Summary
MonChassis is a versatile and efficient biotechnological monoterpenoid production platform with the potential to replace the current wasteful production processes. To verify our concept, we focused on the production of two specific molecules, the monoterpenoids α-pinene and verbenone. We were able to produce notable amounts of our desired molecules, α-pinene (5.3 mg/l) and verbenone (12.8 mg/l) within different optimized Saccharomyces cerevisiae strains. This was successfully achieved both within the cytosolic and peroxisomal approach through our extensive and successful engineering process. Ultimately proving the viability and functionality of our system for the production of monoterpenoids in varying states of oxidation. The relatively simple introduction of the ApL3H, which led to the production of verbenone highlights the modularity and adaptability of our proposed approaches. The evident efficacy together with its versatility proves the great potential of MonChassis.
Saccharomyces cerevisiae: cytosolic α-pinene synthesis
For the cytosolic approach, we created the strains S. cerevisiae_MS_2 and S. cerevisiae_MS_3 . which contain the ScERG13, SctHMGR, AgtGPPS2, GgmFPS144 and PptAPS on two level 2 shuttle plasmids. We introduced Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-assotiated protein 9 (CRISPR-Cas9)-mediated mutations within the genomic sequences of ScERG20 and the ScROX1 (Fig. 1). Thereby we optimized these strains for α-pinene synthesis, which we analyzed using gas chromatography coupled mass spectrometry (GC-MS).
After cultivation for 48 h in liquid culture, cells of each strain were harvested and the monoterpenoids were extracted. The GC-MS measurement revealed an α-pinene concentration of 5.3 mg/l in S. cerevisiae_MS_2 and 4.5 mg/l in S. cerevisiae_MS_3, while the wildtype CEN.PK2-1C contained no α-pinene (Fig. 2). In comparison, Dusséaux et al., 2020 reported 0.66 ± 0.02 mg/l α-pinene concentration within their S. cerevisiae strain with a cytosolic pathway, which we could improve by 8-fold. Detailed information can be found here.
Saccharomyces cerevisiae: peroxisomal α-pinene synthesis
For the peroxisomal approach, we created the strains S. cerevisiae_MS_8 and S. cerevisiae_MS_9. Both strains contain all necessary genes for the mevalonate pathway inside the peroxisome, as well as the SltNPPS1 and the PptAPS. The S. cerevisiae_MS_8 strain is optimized for α-pinene, while the S. cerevisiae_MS_9 strain additionally expresses ApL3H which converts α-pinene to verbenone (Fig. 3).
After cultivation for 48 h in liquid culture, cells of each strain were harvested and the monoterpenoids were extracted. By GC-MS analysis a concentration of 3.6 mg/l α-pinene was measured in S. cerevisiae_MS_8. The sample of the S. cerevisiae_MS_9 strain, which contains the additional ApL3H produced 12.8 mg/l verbenone (Fig. 4). Further information can be found here.
In conclusion, we created several different optimized S. cerevisiae strains, and achieved a production of α-pinene and verbenone of 5.3 mg/l and 12.8 mg/l respectively. The production of verbenone through the introduction of just one easily replaceable protein shows how customizable our platform is. Using our NPP or GPP optimized strains as platforms, production of other high-value monoterpenoids like camphene or thymol are possible. Therefore, we could prove that MonChassis, our approach to a biotechnological monoterpenoid production, is not only viable but versatile.
References
Dusséaux, S., Wajn, W. T., Liu, Y., Ignea, C., & Kampranis, S. C. (2020). Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids. Proceedings of the National Academy of Sciences of the United States of America, 117(50), 31789–31799. https://doi.org/10.1073/pnas.2013968117