RESULTS
Erythritol, a polyol sweetener, exhibits non-cariogenic properties, hypoglycemic effects, and negligible caloric value. This functional sweetener serves as an ideal sucrose alternative for food product development. In current manufacturing methodologies, chemical synthesis prove economically unviable, whereas microbial biosynthesis predominates in industrial production(Liu F etc, 2024; Thuy etc, 2024).We aim to develop a novel and eco-efficient bioprocess for erythritol production while enhancing its yield. Our project involves the synthesis of erythritol using two substrates, which are glycerol and glucose. The pathway for erythritol synthesis is as follows:
Pathway1(Thuy etc, 2024): Synthesizes erythritol using glucose as the substrate, utilizing phosphoketolase(PK), erythritol-4-phosphate dehydrogenase(EPDH) and phosphatase(PTase). Pathway 2: Synthesizes erythritol using glycerol as the substrate. It first utilizes encoding glycerol kinase(GUT1), Encoding glycerol-3-phosphate dehydrogenase(GUT2) and Encoding triosephosphate isomerase(TPI1) to produce fructose-6-phosphate. This fructose-6-phosphate is then further converted into erythritol by the enzymes of Pathway 1(Figure 1).
Figure 1. Erythritol Biosynthetic Pathway
We first designed expression elements containing the T7 promoter, divided them into two vectors, named as pCDF Duet-PK-EPDH-PTase and pET Duet-GUT1-GUT2-TPl1, and constructed them into Escherichia coli BL21. Then, we used Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE) and Western Blot(WB) to verify the expression of functional proteins, and High Performance Liquid Chromatography(HPLC) to detect the yield of erythritol (Figure 2). Based on the test results, we explored the influence of cultivation conditions on the yield and iteratively optimized the process to improve the production efficiency of erythritol. So the purpose of our research is to increase the yield of erythritol and improve the utilization rate of glycerol.
Figure 2. The engineering design schematic diagram.
Results of your work
1. The construction of a plasmid
Currently, we have selected three DNA coding sequences from the National Center for Biotechnology Information (NCBI), namely phosphoketolase (PK), erythritol-4-phosphate dehydrogenase (EPDH), and phosphatase (PTase). These DNA coding sequences were optimized and synthesized according to the codon preference of Escherichia coli. To construct the expression plasmids for PK, EPDH, and PTase, 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 3A, 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 3B shows that the EPDH gene was located between the 1000 bp and 1500 bp markers, while 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 3A. The electrophoresis verification of the target fragment pCDFDuet-Ptase;Figure 3 B. The electrophoresis verification of the target fragment EPDH and PK; Note: phosphoketolase(PK) -2391bp; Erythritol-4-phosphate dehydrogenas(EPDH)-1509bp; pCDFDuet-phosphatase-5369bp
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 4A 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 4B, the growth of monoclonal colonies with SmR(100ng/ml)was observed, indicating that the transformation experiment was initially effective. Figure 4C is the sequencing report returned by the sequencing company. 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 sequencing results confirm that the target gene fragment has been correctly linked to the vector, further verifying the successful construction of the recombinant plasmid.
Figure 4A. Monoclonal colony verification gel plate; Figure 4B. Monoclonal colony verification petri dish diagram in E. coli DH5α ; Figure 4C. Monoclonal colony verification sequencing plot
Three DNA sequences corresponding to GUT1, GUT2, and TPL1, respectively, were selected from the NCBI database for this study. These sequences were codon-optimized based on the codon preference of Escherichia coli before synthesis. To construct an expression plasmid containing glycerol kinase (GUT1), glyceraldehyde-3-phosphate dehydrogenase (GUT2), and isomerase (TPL1), each target gene was amplified polymerase chain reaction (PCR) using specific primers, and the resulting amplification products were then inserted into the linearized pET Duet 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 5A, the GUT1 and GUT2 amplification products migrated between the 1500 bp and 3000 bp markers. Similarly, Figure 5B showed that showed that the TPL1 amplification product was located between the 500 bp and 1000 bp markers, and Figure 5C indicates that the pET Duet exhibited a distinct band about 5000 bp. The observed lengths of all amplified genes were consistent with their respective coding sequences, validating the success of the amplification process.
Figure 5A. The electrophoresis verification of the target fragment pET Duet(5462bp). Figure 5B. The electrophoresis verification of the target fragments GUT1(2130bp)and GUT2(1950bp).Figure 5C. The electrophoresis verification of the target fragment TPL1(747bp) .
