Name：phosphoketolase (PK),BBa_250HMEK7

Length : 2391bp

Origin: Clostridium acetobutylicum

Properties:

Phosphoketolase is a key metabolic enzyme primarily involved in the sugar metabolic pathways of microorganisms, particularly functioning in the phosphoketolase pathway (Bao et al., 2011). This enzyme catalyzes the cleavage of phosphoketoses (fructose-6-phosphate etc.) to generate smaller phosphorylated products, including glyceraldehyde-3-phosphate (G3P) and acetyl phosphate. This cleavage process is accompanied by the formation of high-energy phosphate bonds. For instance, acetyl phosphate can serve as an energy carrier and directly participate in ATP synthesis (Henard CA et al., 2015).

Usage and Biology:

Marco Sonderegger demonstrated that the functional phosphoketolase pathway was successfully constructed in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c through heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase combined with native phosphoketolase, resulting in a 20% increase in ethanol production yield compared to the parental strain(Marco Sonderegger, etc.2004). Phosphoketolase breaks down xylulose-5-phosphate or fructose-6-phosphate to produce acetyl phosphate, which serves as the initial compound for acetic acid formation.Xiong discovered that disrupting the predicted phosphoketolase gene in Synechocystis wild-type strains resulted in defective acetate synthesis during dark conditions, demonstrating that the phosphoketolase pathway supports heterotrophic metabolism(Xiong, etc. 2016).

Experimental data:

We have selected the DNA coding sequences from the National Center for Biotechnology Information (NCBI), namely phosphoketolase (PK), . These DNA coding sequences were optimized and synthesized according to the codon preference of Escherichia coli. And we employed the polymerase chain reaction (PCR) to amplify the coding genes using primers as templates. Figure 1 shows that the PK gene was between the 1500 bp and 3000 bp markers. The amplified lengths of the genes were consistent with the DNA coding, suggesting successful amplification.

Figure 1. The electrophoresis verification of the target fragment PK; Note: phosphoketolase(PK) -2391bp

Name: Erythritol-4-phosphate dehydrogenas(EPDH),BBa_25D5G4JM

Length : 1509bp

Source: Brucella melitensis

Properties:

Erythritol-4-phosphate dehydrogenase (E4PDH) serves as a key enzyme in the erythritol biosynthetic pathway. It catalyzes the dehydrogenation of erythritol-4-phosphate (E4P), converting it into erythrulose-4-phosphate (E4PuP), accompanied by the concomitant reduction of the cofactors NAD⁺/NADH or NADP⁺/NADPH.(T. Barbier, etc.2014).

The equation below shows the reaction: Erythritol-4-phosphate+NAD⁺⇌Erythrulose-4-phosphate+NADH⁺+H⁺

Usage and Biology:

Yun et al. investigated the activity of erythritol-4-phosphate dehydrogenase (EPDH), which exhibited high specificity toward erythrose-4-phosphate with a K_m value of 1.07 mM and an isoelectric point of 4.6. EPDH demonstrated optimal activity at pH 7.0 and 30 °C. The enzyme remained stable within a pH range of 4.0 to 9.0 and at temperatures below 40 °C. Its activity was completely inhibited by 1 mM Hg²⁺ and 1 mM Cu²⁺, but was not significantly affected by other metal cations.(Yun, Na-Rae, etc.2009).

Experimental data:

The DNA coding sequences were optimized and synthesized according to the codon preference of Escherichia coli. And we employed the polymerase chain reaction (PCR) to amplify the coding genes using primers as templates. Figure 2 shows that the EPDH gene was located between the 1000 bp and 1500 bp markers. The amplified lengths of the genes were consistent with the DNA coding, suggesting successful amplification.

Figure 2. The electrophoresis verification of the target fragment EPDH;Note:Erythritol-4-phosphate dehydrogenas(EPDH)-1509bp

Name: phosphatase (PTase),BBa_25F5SO4J

Length : 1665bp

Source: Oenococcus oeni

Properties:

Phosphatase operates as an enzyme that removes phosphate groups from its substrates by breaking down phosphate monoesters which results in phosphate ions and free hydroxyl groups(Wang Qiuying etc; 2011).

