Team:USTC

Composite parts

    Except basic parts, we also add some composite parts on the registry. At the same time, targeting on the main parts, we made a improvement for the certain part.

Table 1. All the composite parts we added this year.

Parts name	Type	Description
BBa_K4231016	Composite	Oleic acid inducible pTEF1
BBa_K4231017	Plasmid	pUC-ERG10-ERG13-URA3
BBa_K4231018	Plasmid	pUC-IDI-NA-URA3-ERG8-ERG19
BBa_K4231019	Plasmid	pUC-Bs3-LEU-ERG12-ERG20
BBa_K4231021	Cell	The Y.lipolytica strain PYB1

Improvement

Regulatory BBa_K4231016 BBa_K4231007 BBa_K4231008 BBa_K4231009

    The length of A1 is 400 bp, the length of R1 is 200 bp, and the length of A3 is 338 bp. The three parts are assembled together to form the UAS sequence of the inducible TEF promoter. Among them, R1 sequence plays the role of oleic acid induction, A1 and A3 sequences play the role of enhancing transcription activity. Considering the limited length and capacity of upstream sequence, we choose a single-repeat R1 sequence to construct UAS sequence. The promoter TEF1 carrying this UAS can be strongly induced by oleic acid, and when the carbon source is glucose, the transcription activity is almost zero.That means that high fold induction exists in the hybrid promoter. Moreover, This mixed promoter induced by oleic acid is nearly three times stronger than the native POX2 promoter and is a powerful tool for regulating and enhancing gene expression.

Figure.1 Promoter strength is determined by expression of hrGFP and measured as mean fluorescence of an equal number of transformed cells. Glucose samples are shown on the left and oleic acid on the right.

Figure.2 Promoter induction using oleic acid as the carbon source relative to glucose as carbon source. Regulatory sequence A1R1A3 present the most suitable fold induction.

Plasimid BBa_K4231017 BBa_K4231018 BBa_K4231019

    In our work, we have constructed three large plasmids, pUC-ERG10-ERG13-URA3，pUC-IDI-NA-URA3-ERG8-ERG19，pUC-Bs3-LEU-ERG12-ERG20.which contain several target gene and tags for Y.lipolytica. In the preparation and transformation of Y.lipolytica competent cells, these three plasmids helped us successfully construct the MVA pathway in Y.lipolytica peroxisomes.

Strain BBa_K4231021

    Our strain, PYB1, was modified from Yarrowia lipolytica strain po1f (ku70+). We transferred the genes of all enzymes of MVA pathway with the peroxisome localization sequence PTS1 into our strain so that it can synthesize GPP through the MVA pathway in the peroxisome. We also transferred the borneol synthase Bs3 with PTS1 into it so that our strain can synthesize borneol from GPP in peroxisomes. Due to the high concentration of acetyl-CoA, the substrate of MVA pathway, in the peroxisome, the concentration of GPP produced by the strain is several times that of po1f. Moreover, the high GPP production capacity also makes the strain have the potential to produce other terpenoids after further modification, which provides ideas and available chassis for subsequent terpenoid production.