Strategies for realizing microbial production of erythritol and xylitol
Our strategy is divided into two main parts: xylitol production by fermentation in Escherichia coli and erythritol production using glucose as a substrate in Yarrowia lipolytica.
Xylitol production by fermentation in Escherichia coli
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.
Figure 1. xylose reductase catalyzes the production of xylitol from xylose
2. Promoter engineering to enhance xylose reductase expression
By replacing promoters 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.
Figure 2. promoter engineering to enhance xylose reductase expression
De novo synthesis of erythritol from glucose as a substrate in Y. lipolytica.
1. 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_K4941020) is an erythritol 4-phosphate phosphatase derived from Lactobacillus helveticus. Based on this, we attempted to overexpress LhYida (BBa_K4941020) and ylER (BBa_K4941019) in Y. lipolytica to enhance the expression of erythrose 4-phosphate (E4P) to erythritol, thus realizing the de novo synthesis of erythritol yield.
Figure 3. The ylER and LhYida co-expression fragments randomly integrated in the genome of Y. lipolytica for the production of erythritol
2. 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.
Figure 4. Construction of a fluorescent reporter system for characterizing erythritol
3. 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. Expression frames are integrated in Y. lipolytica for the production of erythritol. The expression of pylxp-pEYK1-Nluc (BBa_K4941036) was used to assess the erythritol production of each engineered strain by fluorescence intensity.
Figure 5. Screening of Erythrose-4P phosphatase and production of erythritol
Reference
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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.