The total length of the coding GUT1, GUT2, and TPL1 is 4827bp. As shown in the electrophoresis result in figure 6A, 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 6B with Amp(100ng/ml) was observed. And Figure 6C showed that no base mutation occurred at the position marked by the solid red arrow, while a base mutation was present at the position marked by comparing the sequencing file with the target gene sequence. All results 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 6A. Monoclonal Colony Verification Gel Plate; Figure 6B. Monoclonal Colony Verification Petri Dish Diagram in E. coli DH5α; Figure 6C. Monoclonal colony verification sequencing plot
2.1 pCDFDuet-PK-EPDH-PTase
The three-dimensional structures of proteins PK, EPDH, and PTase were predicted using AlphaFold2, and the feasibility of the models was evaluated using the metrics ipTM (interface pTM) and pTM (predicted TM-score). Thefigure 7 shows the three-dimensional structural representation of the protein PK, EPDH, and PTase.
Figure 7. Figure A is the three-dimensional structural of the protein PK;Figure B is the three-dimensional structural of the protein EPDH;Figure C is the three-dimensional structural of the protein PTase.
The recombinant plasmids pCDFDuet-PK-EPDH-PTase were transformed into E. coli BL21 (DE3) for expression. A single colony containing the recombinant plasmid was cultured in medium con-taining antibiotics (100 mg/mL SmR ) at 37oC. When the cul-tures reached optical density (OD600) about 0.6, iso-propy-beta-D-thiogalactopyranoside (0.5mmol and 1mmol IPTG) was added to the broth for induction of the protein in 25oC . And the recombinant His-6 fusion-type PPE protein was purified by Ni2+ affinity chromatography.
As shown in figure 8A, when 0.5 mmol IPTG was used to induce the protein, no PK protein was expressed, but PTase and EPDH were successfully expressed. After being inducted with 1 mmol/L IPTG, both PK, Ptase and EPDH exhibited sig-nificant expression, and the Western blot results showed that both these recombinant prod-ucts could be detected by the anti-His6 antibodies (Figure 8B), suggesting that His6-fused PK, Ptase and EPDH were expressed in E. coli BL21(DE3) successfully.
Figure 8. Optimisation of the culture medium for the soluble expression of the recombinant His6-tagged PK, Ptase and EPDH. (A) SDS-PAGE analysis of the products fermented in media. (B) Western blot analysis of the products fermented in media; Note the Pk protein is 90.6kda. The PTase protein is 59.8kda. The EPDH protein is 56.1kda
The three-dimensional structures of proteins GUT1,GUT2 and TPl1 were predicted using SWISS-MODEL, and the feasibility of the models was evaluated using the metrics GMQE (Global Model Quality Estimation) and QMEAN. The figure 9 shows the three-dimensional structural representation of the protein GUT1,GUT2 and TPl1.
Figure 9. Figure A is the three-dimensional structural of the protein GUT1;Figure B is the three-dimensional structural of the protein GUT2;Figure C is the three-dimensional structural of the protein TPL1.
The recombinant plasmids pET Duet-GUT1-GUT2-TPl1 were transformed into E. coli BL21 (DE3) for expression. A single colony containing the recombinant plasmid was cultured in medium con-taining antibiotics (100 mg/mL Amp) at 37oC. The other protein expression conditions are consistent with pCDFDuet-PK-EPDH-PTase. The figure 10A shown that the TPL1,GUT2 and GUT1 exhibited sig-nificant expression after being inducted with 0.5 and 1 mmol/L IPTG. And the Western blot results showed that both these recombinant prod-ucts could be detected by the anti-His6 antibodies (Figure 10B), suggesting that His6-fused TPL1,GUT2 and GUT1 were expressed in E. coli BL21(DE3) successfully.
Figure 10. Optimisation of the culture medium for the soluble expression of the recombinant His6-tagged TPL1,GUT2 and GUT1. (A) SDS-PAGE analysis of the products fermented in media. (B) Western blot analysis of the products fermented in media; NoteThe size of the GUT1 protein is 79.8kda. The size of the GUT2 protein is 72.7kda. The size of the TPI1 protein is 26.8kda
3．Functional test
The plasmid pCDFDuet-PK-EPDH-PTase are abbreviated as PPE; The plasmids pET Duet-GUT1-GUT2-TPl1 are abbreviated as GGT. The plasmids PPE and GGT were transfed to Escherichia coli BL21. Monoclonal colonies were screened using 100 mg/ml Ampicillin (Amp+) and Streptomycin Resistance (SmR) antibiotics. In figure 11, single colonies have grown.
