Plasmid

Part:BBa_K4941104

Designed by: Xinyu Chen   Group: iGEM23_ZJFH-Nanjing   (2023-10-12)


pylxp-ylER-EcYida

ylER and EcYidA expression plasmids for the de novo synthesis of erythritol by Yarrowia lipolytica. The plasmid carries the Amp resistance gene, URA screening marker and Leu screening tag for screening of engineered strains.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 6898
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 6898
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1788
    Illegal XhoI site found at 2186
    Illegal XhoI site found at 5315
    Illegal XhoI site found at 5348
    Illegal XhoI site found at 10441
    Illegal XhoI site found at 10470
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 6898
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 6898
    Illegal NgoMIV site found at 5503
    Illegal NgoMIV site found at 11327
    Illegal AgeI site found at 2665
    Illegal AgeI site found at 4445
    Illegal AgeI site found at 6273
    Illegal AgeI site found at 9856
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1634
    Illegal BsaI site found at 9035
    Illegal BsaI site found at 10612
    Illegal BsaI.rc site found at 1049
    Illegal BsaI.rc site found at 2988
    Illegal BsaI.rc site found at 4768
    Illegal BsaI.rc site found at 10527
    Illegal BsaI.rc site found at 10810
    Illegal SapI site found at 1936
    Illegal SapI.rc site found at 757
    Illegal SapI.rc site found at 10058

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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