Results

Result 1. Screening of xylose reductases

Xylose reductase (XR) is responsible for encoding xylose reductase and can increase the expression of xylose reductase. It can realize the conversion from xylose to xylitol. Therefore, we chose three different sources of XR to construct pET28a-PsXR (BBa_K4941100), pET28a-DnXR (BBa_K4941099) and pET28a-KsXR (BBa_K4941098). They were transformed into E. coli BL21. And xylose reductase was expressed to catalyze the reaction of xylose to xylitol in TB medium supplemented with 5 g/L xylose precursor, using glycerol as the carbon source. The xylitol yield in the fermentation broth was detected by HPLC.

The correctly sequenced xylose reductase expression plasmids pET28a-KsXR-kana (BBa_K4941034), pET28a-PsXR-kana (BBa_K4941033), pET28a-DnXR-kana (BBa_K4941035)), and T7 RNAP expression plasmids (BBa_K4941063) were transferred into E. coli BL21. The transformants were selected and transferred into the liquid medium and cultured in a shaker at 37 ℃ and 220 rpm for 12 hours. The seed solution was then transferred into 30 ml TB medium and cultured at 37 ℃ and 220 rpm for 3 hours. After that, the expression of xylose reductase was induced by adding 30 ul of IPTG inducer and the culture was continued at 28 ℃ and 160 rpm for 16 hours. Subsequently, xylose was added to a concentration of 5 g/L, and the reaction was carried out at 30 ℃ and 220 rpm for 36 hours. Samples were taken every 12 hours for HPLC detection.

The results showed that BL21-pET28a-PsXR catalyzed the production of 4.6 g/L xylitol from the substrate 5 g/L xylose after 36 hours. It was demonstrated that the xylose reductase derived from Pichia stipitis has the ability to catalyze xylose to produce xylitol in the E. coli BL21 protein expression system, and xylitol production through fermentation in E. coli is achieved.

Result 2. promoter engineering to enhance xylose reductase expression

By replacing promoters of T7RNAP with different strengths, the regulation of protein expression levels can be realized. Based on promoter engineering, the optimal promoter is selected to further enhance the expression of xylose reductase, resulting in more efficient catalytic efficiency.

Three transformants containing different lacUV5 promoters were selected and transfected into 96-well culture plates containing LB liquid medium. The plates were then cultured in a shaker at 37 °C and 220 rpm for 12 hours. The seed solution was further transferred into 48-well culture plates containing 2 ml of fermentation medium and cultured at 37 °C and 220 rpm for 3 hours. Afterwards, 30 μl of IPTG was added as an inducer, and the culture was continued at 28 °C and 160 rpm for 16 hours to allow for the expression of xylose reductase. For xylose reductase expression, the plates were incubated at 16°C and 160 rpm for 16 hours. Subsequently, xylose was added to a final concentration of 8 g/L, and the reaction was carried out at 30°C and 220 rpm for 36 hours. HPLC detection was performed to analyze the reaction.

The results showed that MB7-PsXR successfully catalyzed the production of 6.8 g/L of xylitol from the substrate of 8 g/L xylose after 36 hours. This outcome demonstrates the feasibility of the strategy employed, which involved optimizing the pET expression system through the construction of a promoter library for T7RNAP expression.

Result 3. De novo synthesis of erythritol in Y. lipolytica

A natural erythritol synthesis pathway exists in Y. lipolytica. E4PP/Yida is the gene encoding 4-phosphate erythritol phosphatase, which catalyzes the conversion of erythrose 4-phosphate to erythrose, and ER is the gene encoding erythritol reductase, which catalyzes the conversion of erythrose to erythritol. However, the yield of erythritol was too low to be detected under HPLC conditions. It was speculated that the expression levels of erythritol 4-phosphate phosphatase (Yida) and erythritol reductase (ER) might be too weak, resulting in undetectable erythritol production. LhYida (BBa_K4941041) is an erythritol 4-phosphate phosphatase derived from Lactobacillus helveticus. Based on this, we attempted to overexpress LhYida (BBa_K4941041) and ylER (BBa_K4941037) in Y. lipolytica to enhance the expression of erythrose 4-phosphate (E4P) to erythritol, thus realizing the de novo synthesis of erythritol yield.

We proceeded to integrate the LhYida-ylER expression fragment (BBa_K4941106) into the genome of Y. lipolytica po1g in order to achieve de novo synthesis of erythritol. For successful integration, we employed the lithium acetate transformation method. Subsequently, we selected positive transformants for fermentation experiments. The strain was transferred to YNB-URA medium and cultured for 48 hours to obtain the seed liquid. Samples were taken after transferring 500 µL of seed liquid to 30 mL of fermentation medium, which was then cultured at 30°C and 220 rpm on a shaker for 120 hours. The content of erythritol was detected using HPLC. The results revealed that the engineered strain po1g-1 overexpressing LhYida and ylER achieved a yield of 2.1 g/L of erythritol, thus successfully realizing de novo synthesis of erythritol in Y. lipolytica.

