Compared with the chemosynthetic method and fermentation using sporocarps, we thought it better to utilize engineered yeast to obtain COR. Therefore, we cloned the gene cluster (Figure. 1) for COR and PTN production in C. militaris into S. cerevisiae.

In this pathway (Figure. 1), three enzymes, Cns1, Cns2, and Cns3, are of vital importance. Cns1 and Cns2 can catalyze the formation of COR. And Cns3 produces PTN which prevents the deamination of COR by adenosine deaminase. Using S. cerevisiae engineered by 2019 SCU-China, we can produce both PTN and COR at the same time, as well as guarantee the yield thanks to the PTN’s protection to COR.

Figure. 1 The pathway of cordycepin and pentostatin.

A large population of yeast cells is essential for a desirable yield. Generally, it is fair to utilize inducible promoters to activate COR production after a growth phase has been completed. However, a midterm induction is disturbing and inappropriate for large-scale industrial production. Why don't we make S. cerevisiae produce COR automatically in the midterm?

From this perspective, we designed the delay expression system (Figure. 2). We utilized methionine and a serial transcription unit containing pMET3 and pGAL1 to realize delayed expression of Cns1 and Cns2 to synthesize COR.

Figure. 2 Three kinds of delay system designs. (a) We plan to use pGAL1 independently to acheive the delay because its function can be repressed by the glucose and induced by galactose. (b) We plan to use pMET3 independently to acheive the delay because its function can be repressed by the methionine. (c) We combine the pGAL1 and pMET3 delay expression systems to constructing the integrated delay expression system achieving a more accurate and flexible time regulation.

Cheap Raw Materials

Once the strain has been constructed, the cost for producing COR and PTN was low was. The price (in RMB) was shown as follows. More details.

Automization

Thanks to our delay expression system, we can flexibly regulate Cns1/Cns2 expression, avoiding adding inducers at the middle of fermentation which is inconvenient and may cause contamination.

Byproduct: PTN

In this project, we can get both PTN and COR at the same time. And PTN is also a valuable component that has already been used by FDA to treast leukemia.

During the brainstorm of our winter training (details of this training), our team member Fengxi Yu came up with the idea of CORegulaTIN. The story was about his mother. Several years ago, C. militaris uptake alleviated his mother’s cancer symptoms. Inspired by this experience, Yu decided to seek potential medicinal components of C. militaris. His answer was cordycepin (COR), an adenosine analog, which is a metabolite in C. militaris widely used in both health care and scientific research. What's more, an increasing number of articles have verified the clinical value of COR. It is reported that a phase III clinical trial of COR is now in practice.

It has been demonstrated in a previous studie (Xia Y et al., 2017) that Cns1, Cns2, and Cns3 are key enzymes in the synthesis of COR and PTN, but only Cns1 and Cns2 were successfully expressed and functional in the S. cerevisiae. And the functionality of Cns3 in S. cerevisiae required further verification. 2019 SCU-China iGEM team is the first research group to prove that Cns3 functions normally in S. cerevisiae.

In addition, we investigated the effect of linkers in the fusion protein of Cns1 and Cns2. We established a linker library for fusion proteins with different linkers, and selected the best linker to connect Cns1 with Cns2 in experiments.

As for Cns3, we wanted to express the HisG domain of Cns3 independently to reduce the burden for yeast. The expression of HisG domain of Cns3 in yeast has been rarely reported till now.

What's more, we designed a delay expression system to achieve the automatic and labor-saving fermentation.

Xia, Y., Luo, F., Shang, Y., Chen, P., Lu, Y., & Wang, C.. (2017). Fungal cordycepin biosynthesis is coupled with the production of the safeguard molecule pentostatin. Cell Chemical Biology, S2451945617303276.