Part:BBa_K4941107
pylxp-ylER-StYida
The ylER and StYida expression plasmids for the de novo synthesis of erythritol. The plasmid carries the Amp resistance gene, URA screening marker and Leu screening tag for screening of engineered strains.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 4319
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Illegal XhoI site found at 10467 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 4319
Illegal PstI site found at 6895 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 4319
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Screening of Erythrose-4P phosphatase
Design
The successful construction of the fluorescent reporter system played a crucial role in our ability to effectively screen erythritol-producing strains. In order to enhance the expression of Erythrose-4P phosphatase (Yida) for increased erythritol production, we selected this enzyme from various sources. These selected enzymes were then integrated into Y. lipolytica for erythritol production. We utilized the expression of pYLXP-pEYK1-Nluc (BBa_K4941036) to evaluate the erythritol production of each engineered strain based on fluorescence intensity.
Build
We selected EcYida (BBa_K4297032) from E. coli [1] and StYida (BBa_K4297028) from Streptococcus thermophilus. Through PCR, we obtained the target fragments EcYida (BBa_K4941018) and StYida (BBa_K4941028). Reference to the construction of pylxp-LhYida-ylER (BBa_K4941043), we obtained two expression plasmids, pylxp-EcYida-ylER (BBa_K4941032) and pylxp-StYida-ylER (BBa_K4941046).
- Fig.1: a. pylxp-EcYida-ylER and sequencing results, b. pylxp-StYida-ylER and sequencing results
Result
Next, we proceeded to transform the expression fragments EcYida-ylER (BBa_K4941031) and StYida-ylER (BBa_K4941048) 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 9. The HPLC assay confirmed that the erythritol yield reached 5.9 g/L.
- Fig.2: a. Screening of Erythrose-4P phosphatase, b. Fluorescence results of high throughput screening, c. Erythritol yields of control po1g-3 after 120h.
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