Figure 11. Monoclonal colony culture dish diagram in Escherichia coli BL21
As shown in figure 12, lanes 1 to 8 correspond to the amplification products of PPE1, GGT1, PPE2, GGT2, PPE3, GGT3, PPE4, and GGT4. The results indicate that the amplification bands of PPE4 and GGT4 are present within the molecular weight range of 2000 bp to 4000 bp. Moreover, their actual sizes are exactly consistent with the theoretically expected lengths, thereby confirming that these two target fragments have been successfully amplified.
Figure 12. Monoclonal colony verification. note:The length of GGT is 3726 bp, and the length of PPE is 3504 bp
3.2 Growth Curve
The plasmids PPE and GGT were transfed to Escherichia coli BL21. The OD600 values were measured three times at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, 20 hours, 24 hours, 30 hours, and 48 hours, respectively. As can be seen from figure 13, from 0 to 12 hours, the OD600 values of both CK(Blank control) and co-transformation strains increased linearly. From 12 hours to 48 hours, the OD600 values gradually stabilized. In figure 13, the CK groups were not significant difference to compared with bacterial strain pCDFDuet-PK-EPDH-PTase and pET Duet-GUT1-GUT2-TPl1 . The coding genes PK, EPDH, PTase, GUT1, GUT2, and TPl1 don’t affect the growth of the strain.
Figure 13. The growth curve of pCDF Duet-PK-EPDH-PTase+ pET Duet-GUT1-GUT2-TPl1 and CK. Note:The plasmid pCDFDuet-PK-EPDH-PTase are abbreviated as PPE; The plasmids pET Duet-GUT1-GUT2-TPl1 are abbreviated as GGT
3.3 Determination of erythritol production by HPLC
We prepared five different fermentation media with varying substrate compositions and ratios (fermentation conditions: 37°C, pH 7-8), as follows: Glucose:Glycerol = 10:10 (g/L), Glucose:Glycerol = 20:10 (g/L), Glucose:Glycerol = 10:20 (g/L), Glucose:Glycerol = 20:0 (g/L), Glucose:Glycerol = 0:20 (g/L). Samples were taken at different time points (5h, 16h, 24h, 48h) to measure the concentrations of glucose, glycerol, and erythritol.The concentrations of products and substrates were determined by high-performance liquid chromatography (HPLC). After sampling, centrifugation was performed to collect the supernatant (at 8000 r/min for 5 minutes), and it was filtered through a 0.22 μm membrane. The Aminex HPX-87H analytical column (300 mm ×7.8 mm) (from Bio-Rad, USA), 2414 differential refractive index detector (from Waters, USA). The detection conditions were column temperature 35 °C, injection volume 10 μL, mobile phase 5 mmol/L sulfuric acid, flow rate 0.6 mL/min. All the diagrams were completed using GraphPad.
According to figure 14A, the glucose concentration gradually declines with time in Glucose: Glycerol = 20:0 g/L. The concentration of erythritol increased over time, while no erythritol was produced in the control group(CK-E.coli BL21). This indicates that genes PK, EPDH, and PTase were successfully expressed, and the engineered strain demonstrated the capability to utilize glucose as a carbon substrate for fermentative production of erythritol.
In figure 14B, The concentration of glycerol gradually decreased, while the concentration of erythritol showed an upward trend over time in the Glucose: Glycerol = 0:20 g/L. However, no erythritol was produced in the control group. This indicates that the genes PK, EPDH, PTase, GUT1, GUT2, and TPI1 were successfully co-expressed in the engineered strain, and the glycerol and glucose metabolic pathways were properly established. Furthermore, it demonstrates that the strain can utilize a complete glycerol-based medium for fermentative erythritol production.
Figure 14. The concentration of erythritol, glucose and glycerol (A). The relationship graph of glucose, glycerol, and erythritol with time in Glucose: Glycerol = 20:0 g/L. (B) the relationship graph of glucose, glycerol, and erythritol with time in the Glucose: Glycerol = 0:20 g/L.
