This composite part is used to realize the synthesis and excretion of glycerol. Fps1 is the glycerol facilitated diffusion channel protein. Glycerol-3-phosphate dehydrogenase (Gpd1) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. This intermediate is then dephosphorylated by glycerol-3-phosphatase (Gpp2) to yield glycerol. To enhance the catalytic efficiency of this two-step conversion, gpd1 and gpp2 were fused into a single open reading frame using a flexible (GGGGS) linker. Additionally, the glycerol transporter Fps1 is included to facilitate glycerol export to the extracellular space. The expression of this tri-gene construct is driven by the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Ptac promoter (BBa_K180000), with each gene preceded by a high-efficiency ribosome binding site (RBS) (BBa_J34801).

Figure 1 The metabolic pathway from glucose to glycerol in Saccharomyces cerevisiae^[1]

Promoter: An IPTG-inducible promoter that facilitates controlled gene expression. In our experiments, we utilize the Ptac promoter, which enables the subsequent genes to be expressed effectively.
RBS: Ribosome binding site for highly efficient translation. In this project, we utilize BBa_J34801.
fps1: Encodes the Glycerol Facilitated Diffusion Channel Protein.
gpd1-gpp2: Encodes a fusion protein involved in glycerol synthesis.
Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator consisting of rrnB T1 terminator and T7Te terminator in our experiment.
The gene circuits of the part is shown in Figure 2.

Figure 2 The gene circuit of glycerol synthesis and transport. The fragment of ‘Ptac-fps1-gpd1-gpp2fus’ was inserted into expression vector pYDT

Figure 3 Construction of plasmid pYDT-Ptac-fps1-gpd1-gpp2fus

We introduced the plasmid into E. coli BL21 and performed heterologous expression, followed by colony PCR analysis. The results confirmed the successful integration of the target gene into E. coli BL21.

Figure 4 PCR results of E. coli BL21 colonies

The fps1 and gpd1-gpp2fus genes was all sourced from Saccharomyces cerevisiae S288C.

The glycerol assay is based on a coupled chemical reaction sequence involving the Malaprade reaction and the Hantzsch reaction. The reactions are carried out in a 96-well plate, and kinetic measurements are monitored using a microplate reader. Absorbance at 410 nm is recorded over a period of 25 minutes. Glycerol concentration is calculated according to a glycerol standard curve using Equation 1^[2] (detailed principles and procedures for glycerol measurement are available on the protocol).

To evaluate whether the introduced genes can successfully synthesize glycerol and enable glycerol efflux, we first conducted studies using E coli BL21 as the model strain. We set up different IPTG concentrations (0 mM, 0.1 mM, 0.5 mM, and 1 mM), cultured the strain in M9 medium, and plotted the growth curve.
When IPTG concentration was 0 mM, there was no effect on the growth of either strain, providing a control for other experimental groups. At different IPTG concentrations, the growth of the pYDT strain was generally better than that of the pFps1-fus strain. This indicates that a certain concentration of IPTG inhibits the growth of the engineered bacteria, and the inhibitory effect becomes more pronounced as the IPTG concentration increases. Comprehensive comparison showed that when IPTG concentration was 0.5 mM, it was not only beneficial for the expression of the target genes but also favorable for the growth of the engineered bacteria.

Figure 5 (A) Growth curve when IPTG = 0 mM. (B) Growth curve when IPTG = 0.1mM (C) Growth curve when IPTG = 0.5mM. (D) Growth curve when IPTG = 1mM. Experiments were conducted in triplicate and the error bar represent SD.

To further investigate glycerol production capacity and the impact of IPTG, thereby identifying the optimal induction level, we quantified glycerol yields in both strains. The corresponding glycerol standard curve is shown in Figure 6.

Figure 6 Glycerol Standard Curve. An R-squared value of 0.9914 indicates a good fit of the curve.

Experimental results demonstrated that the highest glycerol yield was achieved in the engineered strain at 0.5 mM IPTG, showing a statistically significant increase over the empty vector control. Peak glycerol accumulation consistently occurred during the late logarithmic phase. Furthermore, comparison of glycerol yields at different time points revealed higher production at 11 hours compared to 23 hours. This pattern is explained by the fact that around 11 hours—corresponding to the late logarithmic phase—bacterial colonies had largely completed active growth and had extensively consumed available nutrients. Subsequently, growth rates declined, and the remaining nutrients in the culture were likely only sufficient to support basic cellular activities, leaving no surplus for additional glycerol synthesis.

Figure 7 (A)Glycerol production at different IPTG concentrations at 11h (B)Glycerol production at different IPTG concentrations at 23h (C)Comparison of glycerol production by pYDT at different concentrations between 11h and 23h (D)Comparison of glycerol production by fps1at different concentrations between 11h and 23h Experiments were conducted in triplicate and the error bar represent SD. Two-sided Student’s t-test was used to analyze the statistical significance (*0.01< P < 0.05)

Building on the CUG-China 2024 project, we aim to explore further synthetic biology applications of Acidithiobacillus ferrooxidans in 2025.Given glycerol’s significant industrial utility and the relative maturity of microbial metabolic engineering for its large-scale production,our 2025 project proposes the introduction of an exogenous glycerol synthesis pathway to redirect carbon flux toward glycerol production.
To achieve efficient glycerol synthesis and extracellular secretion, we engineered Gpd1 and Gpp2 into a fusion protein to facilitate the continuous intracellular synthesis of glycerol in the engineered bacteria. Subsequently, the fps1 gene was introduced, enabling the efficient transport of intracellularly synthesized glycerol to the extracellular environment via channel-mediated facilitated diffusion. This part may inspire other iGEM teams in the community working on carbon fixation and the synthesis of chemical products, and we are pleased if it can be of any help to other future iGEMers.