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We consistently adhere to the Design-Build-Test-Learn cycles to advance the project. This year's project can be broadly categorized into three cycles. In the first cycle, we completed the construction and testing of the tagatose synthesis pathway within an E. coli chassis. In the second cycle, we successfully knocked down the competing pathway involved in tagatose synthesis. Finally, in the third cycle, we designed various bioreactors to optimize the performance of the engineered strain for tagatose production.

Figure 1: Engineering cycle diagram of our project

Since Escherichia coli lacks the pathway for synthesizing D-tagatose, we synthesized the gatz gene from Caldilinea aerophila and the pgp gene from Archaeoglobus profundus, and constructed the gatz and pgp genes into the pYB1c vector to obtain the pYB1c-Gatz-PGP plasmid. Considering that enhancing the conversion from G6P to F6P might be beneficial for D-tagatose production, we also overexpressed the pgi gene from Thermus thermophilus and constructed the pYB1c-Gatz-PGP-PGI plasmid.

Figure 2: Schematic diagrams of plasmids. A: Schematic diagram of the pYB1c-Gatz-PGP plasmid. B: Schematic diagram of the pYB1c-Gatz-PGP-PGI plasmid.

We used in step in fusion clone technology. In this way, two or three genes could be assembled into plasmed efficiently.

pYB1c-Gatz-PGP:

A、PCR amplification of target genes
B、Colony PCR verification
C、Plasmid digestion verification
D、DNA sequencing verification

Figure 3:PCR Results Figure. A: Gel electrophoresis image of PCR amplification for pYB1c, Gatz, and PGP. B: Gel electrophoresis image of colony PCR performed on 10 randomly picked colonies from the plate. C: Gel electrophoresis image of plasmid digestion with KpnI and EcoRI enzymes. D: Sequencing of the correct plasmids verified by colony PCR and enzyme digestion.

pYB1c-Gatz-PGP-PGI：

A、PCR amplification of target genes
B、Colony PCR verification
C、Plasmid digestion verification
D、DNA sequencing verification
Figure 4:PCR Results Figure. A: Gel electrophoresis image of PCR amplification for pYB1c, Gatz, PGI,and PGP. B: Gel electrophoresis image of colony PCR performed on 10 randomly picked colonies from the plate. C: Gel electrophoresis image of plasmid digestion with KpnI and EcoRI enzymes.D: Sequencing of the correct plasmids verified by colony PCR and enzyme digestion.
1、Mapping the D-tagatose standard curve
First, we developed a tagatose assay method. By preparing different concentrations of tagatose standards and mixing them with resorcinol solution, the mixture was incubated at 100°C for 20 minutes. The absorbance was then measured using a microplate reader. A standard curve was constructed by plotting the absorbance on the Y-axis against the concentration of tagatose standards on the X-axis.The resulting standard curve equation is y = 0.0023x + 0.058. The regression constant R²=0.9961. This indicates that our detection method is very effective and highly sensitive for the detection of tagatose.

Figure 5:Determine the color change and absorbance values after the reaction of different concentrations of tagatose with resorcinol.

2、D-Tagatose concentration assay
The plasmids pYB1c-Gatz-PGP and pYB1c-Gatz-PGP-PGI were transformed into competent BW25113 cells to obtain the strains pYB1c-Gatz-PGP/BW25113 and pYB1c-Gatz-PGP-PGI/BW25113. Then, after the addition of 0.2% arabinose, protein expression was induced at 30°C, and 12 hours later, the induced bacterial culture was transferred into M9 fermentation medium containing 20 g/L glucose for the biosynthesis of tagatose. The D-tagatose yield in the fermentation broth was measured. The yield detection results show that the BW strain did not produce D-tagatose. The strain overexpressing PGP and GatZ reached a tagatose concentration of 191.01 mg/L after 12 hours of fermentation. Subsequently, on this basis, we further overexpressed the pgi gene, and its tagatose production reached 197.33 mg/L, which is a 197.3-fold increase in yield compared to the BW25113 strain, indicating that our construction and strengthening of the pathway play an important role in the synthesis of tagatose. It is worth noting that further enhancement of PGI on the basis of GatZ and PGP does not significantly improve the yield of tagatose.

Figure 6:Changes in D-tagatose production in strains overexpressing gatz, pgp and PGI genes compared to strain BW25113

A D-tagatose biosynthesis pathway was successfully established in E. coli. Although the overexpression of the pgi gene did not significantly improve the yield compared to the non-overexpressing strain, the pYB1c-Gatz-PGP-PGI/BW25113 strain was selected for subsequent experiments. The D-tagatose concentration in this strain increased by 197.3 -fold compared to the wild-type BW25113.