The figure 15A and B demonstrate that no erythritol was produced in the CK group (blank control), while the glucose concentration showed a decreasing trend over time in both the Glucose:Glycerol = 20:10 g/L and Glucose:Glycerol = 10:20 g/L. The glycerol concentration exhibited no significant change from 5h to 24 h, followed by a decrease between 24 and 48 hours. At Glucose:Glycerol = 20:10 g/L and Glucose:Glycerol = 10:20 g/L, erythritol concentration accumulation increased with time.
These results indicate that the strain likely preferentially utilizes glucose as the primary substrate during the 5-24 hour period at glucose and glycerol ratios of 2:1 and 1:2. When the glucose concentration becomes sufficiently low, the strain further metabolizes glycerol as a secondary substrate for erythritol production. This confirms that our engineered strain can utilize dual substrates (glucose and glycerol) as carbon sources for fermentative erythritol biosynthesis.
Figure 15. The concentration of erythritol, glucose and glycerol. (A). The relationship graph of glucose, glycerol, and erythritol with time in Glucose: Glycerol = 20:10 g/L. (B) the relationship graph of glucose, glycerol, and erythritol with time in the Glucose: Glycerol = 10:20 g/L.
According to figure 16, the concentration of glucose gradually decreases over time in Glucose: Glycerol = 10:10 g/L, but the concentration of glycerol shows no significant trend from 5 to 24 hours, and then decreases from 24 to 48 hours in Glucose: Glycerol = 10:10 g/L. The concentration of erythritol increases over time. These results indicate that glucose is preferentially utilized as the carbon source at glucose and glycerol ratios of 1:1, 1:2 and 2:1, and erythritol production occurs under all these conditions.
Figure 16. The concentration of erythritol, glucose and glycerol in Glucose: Glycerol = 10:10 g/L.
To enhance erythritol production and investigate the effects of varying glucose-to-glycerol ratios in the culture medium on erythritol yield, we utilized MATLAB software to perform 3D modeling predictions based on existing data. This model predicts erythritol production levels after fermentation using different proportional combinations of glucose and glycerol as carbon sources.A three-dimensional model was constructed by performing surface fitting on each discrete data point, and based on this model, the prediction analysis of the highest and lowest points was realized.
The Y-axis is labeled as "Time (h)" in the figure 17. The X-axis is labeled as "Glucose: Glycerol", displaying different ratio values such as 0:20, 10:10, 20:0, etc. The Z-axis is labeled as "Erythritol (g/L)". The figure 17 shown that the highest value is (1.7676, 39.7474, 3.403643), and the lowest value is (0; 0; 0). At the highest value, the ratio of glucose concentration to glycerol concentration was 17:10. After the initial measurement was completed, the samples were subjected to a secondary fermentation process, and then the highest values were measured using high-performance liquid chromatography (HPLC) technology.
Figure 17. 3D modeling of the production volume of erythritol
In figure 18, the concentration of glucose gradually decreases over time, but the concentration of glycerol shows no significant trend from 5h to 24h, and then decreases from 24 to 48 hours in Glucose: Glycerol = 10:10 g/L. The concentration of erythritol increases over time. These results indicate that glucose is preferentially utilized as the carbon source at glucose and glycerol ratios of 1:1, 1:2, 2:1and 1.7:1, and erythritol production occurs under all these conditions.
Figure 18. the relationship graph of glucose, glycerol, and erythritol with time atGlucose: Glycerol = 17:10 g/L
After 48 hours of fermentation in media with varying ratios of glucose to glycerol, the consumption of glucose and glycerol, as well as the erythritol production yield and conversion rate, were calculated based on data determined by HPLC. After 48 hours, the strain consumed 13.13g glucose , and produced 2.33g erythritol with a conversion rate of 17.74% at Glucose: Glycerol = 20:0 g/L. At Glucose: Glycerol = 20:10 g/L, the strain consumed18.73g glucose and5.03g of glycerol, and produced 2.82g erythritol with a conversion rate of 11.85% . The experimental data for other groups are presented in Table 1. The table 1 shows that the experimental group with the highest erythritol yield was Glucose:Glycerol = 17:10 g/L, followed by Glucose:Glycerol = 10:20 g/L. The group with the lowest erythritol yield was Glucose:Glycerol = 20:0 g/L. In Table 1, the highest erythritol conversion rate was 20.99% in the Glucose:Glycerol = 0:20 g/L, while the lowest was 11.85% in the Glucose:Glycerol = 20:10 g/L .