Result 4. Construction of the fluorescent reporter system for characterizing erythritol

The fluorescent reporter system has been widely used to characterize the intensity of protein expression [1] [2]. Inducible promoters produce different intensities depending on the concentration of the inducer, thus altering the level of protein expression [3]. Jean Marc Nicaud [4]combines an erythritol-inducible promoter with a luciferase reporter system that couples fluorescence intensity to erythritol concentration. Thus, the erythritol concentration was obtained by a more intuitive and simple method. Based on this, we used an erythritol-inducible promoter to express the luciferase-encoding gene. We attempted to build an erythritol-luciferase reporter system in Y. lipolytica, and characterized the erythritol yield using fluorescence intensity.

We proceeded to transform the pylxp-pEYK1-Nluc (BBa_K4941036) plasmid into the Y. lipolytica po1g strain. The transformation was successful, resulting in the creation of the engineered strain po1g pylxp-pEYK1-Nluc (po1g-2). Subsequently, we proceeded to select positive transformants for fermentation experiments. The strain was transferred to YNB-leu medium and cultured for 48 hours to obtain the seed liquid. Then, 500 μl of the seed liquid was transferred to 30 ml of fermentation medium with varying concentrations of erythritol (0 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L). The samples were taken after 48 hours of culturing in a shaking bed at 30°C and 220 rpm. The luciferase substrate was added to initiate the reaction, and a kinetic assay was performed to compare the fluorescence intensity of the fermentation broths with different erythritol concentrations.

The results demonstrated a notable variation in the fluorescence intensity of the fermentation broth upon the addition of different concentrations of erythritol, as depicted in Figure 5. The fluorescence intensity provided a visual representation of the varying erythritol concentrations within the fermentation broth.

Result 5. Screening of Erythrose-4P phosphatase and construction of the optimal strain

The successful construction of the fluorescent reporter system for characterizing erythritol production helped us to better screen erythritol-producing strains. To further enhance the expression of E4P to erythritol, we selected Erythrose-4P phosphatase (Yida) from different sources. We selected EcYida (BBa_K4297032) from E. coli [1] and StYida from Streptococcus thermophilus (BBa_K4297032). The expression of pylxp-pEYK1-Nluc (BBa_K4941036) was used to assess the erythritol production of each engineered strain by fluorescence intensity. we proceeded to transform the expression fragments EcYida-ylER (BBa_K4941103) and StYida-ylER (BBa_K4941108) into the genome of Y. lipolytica po1g, enabling integration of their expression [1, 2]. In our project, the selection marker used was Uracil. Consequently, we successfully integrated the EcYida-ylER and StYida-ylER expression fragments into the genome of Y. lipolytica po1g, resulting in the engineered strains po1g-EcYida-ylER plate (po1g-3) and po1g-StYida-ylER plate (po1g-4).

Subsequently, we utilized the lithium acetate transformation method to introduce pylxp-pEYK1-Nluc into the mixed bacteria of po1g-1, po1g-3, and po1g-4 separately. The strains were then transferred to YNB-URA medium and cultured for 48 hours to obtain the fermentation broth. The fermentation broth was cultivated at 30°C and 220rpm. In order to initiate the reaction, the luciferase substrate was added, and a kinetic assay was performed to compare the fluorescence intensity of the fermentation broths of the three engineered strains. The strain with the highest yield was selected for further analysis. The chosen engineered strains were subsequently transferred into YNB-leu fermentation broth and allowed to ferment for 120 hours. The fermentation broth was then collected for HPLC detetion. According to the results, strain po1g-3, expressing EcYida-ylER, exhibited the highest erythritol yield, as depicted in Figure 6. The HPLC assay confirmed that the erythritol yield reached 5.9 g/L.

Conclusion

Finally, we produced xylitol by fermentation in E. coli. The optimal promoter mutant MB7 was utilized to initiate the expression of T7RNAP, which directed xylose reductase transcription. the MB7-PsXR strain was fermented for 36h in TB medium with 8g/L xylose as substrate to obtain 6.8g/L xylitol. We also successfully constructed the erythritol de novo synthesis pathway in Y. lipolytica po1g. A fluorescent reporter system for characterizing erythritol was constructed to screen the source of Erythrose-4P phosphatase. The engineered strain po1g-3 (po1g- EcYida-ylER) was fermented in YNB-Leu medium for 120 h. The yield of erythritol in the fermentation broth was 5.9 g/L by HPLC.

Reference

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2. Wang, X., et al., Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nat Commun, 2021. 12(1): p. 1411.

3. Qiu, C., H. Zhai, and J. Hou, Biosensors design in yeast and applications in metabolic engineering. FEMS Yeast Res, 2019. 19(8).

4. Trassaert, M., et al., New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica. Microb Cell Fact, 2017. 16(1): p. 141.