G6P is generated in the second catalytic reaction of the D-tagatose biosynthesis pathway. In recombinant E. coli, G6P can be converted by glucose-6-phosphate dehydrogenase (G6PDH), encoded by the zwf gene, into 6-phosphoglucono-δ-lactone, diverting part of the G6P flow into the pentose phosphate pathway. F6P and T6P are produced in the third and fourth catalytic steps of the D-tagatose biosynthesis pathway, respectively. In recombinant E. coli, F6P may be converted by 6-phosphofructokinase I, encoded by the pfkA gene, and the resulting product flows into the tricarboxylic acid (TCA) cycle. To reduce the loss of intermediate products in the D-tagatose biosynthesis pathway during the metabolic processes in recombinant E. coli, without causing growth defects, we utilized CRISPRi technology to modify the competitive pathways in recombinant E. coli, thereby reducing the consumption of intermediate products by competitive pathways and improving the D-tagatose production yield.
First, we verified the D-tagatose yield of strains with individual inhibition of pfkA or zwf:

Figure 7:To assess the effect of CRISPRi-mediated silencing of the zwf and pfkA genes on D-tagatose production.

It was found that individually inhibiting zwf or pfkA could increase D-tagatose production; however, it is worth noting that bacterial growth was severely affected. Therefore, in the next step, we simultaneously inhibited pfkA and zwf, and introduced mutations at the 7th and 8th bp of their sgRNAs to generate a mutant library. From this mutant library, we screened for a mutant that did not affect bacterial growth but could increase the yield of D-tagatose. The R6k-dcas9-pfkA-zwf-M plasmid was constructed.

Figure 8:Schematic diagram of the R6k-dcas9-pfkA-zwf-M plasmid.

We used Golden Gate to assemble sgRNA-pfkA and sgRNA-zwf with a R6K-dCas9 backboen.
1、PCR amplification of target genes
2、Colony PCR verification
3、Validation of plasmid digestion
4、Send Sequencing Verification

Figure 9:PCR Results Figure. A: Gel electrophoresis image of PCR amplification for sgRNA-pfkA-M, sgRNA-zwf-M. B: Gel electrophoresis image of colony PCR performed on 10 randomly picked colonies from the plate. C: Gel electrophoresis image of plasmid digestion with KpnI and XhoI enzymes.D: Sequencing of the correct plasmids verified by colony PCR and enzyme digestion.

Subsequently, the plasmids R6k-dcas9-pfkA-zwf-M and pYB1c-Gatz-PGP-PGI were co-transformed into BW25113. Ninety-five colonies were randomly selected for fermentation experiments using glucose as the substrate, and their growth and production yields were screened.

Figure 10:The fluorescence value detected by the Microplate reader.


Figure 11:The blue bar indicates the yield of Tagatose; the yellow circle indicates OD600


Figure 12: Comparative yield graph after rescreening

A good mutant was identified and named DT-D9. Compared to the non-inhibited strain DT1, the tagatose yield of DT-D9 increased by 3.34-fold, reaching a D-tagatose yield of 634.66 mg/L.
Through sequencing the mutant, we found that the 7th and 8th base pairs of sgRNA-pfkA mutated from GG to TG, and the 7th and 8th base pairs of sgRNA-zwf mutated from AT to CT. By altering the 7th and 8th base pairs of the sgRNA, the binding efficiency of dcas9 to the target gene can be modified, thereby achieving varying degrees of inhibition. The best mutant, DT-D9, showed good growth and higher yield, proving that our strategy was effective.

Figure 13: The sequencing results of the mutations at the 7、8bp position in sgRNA7 of DT-D9

We aim to create a small, simple bioreactor using inexpensive and readily available materials which high school students could be easily used. This bioreactor should have features such as stirring, temperature control, feeding, and pH adjustment, with a capacity for 1.8 L of culture.
For temperature control, we placed the bioreactor on a heating plate combined with a magnetic stirrer, which could be used for temperature control and blending the culture mixature. At the same time, we used an electrode controller that can automatically control the timing of glucose, acid, and base additions. It is also equipped with an air pump to regulate the airflow into the reactor. Additionally, we purchased a pH electrode to measure the pH value inside the reactor and display accurate readings on the controller's screen.

Figure 14: Exhibition of Various Components of a Small-Scale Fermentation bioreactors.
We cultured the optimal strain DT-D9 through primary and secondary seed cultures, eventually inoculating it into the 1.8L bioreactor. After 8 hours of cultivation, the culture became noticeably turbid, proving that the device is functional. We adjusted the bioreactor pH to around 7, temperature to 37°C (However, it had a very bad temperature control), and stirring speed to 450 rpm.

Figure 15: The growth condition of the strain at two hours and eight hours during fermentation.

After adding the final concentration of 0.1mMol/L IPTG and 0.2% arabinose, take a sample every two hours. Centrifuge samples and place them in an microplate reader to detect tagatose concentration. The final tagatose concentration reached 1083.01 mg/L after 26h fermentation.

Figure 16: Time carve of tagetose concentration in fermentation.
By designing a small, simple bioreactor, we successfully scaled up the cultivation of the optimal strain and achieved D-tagatose production in a simple fermentation device. In future experiments, we can further optimize this small, simple device, such as increasing the stirring speed, enhancing airflow, and using 3D printing technology to further improve the controller.