Flavocytochrome c Sulfide Dehydrogenase (FCSD) is a heterodimeric bacterial enzyme exclusively present in purple sulfur phototrophs such as Allochromatium vinosum. It oxidizes toxic H₂S (H₂S) to elemental sulfur (S⁰), utilizing cytochrome c as its sole electron acceptor. The enzyme comprises a flavoprotein subunit with covalently-bound FAD and a cytochrome c subunit housing heme groups, associating peripherally with membranes via hydrophobic interactions. Functionally, FCSD channels electrons directly to cytochrome c to drive anoxygenic photosynthesis while converting H₂S into inert sulfur globules for cellular storage. This mechanistically distinct enzyme is evolutionarily conserved solely in sulfur-oxidizing bacteria and is absent in mitochondria, fungi, or animals.

Figure 1. The Map of the FCSD Gene

Cultivation, Purification, and SDS-PAGE

To achieve the targeted cloning of the FCSD fragment into the pET-28a expression vector, a pair of gene-specific primers was designed. Subsequently, the FCSD fragment was amplified via polymerase chain reaction (PCR) using these primers, yielding an insert DNA compatible with the linearized vector ends. The complete nucleotide sequences of the oligonucleotide primers used in this study are detailed in Table 2.

Table 2. The sequence of the primers

Subsequently, we successfully amplified the coding region of the FCSD gene using PCR technology. Agarose gel electrophoresis confirmed the PCR product size was approximately 1293 bp, consistent with the expected length. Subsequently, we purified the specific amplification band using a gel extraction kit, successfully obtaining a high-purity FCSD gene fragment. This prepared the fragment for subsequent cloning into a linearized vector via homologous recombination.

Figure 2. The Map of FCSD Electrophoresis Results

Construction Design:

In order to construct the recombinant plasmid pET28a-SQR-His, we used homologous recombination to insert the SQR gene, which codes for an eukaryotic enzyme that breaks down H₂S into the vector pET28a. SQR catalyzes the degradation of H₂S and thereby reduces H₂S levels. In our project, an orally delivered SQR capsule would act in the intestinal environment to degrade excess H₂S produced by microbes. This reduction would restore GLP-1 activity, which can promote fat metabolism and suppress appetite.

This plasmid includes several key units to ensure successful and efficient expression and purification. The backbone includes a kanamycin resistance gene (KanR) for antibiotic selection, as well as LacI and the Lac operator for IPTG-induced expression, and a His-tag attached to a T7 promoter to enable protein purification and initiation of transcription.

By integrating the SQR gene into this plasmid, we created a system capable of producing functional SQR enzyme in E.coli. Once the protein is expressed and purified, this enzyme would degrade excess H₂S in the gut, therefore lowering H₂S levels and reactivating the GLP-1 signaling pathway, which is a key regulator of appetite and fat metabolism. This offers a potential therapy for obesity and type II diabetes.

Figure 3. The plasmid map of pET28a-SQR-His

Experimental Approach:

We successfully amplified the SQR gene sequence using PCR technology. The amplified product was verified by agarose gel electrophoresis, as shown in Figure 4A. A clear, specific band was observed at the expected size position, with no non-specific amplification products or primer dimers, confirming successful amplification of the SQR gene. Simultaneously, to construct the recombinant vector, the pET28a empty vector was double-digested with XhoI and BhoI restriction enzymes for linearization. The digestion products were analyzed by agarose gel electrophoresis (Figure 4B). The results showed only a single, clear band, indicating complete linearization of the vector with no uncut or partially digested products remaining, confirming successful linearization. Building upon this, the purified SQR gene fragment was ligated with the linearized pET28a vector via homologous recombination, successfully inserting the SQR sequence into the vector backbone. This laid the foundation for subsequent recombinant plasmid construction and protein expression studies.

