Part:BBa_K4941105

pylxp-ylER-LhYida

ylER and LhYida expression plasmids for the de novo synthesis of erythritol. The plasmids carry 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 6901
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 3846
  Illegal PstI site found at 6901
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1788
  Illegal XhoI site found at 2186
  Illegal XhoI site found at 5318
  Illegal XhoI site found at 5351
  Illegal XhoI site found at 10444
  Illegal XhoI site found at 10473
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 6901
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 6901
  Illegal NgoMIV site found at 5506
  Illegal NgoMIV site found at 11330
  Illegal AgeI site found at 2665
  Illegal AgeI site found at 4224
  Illegal AgeI site found at 4448
  Illegal AgeI site found at 6276
  Illegal AgeI site found at 9859
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1634
  Illegal BsaI site found at 9038
  Illegal BsaI site found at 10615
  Illegal BsaI.rc site found at 1049
  Illegal BsaI.rc site found at 2988
  Illegal BsaI.rc site found at 4771
  Illegal BsaI.rc site found at 10530
  Illegal BsaI.rc site found at 10813
  Illegal SapI site found at 1936
  Illegal SapI.rc site found at 757
  Illegal SapI.rc site found at 10061
  

Design

        A natural pathway for erythritol synthesis exists in Y. lipolytica, with the genes Yida and ER playing important roles. Yida encodes the enzyme 4-phosphate erythritol phosphatase, which converts erythrose 4-phosphate to erythrose. ER, on the other hand, encodes erythritol reductase, which converts erythrose to erythritol. However, the yield of erythritol was too low to be detected using HPLC conditions. It was hypothesized that the expression levels of Yida and ER might be insufficient, leading to undetectable erythritol production. To address this issue, we attempted to overexpress LhYida (BBa_K4941041), which is an erythritol 4-phosphate phosphatase derived from Lactobacillus helveticus, as well as ylER (BBa_K4941019) in Y. lipolytica. The goal was to enhance the expression of erythrose 4-phosphate (E4P) and facilitate the de novo synthesis of erythritol, thereby increasing its yield.

Build

        To enable the de novo synthesis of erythritol in Y. lipolytica, we selected the Yida and ER genes from Y. lipolytica and constructed the LhYida-ylER expression plasmid pYLXP’-LhYida-ylER (BBa_K4941043). We also constructed single gene expression plasmids, namely pYLXP’-LhYida (BBa_K4941041), pYLXP’-ylER (BBa_K4941037), and pYLXP’-ura (BBa_K4941030). Let's take pYLXP’-LhYida as an example:

        i) The target fragments LhYida (BBa_K4941020), pTEF (BBa_K4941012), and XPR2 (BBa_K4941014) were obtained using PCR and designed with 4bp sticky ends for plasmid assembly;

        ii) Plasmid pYLXP' (BBa_K4941039) was digested with nuclease BsaI to obtain linearized pYLXP' (BBa_K4941039);

        iii) The resulting reaction mixture from the golden gate assembly was transformed into E. coli DH5α.iv) The colonies were verified by PCR and sent for sequencing by Sangon Biotech (Shanghai, China) to confirm the correct sequence.

        After obtaining the single gene expression plasmids, we proceeded to construct the pYLXP’-LhYida-ylER expression plasmid:

        i) Firstly, plasmids were digested with AvrII and NotI to obtain PTEF1-LhYida-XPR1 (BBa_K4941044) and PTEF-URA-XPR2 (BBa_K4941026) fragments for plasmid assembly;

        ii) Plasmid pYLXP-ylER (BBa_K4941037) was digested with NheI and NotI to obtain linearized pYLXP-ylER0.

        iii) The linearized pYLXP-ylER and the two standardized fragments were ligated using T4 ligation;

        iv) The resulting T4 assembly reaction mixture was transformed into Escherichia coli DH5α.v) Colonies were verified by PCR and sent for sequencing by Sangon Biotech (Shanghai, China) to confirm successful construction and sequence accuracy.

Fig.1: a. T4 DNA Ligase; b. expression plasmid pylxp-LhYida-ylER (BBa_K4941043); c. Validation results of enzymatic digestion of the expression plasmid.

        

Result

        Next, we proceeded to integrate the LhYida-ylER expression fragment (BBa_K4941045) 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, following a standard protocol previously reported [1, 2]. Here is a brief overview of the protocol:

        1. During the exponential growth phase (16-24 hours), 1 mL of the Y. lipolytica culture was obtained from 2 mL of YPD medium (containing yeast extract 10 g/L, peptone 20 g/L, and dextrose 20 g/L) in a 14 mL shaker tube;

        2. The bacterial cells were then washed twice with 100 mM phosphate buffer (pH 7.0);

        3. The cells were resuspended in 105 µL of transformation solution, consisting of 90 µL of 50% PEG4000 solution, 5 µL of lithium acetate (2M), 5 µL of boiled single-stranded DNA (denatured salmon spermatozoa), and 5 µL of DNA products (including 200-500 ng of plasmid, linear plasmid, or DNA fragment);

        4. The mixture was thoroughly mixed and heated, followed by incubation for 1 hour at 37°C in a water bath;

        5. After incubation, the mixture was coated onto selected solid plates. It is important to note that the transformation mixtures needed to be oscillated every 15 minutes for 15 seconds during the incubation at 37°C;

        6. The selected marker for integration in this study was Uracil. As a result, we successfully integrated the LhYida-ylER fragments into the genome of Y. lipolytica po1g, obtaining the engineered strain po1g-LhYida-ylER (po1g-1).

        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.

Fig.2: a. Integration of LhYida-ylER into the genome for erythritol production; b. Erythritol production of po1g-LhYida-ylER (po1g-1).