The number of people suffering from rectal cancer is now increasing worldwide, accounting for 10.7% of the world's total population【1】，we decided to design myrosinase to convert thioglucoside into radicicol, and then use radicol to inhibit the cancer cells. Sulforaphane inhibits cancer cells through cell cycle arrest (G2/M phase), upregulation of pro-apoptotic factors (such as caspase 8, p21, p53, and Bax), and downregulation of anti-apoptotic factors (such as Bcl-2 and Hsp90).

Figure1  The design of the myrosinase.
Firstly, the IL gene was cloned into the pET28a vector. The ligation product was transformed into E. coli Rosetta (DE3) competent cells and sequenced for verification. The engineered E. coli Rosetta strain was cultured in LB medium containing 50 μg/mL kanamycin. Once the bacterial growth reached OD600 = 0.6, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression, followed by overnight incubation at 16°C to minimize protein aggregation and inclusion body formation. The bacterial pellet was then harvested by centrifugation at 10,000 g for 1 min. The pellet was resuspended in 20 mM Tris-HCl buffer (pH 7.4). Cell lysis was achieved using sonication (500W, 1s sonication, 3s  interval, 20 min) under ice bath conditions. The lysate was then centrifuged at 12,000 rpm for 30 minutes at 4°C to obtain the soluble protein supernatant. This supernatant was filtered through a 0.45μm filter to remove cell debris and large particles. The protein was purified using a nickel column (HisTrapTM HP, Ge HealthCare, 5 mL) and concentrated using ultrafiltration tubes (Millipore, 10 KD) and stored at -80°C for future use.
We determined the enzyme activity of myrosinase and found that the Km value of myrosinase was 84.15 μM and the Vmax was 15.51 μM/μM protein at 37°C and pH 7.4 (Figure.2). Also, we found that the temperature of 37 degrees Celsius and pH 7.4 were the best reaction conditions for myrosinase (Figure.3).               

Figure2   Gel image of myrosinase.

Figure3  The result of expression of myrosinase. (a,Graph of myrosinase Michaelis-Menten Equation. b and c, Optimal Reaction Conditions Testing for myrosinase.)

To characterize the enzyme activity, we used GraphPad Prism software to fit the Michaelis-Menten equation (Figure.1) to determine the maximum rate (Vmax) and Km value of myrosinase reaction. The results showed that the Km value of myrosinase was 84.15 μM and Vmax was 15.51 μM/μM protein at 37°C and pH 7.4.
What's more, we prepared reaction buffers (with a fixed substrate concentration of 100 μM) by preparing the reaction buffers (with a fixed substrate concentration of 100 μM) at different pH values (6.3, 7.4, and 8.2) and at different reaction temperatures (25 °C, 37 °C, and 42 °C) to test the best reaction conditions of the enzyme (Figure.3), and the results showed that the temperature of 37 degrees Celsius and pH 7.4 were the best reaction conditions for myrosinase.

Curcumin is a bright yellow chemical produced by plants of the curcuma longa species. It is often used as a condiment in food or traditional herbs, and it has attracted lots of attention by researchers in the aspect of cancer treatment. However, considering that the taste of ginger is hardly acceptable by many and even if it is accepted, the intake of ginger as a seasoning is low. Thus, we decided to design a system using Diketide-CoA synthase (DCS) and Curcumin synthase (CURS) genes which helps to catalyze the production of curcumin

Figure1  The design of the DCS and CURS.
Bacteria transformed with DCS and CURS were cultured in LB medium containing appropriate antibiotics.   When OD600 reached 0.6-0.8, 0.5 mM IPTG was added to induce protein expression.   The bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4).   Crude enzyme solution was obtained by sonication under ice bath conditions.   Protein concentration was determined using the Bradford assay kit.   For analysis of curcumin production, a reaction was conducted in 200 μL 100 mM PBS (pH 7.4) containing 100 μM feruloyl-CoA (Yuanye, Shanghai, S27603), 100 μM malonyl-CoA (Merck, 63410), and 40 mg/L crude enzyme solution.   After a 1-hour reaction at 37°C, the samples were extracted with ethyl acetate and the organic layer was collected, dried, and resuspended in methanol.   The wavelength of 420 nm, which is the maximum absorption wavelength of curcumin, was set on a spectrophotometer, and methanol was used as a blank for baseline correction.   The sample solution was placed in a cuvette and its absorbance was measured.   Finally, the curcumin concentration of the sample was calculated based on its absorbance using a standard curve prepared with known concentrations of curcumin standard as shown in Figure 3b and 3c.The Figure3A is principle schematic diagram of DCS and CURS.The results showed a significant increase in the production of curcumin by the engineered strains.

Figure2   Gel image of DCS and CURS.

Figure3  The result of expression of DCS and CURS.

Because we  need to achieve sufficient efficacy without causing harm to Escherichia coli (E. coli), we have decided to design a targeted suicide gene strategy focused on the bacterial cell wall. The suicide gene system we are utilizing comprises a series of linked genes known as SRRz. The products of the R and RZ genes within this system are primarily responsible for degrading the bacterial cell wall. Furthermore, since this gene chain exclusively targets cell wall degradation, it poses no harm to human cells, thereby aligning with biological safety requirements.

