Team:SCU-China/Demonstrate
PROJECT
DEMONSTRATION
2019 SCU-China tries to ferment cordycepin and pentostatin together in S.cerevisiae. During our fermentation, Cns3 is constitutively expressed and catalyzes the production of pentostatin (PTN), repressing adenosine deaminases (ADA) gradually. The expression of Cns1 and Cns2 is supposed to be activated automatically when the yeast's growth turns to plateau stage. At plateau stage, the population of cells is adequate, and endogenous ADA has already been inhibited by PTN, which is the best time for COR production.
As the design for co-fermentation of COR and PTN has never been constructed and used in S.cerevisiae before, one of the most important task for demonstration to is prove that our design can work in S.cerevisiae.
There are three parts in our design: Cns1 and Cns2 for COR production, Cns3 for PTN production and the delay expression system.
Cns1 and Cns2 for COR production
COR production is the core of our project. We completed the molecular cloning in E.coli DH5α and S.cerevisiae BY4741. And favorable colony PCR results for modified BY4741 are shown below. (Figure. 1):
Figure.1 Colony PCR of cns1 and cns2.
(a) Colony PCR of pYES2-Cns1. Lane1 and lane 3-8 show the positive results. (b) Colony PCR of pYES2-Cns2. Lan5 and lane8 show the positive results.
We did HPLC to verify the synthesis of COR. Thorough overcoming many difficulties, we successfully detected the existence of COR (Figure. 2).
Figure. 2 HPLC analysis of COR
Top: Standard sample of 5mM COR.
Middle: Supernatant fraction of Cns1-linker-Cns2 sample.
Bottom: Supernatant fraction of BY4741 sample. The strain BY4741 was culturing in YPDG media for 3 days.
Because of the step-by-step verification of the experiments, we could confirm that the Cns1 and Cns2 can work in the S.cerevisiae. Besides, the fusion protein we designed could worked nicely as well.
Cns3 for PTN production
PTN is a valuable and economic product of our project. Besides, it's also the protector in our project, preventing COR from deamination. We successfully constructed the plasmids of Cns3 and its HisG domain, then we verified their expression and function via RT-qPCR (Figure. 3) and HPLC (Figure. 4). The PTN was detected by HPLC.
Overexpression of Cns3 may consume too many resources and affect the growth of yeast. However，we also need enough PTN to ensure the inhibition of adenosine deaminase. We selected several constitutive promoters that have different strengths for Cns3. To test the function of these sequences gained from the genome, we chose yeGFP as a reporter (Figure. 6). These promoters could work as expected because of the successful expression of yeGFP.
Figure. 3 RT-qPCR of Cns3-HisG-mf/Cns3-HisG-only. No-load is the S. cerevisiae without transformed plasmids. The reference gene is PGK1. The RNA of cns3 is detected successfully. The bars are SE.
Figure. 4 HPLC analysis of PTN
a. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of Cns3-hisG-MF sample. The strain BY4741 was culturing in YPDG media for 3 days.
b. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of Cns3-hisG ONLY sample. The strain BY4741 was culturing in YPDG media for 3 days.
c. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of BY4741 sample. The strain BY4741 was culturing in YPDG media for 3 days.
We also constructed the constitutive promoters with cns3 and tested their function. The results are very nice as well.
Figure. 5 RT-qPCR of constitutive promoters (pPDA1/pTEF1/pTPS1) – Cns3. NC is the S. cerevisiae without transformed plasmids (constitutive promoters – Cns3). The reference gene is PGK1. The bars are SE.
Delay expression system
pGAL1 and pMET3 are two basic parts of our delay system. We utilized yeGFP as a reporter to verify their function.
The results shows pGAL1 works very well in S. cerevisiae under the induction of galactose. (Figure. 6).
Figure. 6 Green fluorescence of S.cerevisiae BY4741 transformed pYES2-yeGFP which was induced by galactose. (a) Fluorescence observation of S.cerevisiae BY4741 transformed pYES2-yeGFP. Series 1: BY4741 WT in YPD medium, Series 2: BY4741 WT in YPG medium, Series 3: BY4741 transformed BY4741 in YPD medium, Series 4: Transformed BY4741 in YPG medium. Visible fluorescence in Series 4. (b) The fluorescence measurement of S.cerevisiaeBY4741 transformed pYES2-yeGFP. YPD: YPD medium, YPG: YPG medium, BY4741 WT: S.cerevisiae, BY4741 wildtype, BY4741 transformed: S.cerevisiae, BY4741 transformed plasmid pYES2-yeGFP, excitation light is 488nm and emission light is 525nm，***: P<0.001.
As for pMET3, we pulled down this promoter from the genome of S. cerevisiae BY4742. To confirm its function, we introduced this promoter with a yeGFP downsteam into S. cerevisiae YM4271 and cultured them in the methionine dropout medium. Compared with the complete medium group, It is obvious that pMET3 can work perfectly in the YM4271 (Figure. 7).
Figure. 7 The green fluorescence of S. cerevisiae YM4271 transformed pYES2-pMETHIONINE3-yeGFP.
For the further works of our delay system, we have already constructed the whole gene circuit: pYES2-pMET3-GAL4-pGAL1-yeGFP that and has passed the sequencing verification (Figure. 8). We will test the gene circuit in S. cerevisiae YM4271. For the preparation of the experiment, we made a growth curve of YM4271 (Figure. 9).
Figure. 8 The double enzyme digestion verification result of pYES2-pMET3-GAL4-pGAL1-yeGFP. Marker: Takara DL10000 and Takara DL15000, Lane 1: the plasmid pYES2-pMET3-GAL4-pGAL1-yeGFP (10384bp), Lane2: the plasmid pYES2-pMET3-GAL4-pGAL1-yeGFP after digesting by BamHI (10384bp).
Figure. 9 Data fitting of experimental data and logistic model and parameter estimates of the model. In the figure, b is the opposite number of growth rate, d is the asymptote and e is the x-axis skewing.
In conclusion, our design is considered to work successfully in S. cerevisiae. And SCU-China is proud to be the first research group that verified Cns3 and its HisG domain could work in S. cerevisiae, and Cns1-linker-Cns2 fusion protein can function in S. cerevisiae. We have also perfomed many preparations for future work.