Usage and Biology:

Alkaline phosphatase (ALP) refers to a group of non-specific phosphomonoesterases that demonstrate maximal activity under alkaline conditions(Simopoulos TT, etc.1994). It is directly involved in the transfer and metabolism of phosphate groups in living organisms and contributes to protein secretion, bone formation, and lipid metabolism (Moss AK,etc; 2013). Purified ALP is commonly utilized in nucleic acid analysis and serves as a labeling enzyme in electrochemical enzyme immunoassays and immunosensors (Mukaiyama K, Ooi K , Sardiwal S, Shi Y, etc. 2015, 2007, 2013,2018). Phosphatases can also be applied as herbicides to enhance crop yield and improve plant stress resistance (Shi Y, etc.2018), or used as an indicator for microbial detection in protein-rich foods (Ma CB, Ram B, etc. 2012, 2019). One study isolated and screened lactic acid bacteria from traditional fermented mare’s milk in Mongolia, leading to the identification of a Lactobacillus strain capable of producing ALP with potential probiotic properties. Subsequent work involved the purification and enzymatic characterization of this ALP (DUAN Xiao-Xia, etc.2020).

Experimental data:

We synthesized the PTase gene and inserted it into the pCDFDuet vector with bio-technology company, and the codon usage of phosphatase (PTase) was optimized for expression in Escherichia coli .We employed the polymerase chain reaction (PCR) to amplify the coding genes using primers as templates. The obtained PCR products were inserted into the linearized plasmid pCDFDuet.

As shown in Figure 3, the pCDFDuet-PTase gene was prominently displayed between the 5000 bp and 7500 bp markers, which was in complete agreement with the theoretical length, indicating successful amplification.

Figure 3. The electrophoresis verification of the target fragment pCDFDuet-Ptase;Note:pCDFDuet-phosphatase-5369bp

Name：Glycerol kinase(GUT1),BBa_253DGJES

Length : 2130bp

Source: Saccharomyces cerevisiae(strain ATCC 204508 / S288c)

Properties:

Glycerol kinase catalyzes the conversion of glycerol into glycerol-3-phosphate. In adipocytes, due to the absence of glycerol kinase, glycerol generated from lipolysis cannot be utilized directly. Instead, it must be transported via the bloodstream to tissues such as the liver, kidneys, and intestine for further metabolism(Guo L, etc. 2010).

Usage and Biology:

Currently, numerous studies focus on enhancing glycerol production by overexpressing glycerol kinase and knocking out key genes involved in its inhibitory regulation. Glycerol kinase deficiency (GKD) is an X-linked recessive disorder caused by mutations in the glycerol kinase (GK) gene, leading to hyperglycerolemia, hyperglyceroluria, and pseudohypertriglyceridemia. The role of lipid metabolism in tumorigenesis and progression has garnered significant attention (Guo L, etc. 2010). A study employing immunohistochemical comparative analysis of clinical ESCA tumor samples and normal tissues demonstrated upregulated GK expression in ESCA. Elevated GK expression in ESCA is closely associated with poor prognosis and increased immune cell infiltration, highlighting its potential as both an independent prognostic biomarker and a viable therapeutic target (ying F, etc. 2024).

Experimental data:

Each target gene was amplified polymerase chain reaction (PCR) using specific primers, and separated DNA fragments using 1% agarose gel electrophoresis.In Figure 8A, the length of GUT1 is 2130bp.The observed lengths of all amplified genes were consistent with their respective coding sequences, validating the success of the amplification process.

Figure 4. The electrophoresis verification of the target fragments GUT1(2130bp).

Name：glycerol-3-phosphate dehydrogenase(GUT2),BBa_25VKCZ7O

Length : 1950bp

Source: Saccharomyces cerevisiae(strain ATCC 204508 /S288c)

Properties:

Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is an NAD-dependent oxidoreductase with phosphorylating activity. It catalyzes the oxidation (dehydrogenation) and phosphorylation of glyceraldehyde-3-phosphate to form 1,3-bisphosphoglycerate. As this reaction represents a central step in sugar metabolism, GAPD plays a critical role in glycolytic pathways(Colell A, etc. 2009).

