The generation of highly-expressing, mutation-resistant yeast promoter sequences bears significant scientific implications. Particularly, the development of such mutation-resistant yeast promoter sequences holds great promise in various areas.

Yeast, as a widely used eukaryotic microorganism in biotechnology and biopharmaceutical applications, relies on efficient gene expression for optimal yields. As a sustainable production host capable of undergoing multiple fermentation cycles, accumulated mutations within the promoter region can introduce variations among production strains, which hinders large-scale manufacturing and industrial applications. By employing mutation-resistant promoter sequences, the risk of mutations that may disrupt or diminish gene expression can be mitigated, ensuring stable and high-level expression of target genes in yeast.

Additionally, the utilization of mutation-resistant yeast promoter sequences addresses safety concerns associated with LTB production. LTB serves as a potential vaccine candidate and finds application in vaccine development. However, mutations in the promoter region can potentially lead to increased toxicity or undesirable side effects. By employing mutation-resistant promoter sequences, the risk of such mutations can be mitigated, ensuring stable and efficient expression of LTB in yeast. This, in turn, contributes to the overall success and safety of vaccine development efforts.

Through our wet lab experiments, we manage to validate that our Pymaker generated promoters attain the ability to drive extremely high expression of multiple targeted protein, especially comes to the expression of LTB.

1. Yeast plasmid extraction success

The figure illustrates the plasmid extracted from the targeted microorganisms.

2. Yeast transformation success

Ampicillin has been added to the dishes, and only the yeasts that has been successfully transformed with our plasmids can grow on it.

3. Dual-fluorescence reporter system function success

The figure illustrates that the yeasts can successfully express two different kind of fluorescence, which means our reporter system is functional in yeasts.

The figure illustrates that by using flow cytometry, the reporter system remains steady in a great yeasts’ population, which means our reporter system can be used to directly show the relative expression rate driven by designed promoters, without being influenced by the growing condition of yeasts [1].

We further synthesized the dual-fluorescence reporter plasmids where 15 promoters sequences (8 High expression promoters and 4 Low expression promoters, which both are generated by Pymaker, plus 3 natural high expression promoters: pGAP, pTEF, pADH) are already placed in as we described in registry pages in detail.

We transform the plasmids into targeted yeasts and use fluorescence-activated cell sorting (FACS) strategy [2] and record the fluorescence density through a flow cytometer. The figure below shows the result.(more detailed result of every promoter sequence will be displayed on the registry pages)

1. Plasmid Assemble Process Design

We design to extract promoter sequences from synthesized whole dual-fluorescence reporter plasmids, and then insert them into the LTB-eGFP expression plasmids.

2. Promoter extraction success

We select and extract 10 promoter sequences(7 high promoter sequences and 3 Low promoter sequences, 2 are expired because of failure in getting data from flow cytometry) from our dual-fluorescence reporter plasmids, using colony PCR with designed primers (primers sequences are shown in the picture) [3].

Sine the natural promoters are not inserted into the ‘pA-pT’ framework to maintain their real expression rate, we design to substitute the reporter system in the plasmids with the LTB-eGFP template sequence, rather than doing the same process of AI generated promoters. The below figure illustrates that we selectively clones the sequence.

3. Framework preparation success

We prepare the LTB-eGFP expression plasmids with enzyme digestion assay, where the space will be filled by AI generated promoters.
Meanwhile, we prepare the dual-fluorescence reporter plasmids with double enzyme digestion assay, and the space will be filled by LTB-eGFP template sequences.

4. Ligation success

We use ligation assay to combine our AI generated promoters with LTB-eGFP expression plasmids. The ligation result is shown below.

5. Transformation success

Ampicillin has been added to the dishes, and only the yeasts that has been successfully transformed with our plasmids can grow on it.

1. Expression

Western blot using rabbit anti-GFP antibody shows that LTB-eGFP fusion protein is successfully expressed in yeasts.

We intend to use flow cytometry to record the GFP density, while we come across a failure in getting data from the flow cytometer, which may result from too much cell fragments that block the cytometer. However, the remain result we get is in correspond with that of our western blot result.

2. Quantification

Using GAPDH as an internal control ,we quantify the expression intensity of LTB-eGFP as Intensity[LTB-eGFP]/intensity[GAPDH]. The below figure illustrates that expression driven by our Pymaker generated promoter is significantly higher than natural promoters(p = 0.016), and our Pymaker-promoters-driving expression is up to 3 times higher than natural promoters.

We then checked the quantitative gene expression levels using quantitative RT-PCR, and the results indicated that our generated promoters drive a much higher transcript accumulation than natural promoters. The result gives a strong validation that it is our generated promoters that play a fundamental role in driving a extremely high promoter sequences.

Dengue fever is wide spread over the whole world, causing over 50,000 death one year [4]. However, in spite of its tremendous influence all over the world, few effective vaccine have been invented and put on markets. EDIII2 stands for Domain III of the Envelope protein of dengue virus type 2, which is the active antigen of dengue virus type2 (DV2), functioning as the recognition protein of the virus [5]. We hope to produce the key component of the mucosal vaccine of dengue virus type 2, using our yeast expression system, reaching steps forward to utilize our promoters in real challenges. It has been a popular way to link LTB with antigen to enhance immune response, since LTB is an efficient mucosal adjuvant and carrier molecule for the generation of mucosal antibody responses and induction of systemic T-cell tolerance to linked antigens. So we use it to enhance the immune response to dengue virus type II.

We have designed future process to express LTB-EDIII2 as a fusion protein using the same linker as the fusion protein of LTB-eGFP，we have already designed the LTB-EDIII2 plasmids with empty promoter ‘pA-pT’ scaffold, and we will insert our best performing promoters into the plasmids and using Western Blot to purify and quantify the density of the product if time permits. To achieve that, we have inserted his-tag in the fusion protein to favor Western Blotting.

[1]Vaishnav, E.D., et al., The evolution, evolvability and engineering of gene regulatory DNA. Nature, 2022. 603(7901): p. 455-463.

[2]de Boer, C.G., et al., Deciphering eukaryotic gene-regulatory logic with 100 million random promoters. Nature Biotechnology, 2019. 38(1): p. 56-65.

[3]So, K.K., et al., Improving expression and assembly of difficult-to-express heterologous proteins in Saccharomyces cerevisiae by culturing at a sub-physiological temperature. Microb Cell Fact, 2023. 22(1): p. 55.

[4]Evaluation., I.o.H.M.a. Global Health Data Exchange (GHDx). [cited 2023 11 Oct]; Available from: https://vizhub.healthdata.org/gbd-results/

[5]Whitehead, S.S., et al., Prospects for a dengue virus vaccine. Nature Reviews Microbiology, 2007. 5(7): p. 518-528.