Figure 4. The gel electrophoresis validation of SQR and pET28a linear

Based on the above, the purified SQR gene fragment was ligated with the linearized pET28a vector via homologous recombination, successfully inserting the SQR sequence precisely into the vector backbone to construct a recombinant plasmid. This recombinant plasmid was designated pET28a-SQR-His and transformed into E.coli DH5α competent cells. The transformed bacterial suspension was spread onto LB plates containing the corresponding antibiotic. As shown in Figure 5A, uniformly distributed, well-formed single-colony colonies were observed on the plates, preliminarily indicating successful transformation. Subsequently, several single-colony colonies were randomly selected for further validation via colony PCR and sequencing. Colony PCR results showed distinct amplification bands (Figure 5B), preliminarily confirming that the recombinant plasmid had successfully transferred into the host bacteria and possessed amplification capability. Final sequencing results (Figure 5C) confirmed that the SQR gene sequence was correct and the insertion read-through was accurate, indicating the successful construction of the recombinant plasmid pET28a-SQR-His.

Figure 5. Map for pET28a-SQR-His Transformation Verification

For protein expression, the validated recombinant plasmid was transformed into E.coli BL21(DE3) competent cells. The transformed culture was spread onto LB solid plates containing kanamycin and incubated overnight at 37°C. The results are shown in Figure 6, where uniformly distributed single colonies are visible on the plates, indicating successful transformation.

Figure 6. pET28a-SQR-His Colony Map

Subsequently, we selected a single monoclonal colony and inoculated it into LB liquid medium. After incubating at 37°C with shaking until the logarithmic growth phase (OD600 ≈ 0.6–0.8), we added IPTG to a final concentration of 0.5 mM. Expression was then induced at 16°C for 16 hours to promote soluble protein formation. After induction, bacterial cells were harvested by centrifugation. Cells were lysed using an ultrasonic cell disruptor, and the supernatant was used. Using the histidine tag (His-tag) at the C-terminus of the SQR protein, we purified the protein via nickel affinity chromatography. Four key fractions were collected: crude lysate, flow-through, wash fraction, and elution fraction. Analysis of each fraction by SDS-PAGE gel electrophoresis yielded the results shown in Figure 7. A single, dense protein band appeared at the expected molecular weight (approximately 50 kDa) in the elution fraction, while this band was faint or absent in the crude lysate, flow-through, and wash fractions. Indicating that the target protein SQR-His successfully bound specifically to the nickel column and was efficiently eluted. This confirms the successful expression of the recombinant protein in the E.coli system and the effective purification achieved through the nickel affinity chromatography step. The collected material was used to obtain the final product.

Figure 7. The SDS-PAGE protein gel of samples from different purification stages

Function Test

1. ELISA assay

In this experiment, we employed the enzyme-linked immunosorbent assay (ELISA) to quantitatively analyze the content of sulfquinone reductase (SQR) in different eluate fractions. The experiment began by pre-coating SQR antibodies onto microplate wells. Standard samples, test samples, and HRP-labeled detection antibodies were sequentially added. After incubation and thorough washing, the TMB substrate was introduced for color development. Under peroxidase catalysis, TMB initially turned blue and then converted to yellow upon acid termination. The color intensity correlated positively with SQR concentration in the samples. Absorbance (OD value) was measured at 450 nm, and SQR concentrations in each sample were calculated using the standard curve.



Figure 8. Assaying the Standard Curve for ELISA

Results showed significantly higher SQR enzyme concentrations in the eluate fraction compared to other fractions, indicating this fraction primarily enriched the target protein. A good linear relationship was observed between standard concentrations and OD450 values, with a high standard curve fit, confirming reliable detection results.



Figure 9. ELISA assay showing enzyme concentration

2. DUQ assay

This study employed a spectrophotometric method based on DUQ reduction to evaluate SQR enzyme activity. The principle relies on SQR catalyzing the oxidation of sulfides and transferring electrons to the ubiquinone analog DUQ, resulting in DUQ reduction and a characteristic decrease in absorbance at 275 nm. By monitoring this absorbance change in real time, the enzymatic reaction rate can be quantitatively reflected.