Usage and Biology:

Recent studies have revealed that GAPDH possesses multiple non-canonical functions (Colell A, etc. 2009). These include involvement in membrane fusion, cytoskeletal dynamics, DNA repair, and RNA export. Researchers propose that these diverse functions are likely regulated through post-translational modifications and alterations in subcellular localization (Schuppe-Koistinen I, etc. 1994). GAPDH has been identified as an intracellular and extracellular cellular stress sensor, capable of activating multiple pathways to facilitate recovery from damage or alternatively initiating cell death signaling pathways (Colell A, Schuppe-Koistinen I, etc. 2009, 1994). Its aberrant expression has been observed in cancers, neurodegenerative disorders (such as Alzheimer's disease and Parkinson's disease), and infectious diseases, suggesting its potential as a diagnostic or prognostic biomarker(Oh S, etc. 2024).

Experimental data:

The GUT2 was amplified polymerase chain reaction (PCR) using specific primers, and the resulting amplification products were then inserted into the linearized pETDuet vector. The target fragment was ligated to the vector by homologous recombination, transformed into E. coli DH5α, and the expression plasmids were verified. In Figure 5, the GUT2 amplification products migrated between the 1500 bp and 3000 bp markers.The observed lengths of all amplified genes were consistent with their respective coding sequences, validating the success of the amplification process.

Figure 5. The electrophoresis verification of the target fragments GUT2(1950bp).

Name: Encoding triosephosphate isomerase(TPI1),BBa_25QUT01A

Length :747bp

Source: Saccharomyces cerevisiae(strain ATCC 204508 / S288c)

Properties:

Triosephosphate isomerase (TPI) is a key enzyme in the glycolytic pathway, catalyzing the reversible isomerization between dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP), and ensuring the efficient progression of glycolysis. (Wang Xiaobin, etc. 2021).

Usage and Biology:

Triosephosphate isomerase (TPI) is ubiquitously present across all living organisms and plays significant roles in basic research, medicine, industry, and synthetic biology. Certain pathogens (e.g., Plasmodium and Trypanosomes) depend on TPI to sustain glycolytic metabolism, and inhibition of its enzymatic activity can disrupt their energy supply (G cb, etc. 2022). Our data indicate that TPI1 functions as a tumor suppressor in hepatocellular carcinoma (HCC) and may represent a potential therapeutic target for HCC treatment (Hao J, etc. 2016). In in vitro multi-enzyme catalytic systems, TPI helps maintain the balance of metabolic intermediates and prevents cytotoxicity induced by dihydroxyacetone phosphate (DHAP) accumulation (Hao J, etc. 2016).

Experimental data:

Each target gene was amplified polymerase chain reaction (PCR) using specific primers, and separated DNA fragments using 1% agarose gel electrophoresis. In Figure 6 showed that the TPL1 amplification product was located between the 500 bp and 1000 bp markers.The observed lengths of all amplified genes were consistent with their respective coding sequences, validating the success of the amplification process.

Figure 6.The electrophoresis verification of the target fragment TPL1(747bp) .

Engineering Principle:

Therefore, we enhanced and integrated the erythritol biosynthesis pathway in bacteria through molecular biology techniques. And we not only utilize glucose as a substrate but also incorporate the glycerol metabolic pathway. This approach reduces by-product formation, expands the utilization of low-cost substrates (glycerol), and redirects metabolic flux toward erythritol production. Specifically, we engineered two distinct biosynthetic pathways for erythritol synthesis in Escherichia coli (Figure 7). Only when plasmids pCDFDuet-PK-EPDH-PTase and pETDuet-GUT12-TPI1 are co-transformed into Escherichia coli can erythritol be synthesized using glycerol as the substrate.

Pathway 1 - Metabolic pathway of erythritol synthesis with glucose as substrate(Liu F etc, 2024; Thuy etc, 2024):

Step 1: Glucose Phosphorylation

The enzyme hexokinase catalyzes the phosphorylation of glucose at the C6 position, forming glucose-6-phosphate.

Step 2: Isomerization to Fructose 6-phosphate

Glucose-6-phosphate is isomerized to fructose-6-phosphate.

Step 3: Phosphoketolase Cleavage

Fructose-6-phosphate is cleaved by phosphoketolase (PK) to produce acetyl-phosphate and erythrose-4-phosphate.

Step 4: Reduction to Erythritol 4-phosphate

Erythrose-4-phosphate is converted to erythritol-4-phosphate by erythritol-4-phosphate dehydrogenase(EPDH).

Step 5: Dephosphorylation to Erythritol

The phosphate group is removed from erythritol-4-phosphate by phosphatase(PTase), yielding the final product erythritol.