Table 1. Under different ratios of glucose and glycerol, the concentrations and conversion rates of glucose, glycerol, and erythritol
|
Glucose: Glycerol(g/L) |
ΔGlucose |
Δglycerol |
Erythritol(g/L) |
Percent conversion |
|
20:0 |
13.13 |
0.00 |
2.33 |
17.74% |
|
20:10 |
18.73 |
5.03 |
2.82 |
11.85% |
|
17:10 |
16.37 |
6.63 |
3.42 |
14.86% |
|
10:10 |
9.04 |
6.75 |
2.61 |
16.55% |
|
10:20 |
8.92 |
10.01 |
3.11 |
16.41% |
|
0:20 |
0.00 |
12.20 |
2.56 |
20.99% |
Further Discussion
In Table 1, the highest erythritol conversion rate was 20.99% in the Glucose:Glycerol = 0:20 g/L, while the lowest was 11.85% in the Glucose:Glycerol = 20:10 g/L . The erythritol conversion rate was not significant difference in the Glucose:Glycerol = 10:10 g/L and 10:20 g/L. According to figure 16, the concentration of glucose gradually decreases over time in Glucose: Glycerol = 10:10 g/L, but the concentration of glycerol shows no significant trend from 5 to 24 hours, and then decreases from 24 to 48 hours in Glucose: Glycerol = 10:10 g/L. The concentration of erythritol increases over time. These results indicate that glucose is preferentially utilized as the carbon source at glucose and glycerol ratios of 1:1, 1:2 and 2:1, and erythritol production occurs under all these conditions. Based on our experimental data, the strain preferentially utilizes glucose and continues to consume glycerol when glucose concentration becomes low. When glucose is present, it inhibits the ability of Escherichia coli to utilize other secondary carbon sources (such as glycerol, lactose, arabinose, etc.).The metabolism of glucose reduces the intracellular concentration of a key signaling molecule—cAMP (cyclic adenosine monophosphate). cAMP must bind to CRP (cAMP receptor protein) to form a complex (cAMP-CRP)l(Jacqueline, etc, 2022). This complex is an essential transcription factor required for activating the expression of many operons involved in the metabolism of other carbon sources, such as those for lactose, arabinose, and glyceol(Jacqueline, etc; 2022).
Therefore, subsequent optimization can focus on testing ratios ranging from 10:20 to 0:20 (Glucose:Glycerol) to identify a fermentation medium that achieves both higher conversion rate and higher yield(Kaika, etc,2022; Thuy,etc; 2024 ). Additionally, the effects of varying fermentation temperatures and pH levels on erythritol production yield should be investigated(Heghine, etc; 2022).
The current 3D model is limited by insufficient data points, which compromises its ability to accurately capture overall trends and increases susceptibility to prediction deviations. Moreover, the model does not include the erythritol conversion rate as a key metric. Converting predicted outputs into percentage format would significantly enhance its practicality for observational analysis and decision-making.
Reference
Heghine G ,Satenik K ,Anait V , et al.Metabolic pathways and ΔpH regulation in Escherichia coli during fermentation of glucose and glycerol in the presence of formate at pH the role of FhlA transcriptional activator.[J].FEMS microbiology letters,2022,369(1):
Liu F, Tian JT, Wang YT, Zhao L, Liu Z, Chen J, Wei LJ, Fickers P, Hua Q. Improving an Alternative Glycerol Catabolism Pathway in Yarrowia lipolytica to Enhance Erythritol Production. Yeast. 2024 Oct;41(10):605-614. doi: 10.1002/yea.3980. Epub 2024 Sep 11. PMID: 39262092.
Jacqueline Plumbridge.Regulation of gene expression in the PTS in Escherichia coli : the role and interactions of Mlc[J] . Current Opinion in Microbiology . 2002 (2)
Kaikai W ,Xiaolu W ,Huiying L , et al.Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli[J].International Journal of Molecular Sciences,2022,23(2):773-773.
Thuy Le H, Vu YT, Duong GH, Le TK, Dang MK, Pham DD, Pham NK, Sichaem J, Nguyen NH, Duong TH. Bio-Guided Isolation of Alpha-Glucosidase Inhibitory Compounds from Vietnamese Lichen Roccella Montagnei. Chem Biodivers. 2024 Jul;21(7):e202400438. doi: 10.1002/cbdv.202400438. Epub 2024 May 28. PMID: 38581153.