Results showed that after adding SQR enzyme solution, the absorbance at 275 nm decreased significantly over time, indicating successful catalytic reduction of DUQ by the enzyme and confirming its high catalytic activity. Moreover, the established spectrophotometric method effectively and specifically detects SQR enzyme activity. The results clearly demonstrate the enzyme's high catalytic efficiency under the specified conditions.



Figure 10. spectrophotometric determination of SQR enzyme activity based on DUQ reduction

3. Lead Acetate Test Strip

To assess SQR activity visually, we used lead acetate test strips, which turn black when exposed to H₂S. For this purpose, Escherichia coli MG1655 was cultured in media containing different concentrations of L-cysteine (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) to induce H₂S production. The generation of H₂S was verified by the blackening of lead acetate paper strips. Simultaneously, we further analyzed H₂S yield using ImageJ. Results demonstrated that H₂S production progressively increased with rising L-cysteine concentrations.



Figure 11. Different L-cysteine derivatives induce varying amounts of H₂S production

When SQR was introduced into the testing system, a pronounced concentration-dependent response was observed: the color intensity of the test strip gradually weakened as the amount of added SQR increased. This visual change was further confirmed by grayscale analysis—grayscale values exhibited a corresponding decrease with rising enzyme concentration, verifying the quantitative reduction of H₂S content in the sample.

Notably, when the SQR concentration reached 10 mg/L, H₂S levels decreased significantly, approaching concentrations observed without L-cysteine induction. This result strongly demonstrates that the SQR enzyme efficiently and rapidly catalyzes H₂S degradation, highlighting its potential application in systems requiring H₂S control.



Figure 12. Reduction in greyscale of lead acetate test strips with increasing SQR concentration

4. Hardware Validation

We further employed sensor-based detection equipment to quantitatively validate the enzymatic activity of SQR in real time, enabling real-time quantitative monitoring of hydrogen sulfide (H₂S) concentration. In samples without enzyme treatment, sensor readings consistently remained at elevated levels (Figure 13A). Conversely, in the SQR-treated experimental group, sensor signals exhibited a significant decline (Figure 13B). This result clearly demonstrates that SQR efficiently catalyzes H₂S decomposition, confirming its strong hydrogen sulfide degradation capability.



Figure 13. H₂S concentration with corresponding sensor display

The results above demonstrate that pET28a-SQR-His is a novel functional construct capable of efficiently expressing active sulfquinone reductase (SQR). This construct exhibits excellent biological functionality and practicality, enabling efficient H₂S degradation. It provides a key technological and theoretical basis for subsequent indirect regulation of the GLP-1 signaling pathway.

5. GLP-1 Pathway Detection

To further investigate the effects of H₂S on intestinal endocrine cell function, we established the following in vitro model: the supernatant from L-cysteine-induced E.coli MG1655 was added to the NCI-H716 cell culture system. This supernatant, rich in H₂S produced by bacterial metabolism, significantly inhibited the synthesis and secretion of glucagon-like peptide-1 (GLP-1) by NCI-H716 cells. Results indicate that H₂S intervention disrupts GLP-1-mediated metabolic regulatory pathways, leading to downregulation of GLP-1 gene expression. This process mimics the potential regulatory role of microbial metabolites in the intestinal microenvironment on host cell endocrine function.

Figure 14. Map of GLP-1 Gene Expression Level Changes

NCI-H716 cells were seeded at a density of 5 × 10⁵ cells per well in a 12-well plate. After the cells adhered and reached 70–80% confluence, they were treated with different drugs: One group received supernatant from the L-cysteine-induced Escherichia coli MG1655 strain (Negative group), another group received supernatant from the L-cysteine-induced strain pretreated with SQR protein (SQR-treated group), with corresponding control groups established. After co-incubation for 4 hours, the supernatant was aspirated, cells were washed with PBS to remove drug residues, and fresh medium was added for continued culture for 24 hours, respectively. Following culture, total cellular RNA was extracted, and the expression level of glucagon-like peptide-1 (GLP-1) mRNA was detected using RT-qPCR.

