Engineering Yeast

Down-Regulation

Even though creating a bifunctional protein that can facilitate the last two steps of (+)-Nootkatone synthesis is key to the synthesis process, it is only half of the battle. Because the synthesis process utilizes intermediates of Saccharomyces cerevisiae’s MVA pathway, the innate usage of these intermediates must be downregulated in order to divert the substrate to the production of (+)-Nootkatone.

Figure 1 - MVA Pathway is Saccharomyces cerevisiae with alternate nootkatone pathway created with BioRender.com.

As seen in Figure 1 above, the primary alternate pathway is the production of squalene via ERG9. Using similar tactics to those used by Meng et al., 2020 (1), ERG9 must be downregulated by using a knock-out of its promoter to decrease the transcription and subsequently, translation of the gene. This should cause a build-up of FPP.

Though Meng et al., 2020 (1), created a fusion protein consisting of ERG20 (FPP Synthase) and CnVS (Valencene Synthase) and separately overexpressed HPO and ATR1 (step 9 in Figure 1), our team found that it would be beneficial to create a fusion protein consisting of CnVS and CYP706M1. CYP706M1 is the gene for Valencene Oxidase that has been specifically isolated from the Alaskan Yellow Cedar (Callitropsis nootkatensis), though many cytochrome P450’s are capable of catalyzing this reaction. This is also because ERG20 is natively found in in yeast cells while CnVS and CYP706M1 are not, by creating the fusion protein using these two genes, transformation, regulation and expression become much simpler.

The fusion protein must be present in high quantities in the cell as to maximize the production of nootkatone and thus, a strong promoter preceding the fusion sequence is added. Our team chose to use the lacZ promoter to have selective regulation of gene expression using galactose of lactose (2).

Figure 2 - Yeast cell graphic with budding occurring, made with BioRender.com.

Figure 3 - Plated yeast graphic.

References

1. Meng, X., Liu, H., Xu, W., Zhang, W., Wang, Z., and Liu, W. (2020) Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone. Microb. Cell Fact. 10.1186/S12934-020-1295-6

2. Barnes, C. A., Johnston, G. C., and Singer, R. A. (1991) Expression of lacZ gene fusions affects downstream transcription in yeast. Gene. 104, 47–54

Down-Regulation References Go to Top

Team:Queens Canada/metabolicEngineeringInYeast