Team:USTC
New Basic Parts
MVA pathway BBa_K4231000——>BBa_K4231006
All the enzyme gene is coding the MVA pathway from acetyl-coenzyme A to monoterpene. Moreover, in C-terminal of each enzyme, we design to add the PTS1 signal sequence in order to help them locate in peroxisome successfully. Among them， we choose the promoter pTEF1 for ERG8, ERG12, ERG13 and IDI1 and we select the promoter pGAP for ERG10, ERG19, ERG20 and NADH-HMG. In this way, We can obtain the appropriate enzyme ratio according to the strength of the promoter to achieve higher yield. In addition, we optimized the codons of each enzyme gene for Y.lipolytica so that higher translation efficiency can be achieved in our strain.
Promoter BBa_K4231010 BBa_K4231012 BBa_K4231013
In this part, we choose to understand several promoters in Yarrowia lipolytica. Depending on their transcriptional intensity, it is more convenient to adjust the proportion of each enzyme in the metabolic pathway so that the intermediates do not accumulate. Among them, GAP promoter has the strongest transcriptional activity, ACL2 promoter is the second, and ICL1 promoter is the lowest. In the future modification of metabolic engineering, promoters with different strengths can provide diverse choices in promoter optimization.
Tag BBa_K4231020
HUH is a special select tag in Yarrowia lipolytica. Two HisG homologous arms is connected at both ends of YlURA3, which gives this tag high homologous recombination efficiency. The tag's total length is 3715 bp, of which HisG is 1150 bp, and URA3 is 855 bp. This modification makes it easy for Yarrowia lipolytica to knock out URA3 spontaneously by homologous recombination, which allows experimenters to reuse URA select tag in Yarrowia lipolytica. In addition, the total length of the HUH we build is shorter than most select tags of the same type, which makes the tag much easier to be reused.
Knock-out box for URA3 BBa_K4231011
The knockout box is a 400 bp DNA sequence, which mainly contains the promoter and terminator of the URA3 gene. Inspired by the application of the knockout box in Kluyveromyces lactis, we moved the knockout box to Y.lipolytica and successfully applied it. Using the homologous recombination mechanism of Y.lipolytica, the knockout box can undergo homologous recombination at both ends of the URA3 gene, thereby replacing the URA3 gene and achieving the purpose of knocking out the screening tag URA3. In addition, multiple use of the knockout cassette plus forward screening with FOA medium yields better and more reliable results than traditional HUH screening tags.