Table 3 qPCR detection of GLP-1 mRNA expression levels in cells following SQR administration

Results demonstrated that GLP-1 mRNA expression was significantly upregulated in the SQR-treated group compared to the Negative group (p < 0.05). This indicates that SQR protein effectively mitigates H₂S-induced suppression of GLP-1 gene expression by degrading H₂S in bacterial supernatant, thereby restoring GLP-1 transcriptional levels.

Figure 15. The map of GLP-1 gene expression level changes under different treatments

Construction Design:

To build the recombinant plasmid pET28a-FCSD-His, we used homologous recombination to introduce the FCSD gene—which encodes a Formyl-CoA Sulfhydrylase Domain protein involved in sulfur metabolism—into the pET28a vector. This plasmid comprises key functional elements to guarantee efficient expression and purification. Its backbone incorporates a kanamycin resistance gene (KanR) for antibiotic-based selection, along with LacI and the Lac operator to enable IPTG-induced expression. A His-tag connected with a T7 promoter is included to facilitate protein purification and start transcription. Through introducing the FCSD gene into this plasmid, we set up a system that can yield a functional FCSD enzyme in E. coli. After being expressed and purified, this enzyme plays a role in sulfur compound metabolism, helping to break down specific sulfur-containing substrates in the gut environment. This aids in regulating intestinal sulfur balance, which is essential for preserving metabolic homeostasis.

Figure 16. Plasmid map of pET28a-FCSD-His

Engineering Principle

We developed and assembled a new composite element, pET28a-FCSD-His, engineered to encode the Formyl-CoA Sulfhydrylase Domain protein (FCSD) with an appended His-tag. This tag makes it possible to purify the protein efficiently through nickel-affinity chromatography during the purification process. Within the pET28a vector, the T7 promoter and Lac operator work together to control protein expression; notably, the Lac operator allows for IPTG-inducible expression in Escherichia coli BL21.

FCSD acts as a catalyst for reactions involving formyl-CoA and sulfhydryl groups, and it plays a central role in sulfur metabolism. In our project, FCSD capsules delivered orally function in the intestinal environment to regulate the processing of sulfur compounds, supplementing SQR’s function of degrading H₂S. This joint action helps keep sulfur levels in an optimal range, which in turn supports the balance of gut metabolism.

Experimental Approach

The FCSD gene sequence underwent PCR amplification. Figure 17A displays a distinct band at the anticipated size, confirming that amplification was successful. Meanwhile, the pET28a vector was linearized through double enzymatic digestion with XhoI and BamHI. Figure 17B exhibits a single band, verifying successful linearization.

Figure 17. Gel electrophoresis of PCR-amplified FCSD gene and pET28a linear

The recombinant plasmid pET28a-FCSD-His was transformed into E. coli DH5α competent cells. The transformed culture was spread onto LB plates containing kanamycin and incubated overnight at 37°C to obtain a single colony. Figure 18A shows a distinct single colony formed on the plate, indicating preliminary success of the transformation experiment. Subsequently, multiple positive clones were randomly selected and validated via colony PCR and sequencing. Figure 18B shows that the FCSD sequence matches the expected target sequence perfectly, further confirming the correct construction of the recombinant plasmid.

Figure 18. Map for pET28a-FCSD-His Transformation Verification

For protein expression purposes, the validated recombinant plasmid was transformed into E.coli BL21. As seen in Figure 19, single colonies grew on the plate, proving the transformation worked.