Pathway 2-Metabolic pathway for the synthesis of erythritol with glycerol as substrate(Liu F etc, 2024; Thuy etc, 2024)：

Step 1: Glycerol Phosphorylation

Glycerol is converted to glycerol 3-phosphate by glycerol kinase (GUT1), consuming 1 molecule of ATP.

Step 2: Dehydrogenation to Dihydroxyacetone Phosphate

Glycerol 3-phosphate is oxidized to dihydroxyacetone phosphate (DHAP) by glycerol-3-phosphate dehydrogenase (GUT2), with the concomitant reduction of 1 NAD⁺ to NADH.

Step 3: The enzyme triosephosphate isomerase (TPI1) catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (G3P). Part of GA-3-P flows into the tricarboxylic acid cycle ( TCA cycle) through pyruvate, providing raw materials, energy and cofactors for various life activities of cells. Another part of GA-3-P produced fructose-6-phosphate ( F-6-P ) under gluconeogenesis and entered the pentose phosphate pathway ( PPP ). The gluconeogenesis pathway includes the intermediates F-6-P and GA-3-P produced by the PPP pathway, which flow to the synthesis of erythritol in pathway 1.

Figure 1.Synthesis pathway of erythritol

Figure 7.Synthesis pathway of erythritol

(Note: PK: phosphoketolase; EPDH: erythritol-4-phosphate dehydrogenase; PTase: phosphatase; GUT1: Encoding glycerol kinase; GUT2: Encoding glycerol-3-phosphate dehydrogenase; TPI1: Encoding triosephosphate isomerase)

2.1 pCDFDuet-PK-EPDH-PTase BBa_25VEGQXC

Construction Design

Selection of phosphoketolase (PK), erythritol-4-phosphate dehydrogenase(EPDH), and phosphatase(PTase)were conducted in NCBI database. The genes fused with 6×His-tag at its C-terminus were optimized, and optimized codons. The plasmid map was constructed by snapgene software (Figure 8). and inserted into the expression vector pCDFDuet through homologous recombination, the recombinant plasmids pCDFDuet-PK-EPDH-PTase is composed of phosphoketolase (PK), erythritol-4-phosphate dehydrogenase(EPDH) and phosphatase(PTase)and are further transformed into E.coli DH5α.

Figure 8.The plasmid map

Experimental Approach

Next, we cloned three coding DNA (PK, EPDH, and PTase) into the pCDFDuet vector using homologous recombination, and transformed it into E. coli DH5α competent cells. The total length of the three DNA coding is 5565 bp. The electrophoresis result shown in Figure 9A indicates that the amplified product band is located within the range of 5000-7000 bp, which is consistent with the expected fragment size, confirming that the target gene fragment has been successfully amplified. In Figure 9B, the growth of monoclonal colonies with SmR(100ng/ml)was observed, indicating that the transformation experiment was initially effective.By comparing the sequence file with the target gene sequence, the results show that there is no base mutation at the location marked by the red solid arrow, and a base mutation exists at the location marked by the dotted line arrow. The Figure 9C confirm that the target gene fragment has been correctly linked to the vector, further verifying the successful construction of the recombinant plasmid.

Figure 9A. Monoclonal colony verification gel plate; Figure 9B. Monoclonal colony verification petri dish diagram in E. coli DH5α ; Figure 9C. Monoclonal colony verification sequencing plot

2.2 pETDuet-GUT12-TPI1 BBa_25XX8745

Construction Design

The pETDuet-1 vector harbors the ColE1 replicon, LacI gene and ampicillin resistance gene. This vector can be used in combination with the pCDFDuet vector to achieve co-expression of six target genes in our project within a suitable host strain.The three codeing DNA are inserted into the expression vector pETDuet-1 through homologous recombination (Figure 10).

Figure10.The plasmid map

The total length of the coding GUT1, GUT2, and TPL1 is 4827bp. As shown in the electrophoresis result in figure 11A, the amplified product presents a clear band at approximately 5000 bp, which is highly consistent with the expected fragment size. The growth of monoclonal colonies figure 11B with Amp(100ng/ml) was observed. And Figure 11C confirmed that the target gene fragment has been correctly linked to the vector, further verifying the success of the construction of the recombinant plasmid.

Figure 11A. Monoclonal Colony Verification Gel Plate; Figure 11B. Monoclonal Colony Verification Petri Dish Diagram in E. coli DH5α; Figure 11C. Monoclonal colony verification sequencing plot.