stage of secondary vector construction.

Stage 2:

Secondary Vector Construction and Protein Expression Preparation: Building upon the successful construction of pRSFDuet-PDI in stage 1, the Thaumatin gene isoforms were cloned into this vector. The recombinant plasmid pRSFDuet-PDI was re-extracted from the positive culture obtained in the previous stage using a plasmid extraction kit. The extracted pRSFDuet-PDI plasmid was linearized via double digestion using the restriction enzymes Xho I and Nde I, creating a new linearized vector ready for insertion of the Thaumatin genes. The digestion setup, verification, and fragment recovery proceeded similarly.

Construction of pRSFDuet-PDI-ThaumA/B/C/D via Homologous Recombination: The purified linearized pRSFDuet-PDI vector was individually ligated with each of the four amplified Thaumatin (A/B/C/D) gene fragments using a 2× CE recombinase at 50°C for 30 minutes, forming the final four sets of recombinant plasmids: pRSFDuet-PDI-ThaumA/B/C/D.

Transformation into Host Strains: The four recombinant products (pRSFDuet-PDI-ThaumA/B/C/D) were separately transformed into two types of competent cells: the cloning host E. coli DH5α and the expression host E. coli BL21(DE3) (which carries the T7 RNA polymerase gene for subsequent IPTG-induced expression). Transformation, plating (Kan+ plates), and culture followed similar procedures as before.

Colony PCR Screening (Secondary Vectors): Colonies of pRSFDuet-PDI-ThaumA/B/C/D growing on transformation plates for both DH5α and BL21(DE3) were again screened using colony PCR. Positive clones were confirmed by agarose gel electrophoresis. Positive pRSFDuet-PDI-ThaumA/B/C/D strains identified in DH5α and Origami 2(DE3) were subjected to large-scale expansion culture in a shaking incubator, preparing them for subsequent plasmid extraction (from DH5α) or protein induction (in Origami 2(DE3)/BL21(DE3)).

Stage 3:

Protein Expression, Purification, and Analysis: The focus shifted to obtaining and validating the recombinant Thaumatin protein.

Plasmid Extraction and Verification: The final four recombinant plasmids were extracted from the expanded DH5α cultures (carrying pRSFDuet-PDI-ThaumA/B/C/D) using a plasmid extraction kit. Plasmid integrity was potentially verified by agarose gel electrophoresis and/or restriction digestion.

Large-Scale Culture for Induction: BL21(DE3) or Origami 2(DE3) strains carrying pRSFDuet-PDI-ThaumA/B/C/D were subjected to large-scale expansion culture to prepare sufficient cell mass for subsequent induction and expression.

Cell Harvest and Ultrasonic Lysis: Expression culture broth was harvested by centrifugation; the supernatant was discarded to obtain the cell pellet. The pellet was resuspended in PBS buffer. Cells were lysed under ice-bath conditions using an ultrasonic cell disruptor. After centrifugation, the supernatant was collected as the crude protein extract.

Ni-NTA Affinity Chromatography Purification: Utilizing the recombinant protein’s characteristics (His-Tag), purification was performed using Ni-NTA affinity chromatography. The crude protein extract was incubated with Ni-NTA resin at 4°C for 2 hours to allow His-tagged protein binding. A gravity flow column was used. The flow-through was collected after sample loading. The column was washed with wash buffer to remove non-specifically bound contaminating proteins. Bound protein was eluted using elution buffer, and the eluate was collected. This constituted the preliminary purified Thaumatin protein.

SDS-PAGE Analysis: Purified protein samples were mixed with Loading Buffer and boiled for denaturation. They were then separated by SDS-PAGE electrophoresis. After electrophoresis, gels were stained with Coomassie Brilliant Blue, followed by destaining solution overnight. The target protein band's size and purity were observed to assess purification efficiency and confirm the expected molecular weight.

Stage 4:

Enzymatic Activity Assay: The purified recombinant Thaumatin protein was functionally validated by assaying its β-1,3-glucanase activity.

Reaction Setup: In sodium phosphate-citrate buffer (pH 6.0), using 1% Laminarin as the substrate, an appropriate dilution of the enzyme solution was added.

Incubation: The reaction mixture was incubated at 40°C for 20 minutes.

Reaction Termination and Color Development: The reaction was stopped by adding DNS reagent and mixing thoroughly, followed by boiling in a water bath for 10 minutes to develop color.

Detection: After centrifugation to remove precipitate, the absorbance of the supernatant was measured at a wavelength of 540 nm.

Our project outcomes could be developed into a variety of product categories, meeting people’s health needs from multiple dimensions and providing tangible support for the general public to maintain good health. The products planned for future sustainable development are listed in the table below.