Figure 19. pET28a-FCSD-His single colony map

Cultures were induced with 0.5 mM IPTG at 16°C, and cells were disrupted by ultrasonication to release the target proteins. Purification was carried out using nickel-affinity chromatography, leveraging the His-tag. Four fractions were collected: crude lysate, flow-through, wash, and elution. SDS-PAGE analysis revealed a clear band in the elution fraction at the anticipated molecular weight of FCSD (~43 kDa), confirming that expression and purification were successful.

Figure 20. The SDS-PAGE protein gel of samples from different purification stages

Function test

1. Lead Acetate Test Strips

To assess SQR activity visually, we used lead acetate test strips, which turn black when exposed to H₂S. For this purpose, Escherichia coli MG1655 was cultured in media containing different concentrations of L-cysteine (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) to induce H₂S production. The generation of H₂S was verified by the blackening of lead acetate paper strips. Simultaneously, we further analyzed H₂S yield using ImageJ. Results demonstrated that H₂S production progressively increased with rising L-cysteine concentrations.



Figure 21. Different L-cysteine derivatives induce varying amounts of H₂S production

When FCSD was introduced into the testing system, a pronounced concentration-dependent response was observed: the color intensity of the test strip gradually weakened as the amount of added FCSD increased. This visual change was further confirmed by grayscale analysis—grayscale values exhibited a corresponding decrease with rising enzyme concentration, verifying the quantitative reduction of H₂S content in the sample.

Notably, when the FCSD concentration reached 5mg/L, H₂S levels decreased significantly, approaching concentrations observed without L-cysteine induction. This result strongly demonstrates that the FCSD enzyme efficiently and rapidly catalyzes H₂S degradation, highlighting its potential application in systems requiring H₂S control.



Figure 22. Reduction in greyscale of lead acetate test strips with increasing FCSD concentration

2. Hardware Validation

We subsequently verified the activity of FCSD using sensor-based devices for real-time monitoring of sulfur metabolites. In untreated samples, sensor measurements for target metabolites were high(Figure 23A). By contrast, samples treated with FCSD showed reduced sensor readings, which confirms that the metabolites were processed effectively(Figure 23B).



Figure 23. Sensor display when FCSD is added

3. GLP-1 Pathway Detection

To further investigate the effects of H₂S on intestinal endocrine cell function, we established the following in vitro model: the supernatant from L-cysteine-induced E.coli MG1655 was added to the NCI-H716 cell culture system. This supernatant, rich in H₂S produced by bacterial metabolism, significantly inhibited the synthesis and secretion of glucagon-like peptide-1 (GLP-1) by NCI-H716 cells. Results indicate that H₂S intervention disrupts GLP-1-mediated metabolic regulatory pathways, leading to downregulation of GLP-1 gene expression. This process mimics the potential regulatory role of microbial metabolites in the intestinal microenvironment on host cell endocrine function.



Figure 24. Map of GLP-1 Gene Expression Level Changes

NCI-H716 cells were seeded at a density of 5 × 10⁵ cells per well in a 12-well plate. After the cells adhered and reached 70–80% confluence, they were treated with different drugs: One group received supernatant from the L-cysteine-induced Escherichia coli MG1655 strain (Negative group), another group received supernatant from the L-cysteine-induced strain pretreated with FCSD protein (FCSD-treated group), with corresponding control groups established. After co-incubation for 4 hours, the supernatant was aspirated, cells were washed with PBS to remove drug residues, and fresh medium was added for continued culture for 24 hours, respectively. Following culture, total cellular RNA was extracted, and the expression level of glucagon-like peptide-1 (GLP-1) mRNA was detected using RT-qPCR.

Table 4 qPCR detection of GLP-1 mRNA expression levels in cells following FCSD administration

Results demonstrated that GLP-1 mRNA expression was significantly upregulated in the FCSD-treated group compared to the Negative group (p < 0.05). This indicates that FCSD protein effectively mitigates H₂S-induced suppression of GLP-1 gene expression by degrading H₂S in bacterial supernatant, thereby restoring GLP-1 transcriptional levels.

Figure 25. The map of GLP-1 gene expression level changes under different treatments