Part:BBa_K4941103
ku70UP-ylER-EcYida-URA-ku70dw
Three expression frames (EcYidA expression frame, ylER expression frame, and URA screening marker) initiated by the promoter pTEF and terminated by XPR2 were expressed on one plasmid.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 6663
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 6663
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1553
Illegal XhoI site found at 1951
Illegal XhoI site found at 5080
Illegal XhoI site found at 5113 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 6663
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 6663
Illegal NgoMIV site found at 5268
Illegal AgeI site found at 2430
Illegal AgeI site found at 4210
Illegal AgeI site found at 6038 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1399
Illegal BsaI.rc site found at 814
Illegal BsaI.rc site found at 2753
Illegal BsaI.rc site found at 4533
Illegal SapI site found at 1701
Illegal SapI.rc site found at 522
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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