Figure2   Gel image of Tcl42-pR-pL and SRRz.

Figure3  The result of expression of Tcl42 and SRRz.

To validate the functionality of the Tcl42 temperature-sensitive promoter, we first coupled it upstream of the mRFP reporter gene and cloned it into the pSB1A3 plasmid. Subsequently, we transformed the recombinant plasmid into E. coli DH5α.    The transformed strains were cultured at 37°C and 42°C for 12 hours. We measured the initial mRFP fluorescence intensity at these temperatures and normalized it based on OD600 to evaluate the activity of the Tcl42 promoter.As shown in Figure 3A~D, the Tcl42 temperature-sensitive promoter exhibits the best expression at 42°C.

We placed Tcl42 upstream of the bacterial lysis gene SRRz. Firstly, we cloned the Tcl42 promoter together with the SRRz gene into the pSB1A3 plasmid. Subsequently, we transformed this recombinant plasmid into E. coli DH5α bacteria using a heat shock method and selected it on LB agar plates containing 100 µg/mL Ampicillin.To assess the expression of the SRRz gene at different temperatures, we cultured the transformed bacteria at 25°C, 37°C, and 42°C for 12 hours, measuring their OD600 values.  Additionally, we included wild-type DH5α and DH5α carrying only the pTcl42 pSB1A3 plasmid as controls.  We observed in the results from Figure 3E that the bacterial survival rate under the Tcl42-SRRz condition was the lowest,approximately five times lower than the other two conditions.
Furthermore，to assess the effectiveness of the Tcl42 promoter in driving the SRRz gene at 42°C, we cultured the samples in a 42°C shaking incubator for 12 hours.             Every 2 hours, we took out 500µL samples and measured their OD600 values to evaluate bacterial growth.  Under the same conditions, we also cultured DH5α strains carrying only the pTcl42 pSB1A3 plasmid and wild-type DH5α as controls.（figure3F）The results indicate that under the influence of the pTcl42 pSB1A3 plasmid-carrying DH5α and wild-type DH5α, bacterial survival rates range from 0.4  to 0.5 .             However, under the Tcl42-SRRz condition, the survival rate drops significantly to 0.1  which indicate Tcl42-SRRz is the most efficient choice.

1. In vitro anti-cancer activity and time dependency of myrosinase
CT26 colon cancer cell line in mice was seeded at a density of 5000 cells per well in a 24-well plate. The complete culture medium consisted of DMEM basal medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were cultured at 37°C in a 5% CO2 environment until they reached 60-70% confluency. Myrosinase produced by the engineered bacterial strain was incubated with 100 μM sinigrin (black mustard glycoside). Samples were taken at 3, 24, and 72 hours after treatment, and cell viability was assessed using the CCK8 assay. An aliquot of 100 µL of CCK8 solution was added to each well and incubated at 37°C for 3 hours. The absorbance at 450 nm was measured using an Microplate reader. The average absorbance and standard deviation of each group were calculated, and data analysis and plotting were performed using GraphPad Prism. One-way ANOVA was used to analyze the statistical differences in the data, followed by Tukey's post hoc test. A P-value less than 0.05 was considered statistically significant. The results, as shown in Figure 4AB, indicated a significant decrease in CT26 cell viability after co-expression of myrosinase and sinigrin, demonstrating significant in vitro anti-cancer activity of myrosinase that persisted within 72 hours.

2. In vitro anti-cancer activity of curcumin
Due to the poor solubility of curcumin in water, a stock solution of 10 mM was prepared using DMSO (dimethyl sulfoxide) as an organic solvent. Specifically, under sterile conditions, an appropriate amount of curcumin powder was added to either DMSO or ethanol and thoroughly mixed until completely dissolved. The solution was then aseptically filtered through a 0.22 μm membrane and aliquoted into sterile 1.5 ml centrifuge tubes, stored at -20°C to maintain stability. To test the effect of curcumin on the CT26 colon cancer cell line in mice, CT26 cells were seeded at a density of 5000 cells per well in a 24-well plate. The complete culture medium consisted of DMEM basal medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were cultured at 37°C in a 5% CO2 environment until they reached 60-70% confluency. Curcumin at concentrations of 0, 5, and 10 μM (Solarbio, C7090, China) was used. Cell viability was assessed using the CCK8 assay at 24 hours after treatment. An aliquot of 100 µL of CCK8 solution was added to each well and incubated at 37°C for 3 hours. The absorbance at 450 nm was measured using an Microplate reader. The results, as shown in Figure 4C, indicated a significant decrease in CT26 cell viability after treatment with a curcumin concentration of 10 μM, demonstrating significant in vitro anti-cancer activity of curcumin.

 Figure4      The results about in vitro anti-cancer activity of myrosinase and curcumin

【1】 Worldwide cancer data | World Cancer Research Fund International (wcrf.org)